Biochar enhanced the nitrogen removal performance of heterotrophic nitrication-aerobic denitrication strain pseudomonas uorescent sp. Z03 for insucient carbon source wastewater under low temperature

In this study, biochar BC400 and BC700 were prepared, characterized and coupled with heterotrophic nitrication-aerobic denitrication (HNAD) strain Z03 for nitrogen removal experiments. The characterization results showed that BC700 has a higher specic surface area and a more complex multilayered pore structure, with increased aromatic condensation and higher crystallinity. BC400 and BC700 both have good redox activity, while BC400 has stronger electron donor capacities and BC700 owns better electron transfer properties. In addition, both BC400 and BC700 contain relatively high levels of dissolved organic carbon (DOC), reaching at 62.95 and 51.617mg/g respectively. BC400/BC700 coupled with strain Z03 can signicantly improve the NH 4+ -N removal performance of low-temperature and low C/N wastewater compared with the control group. At a dosage of 4.0 g/L, the removal rate of NH 4+ -N reached to 95.16% (BC400 + Z03) and 84.37% (BC700 + Z03) within 72h, respectively. Higher than the sum of adsorption by BC400/BC700 (16.19%/18.85%) and microbial degradation (41.03%). Besides, the BC400 + BC700 + Z03 NH 4+ -N removal systems provide higher nitrogen removal eciencies than BC400/BC700 + Z03 nitrogen removal systems. When the dosage (BC400 + BC700, mass ratio 5:1) reaches 3.0g/L, it can achieve more than 90% NH 4+ -N removal rate within 48h. The reasons for the promotion of biochar on microbial denitrication were analyzed as follows: 1) DOC can provide an additional carbon source for microorganisms; 2) biochar, as a pH buffer, can neutralize the acidity due to nitrication; 3) BC400 and BC700, as materials with good redox activity, may play a role in promoting the activity of electron transfer system and enzyme activity.


Introduction
Nitrogen pollution in China's water bodies has been a long-standing problem, and human activities have fundamentally altered the nitrogen cycle (Galloway et al., 2008).The current situation of nitrogen pollution in China is directly related to the rapid development of agriculture and various manufacturing industries.
China's current nitrogen fertilizer consumption accounts for about 32% of the world's total nitrogen fertilizer consumption, and the massive use of nitrogen fertilizer and unreasonable irrigation methods result in urea, NH 4 + -N and NO 3 --N entering surface water bodies through surface runoff and other means.
In addition, livestock waste water, domestic sewage and industrial wastewater are also the main sources of nitrogen in water bodies (Liu and Wang, 2019). Agricultural systems in China account for 59% of current surface water nitrogen emissions, while the other 39% comes mainly from domestic waste (13% from urban sewage, 8% from rural sewage and 18% from organic waste) and the remaining 2% from industrial waste. Excessive nitrogen content in water bodies is one of the main causes of eutrophication and marine red tides, and the nitrite accumulated during the nitrogen cycle is toxic and carcinogenic to humans. (Liu and Wang, 2019; C. Q. Yu et al., 2019), Nitrogen pollution in water bodies also increases the risk of methemoglobinemia (Huang et al., 2012), bladder cancer and lung cancer in humans (C. . The studies (C. Q. Yu et al., 2019;Zhang et al., 2015) points out that if the total nitrogen concentration in the e uent of domestic wastewater treatment plants in China is reduced from 15mg/L to 5mg/L, the nitrogen discharge from water bodies can be reduced by 5%-10%.
Although the research on biological denitri cation has gradually become systematic, many urban domestic sewage treatment plants still have di culties in meeting the increasingly stringent e uent standards due to the interference of various problems in practical application. Microbial degradation is often limited in practice by slow cell growth, stimulation of substrate concentration and environmental conditions (Chen et al., 2016;Gao et al., 2011;Yuan et al., 2011). Both practical results and existing studies have shown that low C/N and low temperature conditions can signi cantly inhibit the biological nitrogen removal effect (Chen et al., 2019;Li et al., 2015;C. Wang et al., 2019). Under the strategy of "energy saving and emission reduction", the concentration of organic matter in the in uent water of urban sewage treatment plants is gradually reduced, and the problem of insu cient organic carbon sources available for microorganisms is often faced in the process of nitrogen removal. The electron donor is the rate-limiting substrate that controls substrate utilization and biological growth kinetics. Denitri cation process is inhibited under the condition of low C/N, which easily leads to the production of carcinogenic nitrite and affects the total nitrogen concentration in the e uent. Survey data shows that currently 10% of China's wastewater treatment plants require additional carbon sources to meet the demand for denitri cation, and about 50% of urban wastewater treatment plants in southern areas with high rainfall, such as the Taihu Lake basin (Jie and He, 2018). Organic electron donor is the most commonly exogenous electron donor in denitri cation process. However, the effect of electron donor dosing is easily affected by the uctuation of in uent volume and water quality, and the unsuitable dosing is likely to lead to the increase of sludge and nitrate accumulation, so the selection of carbon source type and accurate dosing is one of the main problems facing biological denitri cation at present.
