Optimizing Winter Composting of Swine Manure Through Housefly Larva Bioconversion: Mechanisms of Protein Recovery and Enzymatic Nitrogen Regulation

Abstract

Sustainable manure recycling in cold climates faces low efficiency and nutrient loss. This study evaluated housefly larva-pretreated manure (HL) for winter swine manure composting in East China, comparing it to sawdust-conditioned (CK2) and untreated manure (CK1). Larval pretreatment converted 12.71% of manure weight into biomass, assimilating 10.69% C, 30.55% N, 8.54% P, and 11.53% K. Harvested larvae contained 53.35% crude protein, with amino acids matching/exceeding fishmeal and soybean meal, while heavy metals were below safety limits. Theoretical annual larval protein yield per unit area (29,530 kg·mu⁻¹·year⁻¹) was 206.5 times higher than soybean crops. During composting, the HL treatment promoted early protease and catalase activation. This enzymatic synergy accelerated organic matter degradation and maturation, achieving a germination index of 147.67% by day 51. Coordinated nitrate and nitrite reductase activity in HL facilitated efficient denitrification, minimizing NO₂⁻ accumulation and N₂O emissions. Consequently, HL composting achieved faster stabilization, enhanced nutrient retention, and greater protein recovery compared to controls. These findings demonstrate that housefly larval pretreatment offers a climate-resilient and scalable strategy for winter manure management and protein valorization, with strong potential for applications in cold and resource-limited agricultural systems worldwide.

1. Introduction

The rapid intensification of livestock and poultry farming worldwide has disrupted the traditional crop–livestock nutrient cycle, placing increasing strains on the environment. In 2024, China produced 96.63 million tons of pork, beef, mutton, and poultry, generating approximately 3.05 billion tons of manure [1,2]. Livestock waste is rich in nitrogen and phosphorus, but its utilization rate remains low at only 60% [3]. Common management methods include land application, anaerobic digestion, and conversion into fertilizer or feed. These practices often cause nutrient loss and environmental risks [4]. Similar challenges are also observed globally. For example, according to the United States Department of Agriculture (USDA), the state of Georgia alone produced ~1.3 billion broiler chickens in 2021, accounting for ~14% of the national total (National Agricultural Statistics Service, 2022). This massive poultry production contributes to the ~1.4 billion tons of livestock and poultry manure generated annually in the United States [5]. These figures highlight the global scale of manure management challenges across Asia, North America, and Europe. In response, many regions are advancing circular economy strategies that couple manure treatment with value-added resources. In 2024, the Ministry of Agriculture and Rural Affairs in China issued a policy encouraging the use of novel protein sources, including insects and algae, thereby creating new pathways for converting manure into high-quality protein and organic fertilizer [6]. The European Union and North America are also investigating insect-based composting and alternative protein production as part of sustainable nutrient recycling [7].

Aerobic composting, which relies on the microbial degradation of organic matter through extracellular enzymes, is the predominant approach for agricultural waste recycling. The thermophilic phase (>50 °C) is critical for pathogen elimination and the stabilization of organic residues [8]. However, in winter, low ambient temperatures hinder the heating of compost piles, extend the mesophilic phase, and lower overall efficiency. This slowdown in turn suppresses microbial metabolism and growth, and in some cases may even force composting to be extended or suspended [9]. To address these challenges, recent studies have highlighted the benefits of material-based interventions. Biochar amendment at low ambient temperatures increased the proportion of heat derived from organic matter degradation from 50.4% to 86.5% and vaporization heat from 29.6% to 73.5%, while reducing heat exchange by nearly two-fold, thereby accelerating temperature rise and prolonging the thermophilic phase [10]. In addition, semi-permeable membrane covering has been reported to create a micro-positive pressure environment that promotes more uniform air distribution, reduces anaerobic zones within the heap, accelerates organic matter decomposition, and ultimately improves compost quality [11]. Other strategies, such as forced aeration, insulation, and supplementation with readily degradable carbon sources, have also been applied in Europe and North America [12,13,14,15]. Despite their benefits, these approaches primarily improve compost process performance without generating additional coproducts, and they often entail high energy or material costs.

In this context, insect-based pretreatment—particularly with housefly (Musca domestica) or black soldier fly (Hermetia illucens) larvae—has attracted growing global attention worldwide. Insect larvae can rapidly degrade fresh manure, reduce moisture and bulk density, and simultaneously generate protein-rich biomass suitable as feed [16,17]. Importantly, larval activity alters substrate porosity and aeration, thereby creating a favorable microenvironment for subsequent composting [17]. Recent studies in Africa, Europe, and Asia have reported enhanced nutrient retention and reduced greenhouse gas emissions when insect pretreatment is combined with aerobic composting [18,19]. However, the synergistic mechanisms linking physical structure modification, extracellular enzymatic activity, and nitrogen transformation under low-temperature conditions remain poorly understood.

