Reactivation and Nitrogen Removal Performance of Idle Anammox Sludge Enhanced by Rape Straw Biochar

Abstract

Low reactivation efficiency of idle anaerobic ammonia oxidation (anammox) sludge hinders its reapplication. To address this issue, rape straw biochar (RSB) was added in the reactivation process of idle anammox sludge, and its effects on the nitrogen transformation and sludge characteristics were investigated, and the mechanism of RSB to enhance the reactivation performance was explored. Results indicated that adding 5 g/L RSB for 35 days successfully reactivated anammox sludge that had been idle for 270 days. The reactivation time was reduced by 34% compared to the control without RSB. During the stable operation period, the average TN removal efficiency reached 90.6%, and the sludge exhibited higher activity. After completion of reactivation, the specific surface area, total pore volume, and average pore diameter of RSB decreased by 59.4%, 66.9%, and 55.2%, respectively, compared with that before reactivation, and the carbon–oxygen functional groups also changed. RSB not only provided a habitat for the enriched growth of nitrogen transforming functional flora but also possessed the potential to supply sufficient electron donors and acceptors for the nitrogen transforming process, which promoted the synergistic removal of nitrate by denitrification, resulting in an effective enhancement of reactivation efficiency and nitrogen removal performance. The addition of RSB provides a novel strategy to enhance the reactivation efficiency of idle anammox sludge, which is of positive significance in promoting its efficient reuse and stable operation.

1. Introduction

Due to the acceleration of urbanization and industrialization, water environment problems caused by nitrogen pollutants discharged into water bodies from living and production processes have become particularly significant [1]. Biotechnology is currently the mainstream nitrogen removal technology in wastewater treatment plants. However, the traditional nitrogen removal biotechnology based on nitrification-denitrification process is highly dependent on the organic carbon source in the wastewater, and the removal of total nitrogen (TN) is poor when treating wastewater with a low carbon source [2]. Anaerobic ammonia oxidation (anammox) technology removes ammonia nitrogen (NH₄⁺-N) by oxidizing it to gaseous nitrogen under anoxic or anaerobic conditions using nitrite (NO₂⁻-N) as the electron acceptor, providing a new strategy to address the drawbacks of the traditional biological nitrogen removal techniques [3]. The process is driven by anammox bacteria (AnAOB), which can eliminate NH₄⁺-N and NO₂⁻-N from wastewater synchronously without the need for an additional organic carbon source, while less sludge production reduces the burden of subsequent treatment [4]. Recently, the process has been used in an increasing number of application cases in wastewater purification, and is considered as a sustainable and low-cost alternative for treating wastewater containing high concentrations of nitrogen and low carbon sources, which holds important application prospects and exploration significance.

Nevertheless, AnAOB is susceptible to the negative impact of external unfavorable environmental factors during operation due to its long generation cycle, sensitivity to the growth environment, and high storage condition requirements, leading to the deterioration of anammox performance [5]. Existing studies have reported the effects of factors such as substrate concentration [6], temperature [7], heavy metal ions [8], antibiotics [9], microplastics [10], and endocrine disruptors [11] on the anammox process, and proposed a series of methods to counteract the unfavorable environmental effects. In addition to these common influencing factors, in actual wastewater treatment projects and laboratory operations, anammox sludge may face the situation of being idle when there are poor operating conditions, such as insufficient hydraulic loading, unsuitable quality of water or environmental conditions, improper operation, equipment shutdown for maintenance, or storage for a long period of time. Due to the lack of necessary nutrient inputs to the anammox sludge, the activity of AnAOB would be inhibited, resulting in its inability to carry out the anammox reaction properly, which ultimately affects the removal of nitrogen from the wastewater. Ji et al. (2024) [12] investigated the response of anammox system to long-term starvation from 31 to 40 days, and showed that the removal of total inorganic nitrogen by the system decreased to 62.16% after starvation, and the nitrogen removal of the system after nutrient restoration was gradually recovered to a level close to the pre-starvation level at a rate of 1.26% per day. Xu et al. (2020) [13] set the TN concentration of the influent water to be only about 1 mg /L and examined the effect of prolonged substrate under-supply on the anammox system, and found that the nitrogen removal dropped sharply from 16.6 × 10⁻³ kgN/(m³·d) to 8.45 × 10⁻³ kgN/(m³·d) by the 54th day of operation, and the dominant bacterial type changed from Candidatus Brocadia to Candidatus Kuenenia. These two types of bacteria are common AnAOBs, both capable of performing the coupled reaction converting NH₄⁺-N and NO₂⁻-N to N₂ via intracellular anaerobic ammonia oxidizing complexes. The low sludge activity and long recovery time of idle anammox sludge make its reuse increase the cost of wastewater treatment. Therefore, exploring a fast, efficient, and cost-effective method for reactivation of idle anammox sludge contributes to the feasibility and convenience of the anammox process in practical applications.