Biochar is a stable carbon-rich by-product synthesized by pyrolysis and carbonization of primary biomass of plants and animals, which is itself an environmentally friendly material for reuse of waste resources.
Its practical applications include carbon sequestration, soil fertility improvement and pollution remediation, etc. (Ahmad et al., 2014a). With the increasing research on biochar, the role of biochar in the eld of pollutant removal has changed from traditional adsorbent to diversi ed occurrence, such as being a microbial immobilization carrier Yang et al., 2020), fertilizer slow release carrier (Li et al., 2020) and electron doner(X. . As a carbon rich material with low toxicity and high porosity, biochar can promote microbial lm growth, enhance microbial adaptation to the environment and provide carbon sources in practical applications (Sima et al., 2017), which can effectively promote biological denitri cation in combination with microbial immobilization and coupled material dosing (Lou et al., 2019). In addition, biochar has a variety of functional groups, and the resulting redox properties enable it to participate in redox reactions in the environment in different ways, thus accelerating its removal of pollutants. Previous studies have shown that differences in carbonized temperatures lead to different pore structures and redox properties of biochar, which in turn lead to different adsorption degradation and mediating mechanisms in the removal of pollutants(X. . In the redox system, the electron donor capacity and electron-mediated ability of biochar can promote the electron transfer and enzymatic activity of the system, thus enhancing the biological denitri cation (Wu et al., 2019a). However, there are few studies on the coupling of biochar with microbial denitri cation at different carbonized temperatures, thus the promotion mechanism of biochar in biological denitri cation process should be investigated in depth.
With these considerations, the aims of this study were thus to: (1) analysis the physicochemical properties of chestnut shell biochar at different carbonization temperatures,(2) investigate the effect of biochar coupled HNAD strain Z03 on the treatment of low C/N wastewater (3) explore the mechanism of biochar-enhanced microbial ammonia nitrogen removal e ciency.

Materials source
The HNAD strain pseudomonas uorescent sp. Z03 was isolated from piggery sewage treatment system in Qufu city, Shandong province, China. The chestnut shells were washed repeatedly with distilled water, drained and dried, crushed and ground, and then passed through a 100-mesh nylon sieve to get the powder.

Preparation and characterization of biochar
The biochar was prepared by oxygen-limited and temperature-controlled carbonization method. An appropriate amount of chestnut shell powder was taken in a porcelain boat and placed in a vacuum tube furnace. After the vacuum pump was pumped to negative pressure, the vacuum tube furnace was heated to 400 ℃ and 700 ℃ at a rate of 10 ℃/min, and then the tube furnace was closed after 1 h of carboning time. The biochar yield was about 32%.
The experiments were carried out to analyze the elemental composition of the prepared BC400 and BC700 by elemental analyzer. The speci c surface area sizes of BC400 and BC700 were determined by speci c surface area analyzer (BET). The shape, surface morphology and material dimensions of BC400 and BC700 were characterized by thermal eld emission scanning electron microscopy (SEM). The infrared absorption spectra of BC400 and BC700 were obtained by Fourier transform infrared spectroscopy (FTIR) in the spectral range of 400 to 4000 cm − 1 . The characteristic wave numbers of speci c bands of the spectra were analyzed to obtain the species of the main functional groups on the surface of the materials to infer the NH4+-N removal mechanism. The zeta potentials of the materials were measured at different pH values to obtain the zero sites, which provide a basis for the study of the adsorption properties and the direction of material modi cation. The structural characteristics and graphitization degree of the synthesized materials were characterized by Raman spectroscopy (RS) to investigate the electrical conductivity of the materials. Analyze the structural changes and crystalline phase structure of the materials by X-Ray diffractomer (XRD). DOC contained in biochar was determined by total organic carbon (TOC) analyzer with reference to sequencing extraction method (Sima et al., 2017).

