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Article

Fe-N-Modified Sludge Biochar for Enhanced Acetic Acid Production from Sludge Anaerobic Fermentation

by
Lingling Wei
,
Jinquan Wan
,
Zhicheng Yan
* and
Yan Wang
College of Environment and Energy, South China University of Technology, Guangzhou 510006, China
*
Author to whom correspondence should be addressed.
Sustainability 2025, 17(7), 3247; https://doi.org/10.3390/su17073247
Submission received: 2 February 2025 / Revised: 19 March 2025 / Accepted: 26 March 2025 / Published: 5 April 2025
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Abstract

:
Sustainable recycling of carbon resources from waste-activated sludge (WAS) is essential for advancing the circular wastewater economy. Anaerobic fermentation provides an eco-efficient pathway for converting organic matter from waste-activated sludge into volatile fatty acids (VFAs). In this study, Fe-N modified biochar was innovatively prepared from WAS for acetic acid yield enhancement, and the system realized the closure of the material cycle. Results show that adding Fe-N-modified biochar (made under the conditions of 0.2M FeCl3 and 10 g/L urea) led to a 38.8% increase in acetic acid yield (1745 mg/L) and a 5.7% increase in its percentage (60.5%) compared to the control. It also improved sludge hydrolysis and hydrolase activity. In addition, Fe-N-modified biochar increased the relative abundance of Chloroflexi, Actinobacteria, and Bacteroidetes, among which Chloroflexi is an electro-active microorganism that promotes the transformation of propionic and butyric acids to acetic acid, while Bacteroidetes is the primary microorganism responsible for VFA production. In summary, Fe-N-modified biochar may serve as an effective material for promoting acetic acid production during the anaerobic fermentation of WAS.

1. Introduction

With the increase in sewage treatment volumes, the production of waste-activated sludge (WAS) in China continues to rise each year, with an annual growth rate ranging from 5% to 13%. By 2020, this production reached approximately 60 million tons [1]. WAS consists of a variety of substances, including microbial cells, various proteins, carbohydrates, lipids, and cellulose, heavy metals, mineralized substances, and other metal salts. It is characterized by a high content of organic matter and is rich in nutrients [2,3]. Therefore, WAS is a potential renewable energy source for the targeted preparation of clean energy (e.g., biogas) and biomaterials (e.g., volatile fatty acids (VFAs)) through anaerobic fermentation processes [4,5], which contributes to the circular economy (CE) [6]. VFAs are very versatile and can be used to synthesize bioplastics (e.g., PHA) [7]; in a new study, VFAs (especially acetic acid) have been suggested to contribute to the degradation of concrete debris in bioleaching processes [8]. Acetic acid is an important component of VFAs and can be used in the synthesis of biofuels [9]. In addition, acetic acid is considered to be one of the most readily utilized carbon source substances by microorganisms during wastewater treatment, so increasing the percentage of acetic acid in the fermentation broth can lead to a higher production value of the fermentation products [10]. At the same time, the use of sludge fermentation solution as a carbon source for biological sewage treatment not only alleviates the cost of sewage treatment but also enables the sustainable recycling of carbon resources.
However, because of the complex composition of WAS, it is extremely inefficient in hydrolysis and difficult to biodegrade under the protection of extracellular polymers (EPSs) [11], resulting in inefficient direct use of WAS to produce VFAs by hydrolytic acidification. Therefore, it is crucial to implement effective methods to enhance the hydrolysis of WAS. Various additives have been developed to improve the solubilization and hydrolysis efficiency of sludge in anaerobic fermentation systems. These include nanoscale nZVI, steel slag, and biochar [12,13,14]. Among these, biochar has gained particular attention in research. It is widely accepted that the incorporation of biochar into anaerobic fermentation systems facilitates electron transfer between microorganisms, enriches functional bacteria, and promotes the degradation and conversion of organic substrates. Yang et al. [15] observed that adding 5.0 g/L activated carbon to a sequencing batch sludge anaerobic digestion system increased the sludge hydrolysis rate from 39.1% to 45.2% and boosted methane production by 17.4%. Zhai et al. [16] added sludge-derived biochar to the sludge anaerobic digestion system, which effectively increased VFA production and enriched acid-forming bacteria, specifically Clostridiales. Nevertheless, carbon-based materials may also exert an inhibitory effect on the anaerobic digestion of organic matter. For instance, Zhang et al. [17] found that adding granular activated carbon (GAC) to a sludge anaerobic fermentation system not only reduced the production of VFAs but also decreased the abundance of all metabolic functions compared to a control system. Salvador et al. [18] found that the addition of carbon nanotubes at 5 g/L inhibited the anaerobic methanogenesis process in pure culture, but at 0.5 and 1.0 g/L, it was able to promote the mass transfer rate between the substrate and microbial cells, which accelerated the methanogenesis process. Furthermore, in a similar study, Li et al. [19] reported that introducing carbon nanotubes at a concentration of 1.0 g/L into an anaerobic digestion system did not significantly affect methane production. The possible reason for this phenomenon is that microbial extracellular polymers in the sludge system cause the nanomaterials to agglomerate, thereby limiting their interaction with the cells. These findings suggest that the type of carbon material, the amount used, and the conditions of anaerobic digestion all influence the function of biochar in the anaerobic fermentation process.
WAS, a major by-product of the wastewater treatment process, is a potential green resource for the production of biochar. Sludge biochar possesses several advantages, including chemical stability, a high specific surface area, a rich porous structure, as well as numerous surface functional groups. These traits can bring great benefits to sludge anaerobic fermentation systems. To optimize the use of sludge biochar, various strategies have been proposed, such as biochar modification, co-pyrolysis, and pre-treatment. However, there is a lack of research exploring how different types of biochar and their embedded trace elements affect process performance. According to existing studies, conductive materials (CMs) (mainly carbon-based CMs and iron-based CMs) can enhance direct interspecies electron transfer (DIET) during AD [20,21]. It has been shown that Fe atoms can form iron oxides (e.g., Fe3O4 or α-Fe2O3) on the surface of biochar, and the metallic conductivity of iron oxides can enhance the conductivity of the material [22]. In addition to this, the introduction of Fe will also change the distribution of functional groups on the surface of biochar and form new active sites [23]. Consequently, in recent years, Fe atoms have been commonly doped into biochar to improve its electrochemical properties and facilitate electron transfer among electroactive microorganisms. However, during the fabrication of magnetically functionalized carbons, metal oxide nanoparticles tend to infiltrate and block the mesoporous channels of biochar substrates [24]. Apart from this, metal dissolution and agglomeration may also diminish the physicochemical properties of the material [25]. In order to cope with this situation, researchers have introduced non-metallic N atoms with high electronegativity into carbonaceous materials. This modification can adjust the surface electronic structure of carbonaceous materials, induce defect formation, and increase the number of nitrogen-containing functional groups [26], which is highly effective in enhancing the stability of the material.
Therefore, in this research, Fe-N-modified sludge biochar was applied for targeted regulation of sludge anaerobic fermentation, with the aim of promoting the production of VFAs and increasing the percentage of acetic acid in VFAs. Firstly, the production of biochar was optimized by preparing functional biochar through a rationally designed process, combined with XRD, SEM, N2 adsorption, FT-IR, and XPS to investigate the structure and properties of the materials. Then, the performance of the sludge anaerobic fermentation system with Fe-N-modified sludge biochar addition was investigated by examining the solubilization of organic matter, acid production, and related hydrolase activities. Finally, the relative abundance and growth status of microorganisms in the acidification phase were examined to comprehensively analyze the structural variation of the microorganisms involved in the biosynthesis of VFAs.