This study systematically investigated the effects of housefly larval pretreatment on swine manure composting under winter conditions in East China. By comparing the housefly larva pretreatment group (HL), sawdust-conditioned composting group (CK2), and untreated control group (CK1) and employing multivariate correlation and network analysis, we clarified the key factors and interactions affecting substrate composition, enzymatic activity, nutrient transformation, and compost maturity at each composting stage. The findings are expected to provide both theoretical insights and practical strategies for efficient, environmentally sustainable animal waste management and high-value protein production in cold or resource-limited regions.

2. Materials and Methods

2.1. Composting Materials

The experiment was conducted in a plastic greenhouse at Huzhou Hongquan Biotechnology Co., Ltd. (Huzhou, China). The main materials included fresh pig manure collected from local large-scale pig farms, extruded manure (solid fraction) obtained from sedimentation tanks containing pig pen rinse water and processed by screw extrusion, and sawdust obtained from nearby wood-processing enterprises as a bulking agent. The essential physicochemical properties of each raw material—including moisture content, total nitrogen (TN), organic carbon (OC), carbon-to-nitrogen (C/N) ratio, and other nutrient indicators—are summarized in

Table 1. Initial characteristics of the composting materials used in the different treatments.

Material	Moisture (%)	Total N (%)	Organic C (%)	C/N	Total K (%)	Total P (%)
Pig manure	73.53	2.42	30.8	12.73	3.235	1.857
Sawdust	15.56	0.14	49.1	350.71	0.361	0.03
Extruded pig manure	56.52	1.02	43.9	43.4	0.28	1.14

Note: Moisture is expressed on a fresh-weight basis; all other parameters are expressed on a dry-weight basis.

. Prior to use, all the materials were homogenized by thorough mixing to minimize variability among replicates.

2.2. Experimental Design and Sampling

All pretreatment and composting operations were conducted in a greenhouse under ambient winter conditions (~10 °C ambient) that lasted until early spring. Physical isolation barriers were established between piles to prevent cross-contamination. During larval pretreatment, sticky traps were installed to monitor potential insect escape or ingress; no such events occurred.

A randomized block design was used, which included three treatments: housefly larva pretreatment (HL), an untreated control (CK1), and sawdust-amended composting (CK2). The water content of all the pig manure was regulated to approximately 73% with extruded manure. In the HL group, the pig manure was first subjected to housefly larval pretreatment. Three field-scale replicates were established, each consisting of approximately 400 kg of pig manure spread on a concrete surface to ~20 cm thickness. Newly hatched housefly larvae (Musca domestica L., ≤24 h old) were introduced at an inoculation rate of ~0.75% (w/w) and were covered with fermenting extruded manure at a thickness of ~1 cm to keep them warm. Each gram of the inoculum consisted of ~2079 larvae. One day later, the mixture of each treatment with housefly larvae was further spread to a thickness of ~7 cm. Over the 7-day pretreatment, larval growth was monitored daily, and no prepupal signs (feeding cessation, cuticle yellowing, wandering) were observed. After pretreatment, all aging larvae were harvested via a light-shield separation device on the basis of larval photophobic behavior, and the residual substrates from three replicates were combined to form a composting pile (2.5 m × 1.8 m × 0.7 m), covered with plastic film for 24 h to stifle possible residue larvae and then entered the normal composting.

To evaluate larval yield and feed potential, a bench-scale validation trial (20 kg of manure per replicate; larval inoculation 0.75% w/w; ~311,850 individuals per replicate) was conducted in parallel. After 7 days, the larvae were harvested and weighed, and harvest rate (%) were calculated. The nutrient composition and feed safety of the harvested larvae were analyzed. The larval harvest rate was calculated via the following equation [20]:

$$\mathrm{Larval} \mathrm{harvest} \mathrm{rate} (\%) = {\displaystyle \frac{M_{larvae}}{M_{substrate}}} \times 100$$

(1)

where $M_{larvae}$ represents the fresh mass of harvested larvae (g), and where $M_{substrate}$ represents the initial fresh mass of the substrate (g).

In the CK1 group, the pig manure was spread on a concrete surface to ~20 cm thickness without larval addition for one day, then further spread to a thickness of ~7 cm and maintained for 6 days, followed by direct composting (3.4 m × 1.8 m × 0.7 m), including composting material covered with plastic film for 24 h as the HL treatment. This treatment served as the blank control.