Con guration of wastewater
The purpose of this study is to investigate the nitrogen removal performance of low C/N wastewater under low temperature conditions. Combined with the actual pollutant concentrations of urban domestic wastewater and industrial wastewater, the initial COD of this experiment is 250 mg/L, the initial NH 4 + concentration is 50 mg/L, and the initial TP is 2.5 mg/L of arti cially simulate and domestic wastewater.

Batch experiments
To investigate the nitrogen removal effect of different biochar coupled strain Z03, the effects of BC400 / BC700 dosage (0.5-4.0 g/L), BC400 and BC700 mixing ratio (1:1-5:1) and BC400 + BC700 at different pH (5.5-10.5) on the nitrogen removal effect of strain Z03 were investigated. Except for the pH experiments, each group was set up in three parallel in 250mL conical asks with 95mL of nitrogenous wastewater at an initial pH of 7.0 and 5mL of Z03 bacterial solution. At the same time, a control group with only the corresponding amount of biochar and Z03 bacterial solution was set up in each group. The above mixed solution was incubated at 120rpm in a full temperature shaker at 10℃, and the OD600 value of the bacterial solution was measured at 12h, 24h, 36h, 48h, 60h and 72h. The content of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in the sample was measured by passing the water sample through 0.45µm lter membrane with a syringe, and the pH value of the water sample was also measured.

Analytical Methods
PH was measured by pH meter (HQ11d, HACH). The OD 600 of the isolate was measured by a double beam spectrophotometer (UV-T9, Puxi, Beijing) at 600 nm. Besides, culture samples ltered by passing the sample through a 0.45 µm lter for chemical analysis. The ammonium concentration was determined by the Nessler's reagent spectrophotometry method, nitrate concentration was determined by the method of phenol disulfonic acid photometry and nitrite concentration was measured according to N-(1naphthalene)-diaminnoethane photometry method.

Biochar characterization
The SEM images and physicochemical properties of BC400 and BC700 are shown in Fig. S1 and Table 1, respectively. Biochar is a carbon rich material with pore size structure and its properties are related to the carbonization temperature (Ahmad et al., 2014b;Guerrero et al., 2008). During the pyrolysis process, the elemental C content of biochar increased with the increasing carbonization temperature. Previous study showed that the DOC content of biochar prepared from different biomasses varies greatly, and the DOC content of biochar decreases as the carbonization temperature increases . BC400 and BC700 contained relatively high DOC contents of 62.95 and 51.617 mg/g, respectively. The pH pzc of BC400 was 3.1 and that of BC700 was 4.3 obtained by zeta potential test (Fig.S2), which may be due to the fact that BC400 contains more oxygenated acidic functional groups (Y. Yu et al., 2019). The pH pzc of both BC400 and BC700 were lower than 4.5, indicating that the biochar was negatively charged in the experimental pH range (5.5-9.5) and could produce electrostatic adsorption of cations. Both BC400 and BC700 have obvious multi-stage pore structure and the pore size type is mainly microporous, but BC700 has signi cantly more pore size structure and the speci c surface area reaches 155.096 m 2 /g. The nitrogen adsorption-desorption isotherms and pore size distributions of BC400 and BC700 are shown in Fig. 1a and Fig. 1b. The adsorption-desorption isotherms of BC400 and BC700 were type III and type I, respectively. The adsorption capacity of BC400 increases gradually with the increase of the partial pressure of nitrogen (P/P 0 ), and there is no obvious hysteresis loop in the curve, which indicates that it contains multi-level pore structure, but the number of pore size is small. The adsorption saturation of BC700 was basically reached in the low-pressure region, indicating that it contains a large number of micropores (< 2 nm). The adsorption amount increased slightly at P/P 0 of 0.4-0.8 and there was an H4-type hysteresis loop, indicating the presence of mesopores (2 ~ 50 nm). The rich pore size structure and high speci c surface area of biochar provide the feasibility of adsorption and carrier.
