Sustainable Bio-Ammonia Recovery from Livestock Wastewater via Biochar-Immobilized Microbial Ammonification

Abstract

Livestock wastewater is an important source of nitrogen pollution, but it also represents a potential feedstock for nitrogen recovery. In this study, longan wood-derived biochar was used as an immobilization carrier in a bio-ammonification system developed with aerobic and facultative ammonifying bacteria isolated from swine wastewater. The system was designed to enhance microbial retention and promote the conversion of organic nitrogen into ammonium concentration (NH₄⁺-N) under oxygenated conditions. Among the tested strains, Lysinibacillus sp. (strain 4-1) showed the highest ammonification activity, reaching an NH₄⁺-N concentration of 340 mg/L in NB medium within 5 days. In sterilized swine wastewater, the biochar-immobilized strain 4-1 achieved 47.17% organic nitrogen removal. The results suggest that coupling microbial ammonification with biochar immobilization may provide a low-carbon approach for nitrogen recovery from livestock wastewater and facilitate decentralized bio-ammonia production.

1. Introduction

Ammonia (NH₃) plays a critical role in both agricultural and industrial sectors because of its versatile reactivity and broad applicability. In agriculture, ammonia is indispensable for producing nitrogen-based fertilizers such as urea, ammonium nitrate, and ammonium sulfate, which are essential for increasing crop yields and ensuring global food security. Approximately 80% of global ammonia production is used in fertilizer manufacturing. In the industrial sector, ammonia serves as a fundamental building block for a wide range of chemical products. It is a precursor for nitric acid, which is used in the manufacture of explosives, synthetic resins, and specialty chemicals. Ammonia also contributes to the production of plastics, synthetic fibers (e.g., nylon and acrylics), dyes, pharmaceuticals, and household cleaning agents, making it indispensable across chemical manufacturing, textile, and consumer goods industries. In addition, ammonia is widely used as an energy-efficient refrigerant (R-717) in food storage, industrial cooling, and air conditioning systems because of its low global warming potential and favorable thermodynamic performance.

Ammonia is gaining attention as a next-generation energy vector. Owing to its high hydrogen density, zero-carbon combustion potential, and established infrastructure for storage and transport, ammonia is being explored as a hydrogen carrier and a clean-burning fuel in decarbonized energy systems [1,2]. However, conventional ammonia production is still dominated by the Haber–Bosch process, which is energy-intensive and strongly dependent on fossil-derived hydrogen, resulting in substantial greenhouse gas emissions [3,4]. Accordingly, the development of alternative and lower-carbon ammonia production pathways has become an important research topic in both environmental and energy fields.

Livestock wastewater is a promising feedstock for biological ammonia recovery because it contains considerable amounts of nitrogen in both organic and ammoniacal forms. If not properly managed, livestock effluents can contribute to eutrophication, odor generation, and secondary nitrogen pollution in surrounding environments [5,6]. At the same time, these waste streams represent an underutilized nitrogen resource that could be converted into value-added ammonia through biological nitrogen transformation. Among the microbial processes involved in nitrogen cycling, ammonification is the key step by which organic nitrogen compounds such as proteins, peptides, and amino acids are mineralized into ammonium (NH₄⁺), thereby producing a recoverable nitrogen form [7,8].

Compared with thermochemical ammonia production, biological ammonification under mild conditions offers the potential for lower energy demand and improved integration with circular waste management systems. Previous studies have demonstrated that ammonia-producing bacteria can convert protein-rich substrates into ammoniacal nitrogen under controlled fermentation conditions [9,10,11]. Several studies have successfully isolated and characterized ammonia-producing bacteria from anaerobic manure pits. Whitehead and Cotta (2004) isolated 40 bacterial strains from swine manure storage pits, several of which produced more than 40 mM ammonia in peptone- and amino acid-based enrichment media [12]. They found the predominant microbial populations of stored swine manure were anaerobic, low-(G + C), Gram-positive bacteria. Few studies have proposed a biological approach to convert protein to ammonia via hyper-ammonia-producing bacteria (HAB) fermentation [13,14,15]. These HAB consortia can utilize nitrogen-containing substrates as their sole nutrient source, enabling simultaneous growth and substantial ammonia production. These studies provide an important basis for developing bio-ammonia routes from biomass-derived nitrogen sources.