2. Materials and Methods

2.1. Sludge and Fe-N-Modified Biochar Preparation

The raw material for the biochar was collected from the WAS in a municipal wastewater treatment plant in Guangdong, China. The sludge was dehydrated, dried, and crushed to 120 mesh. Then, 10.0 g of sludge powder was taken and completely immersed in 100 mL of a FeCI3 solution of a specific concentration (0, 0.1, 0.2, 0.4 M). Then, a specific amount (0, 5, 10, 15 g) of urea was incorporated into the aforementioned mixture. The pH of the solution was set to 10 and sealed for 24 h. The samples were then dried in a vacuum drying oven at 60 °C. Following this, the above materials were pyrolyzed in a tube furnace and protected by a stream of N2 gas. The temperature was increased at a rate of 5 °C/min and maintained at 600 °C for 2 h. After pyrolysis, the materials were washed several times with deionized water and dried at 60 °C to obtain the final Fe-N-modified biochar.
The anaerobic fermentation sludge was obtained from WAS of the same wastewater treatment plant, then filtered and naturally thickened at 4 °C. The supernatant was discarded and stored at 4 °C until use.

2.2. Experimental Setup

Using 250 mL anaerobic flasks as reactors, 200 mL of thickened sludge was introduced to each reactor, accompanied by N2 for 20 min to eliminate oxygen, and then sealed and subjected to a fermentation experiment at 180 rpm and 37 °C in a shaker. The pH was adjusted to 9 every day. Samples of the fermentation broth were collected every 24 h and used to analyze various constituents. All experiments were performed in triplicates.
The first batch of the experiment examined how biochar, prepared with different concentrations of FeCI3 (Fe0–NBC, Fe0.1–NBC, Fe0.2–NBC, and Fe0.4–NBC), affects acid production during anaerobic fermentation. The urea dosage was set to 10 g/L. The dosage of biochar in the above reactor was set to 2 g/L, while the control treatment had no added biochar. In the second batch of the experiment, according to the results derived from the first batch of the experiment, the FeCl3 concentration of 0.2 M was determined to be the optimal modified concentration, followed by investigating the effect of biochar prepared with different urea dosages (Fe-N0 BC, Fe-N5 BC, Fe-N10 BC, Fe-N15 BC) on acid production by anaerobic fermentation. After obtaining optimal modified biochar, the third batch of the experiment focused on the impact of Fe-N-modified biochar on the anaerobic fermentation process. In this batch, the biochar dosage was set to 0, 1, 2, 5, and 10 g/L, and the groups were named as Control, R1, R2, R3, and R4.