The FTIR results of BC400 and BC700 are shown in Fig. 1c. It can be seen that BC400 and BC700 contain various types of functional groups, such as hydroxyl group (-OH, 3440 ~ 3340 cm -1 ), aliphatic C-H bond (2960 ~ 2890 cm -1 ), benzene ring skeleton vibration C = C (1650 ~ 1450 cm -1 ), carboxyl group ( -COOH, 720 cm -1 and 1380 cm -1 ), etc. The series of absorption peaks of C = O from 1850 to 1650 cm -1 indicated the presence of ketone and quinone groups, and the stretching vibration of C-O and the presence of OH bonds indicated the presence of alcohol and phenol groups (S. . FTIR analysis showed that BC400 contains more oxygen-containing functional groups, which indicated that it has the potential to participate in redox reactions (X. ; BC700 contains more conjugated π-structures, which allows electron transfer through the off-domain formed by the hybridization of large π-bond orbitals on thick benzene, thus providing better electron transfer performance (Klüpfel et al., 2014;Wu et al., 2019a;. In order to investigate the structural changes and crystal composition of biochar at different carbonization temperatures, XRD analysis ( Fig. 1d) was performed on BC400 and BC700, respectively.
Results showed two broad and slow amorphous diffraction peaks at 2θ of 24° and 43°, which are graphite microcrystalline d 002 and d 101 diffraction peaks, respectively (Wang et al., 2016). Since the biochar used in the experiment was treated with water washing and almost free of salts, no obvious sharp diffraction peaks were seen. The XRD pattern shows that as the carbonized temperature increases, the peak intensity of BC700 increases compared to BC400, the peak area increases, and the crystallinity increases.
Raman spectroscopy is an effective means to characterize the bond structure of carbon nanomaterials.
the characteristic peaks located at about 1350 cm -1 and 1530 cm -1 in the Raman spectrum correspond to the D and G peaks of the material, which characterize the lattice defects and graphitization of carbon atoms, respectively. The intensity ratio of the D and G peaks, i.e., the value of I D /I G , re ects the degree of defects and graphitization of the carbon nanomaterials structure, and the value is related to The ratio of sp 3 hybridized carbon to sp 2 hybridized carbon (Sima et al., 2017). From the Raman spectrograms of BC400 and BC700 ( Fig. 1e and Fig. 1f), it can be seen that the I D /I G of BC400 (2.51) is higher than that of BC700 (1.59), which indicated that as the carbonized temperature increases, the graphitization of the material increases, the structural defects decrease, and the overall structure becomes more ordered. Feng et al. (Feng et al., 2002) found that the basic units of biochar structure were randomly oriented at temperatures below 700°C, but in the temperature range of 700 to 1500°C they were rearranged and structural defects were removed, resulting in a more ordered structure of the carbonaceous solids. Marsh (Marsh, 1987) found that during biomass heating, the decomposition of ether-type groups led to a decrease in the distance between aromatic layers, and thus the structure of biochar at higher carbonized temperatures became more ordered. Figure 1 Nitrogen adsorption-desorption isotherms of BC400 and BC700 (a); pore size distribution of BC400 and BC700(b); FTIR spectrograms of BC400 and BC700(c); XRD spectrograms of BC400 and BC700 (d); Raman spectrograms of BC400 (e) and BC700 (f).
3.2 Effect of BC400/BC700 on HNAD process 3.2.1 Effect of BC400/BC700 dosage The changes of NH 4 + -N concentration, pH, NO 3 --N and NO 2 --N concentration of BC400/BC700 + Z03 system with different biochar dosages are shown in Fig. 2a and Fig. 2b. The removal effect of NH 4 + -N in both BC400 + Z03 and BC700 + Z03 systems increased with the increase of biochar dosage and reached the best NH 4 + -N removal e ciency at the dosage of 4.0 g/L. When the dosage was greater than 3.0g/L, the concentration of NH 4 + -N in both systems could be reduced to less than 10mg/L within 72h, but the NH 4 + -N removal e ciency of BC400 + Z03 system was slightly better than that of BC700 + Z03 system, and when the dosage was 4.0g/L, the concentration of ammonia nitrogen in both systems could be reduced to 2.42mg/L and 7.81mg/L respectively. In the BC700 + Z03 system, when the BC700 dosage was lower than 3.0 g/L, the NH 4 + -N concentration in each group showed a small rebound from 12 h to 24 h, and then decreased steadily. The adsorption experiments results (Fig.S3) showed that the biochar reached the saturation of ammonia nitrogen adsorption within 12 h, and the NH 4 + -N concentration rebounded with the increase of reaction time, and the ammonia nitrogen desorption, so it was speculated that this phenomenon might be related to the adsorption desorption of biochar. In addition, there was no signi cant accumulation of intermediate products in both systems throughout the NH 4 + -N removal process, and the concentrations of NO 3 --N and NO 2 --N were stable below 1.0 mg/L and 0.15 mg/L, respectively. The pH of BC400 + Z03 system decreased continuously, and nally stabilized at about 4.25.