Despite the growing interest in biological ammonia production from nitrogen-rich biomass, there remains a lack of stable, carrier-assisted microbial ammonification systems specifically designed for livestock wastewater. Previous studies have focused on microbial screening or ammonia production under controlled conditions, whereas fewer studies have addressed the practical limitations of free-cell systems in wastewater treatment, such as biomass washout, low operational stability, and limited reusability. Microbial immobilization has been widely recognized as an effective strategy to improve the stability and performance of biological treatment systems. Immobilized cells can enhance biomass retention, facilitate solid–liquid separation, improve resistance to environmental stress, and support repeated or continuous operation [16,17].

Therefore, the aim of this study is to develop stable and carrier-assisted microbial ammonification systems specifically designed for nitrogen recovery from livestock wastewater. Rather than targeting the complete removal of all wastewater pollutants, this study specifically focuses on the ammonification step as a nitrogen-recovery-oriented unit process within a broader livestock wastewater valorization framework. Accordingly, the objective of this study was to isolate effective ammonifying bacterial strains from livestock wastewater, evaluate their ammonification capacity, and integrate selected strains with porous biochar as an immobilization carrier. This hybrid approach was designed to enhance microbial retention and ammonification stability in order to improve the conversion of organic nitrogen into ammonium in livestock wastewater.

2. Materials and Methods

2.1. Chemicals

Analytical-grade chemicals and nutrient broth medium (NB) components were purchased from Sigma-Aldrich Chemicals, St. Louis, MO, USA.

2.2. Isolation, Enrichment, and Selection of Appropriate Inoculum Concentration

Ammonifying bacterial strains were isolated from anaerobic sludge and digestate (bioliquid) samples collected from livestock wastewater treatment facilities. The samples were serially diluted with sterile water and spread onto nutrient agar (NA) plates. After incubation at 30 °C, morphologically distinct single colonies were selected and repeatedly streaked onto fresh NA plates until pure cultures were obtained.

For enrichment and preliminary screening, purified isolates were cultivated in nutrient broth (NB) medium containing peptone (15 g/L), D(+)-glucose (1 g/L), sodium chloride (6 g/L), and yeast extract (3 g/L) under aerobic conditions at 30 °C. Among the isolated strains, Lysinibacillus sp. (strain 4-1) and Lysinibacillus sphaericus (strain 4-4) were obtained from anaerobic sludge samples, whereas Bacillus nitratireducens (strain 9-5) was isolated from digestate, as summarized in

Table 1. Source, taxonomic identification, and cultivation conditions of the ammonifying bacterial strains used in this study.

Strain Code	4-1	4-4	9-5
Source of isolation	Anaerobic sludge	Anaerobic sludge	Digestate
Nearest strain and similarity (%)	Lysinibacillus sp. (KDP-SUK-M5), 100%	Lysinibacillus sphaericus (BL7), 100%	Bacillus nitratireducens (VITMPAJ1), 100%
Cultivation period (days)	5	5	5
Cultivation temperature (°C)	30	30	30

. These strains were selected based on their ammonification performance and used for subsequent immobilization and reactor experiments. For inoculum preparation, each strain was introduced into the culture medium at an inoculation ratio of 1:150 (v/v). Strain identification was confirmed through 16S rRNA gene sequencing.

All three strains were cultivated under the same conditions at 30 °C for 5 days prior to subsequent evaluation.

2.3. Preparation of Biochar Carrier

Longan wood biochar used in this study, purchased from SunFan Technology Enterprise Co. (Kaohsiung, Taiwan), was produced in a traditional batch-type kiln at temperatures around 500–600 °C in the absence of oxygen. The resulting biochar was ground to particle sizes of 1–2 mm and sterilized by autoclaving at 121 °C for 30 min before use.