2.3. Physicochemical Analysis

The determinations of pH, MLSS, MLVSS and ammonia were based on standard methods [27]. COD was determined by Zhang’s method [28]. VFA content was determined utilizing a gas chromatograph (Shimadzu, Kyoto City, Japan) [29]. Protein concentration was measured using the Protein Content Assay Kit (Bicinchoninic Acid (BCA)). Polysaccharide determination was conducted using a modified anthrone–sulfuric acid colorimetry method [30], with glucose as the standard. On day 5, the activities of ETS, protease and α-glucosidase were measured using Zhong’s method [31]; the detailed operational procedures are provided in the Supplementary Materials (Text S1). The morphology of different materials was examined using scanning electron microscopy (SEM) (Gemini SEM 300, ZEISS, Oberkochen, Germany). The determinations of the specific surface area and pore size distribution were carried out using Micromeritics ASAP 2460 Version 3.01, according to the Barrett–Joyner–Halenda (BJH) method. The functional groups of the various materials were identified through FTIR spectroscopy (Nicolet iS50, Madison, WI, USA). The crystallographic properties of the materials were measured by X-ray diffraction (XRD; EQUINOX 100, Billerica, MA, USA). Xray photoelectron spectroscopy (XPS) spectra were obtained with a Thermo Scientific K-Alpha X-ray (Thermo Scientific K-Alpha, East Grinstead, UK) photoelectron spectrometer (photon energy 1486.7 eV).

2.4. Microbial Community Analysis

At the end of the experiment, sludge samples from Control and R2 were collected and underwent an analysis of 16S rRNA. Specific test methods are demonstrated in the Supplementary Materials (Text S1).

2.5. Statistical Analysis

All experiments were performed in triplicate. ANOVA was used to test the significance of the results, with p < 0.05 considered significant.

3. Results and Discussion

3.1. Optimization of Modification Conditions

3.1.1. Effect of Iron-Doped Biochar on VFAs Production

The effects of different types of biochar on VFAs production are exhibited in Figure 1A,B. The VFAs production of iron-doped biochar groups was greater than that observed in Control (Figure 1A). Among them, Fe0.2–NBC demonstrated the most effective enhancement; its VFAs production was up to 3028.44 mg/L, which was 1.3 times higher than that of Control (2329.4 mg/L). However, as the concentration of FeCl3 increased to 0.4 M, the reinforcing effect of Fe0.4–NBC (2671.38 mg/L) on VFAs production was weakened. This phenomenon may be attributed to the agglomeration of superfluous iron atoms [32] and excessive accumulation of iron groups on the surface of biochar, covering the pore structure and functional groups on the surface of biochar, which obstructs the active sites on the biochar surface [33], thereby diminishing the physicochemical properties of the biochar. Notably, iron-undoped biochar did not significantly promote the production of VFAs (p > 0.05). It has been pointed out that BC or NBC have high adsorption, which can cause the relevant enzymes or organic substrates to become adsorbed and thus ineffective [34]. Consequently, the VFAs yield from Fe0–NBC (2480 mg/L) remained statistically comparable to the control group. In terms of VFAs fractions (Figure S1), acetic acid had the largest share (55–62%) among all groups, followed by propionic acid (9–16%) and n-butyric acid (10–15%). On day 5, the percentage of acetic acid in the reactor with iron-doped biochar increased slightly, from 58% in Control to 62% in biochar reactors, while the percentage of propionic acid decreased slightly, from 14% in Control to 10% in the biochar reactors. Research suggested that Fe3O4, or iron-doped biochar, can act as an electron acceptor to enhance DIET, which promotes the biodegradation of propionic acid [35,36]. The changes in acetic acid corresponded to the VFAs; unsurprisingly, Fe0.2–NBC (1880.44 mg/L) showed the highest acetic acid yield, which was 1.45 times higher than Control (1300.4 mg/L). These results indicate that 0.2 M FeCl3 is the optimal modification concentration for preparing Fe-N-modified biochar.

3.1.2. Effect of Nitrogen-Doped Biochar on VFAs Production

Further research was conducted to determine the influence of nitrogen-doped biochar on VFAs generation. As exhibited in Figure 1B, with the increase in urea dosage, the maximum VFAs production from Fe–N0 BC (2788.4mg/L), Fe–N5 BC (2838.1mg/L), and Fe–N10 BC (3011.6mg/L) exceeded Control (2355.8 mg/L) by factors of 1.18, 1.2, and 1.28, respectively. However, when the urea dosage was raised to 15 g/L, the maximum yield of VFAs in Fe–N15 BC (3005.7 mg/L) was similar to that of Fe-N10 BC. It has been reported that the nitrogen and oxygen composition of biochar is influenced by the initial nitrogen doping amount [37]. N atoms act as electron donors to provide more electrons to the off-domain carbon network, thus increasing the capacity and overall electrical conductivity of biochar [38], which facilitates accelerated electron transfer between electroactive microorganisms. In addition, sludge is rich in N atoms, which reduces the supplementation of external N sources during the preparation process. Therefore, continuing to increase the urea dosage had little effect on the biochar. It is inferred that a urea dosage of 10 g/L is the optimum dosage for the preparation of Fe-N-modified biochar.