In the CK2 group, the pig manure was piled up and direct composting. As pile temperature did not rise significantly after 7 days, sawdust was incorporated at a 9:1 manure-to-sawdust ratio (w/w), resulting in a pile size similar to the HL treatment. This treatment did not include larvae at any stage and functioned as a physical conditioning control.

All piles were turned manually every three days to enhance aeration, and composting continued for 104 days (excluding the 7-day HL pretreatment). Samples were collected every three days during the first month, when physicochemical changes were rapid. After stabilization, samples were collected every six days. At each sampling, three subsamples were taken randomly and composited to form one representative sample per pile.

2.3. Measurement Methodology

Temperature Monitoring: Temperature was measured daily at 8:30 a.m. via a digital thermistor thermometer inserted 30 cm below the pile surface to record both the pile and ambient temperatures (AT). On the basis of temperature trends, the composting process was divided into four stages: mesophilic (<25 °C), heating (gradual rise to 50 °C), thermophilic (≥50 °C), and maturation (decline and stabilization near AT). In accordance with Chinese Standard [21], compost is considered sanitized when the pile temperature exceeds 50 °C for 10 days or 60 °C for 5 days.

Physicochemical analysis: Moisture content, pH, organic matter, total nitrogen (TN), total phosphorus (TP), and total potassium (TK) were measured in accordance with the Chinese Standard for Organic Fertilizer [22].

Protease activity was determined as follows: 25 mL of casein matrix was placed in a 100 mL triangular flask and incubated in a water bath at 30 °C. Then, 0.1 g of sample and 1 mL of toluene were added, mixed gently, and incubated in a constant-temperature incubator at 30 °C for 48 h. After incubation, 25 mL of 10% trichloroacetic acid solution was added and allowed to stand for 30 min to ensure complete protein precipitation. The mixture was filtered through dry filter paper. Subsequently, 1 mL of the filtrate was transferred to a test tube, mixed with 1 mL of 1:3 Folin reagent and 5 mL of 0.55% sodium bicarbonate solution, vortexed thoroughly, and incubated in a 30 °C water bath for 30 min. Absorbance was finally measured at 680 nm. Enzyme activity was expressed as milligrams of tyrosine produced per gram of sample per 48 h.

To determine luciferase activity, 0.1 g of the sample was placed into a 50 mL triangular flask. Then, 15 mL of potassium dihydrogen phosphate buffer (60 mL/L, pH 7.6) was added and gently mixed. Subsequently, 0.2 mL of a 1000 μg/mL fluorescein diacetate stock solution was introduced and thoroughly blended. The mixture was incubated at 30 °C in a constant-temperature shaker operating at 200 r/min for 20 min. Following incubation, 15 mL of a methanol-chloroform mixture was immediately added and vigorously shaken. The entire solution was transferred to a 50 mL centrifuge tube and centrifuged at 2000 r/min for 3 min. Absorbance was measured at 490 nm. Enzyme activity was expressed on the amount of fluorescein generated per gram of sample per 20 min.

Urease, catalase, nitrate reductase (NR), and nitrite reductase (NiR) activities were quantified via colorimetric or titrimetric assays following the reference [6].

2.4. Data Processing and Analysis

All analyses were based on composite samples prepared from three subsamples per pile, with the results expressed as the means ± standard deviations (SDs). Statistical significance was assessed by multifactor analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test via SPSS 26.0 (IBM, New York, NY, USA), with significance set at p < 0.05. Correlation heatmaps for multivariate analysis were generated via Origin 2021 (OriginLab, Northampton, MA, USA) and R 4.2.1. The variations across composting stages and the dominant influencing factors were visualized and interpreted via heatmaps.

3. Results and Discussion

3.1. Larval Bioconversion Efficiency and Nutrient Recovery

This study evaluated the bioconversion efficiency of housefly larvae for fresh swine manure and the nutritional value of the resulting larval biomass as animal feed. As shown in

Table 2. Harvest performance and nutrient conversion efficiency of housefly larvae (Musca domestica L.) during pig manure bioconversion.

Parameter	Value (Mean ± SD)	Unit
Initial pig manure	20.06 ± 0.04	kg (FW)
Larval inoculation density	2079	individuals·g−1 (FW)
Larval inoculation amount	150.00 ± 0.05	g (FW)
Larval biomass harvested	2.55 ± 0.02	kg (FW)
Larval harvest rate	12.71 ± 0.07	% (FW)
C conversion rate	10.69 ± 0.06	% (DW)
N conversion rate	30.55 ± 0.39	% (DW)
P conversion rate	8.54 ± 0.12	% (DW)
K conversion rate	11.53 ± 0.50	% (DW)

Note: FW = fresh weight; DW = dry weight. Larval harvest rate was calculated according to Equation (1).