2.4. Determination of Microbial Growth

Cell biomass concentration was determined using the optical density (OD) method. At various time intervals, the OD of the fermentation media was measured at 660 nm using a UV-Vis spectrophotometer (V-360, JASCO International Co., Ltd., Tokyo, Japan). For suspended cultures, OD₆₆₀ was directly measured in 1 cm cuvettes after dilution, if necessary, with sterile NB medium as a blank reference. In the case of samples containing biochar, background absorbance caused by the biochar particles was accounted for by establishing a calibration curve using sterile biochar suspended in the same culture medium. This correction ensured accurate OD readings reflecting only microbial growth.

2.5. Cell Immobilization Using Biochar

For biochar-based immobilization, selected ammonifying bacterial strains were first cultivated in NB medium. Each strain was inoculated into NB medium at a ratio of 1:150 (v/v) and incubated at 30 °C with shaking at 150 rpm for 24 h to allow active cell growth. Pre-sterilized porous longan wood biochar (particle size: 1–2 mm; pyrolyzed at 500–600 °C) was then added directly to 100 mL of the culture broth at biochar loadings of 2 g and 20 g to evaluate immobilization behavior under different carrier amounts. The mixtures were further incubated under the same conditions (30 °C, 150 rpm) for an additional 5 days to allow sufficient microbial attachment and stabilization on the biochar surface before subsequent application. Based on the growth comparison at 2 g and 20 g per 100 mL, the lower biochar loading was selected and proportionally scaled for the reactor experiment.

2.6. Determination of Ammonium Nitrogen

To measure ammonia production, the cultures were maintained in sealed flasks to allow equilibrium between NH₄⁺ and NH₃, and the ammonium nitrogen (NH₄⁺-N) concentration in the culture broth was determined using a nitrogen–ammonia reagent kit (Hach TNT 832, Hach Company, Loveland, CO, USA) based on the salicylate colorimetric method. Briefly, 2.0 mL of well-mixed and filtered sample was transferred into a prefilled TNT plus vial (TNT 832), mixed thoroughly, and allowed to react according to the manufacturer’s instructions. The vials were then measured using a Hach DR3900 spectrophotometer, Hach Company, Loveland, CO, USA with the NH₃-N program, and the results are expressed as NH₄⁺-N for consistency throughout this study.

2.7. Analysis of Wastewater Quality Parameters

To evaluate the chemical characteristics of livestock wastewater and the performance of the ammonification process, the following water quality parameters were analyzed:

i.

pH was measured by a benchtop pH meter (SUNTEX Instruments Co., Ltd., New Taipei City, Taiwan).

ii.

Electrical conductivity (EC) was measured using a conductivity probe with automatic temperature compensation.

iii.

Oxidation–reduction potential (ORP) was measured using a platinum ORP electrode and reference solution.

iv.

Total nitrogen (TN) was determined using the HACH Test ‘N Tube 10071 LR Persulfate Digestion Method. In this procedure, 2.0 mL of a well-mixed sample was added to a digestion vial, digested at 105 °C for 30 min, cooled, and analyzed colorimetrically using the DR3900 (Hach Company, Loveland, CO, USA).

v.

Nitrate nitrogen (NO₃⁻-N) was determined using the HACH 8039 cadmium reduction method with a HACH DR900 portable colorimeter (Hach Company, Loveland, CO, USA). In this method, nitrate is reduced to nitrite by cadmium, followed by color development and colorimetric measurement according to the manufacturer’s instructions. The nitrate concentration was expressed as mg/L NO₃⁻-N.

vi.

Ammonium nitrogen (NH₄⁺-N) was determined as described in Section 2.6.

vii.

Organic nitrogen (Org-N) was estimated as the difference between total nitrogen and the measured inorganic nitrogen species. Since NO₂⁻-N was assumed to be negligible, Org-N was calculated as follows: Org-N = TN − NH₄⁺-N − NO₃⁻-N.