To enhance the activity of anammox sludge, Sui et al. (2025) [14] investigated the effect of pyrite on an ascending anaerobic sludge bed reactor subjected to starvation inhibition, and found that the addition of pyrite promoted the secretion of extracellular polymeric substances (EPS), which helped to maintain AnAOB metabolic activity, eventually shortening the delayed period of activity recovery by about 8 days and improving the nitrogen removal efficiency by 18.4%. Guo et al. (2024) [15] investigated the performance, recovery and microbial community dynamics of a single anammox process and a partial denitrification/anammox (PD/A) process after 30 days of starvation, and the results showed that the decay rate of AnAOB activity during starvation was 69.59% lower in the PD/A system than that of the single anammox system, and the PD/A system recovered its nitrogen removal performance to the pre- starvation level within 24 h. Li et al. (2023) [16] studied the resilience of biochar addition and hydraulic retention time extension on the performance of anammox sludge stored at room temperature for 68 days and found that 28 days of operation restored its nitrogen removal performance to its original level and 56 days of operation re-granularized it. The addition of biochar enhanced the secretion of EPS to 56.96 mg/gVSS and the abundance of AnAOB increased to 38.76%, making the system more resilient to the risk of environmental change. Supaporn et al. (2017) [17] observed the recovery of suspended and attached-grown anammox sludge after 3 months of starvation and found that attached-grown anammox sludge recovered its activity faster than suspended-grown anammox sludge without the addition of any exogenous restorers and that the short-term addition of acetic acid allowed suspended-grown anammox sludge to recover its activity with higher efficiency within 60 days. From the existing reports, the addition of exogenous reinforcing agents, the adjustment of operating conditions or the improvement of process running mode can have a positive effect on the recovery of idle anammox sludge performance.

Biochar, as a novel environmentally friendly material, features a simple preparation process, low raw material costs, and excellent recyclability [18]. Its use to accelerate sludge performance recovery has been demonstrated to be feasible. Adams et al. (2021) [19] investigated the effects of coconut, peach, and bamboo biochar at different dosages on the start-up of an anammox expanded granular sludge bed. They found that adding 5% coconut biochar achieved the highest start-up efficiency, requiring only 46 days, and enabled more robust nitrogen removal from wastewater. An et al. (2023) [20] demonstrated that magnetic biochar addition effectively promoted rapid anammox activation and long-term stability, reducing start-up time by 15–24 days compared to no biochar addition and by 6–26 days compared to conventional pyrolytic biochar. This performance enhancement was closely linked to microbial community structure and underlying mechanisms. Unlike idle anammox sludge, the inoculum sludge in the above studies was sourced from secondary sedimentation tanks or anaerobic tanks in wastewater treatment plants. Eventually, the goal was to successfully convert fresh anaerobic sludge, whose primary function was not anammox, into anammox sludge. In addition, since the types and preparation processes of biochar are diverse, there remains significant research potential for biochar-based strategies to enhance the activity of idle anammox sludge. The mechanisms underlying this enhancement also require further refinement. Furthermore, in current studies on reactivating idle sludge, the idle period generally was less than 100 days. Little attention has been paid to the recovery of anammox sludge performance after longer periods of inactivity.

In this study, the long-term idle anammox sludge in the laboratory was used as the recovery object, and the rape straw biochar was injected into the reactor, which was reactivated by gradually increasing the influent nitrogen load. Based on the transformation of nitrogen pollutants and the changing rules of sludge and biochar performance, the feasibility of applying rape straw biochar to enhance the activity recovery of idle anammox sludge was analyzed, and its enhancement mechanism was explored, aiming to provide a useful methodology for the efficient reuse and stable operation of idle anammox sludge.

2. Materials and Methods

2.1. Experimental Setup and Operational Conditions

Two sets of anaerobic sequencing batch reactors (ASBRs), numbered A1 and A2, were used to initiate the anammox process, and their main structures are shown in Figure 1. The raw materials of the reactors were sourced from Zhongliansuhua Technology Co., Ltd. (Jiangmen, China) and subjected to custom processing. A single ASBR with an effective volume of 2000 mL was equipped with a water inlet line led to the bottom of the reactor, a water outlet and a sludge outlet at the side wall, a gas outlet at the top, and an internal stirrer with a rotational speed of 180 r/min. The ASBR was surrounded by a black plastic film to prevent the light from affecting the experimental results. The reactor was operated for two cycles per day, each cycle included 15 min of water inlet time, 10 h of reaction time, 30 min of sedimentation time, 15 min of drainage time, and 1 h of idle time. The reactor was operated at a temperature of (35 ± 1) °C. The reactor was placed in a constant-temperature water bath. An external temperature probe provided feedback to control the heating rod’s activation and deactivation, achieving an accuracy of ±0.1 °C. Simultaneously, glass wool insulation was used for thermal retention, with measured temperature fluctuations below ±0.5 °C. Typically, AnAOB exhibits optimal growth under these temperature conditions, thereby minimizing the impact of temperature fluctuations on its activation and allowing for a focus on the effects of biochar addition.

2.2. Influent Quality and Inoculated Sludge

In this study, the used chemicals were bought from Aladdin Biochemical Technology Co., Ltd., Shanghai, China. For reducing the interference of water quality fluctuations on the experimental results, artificial wastewater was used to simulate the effluent of partial nitrification reactor to carry out the experiment, and the concentration ratio of NH₄⁺-N and NO₂⁻-N in the simulated wastewater was controlled to be about 1:1.32. The simulated effluent was based on tap water, and NH₄⁺-N and NO₂⁻-N were supplied by adding appropriate amounts of NH₄Cl and NaNO₂, respectively, and then 500 mg/L of KHCO₃, 10 mg/L of KH₂PO₄, 5.6 mg/L of CaCl₂, 300 mg/L of MgSO₄·7H₂O, 1 mL/L of trace element I and 1 mL/L of trace element II were added [21]. Trace elements I contains C₁₀H₁₆N₂O₈ 5000 mg/L and FeSO₄ 5000 mg/L. Trace elements II contains C₁₀H₁₆N₂O₈ 15000 mg/L, H₃BO₄ 14 mg/L, MnCl₂·4H₂O 990 mg/L, CuSO₄·5H₂O 250 mg/L, ZnSO₄·7H₂O 430 mg/L, NiCl₂·6H₂O 190 mg/L, Na₂MoO₄·2H₂O 220 mg/L, and Na₂SeO₄·10H₂O 210 mg/L. Artificial wastewater was freshly prepared daily to prevent the auto-oxidation of nitrites, the deterioration of other components, or microbial proliferation.