3.2. Characterization of Different Biochar

The different promotion impacts of different biochar on the generation of VFAs are inextricably linked to the unique physicochemical characteristics of the biochar. To further explain the variation in the production of VFAs, three types of biochar, Fe–N10 BC, Fe–N0 BC, and Fe0–NBC, were selected for characterization and named as FNBC, Fe–BC, and NBC, respectively.
SEM (Figure 2A) observation showed that the surface of NBC was rough and contained abundant pores, but the Fe-BC surface was adhered to by a large number of crystals. This may be due to the lack of N modification, where Fe3O4 particles clogged the biochar surface [39]. And after co-modification with Fe-N, the FNBC surface was not only loaded with iron oxide particles but also showed rough and porous morphological features. This is demonstrated in the BET (Table S1) analysis results, where FNBC has a larger surface area (FNBC, 59.8761 m2/g; Fe-BC, 58.1084 m2/g; NBC, 22.1003 m2/g) and pore volume (FNBC, 0.135748 cm3/g; Fe-BC, 0.132859 cm3/g; NBC, 0.125253 cm3/g); this increases the contact area between the biochar and microorganisms, which in turn leads to more microbial interaction sites with the biochar and provides colonization sites for microbial growth [40]. As shown in Figure S2, all three materials are type IV isotherms, indicating that the materials have mesoporous structures [41]. Based on the pore size distribution, the average pore size of FNBC (9.5 nm) was larger than that of Fe-BC (8.8 nm). Evidence suggests that the simultaneous incorporation of Fe and N is more effective in maintaining the porosity of biochar than the sole addition of Fe [24].
The surface functional groups of the biochar were analyzed using FTIR (Figure 2B), and the results show that the characteristic peaks of Fe-O appeared near 569 cm−1 for FNBC and FBC, indicating that Fe was successfully loaded onto the surface of the biochar, which is conducive to accelerated electron transfer [22]. The characteristic peaks observed near 3432 cm−1 for three samples can be ascribed to the O-H stretching vibration. Additionally, the characteristic peaks observed near 1618 cm−1 and 1048 cm−1 for the three samples imply the presence of C=O and C-O. Notably, the intensity of the C=O and O-H characteristic peaks is more pronounced in the FNBC mapping, revealing an increase in the content of C=O and O-H functional groups, which can be confirmed by XPS analysis later. It has been shown that C=O groups can contribute pseudocapacitance through redox reactions, such as quinone–hydroquinone transitions [42], while C=O and O-H are common redox pairs that drive electron transfer [43]. These results indicate that the surface of sludge biochar is abundant in functional groups, which can enhance the capacitance of the biochar and consequently improve the material’s electrochemical properties [44]. XRD (Figure 2C) analysis showed that the spectra of FNBC and FBC showed peaks of Fe3O4 (near-diffraction angles 2θ = 35.42°, 43.2°, 57.1°, 62.42°, corresponding to the standard magnetite PDF card (# 99-0073)), which suggests that Fe atoms are present on the surface of biochar in the form of Fe3O4. Studies have demonstrated that Fe3O4 has semiconducting properties and can act as an electron conductor to accelerate the decomposition of substrates (e.g., polysaccharides and proteins) into VFAs during the acidification phase of hydrolysis [45]. In addition, the spectra of the three samples show peaks of quartz (near-diffraction angles 2θ = 26.5°, 36.6°, corresponding to the standard quartz PDF card (# 79-1906)). As shown by XPS mapping (Figure 2D), FNBC and Fe-BC detected peaks of Fe2p with 4.33% and 3.16%, significantly higher than that of NBC (1.18%). In addition, more pronounced peaks of N1s were detected in FNBC and NBC. The N content of Fe-BC was 3.25%; after N doping, the N content of FNBC (6.4%) was significantly higher than that of Fe-BC, which indicated that the nitrogen was successfully doped into the biochar. The C1s spectra (Figure S3) of the three materials all reveal the presence of graphitic carbon (284.8 eV), C-O (286.5 eV), C=O (289 eV), and O-C=O (293.4 eV) [46]. The difference is that C=O (288.5 eV) is higher in the FNBC, in agreement with the FTIR results. The peaks of the N1s spectrum (Figure S3) at 398.4 and 400.6 eV correspond to pyridine N and pyrrole N, respectively [47], indicating the formation of nitrogen-containing functional groups. Compared to Fe-BC, FNBC exhibited a significant increase in pyrrolic N content. Studies have demonstrated that nitrogen-containing functional groups can preserve the electrochemical activity of metals through coordination with metallic elements, which is critical for enhancing the conductivity of biochar materials [48]. This is corroborated by the Fe 2p spectra (Figure S3), where FNBC shows a higher proportion of Fe 2p1/2 at 711.66 eV (38.25%) compared to Fe-BC (29.09%). These findings indicate that after co-modification with Fe and N, FNBC exhibits more surface functional groups, showing improved electrical capacity and electron transfer efficiency, thereby enhancing electron transfer in anaerobic fermentation systems to varying degrees.
To further clarify the effect of Fe-N-modified biochar on acid production during the anaerobic fermentation process, Fe-N10 BC (FNBC) was selected to continue the following experiments.