In BC700 + Z03 system, when the BC700 dosage was lower than 1.0g/L, the pH decreased continuously, and when the dosage was 2.0 ~ 4.0g/L, the pH increased within 12h, decreased continuously within 12 ~ 48h, but increased again after 48 ~ 72h. The nal pH of each group ranged from 4.0 to 7.5 with the dosage from low to high.
In order to compare the NH 4 + -N removal e ciency of the BC400/BC700 + Z03 system with the control group, the NH 4 + -N removal e ciency and the corresponding pH of each system at 72 h of reaction are shown in Fig. 2c. Strain Z03 inoculated with 5% inoculum to arti cial wastewater could achieve 41.03% NH 4 + -N removal e ciency at 72h, which was higher than the e ciency of biochar adsorption at each dosage, but signi cantly lower than that of biochar coupled with strain Z03. In addition, the removal effect of biochar on NH 4 + -N was not signi cantly correlated with the dosage, and the maximum NH 4 + -N removal e ciency was 31.96% at the dosage of 0.5 g/L for BC400 and 26.60% at the dosage of 2.0 g/L for BC700. The adsorption was the main method. The pH was signi cantly different among the systems after 72 h, and the difference between the groups within the same system was not signi cant. The higher the NH 4 + -N removal e ciency of the system, the lower the pH value at 72 h. The nal pH of BC400 + Z03, Z03 and biochar NH 4 + -N removal systems were around 4.5, 6.0 and 7.5, respectively, but the pH of BC700 + Z03 system increased with the increase of the dosage. In order to further investigate the difference of the NH 4 + -N removal e ciency between BC400 and BC700 coupled strain Z03, the experiments compared the ammonia nitrogen removal rate and pH of three systems, BC400 + Z03, BC700 + Z03 and strain Z03, at 72h of reaction, and the results are shown in Fig. 3.
It can be seen that the NH 4 + -N removal e ciency of biochar coupled strain Z03 NH 4 + -N removal system was higher than that of strain Z03 NH 4 + -N removal system under each dosage, and the NH 4 + -N removal e ciency increased with the increasing dosage. When the dosage was 0.5 g/L, the NH 4 + -N removal e ciency of BC400 + Z03, BC700 + Z03 and Z03 systems were 47.05%, 43.83% and 41.04%, respectively.
When the dosage was 1.0 g/L and 2.0 g/L, the NH 4 + -N removal e ciency of BC700 + Z03 system was higher than the other two systems, and the NH 4 + -N removal e ciency could reach 72.02%. When the dosage was 3.0 g/L and 4.0 g/L, the NH 4 + -N removal e ciency of BC400 + Z03 system was signi cantly higher than the other two systems, reaching 89.74% and 95.16%, respectively, while the NH 4 + -N removal e ciency of BC700 + Z03 system was 82.91% and 84.37%, respectively.
Biochar, as a carbon-rich material with a multilayer pore structure, can slowly release DOC to provide an additional carbon source for strain Z03. The measurement results of DOC contained in BC400 and BC700 showed that BC400 and BC700 contained relatively high DOC content, 62.95 and 51.617 mg/g, respectively, which may be related to the biomass material and the carbonization temperature. It has been shown that biochar can release bioavailable soluble organic carbon and promote denitri cation reactions (Mukherjee and Zimmerman, 2013;Sima et al., 2017;Steinbeiss et al., 2009). Tang et al. (Tang et al., 2016)characterized soluble organic matter in birch and maple biochar by optical analysis and found that carbonization temperature can affect the composition of soluble organic matter in biochar, which in turn affects the bioavailability of dissolved organic matter. This is probably the main reason why the NH 4 + -N removal e ciency of the BC400 + Z03 system is better than that of BC700 + Z03 at high dosing rates, while the adsorption effect of biochar at low dosing rates plays a major role in the NH 4 + -N removal process.
The experimental results revealed that biochar has a pH retarding effect, which can neutralize the acidity due to nitri cation reaction and reduce the inhibitory effect of water pH on enzyme activity. The nitri cation and denitri cation processes are highly susceptible to the in uence of environmental pH. Nitri cation is an acid-producing process, while denitri cation is an alkaline-producing process. Microorganisms are extremely sensitive to the environmental pH, and either too high or too low pH will affect the biological denitri cation effect (Ren et al., 2014;Zhang et al., 2012).