The inoculated sludge (No. A0) was collected from anammox granular sludge that had been idle in the laboratory for up to 270 days, with a mass concentration of about 9.8 g/L. During the idle period, no specific temperature control was carried out, allowing the temperature to fluctuate with seasonal changes within a range of approximately 0–35 °C. Without any nutrient supply, the sludge remained in a prolonged starved state, characterized by loose particles predominantly black and dark brown in color, accompanied by a distinct odor. This condition resulted in poor nitrogen removal efficiency from the wastewater. Preliminary experiments indicated that this idle period represents a stage of severe sludge activity decline yet remains salvageable, making it representative and valuable for engineering reference. The idle anammox sludge was rinsed with 0.1 mol/L phosphate buffer to minimize the interference of contaminants remaining on the sludge for subsequent experiments before dispensing into new ASBRs. The granular sludge was passed through an 18-mesh sieve to screen the granular sludge with a particle size of 1 mm or more. Reactors A1 and A2 were each filled with 500 mL of anammox granular sludge mixture and 1000 mL of simulated wastewater. The influent was pre-ventilated with nitrogen gas to reduce the concentration of dissolved oxygen to less than 0.5 mg/L.

2.3. Preparation of Rape Straw Biochar

Rape straw is produced in large quantities in China but has a low reuse rate. Improper handling can cause secondary pollution. Moreover, the glycoside degradation products and nitrogen-containing heterocyclic compounds within its lignocellulosic structure can form nitrogen-rich and oxygen-rich functional groups during low-temperature pyrolysis. These groups may facilitate electron shuttling and microbial adhesion, making rape straw an ideal raw material for biochar production.

Oxygen limited pyrolysis method was used to prepare rape straw biochar (RSB). The wasted rape straw was used as the raw material, crushed with a ceramic crucible and sent to the muffle furnace chamber, with the appropriate amount of nitrogen gas to exhaust air. The furnace was heated to 400 °C at an elevated rate of 9.5 °C/min and then kept at a constant temperature for 2.5 h. 100 mL of 1.0 mol/L HCl solution was added to every 10 g of pyrolysis product, and the washing was repeated until there was no suspended gray foam in the supernatant. Acid washing could remove ash and surface films, expose pore structures, and reduce pH buffering capacity, thereby preventing a sudden pH surge in the system after biochar addition. The solid was filtered out and dried at 55 °C, and the RSB with particle size less than 0.15 mm was obtained after passing through a 100-mesh sieve. The leachate analysis of RSB obtained through pyrolysis and acid washing revealed dissolved organic carbon (DOC) concentrations of only 0.1–0.35 mg/L, far below the threshold for promoting or inhibiting anammox reactions; Heavy metals Cd, Pb, and Cr were all below detection limits (ICP-MS, <0.01 mg/L). The pH ranged from 7.8 to 8.2, with alkalinity between 45 and 200 mg CaCO₃/L. These conditions align with the pH and alkalinity requirements for anammox, preventing potential inhibition caused by pH shocks.

2.4. Experimental Program

Reactor A1 was used as the control group without adding RSB, while 5 g/L of RSB was added to reactor A2. The dosage here refers to adding 5 g of RSB per 1 L of influent. Preliminary experimental results indicated that when the RSB dosage was too low, the activation effect was unsatisfactory. Conversely, when the RSB dosage was excessively high, the nitrogen removal efficiency showed little improvement. Moreover, the elevated suspended solids in the effluent led to increased SS levels, while simultaneously raising the biochar addition costs. Therefore, the optimal dosage of 5 g/L was selected for the experiments, with subsequent efforts focused on in-depth mechanism comparisons. The water exchange volume for a single operating cycle was 1000 mL. Reactivation of reactors A1 and A2 was carried out by gradient increase in influent nitrogen loading. For analytical purposes, the operational stages were categorized based on nitrogen removal performance before and after RSB addition (

Table 1. Operational stages and related parameters.

Stages	t (Days)		Influent Concentrations (mg/L)			RSB Dosages (g/L)		HRT (h)	pH	T (°C)
	A1	A2	NH4+-N	NO2−-N	TN	A1	A2
I	1~12	1~8	50	66	116	0	5	12	7.5 ± 0.2	35 ± 1
II	13~28	9~18	80	105.6	185.6	0	0	12	7.5 ± 0.2	35 ± 1
III	28~53	19~35	110	145.2	255.2	0	0	12	7.5 ± 0.2	35 ± 1
IV	54~60	36~60	110	145.2	255.2	0	0	12	7.5 ± 0.2	35 ± 1

). Stages I, II, and III were set up as startup periods, with influent NH₄⁺-N concentrations at approximately 50, 80, and 110 mg/L, respectively, and influent NO₂⁻-N concentrations at 1.32 times the corresponding influent NH₄⁺-N concentrations. Once nitrogen removal stabilized, the next operational stage was initiated by sequentially increasing the nitrogen concentrations in the influent. Stage IV was set as the stable operation period, and its influent conditions were consistent with Stage III. The transformation rules of nitrogen pollutants during the reactivation process were investigated, and faster and more efficient reactivation methods were explored based on the changes in nitrogen transformation efficiency, stoichiometric ratio and nitrogen removal rate. Combined with the changes in sludge performance, RSB structure and bacterial distribution, the mechanism of RSB to accelerate the reactivation of idle anammox sludge and enhance its nitrogen removal performance was revealed.