3.3. Effect of Fe-N-Modified Biochar on Anaerobic Fermentation Process

3.3.1. Production and Distribution of VFAs

The level of COD content can reflect the extent of sludge hydrolysis. As shown in Figure 3A, with the addition of FNBC, the COD content in the Control, R1, R2, R3, and R4 groups reached levels of 3697, 4140, 4320, 4078, and 3834 mg/L, respectively. Notably, the COD content in R2 increased by 14.4% compared to Control. Additionally, ammonia nitrogen mainly comes from protein decomposition, making it another useful indicator of sludge hydrolysis to some degree [49]. As the reaction proceeded, the ammonia nitrogen in each reactor showed an overall increasing trend, followed by a gradual stabilization trend, which means that substrate hydrolysis was nearing completion (Figure 3B). The above results indicate that FNBC can effectively promote the solubilization and hydrolysis of WAS.
As exhibited in Figure 3C, the addition of FNBC effectively promoted the production of VFAs, and the changes in VFAs corresponded to the changes in COD. The peak values of the VFAs in the Control, R1, R2, R3 and R4 groups were 2290, 2723, 2886, 2745 and 2389 mg/L, respectively. More importantly, adding FNBC shortened the time to reach the peak of VFAs (5d), while Control reached the peak on day 7. With the increase in FNBC dosage, the VFAs tended to increase and then decrease. The highest VFA production occurred with a biochar dosage of 2 g/L; however, a significant decrease was observed when the dosage was increased to 10 g/L, indicating that excessive FNBC negatively affected VFA production. It has been reported that the leaching of heavy metal ions on the surface of biochar can provide trace elements (such as Fe, Zn, Ni, Cu, Co, etc.) that are beneficial for microbial growth, to some extent [50]. However, the release of large amounts of heavy metal ions can inhibit the activity of acid-producing microorganisms [51]. Furthermore, the massive addition of biochar disrupts microbial communication induced by community sensing (QS) [52], which interferes with the degradation of organic substrates and affects acid production. In terms of VFA composition (Figure 3D), acetic acid was the predominant component across all groups. The maximum peak concentration of acetic acid was observed in R2 (1745 mg/L), which was 38.8% higher than Control (1257 mg/L) and slightly higher than the 34.4% increase achieved by the previous study using magnetite to enhance acid production [53]. On day 5, with the addition of FNBC, the proportions of acetic acid in Control, R1, R2, R3 and R4 were 54.8%, 58.5%, 60.5%, 59% and 56.8%, respectively. This finding indicates that adding FNBC enhances the proportion of acetic acid in the VFAs.
To further explain this phenomenon, we continued to analyze the ratio of acetic acid to propionic acid and to butyric acid. As shown in Figure 4A,B, in Control, R1, R2, R3, and R4, the highest ratio of mass concentration of acetic acid to propionic acid reached 4.29, 4.91, 5.48, 5.21 and 4.69, respectively, and the highest ratio of mass concentration of acetic acid to butyric acid reached 4.57, 6.13, 5.69, 5.53 and 5.47, respectively. Overall, the percentage of acetic acid was higher in all groups where FNBC had been added. It was hypothesized that FNBC may facilitate the conversion of organic substrates (e.g., glucose) into acetic acid by the acid-producing bacteria during the hydrolytic acidification stage.
As the reaction time goes by (1–5 d), the ratio of the mass concentration of acetic acid to propionic acid does not change much during the hydrogen-producing acetic acid phase, whereas the ratio of the mass concentration of acetic acid to butyric acid gradually increases. We further explained this phenomenon from a thermodynamic perspective. As shown in Table 1, the anaerobic oxidation of propionic acid and butyric acid to acetic acid and hydrogen is energy-consuming (Reaction 1), and it can only occur at lower partial pressures of hydrogen and with the mutualism of methanogenic bacteria (Reaction 2). Moreover, the anaerobic degradation process of propionic acid requires more energy, making it one of the most challenging substrates for hydrogenesis and acetogenesis. However, the presence of Fe3O4 is able to break the thermodynamic barrier and promote the degradation of complex organic matter, mainly thanks to the heterogeneous iron reduction process [54]. In addition, biochar is able to adsorb propionic and butyric acid through its porous structure, enriching reciprocal oxidizing and acetic acid-producing bacteria and facilitating the conversion of propionic/butyric acid to acetic acid through the reciprocal oxidizing pathway (demonstrated in 3.4). In this study, setting the pH to 9 resulted in some inhibition of the activity of methanogenic bacteria. Therefore, reaction 2 occurs with difficulty. It is hypothesized that the accumulation of acetic acid is due to the camping of hydrogen-producing acetogenic bacteria with homoacetogenic bacteria (reaction 3). However, electron transfer between syntrophic bacteria has a significant impact on acetate production [55]. Previous studies confirmed that magnetic biochar can enhance extracellular electron transfer [45], which would accelerate the electron flow between co-culture bacteria and provide sufficient electrons for enzyme and ATP synthesis [56] and thus stimulate the conversion of propionic acid and butyric acid to acetic acid. In addition, considering the possibility of residual methanogenic bacteria in the system, propionate-oxidative bacteria and butyrate-oxidating bacteria can establish DIET with hydrogen-consuming methanogenic bacteria via FNBC to promote the biodegradation of propionic acid and butyric acid. In contrast, the lack of electron acceptors in Control made it challenging to oxidize propionic acid and butyric acid to acetic acid. In summary, FNBC may accumulate acetic acid by promoting the conversion of macromolecular organic matter to acetic acid and the conversion of propionic acid and butyric acid to acetic acid via the reciprocal oxidation pathway.