2.8. Reactor Operation for Ammonia Production

To simulate on-farm wastewater treatment scenarios, a laboratory-scale bio-ammonification system was established using sterilized swine wastewater and biochar carriers, as shown in Figure 1. The experimental setup consisted of a 9 L stirred reactor, a thermostatic water bath connected to the reactor to maintain the operating temperature at 30 °C, and a control unit for continuous monitoring and recording of the reactor temperature and oxidation–reduction potential (ORP). During the ammonification experiment, the reactor solution was gently stirred to maintain homogeneous mixing.

The swine wastewater was sourced from a livestock facility operated by Taiwan Sugar Corporation (Taisugar), Tainan, Taiwan—a major state-owned enterprise managing integrated pig farming systems in Taiwan. The sample was collected from the anaerobic effluent stream at a commercial-scale pig farm and transported to the laboratory under cooled, sealed conditions to preserve its chemical and microbial integrity. To eliminate indigenous microbial interference and standardize the testing environment, the wastewater was autoclaved at 121 °C for 20 min before use.

The system was designed to evaluate microbial ammonification under conditions representative of nutrient-rich livestock effluents. Ammonifying bacterial strains were first pre-cultivated in 100 mL of sterilized swine wastewater and incubated at 30 °C for 3 days at 160 rpm in a shaking incubator to allow physiological adaptation and activation.

Following pre-cultivation, the bacterial suspension was inoculated into 2.9 L of fresh sterilized swine wastewater for ammonification experiments. In the biochar-immobilized treatment, 50 g of pre-sterilized longan wood-derived biochar was added to the reactor to serve as the microbial carrier. The reactor was maintained under aerobic conditions with gentle aeration at 30 °C for 5 days to promote organic nitrogen conversion and ammonium accumulation. The experiments conducted in sterilized swine wastewater were performed as a preliminary single-run feasibility test; therefore, the corresponding data are presented descriptively and were not subjected to statistical analysis.

3. Results and Discussion

Biochar, a porous carbonaceous material derived from biomass pyrolysis, has gained considerable attention as a sustainable biomass-derived adsorbent for water treatment. Its high surface area, functional group diversity, and tunable pore structure make it effective for removing a wide range of contaminants, including nutrients (e.g., ammonium, nitrate, and phosphate), heavy metals, organic pollutants, and microbial pathogens [18,19,20]. When integrated into biological processes, biochar improves microbial retention, buffering capacity, and contaminant loading tolerance. Its use not only enhances treatment efficiency but also contributes to carbon sequestration and circular resource recovery, making it a promising material for sustainable environmental remediation.

3.1. Characterization of Biochar-Immobilized Strains

3.1.1. SEM Observation

The morphology of bacterial strains immobilized within the pores of biochar was observed by scanning electron microscopy (SEM), as shown in Figure 2A–C. The strains Lysinibacillus sp. (strain 4-1), Lysinibacillus sphaericus (strain 4-4), and Bacillus nitratireducens (strain 9-5) were observed attaching densely along pore walls and accumulating within large macro- and mesopores (>10 µm). SEM images revealed numerous rounded bacterial cells embedded along pore edges, suggesting active colonization within the pores and early-stage biofilm development [21]. The rough texture and well-developed porous structure of the biochar provided favorable sites for microbial entrapment and immobilization. Such immobilized cells have been shown to offer multiple benefits, including effective spatiotemporal management of biomass within contaminated media, efficient separation of biomass from treated water, prolonged survivability and stability, and reusability without significant loss of activity [22]. These findings highlight the suitability of biochar as a robust carrier material for sustainable bio-ammonification processes. The dense attachment of strains 4-1, 4-4, and 9-5 within the macro- and mesopores of the biochar is consistent with previous findings reported by Lago et al. [21], who demonstrated that porous carrier materials facilitate microbial biofilm formation, enhance biomass retention, and reduce cell washout during wastewater treatment processes. The observed colonization pattern, therefore, supports the role of biochar not only as a physical support but also as a microenvironment favorable for sustained microbial activity.