2.5. Analytical Methods

Nitrogen contents in the effluent were tested with reference to Analytical Methods for Water and Wastewater Monitoring (Fourth Edition). TN concentration was measured as the sum of NH₄⁺-N, NO₂⁻-N, and NO₃⁻-N concentrations. The removal efficiency of NH₄⁺-N (ARE), NO₂⁻-N (NRE), and TN (TNRE) were analyzed based on the changes in nitrogen concentration in the influent and effluent water. The volumetric nitrogen removal load (VNRL) of each stage was analyzed based on the removal of nitrogen per unit volume reactor. The values of the stoichiometric ratios ΔNO₂⁻-N/ΔNH₄⁺-N and ΔNO₃⁻-N/ΔNH₄⁺-N were analyzed based on the consumption of NO₂⁻-N (ΔNO₂⁻-N), the consumption of NH₄⁺-N (ΔNH₄⁺-N), and the production of NO₃⁻-N (ΔNO₃⁻-N). The kinetic analysis of nitrogen removal was based on a proposed first-order kinetic model as shown in Equation (1), and the effluent nitrogen mass concentration data were analyzed at 2 h intervals during the reaction period of a single operating cycle. All parameters were tested in triplicate, with results expressed as the average of repeated measurements.

$$\ln {\displaystyle \frac{{\rho }_t}{{\rho }_0}}=-\mathit{kt} +a$$

(1)

where ρ₀ and ρ_t denote the nitrogen concentration in the influent and effluent water, respectively, mg/L; t denotes the reaction time, h; k denotes the nitrogen removal rate constant, h⁻¹; and a denotes the intercept.

The morphology of sludge was observed by digital camera. Digital camera specifications: Nikon D5600 (Nikon Corporation, Tokyo, Japan), AF-S 40 mm macro lens, aperture f/8, ISO 200, dual LED softbox 5500 K, image resolution 4000 × 6000 pixels, white balance calibrated using a 50% gray card. The concentration of EPS was measured as the concentration of protein (PN) and polysaccharide (PS) combined, and the contents of PN and PS were detected by the colorimetric methods of koammas Brilliant Blue method and sulfuric acid-phenol, respectively. Heme content was determined by pyridine hemoglobin spectrophotometry. Specific anammox activity (SAA) was determined by serum vial method and calculated from the variation curve of matrix mass concentration. During VSS testing, a blank group was established to subtract the contribution of RSB to VSS, ensuring the accuracy of active biomass calculations. Surface morphology characterization of RSB was performed by SEM method using ZEISS Sigma 300 field emission scanning electron microscope (Carl Zeiss AG, Oberkochen, Germany). The specific surface area (SSA), total pore volume (TPV), and average pore diameter (APD) of RSB were analyzed by the BET method using Micromeritics ASAP 2460 (Micromeritics Instrument Corporation, Norcross, GA, USA) fully automated specific surface and porosity analyzer. The BET analysis conditions were as follows: degassing temperature 105 °C, duration 12 h, P/P₀ range 0.05–0.30. The functional group structure of RSB was analyzed by FTIR method using Thermo Scientific Nicolet 6700 Fourier Infrared Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The bacterial colony structure was analyzed by 16S rRNA high-throughput sequencing.

3. Results and Discussion

3.1. Nitrogen Removal in the Reactivation Process

3.1.1. Transformation of Nitrogen Pollutants

The transformation of nitrogen pollutants in the reactivation process of idle anammox sludge is shown in Figure 2.

The overall pattern of nitrogen transformation by A1 and A2 was similar with the increase in operation time. At the beginning of each operation stage, due to the sudden increase of nitrogen concentration in the influent water, the microorganisms in the reactor were shocked and could not metabolize these nitrogen efficiently, resulting in a sudden increase in the concentrations of NH₄⁺-N, NO₂⁻-N, and TN in the effluent water, and a sudden drop in the corresponding nitrogen removal efficiency [22]. After continuing to run for a period of time, the microorganisms adapted to the new influent conditions, and their ability to transform nitrogen gradually improved, thus the concentrations of NH₄⁺-N, NO₂⁻-N, and TN in the effluent water gradually decreased, and the corresponding nitrogen removal efficiency also rebounded [23]. For the A1 reactor, it entered the stabilization period after 53 days of operation, and the average concentrations of NH₄⁺-N, NO₂⁻-N, and TN in the effluent were 5.6, 0.4, and 32.8 mg/L, respectively, and the corresponding mean values of ARE, NRE, and TNRE were 95%, 99.7%, and 87.3%, respectively. For the A2 reactor, the values of ARE, NRE, and TNRE recovered to 96.8%, 100%, and 90.6%, respectively, by the 35th day of operation, which were 21.2%, 23%, and 31.9% higher than the above values of the A1 reactor at the same time point. The average concentrations of NH₄⁺-N, NO₂⁻-N, and TN in the effluent of the A2 reactor at stage IV were 3.8, 0.1, and 23.8 mg/L, corresponding to the average values of ARE, NRE, and TNRE of 96.6%, 99.9%, and 90.8%, respectively. So far, A1 and A2 completed the successful reactivation of idle anammox sludge, and realized the simultaneous removal of NH₄⁺-N and NO₂⁻-N from the wastewater.

The concentration of NO₃⁻-N in the effluent was slowly increasing and then gradually stabilized because of the by-product NO₃⁻-N produced by the anammox process [24]. The average values of NO₃⁻-N concentration in the effluent of A1 and A2 reactors during the stable operation period were 26.8 and 19.8 mg/L, respectively. NO₃⁻-N became the main contributor to effluent TN, but the A2 reactor exhibited a lower effluent NO₃⁻-N concentration, indicating that a richer NO₃⁻-N transformation pathway also existed in this reactor, which also created conditions for its TN removal efficiency.