3.3.2. Variations of Organic Substrates

During anaerobic fermentation of sludge, soluble polysaccharides and soluble proteins are the main substrates for acid production in fermentation. The concentrations of soluble polysaccharides (Figure 5A) and soluble proteins (Figure 5B) increased rapidly in the first 2–3 days. The peak concentrations of soluble polysaccharides in Control, R1, R2, R3, and R4 reached 69.6, 102.4, 115.1, 107.8, and 93.2 mg/L, respectively, and the peak concentrations of soluble proteins reached 188.0, 253.1, 270.4, 260.3, and 215.7 mg/L, respectively. From these results, it can be presumed that FNBC promotes sludge cell breakdown, leading to more organic substrate leaching. During days 3 to 6, these organic substances showed a decreasing trend, as they were utilized by microbial decomposition. The hydrolysis efficiency was calculated from the concentration of the highest peak to the lowest peak, and the hydrolysis efficiencies of soluble polysaccharides were 56.9%, 62%, 62.5%, 61.2% and 59.4%, respectively. The hydrolysis efficiencies of soluble proteins were 45%, 47.4%, 48.2%, 49.6%, and 46.9%, respectively. In line with the changes in VFAs, the hydrolysis efficiency of polysaccharides and proteins was better in R2, or 1.1 and 1.07 times higher than that of Control, respectively. These results further suggest that FNBC can effectively promote the hydrolysis of organic substrates, thereby providing more available organic matter for acid-producing microorganisms and promoting the accumulation of VFAs.
To further explain this phenomenon, two enzymes that are directly related to the hydrolysis of proteins and polysaccharides were measured: α-glucosidase and protease. As shown in Figure 5C, the α-glucosidase activities of R1, R2, R3 and R4 were increased by 11.01%, 20.9%, 15.04% and 2.33%, respectively, and the protease activities were increased by 23.4%, 30.9%, 18.7% and 4.2%, respectively, compared to Control. It suggests that the addition of FNBC may improve the activity of related hydrolases due to the various trace elements present in FNBC, which can function as a biostimulant to stimulate the synthesis of essential enzymes [57], thereby facilitating the hydrolysis of macromolecular organic compounds and providing sufficient substrates for acid fermentation. However, it is important to note that high doses of FNBC can negatively impact hydrolase activity, which explains why the relative enzyme activity of R4 is generally lower.