3.1.2. Screening Microbial Growth in NB Medium Without Biochar as a Carrier

The growth profiles of three ammonifying bacterial strains—strain 4-1, strain 9-5, and strain 4-4—were evaluated in NB medium over a 5-day cultivation period based on OD₆₆₀ measurements, and the results are shown in Figure 3. The OD₆₆₀ readings reflect light scattering by suspended bacterial cells, which increases proportionally with cell biomass, and thus allow for real-time monitoring of growth progression. The blank control (NB medium without inoculation) showed a minimal change in OD₆₆₀ throughout the experiment, confirming that the observed turbidity increases in the inoculated groups were due to bacterial cell growth rather than medium interference. These results indicate differential growth performance among the tested strains under the given culture conditions.

All inoculated groups showed a sharp increase in optical density within the first 24 h. There was no obvious initial lag phase for any of the strains, indicating rapid initial proliferation. A stationary phase was observed from Day 3 onward for strains 4-1 and 9-5. Strain 4-4 showed slower growth under the tested conditions and only moderate biomass accumulation; therefore, it was excluded from further analysis.

3.1.3. The Effect of Biochar Addition on Microbial Growth

Compared to free-living microbial cells, immobilized or encapsulated bacteria offer significant advantages for biological processes. For example, free cells often suffer from low localized biomass density, are prone to washout during treatment, and are more vulnerable to environmental stress. In contrast, immobilized systems enable better spatial and temporal control of microbial activity, facilitate easy separation from treated effluents, and improve the long-term stability, survivability, and reusability of the microbial consortium without substantial loss of activity [23]. In this study, the immobilization of bacterial strains onto the biochar carrier was achieved through physical adsorption, without the use of any chemical adhesives or binding agents. This approach relies on natural interactions between the microbial cell surface and the porous biochar matrix—primarily van der Waals forces, ionic interactions, and hydrogen bonding [21].

Figure 4 illustrates the growth behavior of strain 4-1 and strain 9-5 under free-cell and biochar-immobilized conditions. Biochar derived from longan wood was added at 2 g or 20 g per 100 mL of medium. The immobilization relied entirely on passive adsorption, where microbial cells adhered to the porous carbon surface via physical interactions. The results showed that in the absence of biochar (free-cell conditions), both strains exhibited rapid biomass accumulation, reaching peak OD₆₆₀ values of 1.75 (strain 4-1) and 1.65 (strain 9-5). Moderate biochar addition (2 g) initially resulted in a slight delay in exponential growth, likely due to partial immobilization, but both strains recovered and reached OD levels comparable to the free-cell groups by Day 4. On the other hand, excessive biochar loading (20 g) consistently limited growth, which may be attributed to physical crowding, reduced nutrient diffusion, or adsorption of essential components. By Day 5, all treatments—including those with 2 g and 20 g of biochar—showed OD₆₆₀ values exceeding 1.0, indicating that biochar addition did not hinder bacterial proliferation. Although the immobilized groups showed a slightly slower increase in OD₆₆₀ during the initial stage, their growth profiles became more stable after Day 3 and remained comparable to those of the free-cell treatments. These results suggest that biochar immobilization did not impair microbial activity but instead provided a more stable microenvironment for bacterial growth. Similar behavior has been reported by Mehrotra et al. [22], who indicated that immobilized bacteria generally exhibit improved long-term stability, greater resistance to environmental stress, and more sustained metabolic activity than free-cell systems. Combined with the SEM observations in Figure 2, the results indicate that the addition of biochar at appropriate levels did not inhibit the growth or proliferation of ammonifying bacteria. Moreover, the colonization of bacterial cells on the surface and within the pores of the biochar confirms its function as an effective carrier during the ammonification process. This behavior agrees with the observations of Lago et al. [21], who reported that immobilization on porous carriers improves microbial attachment and minimizes biomass loss from the liquid phase, thereby supporting more stable treatment performance. In this study, 2 g of biochar was used in the subsequent experiments.