From the change in VNRL values, they were also gradually increased and then stabilized, the average VNRL values of A1 and A2 reactors after entering the stabilization period were 0.3465 and 0.3598 kgN/(m³·d), respectively, and the latter was increased by 3.84% compared with the former, which showed that the A2 reactor started by RSB addition could remove more nitrogen pollutants in the same volume and reaction time. In terms of startup time, the A2 reactor took 4, 6, and 8 days less than the A1 reactor in stages I, II, and III, respectively, and the total reactivation time was 34% less than the A1 reactor. In terms of nitrogen removal efficiency, the values of TNRE at the end of the operation of the A2 reactor at stages I, II, and III increased by 1.8%, 2.4%, and 3.5%, respectively, compared to the A1 reactor. Eventually, the removal efficiency of the A2 reactor for NH₄⁺-N, NO₂⁻-N, and TN at stage IV increased by 1.6%, 0.2%, and 3.5%, respectively, compared to the A1 reactor. Therefore, the addition of RSB effectively accelerated the recovery of AnAOB and related microbial activities, leading to a significant reduction in the reactivation time while promoting the nitrogen transformation efficiency of the anammox process.

3.1.2. Changes in Stoichiometric Ratios

The anammox reaction shares a specific stoichiometric ratio and its reaction equation is shown in Equation (2). When the values of ∆NO₂⁻-N /∆NH₄⁺-N and ∆NO₃⁻-N /∆NH₄⁺-N are close to 1.32 and 0.26, respectively, it indicates that the main removal pathway of nitrogen is anammox [25]. However, in the actual biological treatment of wastewater, the pathways of nitrogen transformation are usually diverse and co-existing, which leads to a gap between the actual and theoretical values of stoichiometric ratios for anammox reactors [26].

NH₄⁺ + 1.32NO₂⁻ + 0.066HCO₃⁻ + 0.13H⁺ → 1.02N₂ + 0.26NO₃⁻ + 0.066CH₂O_0.15N_0.15 + 2.03H₂O

(2)

According to the changes in stoichiometric ratios of the idle anammox sludge reactivation process reflected in Figure 3, the values of ∆NO₂⁻-N/∆NH₄⁺-N and ∆NO₃⁻-N /∆NH₄⁺-N gradually went from fluctuating apparently to stabilizing. During the stable operation period, the mean values of ∆NO₂⁻-N/∆NH₄⁺-N for A1 and A2 reactors were 1.3897 and 1.3651, respectively, and both of them were higher than the theoretical value of 1.32, which indicated that the growth of ∆NO₂⁻-N was higher than that of ∆NH₄⁺-N in this stage. In other words, this means that the consumption of NO₂⁻-N was not only limited to the anammox pathway, but may also be transformed through other pathways. For example, NO₂⁻-N could be further oxidized to NO₃⁻-N through the nitrification pathway or reduced to N₂ through the nitrite-type denitrification (nitrite-DN) pathway, resulting in excess removal [27]. The anammox sludge in this study was in an anoxic environment in the ASBR reactor, with a low contribution of aerobic nitrification and a high potential for nitrite-DN to occur. From the values of ∆NO₃⁻-N/∆NH₄⁺-N, their average values in the stable operation period of A1 and A2 reactors were 0.2537 and 0.1850, respectively, which were lower than the theoretical value of 0.26, which indicated that the growth rate of ∆NO₃⁻-N in this stage was lower than that of ∆NH₄⁺-N, confirming that there were other nitrogen transformation pathways coexisting in the reactors in addition to anammox. The reuse of NO₃⁻-N or the excess removal of NH₄⁺-N resulted in a significant deviation of the actual value of ∆NO₃⁻-N/∆NH₄⁺-N from the theoretical value. For example, NO₃⁻-N can be reduced to NO₂⁻-N via the short-range denitrification (short-DN) pathway or further reduced to N₂ via the nitrate-type denitrification (nitrate-DN) pathway for additional removal [28]. NO₂⁻-N produced by the short-DN pathway can be removed by further anammox with the remaining NH₄⁺-N in the wastewater [29]. The coexistence of these nitrogen transformation pathways not only further reduced the NO₃⁻-N content in the effluent, but also provided more reaction substrates for the anammox process, thus promoting the nitrogen removal efficiency. Comparison revealed that the changes in stoichiometric ratios of the A1 and A2 reactors were significantly different, indicating that the addition of RSB in the reactivation process changed the original nitrogen transformation pathways in the reactor, which made the enhanced removal of nitrogen possible.

3.1.3. Changes in Nitrogen Removal Rates

The original sludge A0 and the sludge in the A1 and A2 reactors at the 35th day of operation were selected for the kinetic analysis of NH₄⁺-N and NO₂⁻-N removal, and the results are shown in Figure 4. The reason for selecting this time point is that the A2 reactor has already completed startup at this time, so as a control, the differences in the rates of nitrogen removal between the A1 and A2 reactors under the same startup time and influent nitrogen concentration conditions can be compared.