It is known that in the denitri cation system with only strain Z03, the pH eventually decreased to about 6.0 as the reaction proceeded, and the nitri cation rate of nitrifying bacteria usually slowed down signi cantly at pH below 6.5, which is another limiting factor for its denitri cation effect in addition to the carbon source. In contrast, the pH of all groups with biochar only increased, indicating the presence of alkali in the biochar, which is the same as the previous study by Yuan et al. (Yuan et al., 2011). They studied the morphology of alkali presence in biochar using FTIR, XRD and potentiometric titration, and found that the release of alkali from biochar in the water column could be the effect of organic functional groups such as -COO-and -O-on its surface and the presence of carbonate in biochar, and the content of alkali in biomass char due to carbonate increased with the increase of carbonized temperature, while the effect of organic functional groups on alkali in biomass char was the same as On the contrary, this conclusion is consistent with the results of the present experiment in which the nal pH of the groups in which BC700 was injected under the same conditions was higher than that of the BC400 group. In addition, in the BC400/BC700 + Z03 system, it can be seen that some groups showed a small increase and then decrease in pH within 12 h from the beginning of the reaction, which is due to the fact that strain Z03 is in the adaptation period to the environment, the nitri cation reaction rate is low, and the alkaline material released from biochar makes the pH rise back, and then the pH of the system continues to decrease as the reaction proceeds. After 48 h or 60 h, the rate of denitri cation gradually decreased, and with the decrease of the reaction rate, the pH of each group with higher biochar dosage increased signi cantly, presumably because the concentration of ammonia and nitrogen in the system was too low, and the concentration of nitri cation substrate and carbon source limited the reaction, but the biochar still continued to release alkaline substances, so the pH kept increasing. Therefore, biochar can be used as a pH buffer to neutralize the acidity generated by the nitri cation process and continuously release alkaline components throughout the reaction to prevent the inhibitory effect on biological denitri cation due to low pH. In addition, the variation of pH causes the variation of the adsorption capacity of biochar for ammonia nitrogen, which may be the reason why the adsorption effect of each system material was not signi cantly correlated with the dosage, and some groups obtained the highest adsorption effect at the lowest dosage. Figure 3 Comparison of 72h NH 4 + -N removal rate and pH change between BC400/BC700 + Z03 and strain Z03 systems with different biochar dosages 3.2.2 Effect of BC400 + BC700 on HNAD process Different carbonized temperatures of biochar have different physicochemical properties, and thus have various promotion mechanisms for microbial denitri cation. In order to investigate whether the BC400 + BC700 + Z03 system has different NH 4 + -N removal effect than the BC400/BC700 + Z03 systems, this study designed the BC400 + BC700 coupled Z03 system and investigated the effects of dosage, mixing ratio and pH on the NH 4 + -N removal effect. The changes of NH 4 + -N concentration, pH, NO 3 --N and NO 2 --N concentration of BC400 + BC700 + Z03 system with different biochar dosages mixed at 1:1 mass ratio are shown in Fig. 4a. It can be seen that the NH 4 + -N removal e ciency of the BC400 + BC700 + Z03 system increased with the increase of the biochar dosing amount in 72 h. The NH 4 + -N concentration could be reduced to below 10 mg/L within 48 h when the dosage was 3.0 g/L and 4.0 g/L, and there was almost no accumulation of NO 3 --N and NO 2 --N. The pH of the system showed an overall decreasing trend within 72 h. The pH of the group with the dosing amount over 3.0 g/L increased after 48 h, and nally the pH of each system was in the range of 3.75 ~ 5.0 according to the dosing amount from low to high, respectively.
In order to compare the difference of NH 4 + -N removal e ciency between the BC400 + BC700 + Z03 system and the control group (only BC400 + BC700/strain Z03), the NH 4 + -N removal e ciency of each system at 72 h was compared in Fig. 4b. The results showed that the NH 4 + -N removal e ciency of BC400 + BC700 + Z03 system was signi cantly better than that of the control groups, and the highest NH 4 + -N removal e ciency was 94.36% when the mixed biochar dosage was 3.0 g/L. The e ciency of BC400 + BC700 adsorption was signi cantly lower than that of strain Z03 HNAD e ciency, and the NH 4 + -N removal e ciency were 25.74%, 24.12%, 27.49%, 15.66% and 16.61% for the mixed biochar dosage from 0.5 to 4.0 g/L, respectively. After 72 h of reaction, the nal pH of the system with higher NH 4 + -N removal e ciency was lower. The nal pH of each group with different dosage of BC400 + BC700 did not differ much and was stable at 7.5 ± 0.2. The nal pH of BC400 + BC700 + Z03 system gradually increased from 4.0 to 5.0 with the increase of dosage.