3.2. Effects of Biochar Addition on pH Dynamics and Ammonifying Bacterial Activity

Ammonification is a critical step in the biological mineralization of organic nitrogen, involving the microbial hydrolysis and deamination of nitrogen-containing compounds such as proteins, peptides, and amino acids [8,24]. This process leads to the formation of ammonia (NH₃), which equilibrates with ammonium ions (NH₄⁺) in aqueous environments, depending on the prevailing pH and temperature conditions. Previous studies have shown that several environmental factors influence the ammonification process, including temperature, pH, C/N ratio, and nutrient availability [8,24]. The general reaction can be expressed as follows:

R-CH(NH₂)-COOH + H₂O→R-CH(OH)-COOH + NH₃

(1)

where R represents the organic side chain.

Figure 5 illustrates pH changes in cultures containing biochar only (char), bacterial strains (strain 4-1 and strain 9-5) individually, and combinations of each strain with biochar (4-1+char and 9-5+char), compared to a blank control. All inoculated groups showed a marked increase in pH, reaching above 8.5 by Day 5. The biochar-only group showed a moderate pH rise (max ~8.2) due to its inherent alkalinity. In contrast, the blank control maintained a relatively stable pH near neutral (7.0–7.2), suggesting minimal biological activity. As shown in Figure 6, ammonium nitrogen (NH₄⁺-N) production in NB medium over five days revealed the effects of both microbial inoculation and biochar addition on the activity of nitrogen transformation. The blank and biochar-only groups maintained low and moderate NH₄⁺-N levels throughout the incubation. Taken together, the observed pH variation and ammonium production indicate that microbial ammonification was the primary factor driving both alkalinity increase and nitrogen conversion in the NB medium.

In NB medium, strain 4-1 in the free-cell group showed the highest peak concentration (~340 mg/L on Day 4), followed by a sharp decrease by Day 5, suggesting possible NH₃ volatilization, uptake, or equilibrium shifts. Although the NH₄⁺-N concentration obtained in this study was lower than that reported for hyper-ammonia-producing bacteria isolated from swine manure pits, the present strains still showed substantial ammonification activity under aerobic conditions. Whitehead and Cotta [12] reported ammonia production exceeding 40 mM by anaerobic isolates from swine manure storage pits, whereas the lower values observed here are likely attributable to the use of raw wastewater and aerobic incubation conditions. When biochar was used as a microbial carrier (4-1+char and 9-5+char), NH₄⁺-N production remained consistently high but showed more stable profiles across days, with final concentrations ranging between 180 and 210 mg/L. This indicates that immobilization on biochar maintained ammonification activity while possibly buffering against peak accumulation or loss. Nevertheless, the pH trends confirm that microbial activity—rather than biochar alone—was the primary driver of alkalinity increase, and that biochar-supported immobilization maintained sufficient metabolic function for effective nitrogen transformation.

3.3. Bio-Ammonia Generation in Sterilized Swine Wastewater

Table 2. Comparison of key water quality parameters of crude and treated wastewater after 5 days under different ammonification treatments.

	pH	TN (mg/L)	NH4+-N (mg/L)	COD (mg/L)	ORP (mV)	NO3-N (mg/L)	Org-N (mg/L)	Org-N Removal (%)
Crude wastewater	6.85	808	24	3108	120	22	762	-
Treatments	After 5 days
Blank	7.2	1530	30	4660	155	1.1	1499	23.52%
Char	7.28	960	120	3610	3	1	830	47.15%
Strain 4-1	8.81	1290	220	2270	15	1.7	1068.3	26.32%
4-1+char	8.51	790	160	1870	39	1.3	628.7	47.17%
Strain 9-5	8.72	1310	170	3120	4	1.1	1138.9	1.69%
9-5+char	8.5	910	160	2630	76	1.2	748.8	32.54%

summarizes the key water quality parameters of the crude wastewater and the treated wastewater after 5 days under different ammonification treatments. The crude wastewater exhibited typical characteristics of a nitrogen- and carbon-rich livestock effluent, with an initial pH of 6.85, total nitrogen (TN) of 808 mg/L, NH₄⁺-N of 24 mg/L, COD of 3108 mg/L, and organic nitrogen of 762 mg/L. Only 24 mg/L of the 808 mg/L total nitrogen was initially present as NH₄⁺-N, indicating that approximately 97% of nitrogen remained in organic or other non-mineralized forms. These characteristics suggest that the wastewater provided a suitable and representative matrix for evaluating microbial ammonification performance, particularly with respect to the bioconversion of organic nitrogen into ammonium. Accordingly, the experimental design focused on assessing the ability of selected ammonifying bacterial strains, in both free-cell and biochar-immobilized forms, to promote organic nitrogen mineralization and enhance ammonium recovery under controlled aerobic conditions.