It is usually considered that the rate of nitrogen removal can be reflected by the k value of the kinetic model, and the higher the k value, the higher the rate of nitrogen removal [30]. The coefficient of determination R² of the fitted curves reached a maximum of 0.9924 and a minimum of 0.9667, indicating that the analytical results were of high accuracy. From the removal of NH₄⁺-N and NO₂⁻-N, the original sludge (A0) was dormant or partially dead due to long-term idling without nutrient supply, and the AnAOB was of low activity [31], so the values of its rate constants k for the removal of NH₄⁺-N and NO₂⁻-N were at a low level of only 0.0128 h⁻¹, 0.0138 h⁻¹, respectively. The sludge of A1 reactor was in the late stage II operation, and the anammox sludge had recovered part of its activity, and the k values corresponding to NH₄⁺-N and NO₂⁻-N removal increased to 0.1443 h⁻¹ and 0.1521 h⁻¹, respectively, and their removal rates increased about 10 times compared with those of A0, so the values of ARE and NRE were significantly larger. The recovery rate of anammox sludge activity was further increased compared to A1 due to the addition of RSB. The k values corresponding to NH₄⁺-N and NO₂⁻-N removal reached the maximum of 0.3383 h⁻¹ and 0.7404 h⁻¹, respectively, which corresponded to the nitrogen removal rates of 25.4 and 52.7 times higher than those of A0, and the anammox sludge activity was greatly enhanced, and the values of ARE, NRE values also reached the maximum. The removal rates of NH₄⁺-N and NO₂⁻-N in the A2 reactor were 2.3 and 4.9 times higher than those in the A1 reactor on the 35th day of operation, and the latter had a greater increase in the removal rate. This shows that the addition of RSB can accelerate the removal of nitrogen in the ASBR reactor, thus enhancing the recovery effect of its nitrogen removal performance.

It should be noted that in future studies of continuous-flow steady-state operation, analyses can also be conducted based on the Monod model or second-order kinetic models. By integrating biomass growth and decay rates with substrate inhibition functions, higher-order model validation can be performed using platforms such as AQUASIM or COMSOL to better reflect the kinetic characteristics of microbial reactions.

3.2. Sludge Properties in the Reactivation Process

3.2.1. Changes in Morphology

The morphology of sludge exerts an important influence on nitrogen removal. Red anammox granular sludge had been reported in successfully operated anammox systems, and its morphology and color were related to sludge activity [32]. Figure 5 demonstrates the changes in morphology of sludge from A0 and stabilized sludge operating in A1 and A2 reactors. The original sludge A0 was dominated by black-brown particles, mixed with a small amount of dark red particles, with obvious shrinkage on the surface of the particles and a large proportion of small-sized particles. The sludge in reactor A1 changed to reddish brown in color, with a reduced number of small particles and well-defined particles. The sludge particles in reactor A2 further increased in size and number, and the contact between particles was more compact. The ASBR reactor restarted with the addition of RSB formed a more distinct and stable particle structure, with a higher degree of microbial aggregation and better growth conditions, resulting in superior nitrogen removal.

3.2.2. Comparison of Heme Content

Heme is an iron-containing porphyrin compound, which is widely found in AnAOB bacteria and has important physiological functions. In the anammox process, heme can participate in the electron transfer process, helping to transfer the electrons from ammonia to nitrite, thus promoting the reaction [32]. Heme can act as a cofactor for enzymes and play an important role in catalyzing reactions such as ammonia oxidation and nitrite reduction [33]. Heme can also participate in the regulation of the cellular respiratory chain, helping AnAOB to maintain normal energy metabolism under hypoxic conditions. The color of granular sludge is also closely related to the level of heme content in the bacterium, and higher levels of heme will make the granular sludge show dark red or brick red.

Figure 6a shows the comparative results of heme content in sludge from A0 and A1 and A2 reactors after stable operation stabilization. The heme content in the original sludge was at a low level of 0.46 μmol/gVSS, which showed that the heme content in the anammox sludge was greatly reduced because of the long-term idling, and the activity of AnAOB was decreased, and its brick-red color could not be fully revealed. The heme content of the sludge in the A1 reactor increased to 2.59 μmol/gVSS, which was 4.63 times higher than that of the original sludge, indicating that the sludge heme content could be increased by gradient elevation of influent nitrogen load to obtain good nitrogen removal effect, but this process took a relatively long time. The heme content of the sludge in the A2 reactor increased to 3.18 μmol/gVSS, which was 5.91 and 1.23 times higher than that of the original sludge and the sludge in the A1 reactor, respectively. Hence, the sludge in the RSB-added A2 reactor possessed a higher heme content, and its particle fullness, quantity, and color performance were better after the completion of reactivation. The increased heme content could promote the expression of the key functional genes in AnAOB, which prompted the long-idle and poorly active anammox sludge to return to its optimal state rapidly [34], and the advantages in startup time and nitrogen removal efficiency was even more significant.

3.2.3. Comparison of EPS Content

EPS plays an important role in the formation and maintenance of structural integrity of anammox granular sludge, and the main components include PN and PS. The content of EPS, PN, PS, and the variation in PN/PS values in the sludge before and after restarting are shown in Figure 6b. The contents of EPS, PN, and PS in the original sludge were at a low level of only 23.6, 10.6, and 13 mg/gVSS, respectively, corresponding to a value of PN/PS of 0.82. After the completion of reactivation, the contents of EPS, PN, and PS in the sludge in the A1 reactor increased significantly to 207.5, 136.4, and 71.1 mg/gVSS, respectively, and the corresponding PN/PS values increased to 1.92. The EPS, PN, and PS content in the sludge in reactor A2 increased further to 244.8, 171.6, and 73.2 mg/gVSS, respectively. The PN content in the sludge in reactor A2 increased even more than that in reactor A1, resulting in an increase in the corresponding PN/PS to 2.34. EPS is the microenvironment for microorganisms to carry out their life activities in the waters, which has an important influence on the formation of particles and biofilms [35]. Proteins in EPS have high hydrophobicity, while polysaccharides have strong hydrophilicity [36]. The larger the value of PN/PS, the higher the hydrophobicity of the sludge and the stronger the cohesion of microorganisms with each other, and the stability of the formed particles or biofilm structure is stronger as well [37]. The highest content of EPS and the largest value of PN/PS in the sludge in the A2 reactor provided a favorable basis for obtaining better particle morphology and superior nitrogen removal.