The above results showed that compared to the BC400/BC700 + Z03 system, the BC400 + BC700 + Z03 system was able to reduce the ammonia nitrogen concentration to less than 10 mg/L in a shorter reaction time when the biochar dosage exceeded 3.0 g/L. Figure 4c shows the changes in NH 4 + -N removal e ciency and pH of the BC400/BC700 + Z03 system and the BC400 + BC700 + Z03 systems at 48h and 72h, respectively. As can be seen from Fig. 4c, the NH 4 + -N removal e ciency of the BC400 + BC700 + Z03 system was signi cantly higher than that of the BC400/BC700 + Z03 system in the reaction 48 h at the dosing amounts of 3.0 g/L and 4.0 g/L, which could reach 87.36% and 86.56%, respectively. The mixed biochar system can signi cantly shorten the reaction time under the condition that the e uent ammonia nitrogen meets the standard. The differences in pH values between groups within each system were not signi cant. With the increase of reaction time to 72 h, the difference of NH 4 + -N removal e ciency of each system decreased, and the pH values of BC400 + BC700 + Z03 and BC700 + Z03 systems increased with the increase of biochar dosage. At the dosing of BC400 + BC700 only, the NH 4 + -N removal e ciency of all groups was lower than 30% at different dosing amounts, and the adsorption effect increased with the increase of initial pH. system with different biochar dosages (a); comparison of NH 4 + -N removal e ciency and pH between BC400 + BC700 + Z03 systems and control group at different biochar dosages for 72h (b); comparison of NH 4 + -N removal e ciency and pH between BC400 + BC700 + Z03 and BC400 BC400/BC700 + Z03 systems with different biochar dosages(c). Figure 5a shows the effect of the mixing ratio of BC400 and BC700, showing that when the dosage was 3.0 g/L and the mass ratios of BC400 and BC700 were 1:1, 2:1, 3:1, 4:1 and 5:1, respectively, all groups could reduce the concentration of ammonia nitrogen to less than 10 mg/L within 48 h, and there was no signi cant accumulation of nitrate nitrogen and nitrite nitrogen during the whole reaction process. The accumulation of nitrate and nitrite nitrogen was not obvious during the whole reaction. The pH of each group in the system increased slightly from 0 to 12 h and then decreased continuously. The pH of the 1:1 and 2:1 groups increased continuously after 48 h, and the pH of the other three groups increased slightly after 60 h. The nal pH values of each group from low to high by mass ratio were 6. 33, 5.34, 4.84, 4.64 and 4.55, respectively.
The NH 4 + -N removal e ciency of BC400 + BC700 + Z03 system and the control group at 72h are shown in Fig. 5b.The results indicated that the NH 4 + -N removal e ciency by BC400 + BC700 system at 72h is lower than 10% when the dosage is 3.0g/L, while the removal rate of ammonia nitrogen after coupling Z03 is all located between 93.58% and 95.05%, which is much higher than that of only adding biochar or only strain Z03. At 72 h, the pH of all groups in the BC400 + BC700 system was between 7.5 ± 0.4, and the pH of all groups in the BC400 + BC700 + Z03 system gradually decreased from 6.5 to 4.5 with the increase of the BC400:BC700 mass ratio. system with different biochar mass ratio (a); comparison of 72h NH 4 + -N removal e ciency and pH change between BC400 + BC700 + Z03 system and control group with different biochar mass ratio (b).
Environment pH affects the adsorption performance of biochar and the growth and metabolic capacity of microorganisms. To investigate the effect of initial pH on different NH 4 + -N removal systems, the NH 4 + -N removal e ciency of each system at different initial pH was observed when BC400 and BC700 were and after 48 h the pH of the groups with starting pH of 7.5, 8.5 and 9.5 slightly recovered, while the pH of the remaining groups continued to decrease, and the nal pH of each group was 7.63, 7.31, 6.6, 5.19, 4.51 and 4.47, respectively, according to the starting pH from high to low.
