New insights into the production of volatile fatty acids through low-temperature anaerobic fermentation of sludge enhanced by peracetic acid

Excess sludge (ES), an ancillary product of the biological wastewater treatment process, is characterized by its high water content, resistance to degradation, and propensity to transmit pathogens (Liu et al. 2022a, b). Research has validated that the treatment costs associated with ES constitute approximately 60% of the operational expenses of a wastewater treatment facility (Garrido-Baserba et al. 2018). Furthermore, ES is a rich source of organic matter, including proteins and polysaccharides, which can be transformed into alternative resources for recovery through appropriate processing. Anaerobic fermentation, driven by microbial metabolism, converts organic matter into valuable products such as hydrogen, methane, and volatile fatty acids (VFAs) (Zhao et al. 2018; Yuan et al. 2019; Liang et al. 2021; Kan et al. 2022a). In recent years, the application of ES to anaerobically produce VFAs has garnered considerable attention (Zhang et al. 2023a). This interest stems from the fact that VFAs can serve as a high-quality carbon source for intensifying the removal of nutrients in wastewater and are also a viable raw material for the production of biodegradable plastics (Atasoy et al. 2018; Zhao et al. 2024). Consequently, anaerobic fermentation is regarded as a sustainable and low-carbon strategy for ES management, aligning with the goals of environmental conservation and resource efficiency.

A key factor in the ES anaerobic fermentation for the VFA production is the operating temperature (Cubero-Cardoso et al. 2022). Studies have confirmed that an optimal increase in fermentation temperature benefits microbial metabolism and the expression of key enzyme activities (Rubio et al. 2022; Zhang et al. 2023b). However, in practice, especially in some cold regions, low temperatures limit microbial metabolic activity, leading to insufficient utilization of organic matter within ES. Low temperatures can cause a decrease in enzyme activity, particularly key enzymes involved in the hydrolysis and acidification processes, such as acetate kinase (AK). Therefore, enhancing the hydrolysis and acidification of organic matter during low-temperature digestion of ES is crucial for increasing the accumulation of high-value products like VFAs. To improve the solubilization of organic matter, various pretreatment strategies such as ultrasound, enzymatic hydrolysis, and irradiation have been employed to enhance the ES anaerobic digestion efficiency at the laboratory scale. Among these, enzymatic hydrolysis pretreatment is favored for large-scale application due to its effectiveness and the mild conditions it employs, which do not adversely affect the nutritional value of the ES, and many other types of pretreatment are, at best, experimental and may not be scalable to the full extent for practical applications (Chuenchart et al. 2021; Banu et al. 2021). However, these strategies have the disadvantage of high energy or chemical input. Consequently, there is significant interest in developing economical and efficient methods to improve VFAs production through low-temperature ES anaerobic fermentation.

Peracetic acid (PAA), known for its potent oxidative properties, serves as a versatile disinfectant and bactericide, effectively neutralizing bacteria, viruses, and other microorganisms (Ao et al. 2021). Its applications span across water treatment, food processing, and public health sectors. In recent years, the utilization of PAA has expanded to enhance ES dewatering capabilities, soil remediation, and air purification. For instance, Appels et al. (2011) employed a 15% PAA solution for sludge pretreatment over a period of 24 h, and the findings indicated that PAA treatment significantly facilitated the dissolution of organic matter. However, an excessive dosage was found to inhibit anaerobic microorganisms, leading to a decrease in biogas production. Liu et al. (2022a, b) investigated the impact of PAA on the anaerobic digestion of poultry wastewater, and their findings revealed that PAA exerted a prolonged influence on methane production, particularly affecting the methane generation by acetogenic bacteria. Recently, PAA has emerged as an oxidizing agent to enhance the ES anaerobic fermentation for the recovery of high-value energy substances, such as hydrogen and VFAs. For instance, Li et al. (2022) discovered that an 8.0 mg/g dosage of PAA was optimal for boosting hydrogen production during sludge dark fermentation, achieving a hydrogen yield as high as 14.2 mL/g of volatile suspended solids (VSS). However, an excessively high concentration of PAA was found to diminish the yields of both hydrogen and methane. PAA, in combination with other pretreatment technologies, has also been proven to effectively promote the recovery of carbon from sludge. Sun et al. (2024) reported that the combination of PAA with free ammonia pretreatment can effectively enhance the production of short-chain fatty acids (SCFAs) and improve the subsequent biosynthesis of polyhydroxyalkanoates (PHAs). Li et al. (2023) investigated the combined pretreatment of PAA and ultrasound to increase the accumulation of SCFAs in sludge anaerobic fermentation and found that the PAA pretreatment enhanced by ultrasound-enriched acid-producing microorganisms. These innovative studies provide valuable insights for the application of PAA in the field of sludge resource recovery. However, most of the aforementioned investigations have focused on ambient or high-temperature conditions, and the impact of PAA on the ES low-temperature anaerobic fermentation has rarely been explored. In addition, the influence of PAA on the bioconversion patterns of organic matter in ES and the activity of key enzymes is also not well understood. Elucidating the impact and mechanisms of PAA on the ES low-temperature anaerobic fermentation for the VFAs production provides technical support for the high-value resource utilization of ES.