3.2.4. Comparison of SAA

SAA is a key indicator for assessing the performance of anammox granular sludge, and its value can reflect the nitrogen removal capacity possessed by the sludge [38]. The changes in the SAA values of the original sludge and the sludge after successful reactivation are shown in Figure 6c. The SAA value of the original sludge was only 12.4 mgN/(gVSS·d), whereas it increased to 151.7 mgN/(gVSS·d) for the sludge in the A1 reactor, which was 11.2 times higher than that before startup. The SAA value of the sludge in the A2 reactor reached 179.2 mgN/(gVSS·d), which was 13.5 and 0.18 times higher compared with that of the original sludge and the sludge in A1 reactor, respectively. It can be seen that the gradient increase in influent nitrogen loading can slowly recover the anammox activity of the idle sludge, while the addition of RSB can accelerate the recovery of its activity and make it reach higher values more quickly.

3.3. Changes in Biochar Structure

3.3.1. SEM Analysis

The SEM analysis results of the original RSB and the RSB (RSB-A2) after addition to the A2 reactor after operation and stabilization are shown in Figure 7. RSB was a biochar material with a rough surface covered with a large number of pores of different sizes, which had been proven to have a good adsorption performance for pollutants in the waters in previous studies [39,40,41]. The pore structure of RSB-A2 changed, with its surface becoming relatively smooth due to being tightly covered by sludge, and the number of pores significantly reduced. RSB possesses a well-developed pore structure, which can provide more abundant space for microorganisms to attach and grow during the reactivation process of idle anammox sludge [40], forming a stable bacterial-biochar symbiotic structure.

3.3.2. BET Analysis

Table 2. Comparison of BET analysis results between RSB and RSB-A2.

Biochars	SSA (m2/g)	TPV (cm3/g)	APD (nm)
RSB	45.192	0.136	7.281
RSB-A2	18.335	0.045	3.259

reflects the results of BET analysis of RSB before and after its addition to the A2 reactor. The values of SSA, TPV, and APD of the pristine RSB were 45.192 m²/g, 0.136 cm³/g and 7.281 nm, respectively, which showed a large specific surface area and ample space to provide high quality attachment sites for microorganisms. After completing the reactivation, the values of SSA, TPV, and APD of RSB-A2 decreased to 18.335 m²/g, 0.045 cm³/g and 3.259 nm, which were reduced by 59.4%, 66.9%, and 55.2%, respectively, compared with the above values of RSB. This indicates that a part of the microorganisms within the reaction system transferred and enriched to RSB, forming a biofilm on its surface, and thus the pore structure was reduced, which was consistent with the analytical results of SEM. The structural characteristics of RSB itself make it can be used as an excellent carrier for microorganisms and an efficient adsorbent for pollutants, and its incorporation can provide more attachment sites and more adequate nutrients for the nitrogen transformation functional flora [42], prompting the A2 reactor to obtain a better nitrogen removal effect in a shorter startup time.

3.3.3. FTIR Analysis

Figure 8 reflects the FTIR analysis results of RSB and RSB-A2. The numerous obvious absorption peaks on the curves showed the rich distribution of functional groups on RSB. The absorption peaks at 3465, 2929, 1624, 1435, 1095, and 796 cm⁻¹ on RSB came from the stretching vibration of —OH, the stretching vibration of C—H, the stretching vibration of C=C (C=O), the bending vibration of —CH₃, the stretching vibration of C—O—C, and the bending vibration of C—H, while the positions of these absorption peaks on RSB-A2 were shifted to 3471, 2954, 1633, 1375, 1099, and 793 cm⁻¹, respectively.

From the above analysis, it can be seen that the functional groups on RSB, such as, —OH, C—H, C=O, C=C, —CH₃, C—O—C, were involved in the reactivation of idle anammox sludge in the A2 reactor [43]. The functional groups on the surface of RSB have redox properties, e.g., functional groups —OH, C—H, —CH₃ can provide electrons for the anammox reaction, and functional groups C=O, C=C possess the ability to receive electrons [44]. Therefore, RSB was highly likely to function as electron shuttles (ES) to facilitate extracellular electron transfer in microorganisms. In this study, RSB demonstrated potential as an insoluble ES, undergoing cyclic oxidation and reduction within the ASBR reaction system. This process provided ample electron donors and acceptors for nitrogen transformation, promoted electron transfer, and enhanced anammox metabolic activity [45], thereby improving the restart performance of idle anammox sludge. Further studies may employ cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) to quantitatively determine the redox potential and electron transfer impedance of RSB, thereby validating its electron shuttle capability.

3.4. Analysis of Bacterial Flora Structure

Figure 9 shows the major genus level flora distribution of microorganisms within the sludge before and after anammox reactivation. It can be seen that the genera nor-ank_o__SBR1031 and norank_c__OLB14, which belong to Chloroflexi, had the largest relative abundance in each sludge. Their combined relative abundance in the A2 reactor was 33.85%, which was 19.32% and 2.4% higher than that of the original sludge and the sludge in the A1 reactor, respectively, and their growth was promoted by the addition of RSB. The intertwining of filamentous structures between these two types of bacteria can provide skeleton and support for granular sludge, which was beneficial to maintain the sludge morphology.