Figure 7 shows pH variation during a 5-day incubation in sterilized swine wastewater treated with different ammonifying bacterial strains and biochar carriers. Figure 8 illustrates ammonium nitrogen (NH₄⁺-N) concentration profiles during the same incubation period in sterilized swine wastewater. The temporal profiles of pH and ammonium nitrogen (NH₄⁺-N) concentrations during the 5-day incubation provide complementary insights into the microbial ammonification processes occurring in the swine wastewater system. First, a continuous increase in pH was observed in all microbial treatments, in contrast to the biochar-only control, which showed a limited and transient pH rise. This alkalization trend correlates well with the accumulation of NH₄⁺-N, as ammonia production through microbial deamination typically results in a shift toward more alkaline conditions. These results are consistent with previous studies showing that the optimal pH range for the ammonification process is 6.5–8.5 [25,26]. Among the tested strains, strain 4-1 exhibited the most pronounced effects, with a sharp pH increase to above 8.8 and a peak NH₄⁺-N concentration on Day 4, indicating vigorous ammonification activity. The 4-1+char group followed a similar pattern but with a more gradual rise and slightly lower NH₄⁺-N levels, suggesting that biochar immobilization supported microbial function while moderating ammonium accumulation. In comparison, strain 9-5 and its biochar-immobilized counterpart (9-5+char) demonstrated a more stable and moderate increase in both pH and NH₄⁺-N, reflecting a slower but sustained ammonification performance. Notably, the biochar-only control did not show significant NH₄⁺-N accumulation or sustained pH elevation, confirming that the observed effects were driven primarily by microbial activity rather than abiotic factors. In the present study, the highest NH₄⁺-N concentration obtained with strain 4-1 was approximately 342 mg/L in sterilized swine wastewater, which was markedly lower than the >8000 mg/L reported by Bello and co-workers during anaerobic fermentation of soy meal protein. This discrepancy is likely attributable to the different substrate characteristics and operational conditions [11]. Bello et al. employed a protein-rich substrate under strictly anaerobic conditions specifically optimized for hyper-ammonia-producing bacteria, whereas the present study used raw swine wastewater with a lower readily biodegradable protein content and operated under aerobic conditions.

In addition, the lower NH₄⁺-N concentration observed in the present study may also reflect partial ammonium adsorption by the biochar matrix [27,28]. Accordingly, part of the produced ammonium may have been retained on or within the biochar rather than remaining entirely in the aqueous phase for direct measurement. This interpretation is consistent with the more gradual and stable NH₄⁺-N accumulation observed in the biochar-immobilized treatments. Therefore, the lower NH₄⁺-N concentration observed here likely reflects both process-related differences from previous anaerobic ammonia-production studies and partial ammonium retention by the biochar carrier. The lower measured NH₄⁺-N concentration in the immobilized groups indicates that future optimization may be needed to improve ammonium recovery efficiency. Future optimization of the biochar surface properties, including its hydrophilic–hydrophobic balance, may help reduce excessive ammonium retention on the carrier and improve ammonium recovery in the liquid phase. Nevertheless, the observed ammonification performance indicates that the selected strains were capable of converting organic nitrogen present in livestock wastewater into recoverable ammonium under environmentally relevant conditions.

Figure 9 shows the variation in organic nitrogen concentration (Org-N) in sterilized swine wastewater over a 5-day incubation period under different ammonification treatments and a blank control. In the blank treatment, Org-N remained at a consistently high level throughout the incubation period. In contrast, all microbial treatments resulted in different degrees of organic nitrogen reduction, indicating active nitrogen transformation. Among the free-cell treatments, strain 4-1 showed the greatest reduction, reaching 26.32% by Day 5, which suggests stronger ammonification activity than strain 9-5 under the tested conditions.