The results of NH 4 + -N removal e ciency of BC400 + BC700 + Z03 system and control group at different initial pH at 72 h are shown in Fig. 6b. The gure demonstrated that the NH 4 + -N removal e ciency of each group with biochar only increased with the increase of initial pH, and the highest NH 4 + -N removal e ciency of 31.23% was achieved at the initial pH of 10.5, while the NH 4 + -N removal e ciency of the rest of the groups was lower than 20% within 72 h. The NH 4 + -N removal e ciency in the groups inoculated with strain Z03 only were in the range of 30 ~ 43%, and the alkaline environment had a certain inhibitory effect on microbial HNAD process under the low C/N ratio. All groups of the mixed biochar coupled with strain Z03 nitrogen removal system could achieve more than 95% NH 4 + -N removal e ciency at 72 h,and the removal rate increased slightly with the increase of the initial pH.
BC400 and BC700, as materials with good redox activity, may play a role in promoting electron transfer system activity and enzyme activity during biological denitri cation. Previous studies (Klüpfel et al., 2014;have shown that, the low-temperature biochar has a better electron supply capacity than the high-temperature biochar, and the high-temperature biochar has the role of an electron shuttle mediator that can facilitate electron transfer, and this result is consistent with the FTIR analysis of BC400 and BC700. Wu et al. (Wu et al., 2019b) showed that biochar at 300°C signi cantly enhanced the activity of the electron transfer system of denitrifying microorganisms and promoted their utilization rate of carbon sources, and in addition, compared to high-temperature biochar, low-temperature biochar increased the activity of denitrifying enzymes NAR and NIR, which in turn increased the denitri cation rate. Thus, it is speculated that the high dosage of BC400 + Z03 NH 4 + -N removal e ciency is slightly better than BC700 + Z03 in the denitri cation reaction because BC400 is more effective in promoting the activity of nitrilase and denitrifying enzymes, while the signi cant increase in the reaction rate of BC400 + BC700 + Z03 compared with the previous two may be due to the electron transfer capacity of BC700 which promotes the ability of BC400 as an electron donor to increase the activity of the reaction electron transfer system. The signi cant increase in the reaction rate of BC400 + BC700 + Z03 system compared to BC400/BC700 + Z03 system may be due to the ability of BC400 as an electron donor to enhance the activity of the reaction electron transfer system. However, the difference in the NH 4 + -N removal e ciency of these three systems was signi cantly smaller than that of 48 h after 72 h. Therefore, it was speculated that the mixed biochar system could improve the reaction rate by enhancing the activity of the electron transfer system, and the nal NH 4 + -N removal effect was mainly in uenced by the content of DOC contained in the biochar and the pH retarding effect. Figure 6 Variation of NH 4 + -N concentration, pH, NO 3 --N and NO 2 --N concentration in BC400 + BC700 + Z03 system with different initial pH (a); comparison of 72h NH 4 + -N removal e ciency and pH change between BC400 + BC700 + Z03 system and control group with different initial pH (b).

Conclusion
The biochar BC400 and BC700 were prepared by the oxygen-limited temperature control method, and characterized by SEM, BET, FITR, XRD, RS, DOC, zeta potential and elemental analysis. It was found that with the increase of carbonization temperature, BC700 had higher speci c surface area and more multilayer pore size structure, increased aromatic condensation, higher crystallinity and more ordered structure. Both of them have better redox activity, BC400 has stronger electron donor ability and BC700 has better electron transfer performance. BC400 and BC700 contained relatively high DOC contents of 62.95 and 51.617 mg/g, respectively. BC400/BC700 + Z03 system was able to signi cantly improve the NH 4 + -N removal effect on low temperature low C/N containing wastewater compared to the control group (biochar only/ strain Z03 only i). The BC400 + BC700 + Z03 system has a faster NH 4 + -N removal rate compared to the BC400/BC700 + Z03 system. The promotion of strain Z03 by biochar may be due to the ability of biochar to provide a carbon source for strain Z03, to achieve pH retardation and to promote the activity of electron transfer systems and enzymes in HNAD process.

Declarations
Ethics approval and consent to participate. Not applicable.
Consent for publication. Not applicable.
Availability of data and material. All data generated or analysed during this study are included in this published article [and its supplementary information les].
Competing interests. There are no con icts of interest to declare. Comparison of 72h NH4+-N removal rate and pH change between BC400/BC700+Z03 and strain Z03 systems with different biochar dosages Variation of NH4+-N concentration, pH, NO3--N and NO2--N concentration in BC400+BC700+Z03 system with different biochar dosages (a); comparison of NH4+-N removal e ciency and pH between BC400+BC700+Z03 systems and control group at different biochar dosages for 72h (b); comparison of NH4+-N removal e ciency and pH between BC400+BC700+Z03 and BC400 BC400/BC700+Z03 systems with different biochar dosages(c).