Therefore, this work investigated the feasibility of using PAA to enhance the ES low-temperature anaerobic fermentation for VFAs production and revealed the related mechanisms. First, the influence of PAA content on the VFAs production in enhanced ES low-temperature anaerobic fermentation was systematically explored. Then, the impacts of PAA on the transformation characteristics of organic matter in ES were analyzed, and the effects of PAA on the solubilization and acidification processes during the ES anaerobic fermentation were also researched. Finally, the effects of PAA exposure on key enzyme activities in the ES anaerobic fermentation process were examined. The results of this work offer new ideas for the application of PAA pretreatment strategies in the low-temperature anaerobic production of VFA from ES.

ES was sourced from the sludge return pipe of the secondary sedimentation tank at a wastewater treatment plant located in Huangdao District, Qingdao, China. The inoculum was derived from the anaerobic zone of the A²/O process employed by the same wastewater treatment facility. Upon collection, both the ES and the inoculum were subjected to a 1.0 mm mesh filtration and allowed to settle for 24 h. Subsequently, they were stored at 4 °C in a refrigerator for later use. The main characteristics of the ES and inoculum are presented in Table 1.

Table 1

Main physical and chemical characteristics of the inocula and ES used in the experiment

Item .	Units .	ES .	Inoculum .
pH	/	7.2 ± 0.2	6.8 ± 0.1
Total suspended solids (TSS)	g/L	13.2 ± 0.5	15.8 ± 0.4
Volatile suspended solids (VSS)	g/L	8.6 ± 0.4	12.7 ± 0.7
Total COD (TCOD)	g/L	15.4 ± 0.8	19.5 ± 0.6
Soluble COD (SCOD)	g/L	0.64 ± 0.03	0.52 ± 0.07
Soluble protein (SPN)	mg/L	215 ± 16	226 ± 19
Soluble polysaccharides (SPS)	mg/L	116 ± 6	135 ± 5.8
-N	mg/L	26.3 ± 1.1	29.8 ± 1.6

Item .	Units .	ES .	Inoculum .
pH	/	7.2 ± 0.2	6.8 ± 0.1
Total suspended solids (TSS)	g/L	13.2 ± 0.5	15.8 ± 0.4
Volatile suspended solids (VSS)	g/L	8.6 ± 0.4	12.7 ± 0.7
Total COD (TCOD)	g/L	15.4 ± 0.8	19.5 ± 0.6
Soluble COD (SCOD)	g/L	0.64 ± 0.03	0.52 ± 0.07
Soluble protein (SPN)	mg/L	215 ± 16	226 ± 19
Soluble polysaccharides (SPS)	mg/L	116 ± 6	135 ± 5.8
-N	mg/L	26.3 ± 1.1	29.8 ± 1.6

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The PAA was procured from a bioreagent company located in Nanjing, China. The principal characteristics of the PAA are as follows: it boasts a purity exceeding 97.5%, with the chemical formula C₂H₄O₃, a molecular weight of 76.05, and a structural formula represented as CH₃COOOH, with the CAS Registry Number 79-21-0. PAA is a colorless and transparent liquid, exhibiting weak acidity, high volatility, a strong, pungent odor, and carries a pronounced acetic acid scent.

Sequential batch reactors (SBRs) were utilized to assess the impact of PAA on the ES low-temperature anaerobic fermentation for the VFA production. The experimental setup involved custom-made SBRs with a working volume of 5.0 L, constructed from resin glass in the form of cylindrical vessels. The base diameter, height, and aspect ratio of the SBRs are denoted as 16 cm, 25 cm, and 0.64, respectively. A mechanical stirrer was installed at the base of the SBRs to maintain a rotation speed of 240–300 rpm during the operation, ensuring adequate mixing and contact between the inoculum and ES. Above the SBRs, a 2.0 L dual-valve gas collection bag was positioned and connected to the reactors via rubber tubing.

Five sets of parallel reactors, each comprising three units, were employed to investigate the impact of PAA on the ES low-temperature anaerobic fermentation. Each reactor was inoculated with 3.0 L of ES and 2.0 L of inoculum to ensure a volumetric ratio of digestion substrate to inoculum of 1.5. Subsequently, PAA was added to each reactor to achieve the predetermined content of 0, 3, 6, 9, and 12%, based on the dry weight of ES. The choice of PAA exposure dosage was slightly modified according to the previous literature (Li et al. 2023). Each reactor was then purged with high-purity nitrogen to establish anaerobic conditions, followed by sealing with rubber stoppers. The reactors were subsequently transferred to a temperature-controlled shaker incubator set at 10 °C for the experimental trials. Throughout the anaerobic fermentation of ES, the composition of the fermentation liquid, sludge, and biogas was monitored every 2 days to track the changes in content. This experiment aims to elucidate the impact of PAA on the low-temperature ES anaerobic fermentation by comparing the operational performance of reactors across different PAA exposure groups.