Next in relative abundance were the genera Candidatus Brocadia and Candidatus Kuenenia belonging to Planctomycetes, which are typical AnAOBs with anammox function and simultaneous removal of NH₄⁺-N and NO₂⁻-N. They represented a low percentage of raw sludge, only 7.58% and 1.26%, respectively, and thus their anammox performance was poor. However, their relative abundance share in the A1 reactor increased to 15.91% and 3.48%, respectively, creating conditions for anammox performance in this reactor. After completing the reactivation, the relative abundance of these two genera in the A2 reactor had further increased to a total of 23.49%, which was 14.65% and 4.1% higher than that of the original sludge and the sludge in the A1 reactor, respectively, showing that the addition of RSB promoted the growth of the AnAOBs in the sludge and thus possessed a better nitrogen removal performance.

The genera Denitratisoma and Pseudomonas, which belong to the Proteobacteria, were also detected in the sludge. They are typical heterotrophic denitrifying bacteria with denitrification function, capable of utilizing organic matter produced by the metabolism or death of microorganisms in sludge as a carbon source for nitrate denitrification (Nitrate DN) and nitrite denitrification (Nitrite DN), which reduced NO₃⁻-N and NO₂⁻-N to N₂. The relative abundance of these two genera in the raw sludge totaled 5.24%, which was higher compared to A1 and A2, indicating that the contribution of denitrification in the nitrogen transformation process within the long-term idle anammox sludge system deserved to be scrutinized. Due to the lack of exogenous nutrient input, some of the microorganisms within the original sludge died, and the organic matter produced by the lysis of the dead organisms was in turn utilized to sustain other microorganisms. The addition of RSB promoted the transfer of electrons, which played a positive role in these denitrification processes.

In addition, a small number of Thauera spp. with partial denitrification (Partial DN) functions were detected, which can utilize organic matter to reduce NO₃⁻-N to NO₂⁻-N under anoxic or anaerobic conditions. The generated NO₂⁻-N can be recycled again by the anammox reaction or further reduced to N₂, reducing the nitrogen content of the effluent. The relative abundance of the genus Thauera in the pristine sludge was only 1.21%, and the partial denitrification effect was relatively weak. The relative abundance of the genus Thauera in the sludge of A1 and A2 reactors was 2.17% and 2.29%, which was 0.96% and 1.08% higher than that of the original sludge, respectively. It can be seen that the addition of RSB increased the contribution of partial denitrification effect in nitrogen transformation. In addition, the genera Geobacter and Ignavibacteriae belonging to Proteobacteria were detected and their relative abundance increased after RSB addition. Symbiosis of these genera was often found during the enrichment of AnAOB, and they may participate in the nitrogen cycle by degrading or synthesizing extracellular substances, secreting secondary metabolites and interacting with AnAOB, thus increasing the nitrogen removal efficiency.

3.5. Enhancement Mechanism Analysis

The enhancement mechanism of RSB on the reactivation performance of idle anammox sludge is shown in Figure 10. When idle anammox sludge was reactivated by traditional methods, there were two difficulties, one was that the inoculated sludge itself was of low activity, and the activity of AnAOB recovered slowly, so the reactivation was usually time-consuming and inefficient [46]; and the other was that a single anammox reaction would produce about 11% of by-products NO₃⁻-N, which made it difficult to break through the theoretical removal of TN to 90% [47]. After the addition of RSB, relying on its large specific surface area and porous structure, it provided a more abundant and more interference-resistant habitat for AnAOB and denitrifying bacteria, which was conducive to the synergistic growth of denitrifying bacteria to achieve the sustained consumption and reuse of NO₃⁻-N and to reduce the content of nitrogen byproducts in wastewater [48]. Moreover, relying on its abundant carbon-oxygen functional group structure as an electron acceptor or donor, it was highly likely to participate in the anammox and denitrification electron transfer process, which reduced the dependence of anammox on NO₂⁻-N in influent water and denitrification on carbon source, thus enhancing the metabolic efficiencies of the AnAOB and denitrification flora [49,50], and enabling the A2 reactor to obtain higher reactivation efficiencies and nitrogen removal performance.

4. Conclusions

Rape straw biochar (RSB) added to the ASBR reactor not only shortened the restart time of idle anammox sludge to 35 days but also enhanced nitrogen removal performance, achieving an average TN removal efficiency exceeding 90%. The approach of adding RSB to accelerate the reactivation of idle anammox sludge exhibited good feasibility. After restarting, the granular sludge in reactors supplemented with RSB exhibited superior morphology, heme content, extracellular polymer content, and specific anammox activity compared to untreated sludge, enabling faster conversion of NH₄⁺-N and NO₂⁻-N in wastewater. Beyond anammox reactions, significant denitrification was observed in RSB-amended reactors, enabling more efficient removal of residual NO₃⁻-N and promoting more complete process completion. RSB possessed the potential to simultaneously function as both a carrier and an electron shuttle within the ASBR reactor. Its abundant pore structure facilitated the enrichment and growth of diverse nitrogen-transforming functional bacteria. The rich carbon-oxygen functional group structure may supplement electron donors/acceptors for nitrogen removal reactions, thereby providing conditions to accelerate the reactivation of idle anammox sludge and enhance nitrogen removal performance.

This study demonstrated that RSB could provide a low-cost shortcut for reactivating idle anammox sludge. However, the current approach was limited to laboratory-scale ASBRs, artificial water distribution, and single biochar type and dosage. Its applicability to toxic wastewater, pilot-scale flow conditions, and long-term operational stability requires further validation. Comparing the effects of RSB with non-carbonized straw and commercial carbon materials is also essential. Future work could explore coupling multi-source biochar with electric fields or short-path denitrification to further shorten the reactivation cycle and enhance nitrogen removal efficiency. Furthermore, the pathways for reusing or ultimately disposing of biochar after the process operation concludes also require further exploration. By continuously refining research, expanding the application prospects of RSB, and enhancing its commercial potential.