A marked decrease in Org-N was also observed in the biochar-only treatment, despite the absence of bacterial inoculation. This result suggests that biochar itself contributed to organic nitrogen removal, likely through adsorption of soluble nitrogen-containing compounds and associated surface interactions. Previous studies have shown that porous biochar materials possess high surface area, developed pore structure, and diverse surface functional groups, enabling them to adsorb a wide range of contaminants, particularly nitrogen species, through mechanisms such as surface adsorption, pore filling, and electrostatic interactions [18,19,20,27,28]. This interpretation is consistent with the findings of Wu et al. [27], who reported that biochar can exhibit a strong affinity for dissolved nitrogen compounds, and with Gao et al. [28], who emphasized that biochar in livestock wastewater systems may function not only as a microbial support medium but also as an active material that promotes nitrogen retention and resource recovery. Therefore, the decrease in Org-N observed in the biochar-only treatment was likely attributable, at least in part, to the physicochemical properties of the biochar matrix rather than to microbial ammonification alone.

When biochar was combined with ammonifying strains, different responses were observed depending on the bacterial strain. The 4-1+char treatment exhibited the lowest final Org-N concentration and the highest organic nitrogen reduction among all tested treatments, indicating a favorable interaction between microbial ammonification and the physicochemical properties of the biochar matrix. The temporal profile also showed a more gradual and sustained decrease in Org-N compared with the corresponding free-cell treatment, suggesting a possible synergistic effect between strain 4-1 and biochar. In contrast, the 9-5+char treatment showed improved Org-N reduction relative to the corresponding free-cell treatment, but the extent of improvement was less pronounced than that observed for 4-1+char. These results suggest that the effect of biochar immobilization was strain-dependent.

The present findings are also consistent with previous immobilization studies showing that porous carrier materials can enhance microbial attachment, improve biomass retention, reduce cell washout, and provide a more stable microenvironment for sustained biological activity [21,22,23]. In agreement with these reports, SEM observations in the present study confirmed dense bacterial colonization on the biochar surface and within its pore structure, while the biochar-immobilized treatments showed more stable growth and nitrogen transformation behavior than the corresponding free-cell treatments. Compared with previous studies, the present work further extends the role of biochar from a passive sorptive material to a functional carrier for ammonifying bacteria in livestock wastewater treatment. Overall, the combined performance of the immobilized treatments may be attributed to the dual role of biochar as both a microbial carrier and a reactive sorptive matrix, providing a favorable microenvironment for bacterial activity while also contributing to nitrogen transformation and retention. This interpretation is consistent with the observations of Lago et al. [21], Mehrotra et al. [22], and Martins et al. [23], who highlighted the advantages of immobilized microbial systems for improving treatment stability and biological performance in wastewater applications.

4. Conclusions

This study demonstrates the feasibility of microbial ammonification as a strategy for recovering inorganic nitrogen from nitrogen-rich swine wastewater. Among the tested treatments, the biochar-immobilized strain 4-1 system achieved the highest organic nitrogen removal (47.17%) in sterilized swine wastewater, indicating the benefit of coupling microbial ammonification with a porous carbon support. Biochar functioned as an effective carrier for bacterial colonization and contributed to stable ammonification performance during the treatment period. Collectively, these findings suggest that biochar-immobilized ammonifying bacteria have potential as a low-carbon, modular, and decentralized approach for sustainable nitrogen recovery in livestock wastewater treatment systems.

The present study specifically focused on the ammonification process and ammonia recovery pathway through the conversion of organic nitrogen into NH₄⁺. Because the sterilized swine wastewater experiment was conducted as a preliminary single-run feasibility test without biological replicates, these findings should be interpreted with caution. Future work should therefore include replicated experiments, together with validation under non-sterile conditions and integration with upstream biological pretreatment for COD and lipid reduction. These efforts may improve overall process applicability and support the development of a more comprehensive livestock wastewater valorization framework.