The determinations of total suspended solids (TSS), VSS, protein (PN), polysaccharides (PS), soluble protein (SPN), soluble polysaccharides (SPS), and soluble chemical oxygen demand (SCOD) were conducted in accordance with national standard methods. The measurement of total organic carbon (TOC) was performed using a total organic carbon analyzer (SQ-TOC20A), while pH was assessed with an online pH meter. The determinations of methane and VFAs were conducted using gas chromatography. The specific model of the gas chromatograph used is Shimadzu GC-2010, and the detailed procedure for the analysis can be found in the preceding literature (Aramrueang et al. 2022). The anaerobic digestion of organic matter involves a variety of functional enzymes, including proteases, amylases, AK, butyrate kinase (BK), phosphotransacetylase (PTA), and F420, with detailed methods for their determination extensively presented in previous publications (Zhao et al. 2019; Chen et al. 2021).

The organic matter within ES is primarily composed of PN and PS (Fu et al. 2023; Wang et al. 2024). The presence of PAA similarly significantly elevates the concentrations of SPN and SPS in the fermentation liquid. As shown in Figure 2, both SPN and SPS concentrations exhibit a pattern of initial increase followed by a decrease over the digestion time. In the early stages of digestion, the decline in SPS concentration is due to the fact that SPS is not rapidly solubilized, leading to its utilization by microorganisms, which in turn causes the curve to drop. The second decline is due to the fact that the solubilization of SPS within the sludge has reached its maximum value, and the microorganisms utilize SPS for metabolism, resulting in a decrease in SPS concentration. In the control, the maximum concentrations of SPN and SPS were 944 and 861 mg/L, respectively, corresponding to a fermentation time of 14 and 18 days. When PAA was present, the concentrations of SPN and SPS increased to varying degrees, with higher concentrations of PAA generally leading to more pronounced increased in SPN and SPS concentrations. For instance, when the PAA content was 3%, the maximum concentrations of SPN and SPS were 1,311 and 882 mg/L, respectively, while when the PAA content was 12%, the concentrations of SPN and SPS rose significantly to 2,031 and 1,211 mg/L, markedly higher than in other groups. PAA effectively lyses the particulate matter within ES, thereby increasing the concentrations of SPN and SPS in the fermentation liquid, enhancing the activity of hydrolytic and acidogenic microorganisms, and providing a material basis for the subsequent methanogenic process. PAA can break the carbon–carbon bond and the carbon–hydrogen bond of long-chain organic molecules, thus degrading a macromolecular organic matter into a small molecular organic matter (Maghrebi et al. 2021).

Low temperatures can reduce microbial metabolic activity and the expression of key enzymes, thereby decreasing the extent of sludge resource utilization. In this study, the oxidant PAA was utilized to enhance the fixation of carbon in the form of VFAs during the ES low-temperature fermentation (Kan et al. 2021a, 2022b). The recommended dosage of PAA is 9%, with the maximum accumulation of VFAs reaching 239.5 mg COD/g VSS, approximately 1.47 times that of the control group. As an environmentally friendly oxidant, the application of PAA in sludge pretreatment helps to reduce secondary pollution issues in chemical sludge treatment processes. According to previous literature, the main degradation products of PAA in an anaerobic system are free radicals, acetic acid, and carbon dioxide, all of which are clean and non-toxic products (Chen et al. 2021; Liu et al. 2022a, b). Investigating the degradation pattern of PAA in the anaerobic digestion system and the characteristics of its main products will also be a major focus of future work. The dosage of 3% PAA might be too high for practical engineering applications. We will carry out relevant research on the PAA dosage and enhanced ES resource utilization in future experiments. Although previous studies have reported on the coupling of PAA with other pretreatments such as ultrasound and free ammonia to enhance VFA production (Li et al. 2023; Ren et al. 2024), the exploration of PAA application to strengthen sludge resource utilization under low-temperature operating conditions should be a novel approach, broadening the application of PAA at low temperature.

This study reports a novel strategy for enhancing the accumulation of VFAs through low-temperature sludge anaerobic fermentation with PAA and elucidates the underlying mechanisms. PAA effectively facilitated the sludge disintegration and hydrolysis processes, increasing the concentration of SCOD in the fermentation liquid and enhancing the utilization of ES, thereby providing a material basis for the acidification process. The optimal dosage of PAA was 9%, with the maximum accumulation of VFAs reaching 239.5 mg COD/g VSS, approximately 1.47 times that of the control group. The presence of PAA inhibited the methanation process, reducing the consumption of VFAs and thus enhancing their accumulation.

This work was financially supported by the National Natural Science Foundation (51908305), the Natural Science Foundation in Shandong Province of China (ZR2024ME086), and Special Funding for Postdoctoral Station, the project funded by China Postdoctoral Science Foundation (2023T160349).

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.