3.4. Microbial Community and Growth

Given the positive impact of FNBC addition on the production of VFAs, it is reasonable to assume that FNBC led to changes in the microbial community structure, so Control and R2 were subjected to 16S rRNA gene sequencing. As outlined in Table S1, the Shannon index decreased from 7.44 to 7.21, while the Simpson index increased from 0.03 to 0.05 after the addition of FNBC. This indicates that the addition of FNBC reduced the diversity of bacterial communities but promoted the growth of dominant microorganisms, which is consistent with the results of previous studies [58].
The dominant bacterial communities at the phylum level (Figure 6A) in both reactors were Firmicutes, Proteobacteria, Chloroflexi, Actinobacteria, and Bacteroidetes. The anaerobic Firmicutes play a significant role in the hydrolysis and acid production phases. However, the addition of FNBC decreased the relative abundance of Firmicutes from 37.82% in Control to 34.81% in R2, which is in line with Feng’s results [51]. Phylum Proteobacteria is usually considered as an acid consumer [59]. Its relative abundance decreased from 17.29% in Control to 14.15% in R2, which promoted the accumulation of VFAs. Compared to Control, the relative abundance of phylum Chloroflexi increased from 17.04% to 21.04%. It has been reported that phylum Chloroflexi has DIET ability, which can utilize Fe (III) as an electron acceptor [60], thereby being selectively enriched, while it also participates in the co-trophic metabolism of the anaerobic fermentation process to promote cellulose degradation [61]. In addition, Chloroflexi accelerates the degradation of propionic acid and butyric acid to acetic acid [24], which may be a contributing factor for the increased production of acetic acid. Moreover, Actinobacteriota and Bacteroidota are also well-known electroactive microorganisms that can enhance metabolic activity through Fe-N-modified biochar-mediated electron transfer [45], and thus their abundance was also enhanced. Their relative abundance increased from 10.79% and 6.96% in Control to 14.08% and 8.94% in R2, respectively. They are also important members involved in the production of VFAs. Among them, Bacteroidetes plays a key role in the hydrolysis of large organic molecules (e.g., proteins, lipids, and polysaccharides) during anaerobic fermentation. Bacteroidetes also contains many acetic acid-producing bacteria, which are believed to contribute to the accumulation of acetic acid [62]. Overall, FNBC significantly enriched electroactive microorganisms associated with hydrolysis and acidification, resulting in an increase in the production of VFAs, especially acetic acid.
At the genus level (Figure 6B), the abundance of bacteria related to anaerobic hydrolysis, such as Proteiniclasticum, Proteocatella, Romboutsia, and Guggenheimella, increased significantly in R2. In addition, as expected, homozygous acetic acid-producing bacteria (Acetoanaerobium) were detected. The latter is mainly responsible for converting organic substrates into acetic acid, and its abundance increased from 4.18% in Control to 6.38% in R2.
As shown in Table 2, the bacteria responsible for butyric acid degradation [63] and propionic acid degradation [64] were also detected, as well as a variety of genera that are most likely to have homoacetogen [65], and their abundance in R2 was significantly higher than that in Control. Hydrogen-acetic acid-producing bacteria (e.g., Syntrophobacter, Syntrophomonas) and homozygous acetic acid-producing bacteria have been reported to be able to convert long-chain fatty acids (e.g., propionic acid, butyric acid) to acetic acid through intercalation [58]. No methanogenic bacteria were detected in Control due to the influence of pH, and Methanosarcina was detected in R2 at an abundance of only 0.01%. This suggests that inhibition of methanogenic bacteria leads to acetic acid accumulation. The addition of FNBC significantly enhanced the abundance of these bacteria, and thus, R2 exhibited higher acetic acid production. Additionally, Clostridium belonging to the phylum Firmicutes was detected, which is recognized as a dissimilatory iron-reducing bacterium [66], which can also be selectively enriched by utilizing Fe (III) as an electron acceptor. Clostridium can directly utilize soluble organic compounds via the Wood-Ljungdahl pathway to produce butyric and acetic acids [67], which increased its abundance from 0.26% in Control to 0.37% in R2. Apparently, the addition of FNBC achieved an enrichment of hydrolyzed acid-producing bacteria, hydrogen-producing acetogenic bacteria, homoacetogen bacteria, and some electro-active acid-producing bacteria, all of which contributed to improved acetic acid production performance.

3.5. Variation in ETS Activities

Microbial community changes indicate that Fe/N co-doped biochar (FNBC) can function as an electron acceptor to enhance acetate generation. However, direct evidence illustrating improved electron transfer efficiency remains lacking. Relevant studies demonstrate that electron transport system (ETS) activity effectively characterizes extracellular electron transfer capabilities of microorganisms in anaerobic systems [68]. We therefore compared the ETS activity of sludge between the Control and FNBC-amended groups (R2). As shown in Figure S4, FNBC addition increased ETS activity by 88.3% compared to Control, confirming enhanced extracellular electron transfer capacity post-biochar supplementation. These results further suggest that FNBC acts as an electron shuttle to facilitate interspecies electron transfer among acidogenic microbes, thereby improving system acidification performance.

4. Conclusions

In this research, Fe-N-modified biochar was produced from WAS and used to enhance acid production from sludge anaerobic fermentation. The co-modification of Fe and N improved the physicochemical properties of the biochar, leading to an increased specific surface area and a richer pore structure of FNBC, which provides more attachment sites for microorganisms and enzymes, and further promoting the solubilization and hydrolysis of sludge. Moreover, the excellent electrical conductivity of FNBC facilitates the enrichment of electroactive acid-producing microorganisms (e.g., Chloroflexi, Clostridium), which effectively promotes the accumulation of VFAs. In addition, the enrichment of hydrogen-producing acetogenic bacteria and homoacetogen bacteria accelerated the conversion of propionic and butyric acids into acetic acid, resulting in a higher percentage of acetic acid in the fermentation process. In summary, Fe-N-modified sludge biochar can effectively promote acetic acid production, which is significant for achieving the goal of carbon neutrality in wastewater treatment. Overall, this waste-to-resource strategy improves resource recovery and valorization of wastewater treatment byproducts, contributing to the achievement of environmental sustainability.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/su17073247/s1, Figure S1: Volatile fatty acids concentrations of different Biochar; Figure S2: N2 adsorption-desorption isotherms and pore size distribution of different materials; Figure S3: High-resolution XPS spectra of NBC (a), Fe-BC (b), FNBC (c); Figure S4: Relative activities of ETS in the control and R2; Table S1: Characteristics of different materials; Table S2: The alpha biodiversity analysis of mixture samples for different reactors.

Author Contributions

L.W.: Writing—original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Z.Y.: Writing–review and editing, Resources, Conceptualization. J.W.: Writing—review and editing, Supervision, Funding acquisition, Conceptualization. Y.W.: Writing—review and editing, Supervision, Funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data for this study are currently not publicly available, but can be obtained by contacting the corresponding author if necessary. Readers can contact the corresponding author by email (yanzhicheng@scut.edu.cn), and a relevant agreement is required to obtain the data. We welcome scholars in related fields to further discuss data sharing or collaborative research.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The variation of VFAs under iron-doped biochar (A) and nitrogen-doped biochar (B).
Figure 1. The variation of VFAs under iron-doped biochar (A) and nitrogen-doped biochar (B).
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Figure 2. The SEM image (A) and FTIR spectra (B), XRD pattern (C), and XPS spectra (D) of FNBC, Fe-BC, and N-BC.
Figure 2. The SEM image (A) and FTIR spectra (B), XRD pattern (C), and XPS spectra (D) of FNBC, Fe-BC, and N-BC.
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Figure 3. Changes in COD (A), TAN (B) and VFAs (C,D) in WAS fermentation with FNBC.
Figure 3. Changes in COD (A), TAN (B) and VFAs (C,D) in WAS fermentation with FNBC.
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Figure 4. Acetate to propionic (A) and to n-butyrate (B) mass concentration ratio variation under biochar.
Figure 4. Acetate to propionic (A) and to n-butyrate (B) mass concentration ratio variation under biochar.
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Figure 5. The effects of FNBC on the variation of soluble polysaccharide (A), soluble protein (B) and hydrolases (C).
Figure 5. The effects of FNBC on the variation of soluble polysaccharide (A), soluble protein (B) and hydrolases (C).
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Figure 6. The variation of microorganisms at phylum (A) and genus (B) levels in the presence of FNBC. (Sequences showing percentages of reads > 1%).
Figure 6. The variation of microorganisms at phylum (A) and genus (B) levels in the presence of FNBC. (Sequences showing percentages of reads > 1%).
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Table 1. Relevant reaction equations and standard Gibbs free energy.
Table 1. Relevant reaction equations and standard Gibbs free energy.
ReactionReaction Equations△G0
(KJ/mol)
Reaction 1: hydrogenesis and acetogenesisCH3CH2COOH + H2O → 2CH3COOH + 3H2 + CO2+76.1
CH3CH2CH2COOH + 2H2O → 2CH3COOH + 2H2+48.1
Reaction 2: hydrogen-consuming methanogenic4H2 + CO2 → CH4 + 2H2O−131.7
Reaction 3: homoacetogenesis4H2 + 2CO2 → CH3COOH + 2H2O−55.1
Table 2. The relative abundance of microorganisms at genus levels. (Sequences showing percentages of reads < 1%).
Table 2. The relative abundance of microorganisms at genus levels. (Sequences showing percentages of reads < 1%).
Categories BacteriaControlR2
Genus of homoacetogen bacteriaf__Sporomusaceae_Unclassified0.120.16
f__Holophagaceae_Unclassified0.110.15
Natronincola0.050.09
Holophaga0.010.03
Propionate-oxidative bacteriaSyntrophobacter00.01
Smithella0.010.03
Butyrate-oxidating bacteriaSyntrophomonas0.050.12
MethanogensMethanosarcina00.01
ClostridiumClostridium sensu stricto0.260.37
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MDPI and ACS Style

Wei, L.; Wan, J.; Yan, Z.; Wang, Y. Fe-N-Modified Sludge Biochar for Enhanced Acetic Acid Production from Sludge Anaerobic Fermentation. Sustainability 2025, 17, 3247. https://doi.org/10.3390/su17073247

AMA Style

Wei L, Wan J, Yan Z, Wang Y. Fe-N-Modified Sludge Biochar for Enhanced Acetic Acid Production from Sludge Anaerobic Fermentation. Sustainability. 2025; 17(7):3247. https://doi.org/10.3390/su17073247

Chicago/Turabian Style

Wei, Lingling, Jinquan Wan, Zhicheng Yan, and Yan Wang. 2025. "Fe-N-Modified Sludge Biochar for Enhanced Acetic Acid Production from Sludge Anaerobic Fermentation" Sustainability 17, no. 7: 3247. https://doi.org/10.3390/su17073247

APA Style

Wei, L., Wan, J., Yan, Z., & Wang, Y. (2025). Fe-N-Modified Sludge Biochar for Enhanced Acetic Acid Production from Sludge Anaerobic Fermentation. Sustainability, 17(7), 3247. https://doi.org/10.3390/su17073247

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