, housefly larvae achieved a fresh weight bioconversion rate of 12.71%, recovering 10.69% of the carbon, 30.55% of the nitrogen, 8.54% of the phosphorus, and 11.53% of the potassium from manure. These results highlight Section 3.1. Larval bioconversion efficiency and nutrient recovery [23,24]. This process enhances resource recovery while reducing nutrient losses, particularly those of carbon and nitrogen.

The nutritional composition and feed safety of the harvested larvae are summarized in

Table 3. Safety and nutritional composition of housefly larvae (Musca domestica L.) compared with feed standards.

Category	Parameter	Value	Standard/Reference Value
Heavy Metals	As (mg·kg−1)	0.134 ± 0.000	≤2 [27]
	Pb (mg·kg−1)	0.821 ± 0.037	≤10 [27]
	Hg (mg·kg−1)	0.005 ± 0.002	≤ 0.1 [27]
	Cd (mg·kg−1)	0.149 ± 0.000	≤10 [27]
	Cr (mg·kg−1)	1.231 ± 0.037	≤5 [27]
Nutritional Composition	Crude protein (%)	53.35 ± 0.69	≥50.0 [26]
			≥44.0 [28]
	Crude fat (%)	27.6 ± 1.18	≥22.0 [28]
	Moisture (%)	73.2 ± 0.0	7.85 [25]
Amino Acid Profile	ASP	4.68 ± 0.19	3.76 [25]
	THR	2.16 ± 0.07	1.48 [25]
	SER	2.16 ± 0.07	2.07 [25]
	GLU	6.37 ± 0.23	6.80 [25]
	PRO	2.14 ± 0.11	2.13 [25]
	LYS	3.91 ± 0.11	2.14 [25]
			≥3.0 [26]
	GLY	2.13 ± 0.07	1.35 [25]
	ALA	3.07 ± 0.09	2.14 [25]
	CYS	ND	0.77 [25]
	VAL	2.43 ± 0.11	1.74 [25]
	MET	1.11 ± 0.06	0.49 [25]
	ILE	1.89 ± 0.09	1.74 [25]
	LEU	3.18 ± 0.11	3.06 [25]
	TYR	2.39 ± 0.13	1.40 [25]
	PHE	3.21 ± 0.11	2.15 [25]
	HIS	4.49 ± 0.09	0.95 [25]
	ARG	2.24 ± 0.13	2.94 [25]
	Total (%)	47.55 ± 1.77	37.10 [25]
	17 AA/crude protein (%)	89.1	≥87 [26]
			96.11 [25]
	GLY/total AA (%)	4.47	≤8.0 [26]
			3.64 [25]

Note: Unless otherwise specified, values are expressed on a dry weight (DW) basis. Moisture content is expressed on a fresh weight (FW) basis.

. The crude protein content of larval dry matter reached 53.35 ± 0.69%, exceeding the national standards for fishmeal (≥50.0%) and first-grade high-protein soybean (≥44.0%). Essential amino acid analysis showed that glutamic acid (Glu) and arginine (Arg) levels in larval protein were slightly lower than those in soybean [25], while cysteine (Cys) was not detected. The remaining amino acids were present at levels comparable to or higher than those in soybean. The total amino acid content in larvae (47.55 ± 1.77%) was significantly higher than that in soybean (37.10%). However, the proportion of these 17 amino acids relative to crude protein was 89.1%, slightly lower than soybean (96.11%), possibly due to the presence of nonprotein nitrogen compounds in the larval biomass. Nevertheless, the essential amino acid profile of larval protein met the requirements for special-grade fishmeal [26], and its glycine (Gly) content remained below the upper threshold for feed safety. Importantly, the lysine (Lys) content of larvae (3.91 ± 0.11%) exceeded the minimum standard for Grade III fishmeal, underscoring a notable nutritional advantage for animal feed applications. In terms of feed safety, heavy metal concentrations (As, Pb, Hg, Cd, and Cr) in larvae were well below the regulatory limits set by Hygienical Standard for Feeds [27] (Table 3), confirming the suitability of housefly larvae as a safe protein sources.

To further assess the substitution potential of larval protein, annual protein yields from soybeans and housefly larvae were compared per unit land area in Zhejiang Province. According to 2024 grain crop statistics, soybeans are cultivated in both spring and autumn, with an average yield of 176.4 kg·mu⁻¹ per season, corresponding to352.8 kg·mu⁻¹ annually. Based on a moisture content of 7.85% [25] and a minimum protein content of 44% [28], the annual protein yield from soybeans was 143 kg·mu⁻¹. In this study, every 20 kg of pig manure yielded 0.36 kg of larval protein. Covering one mu (approximately 666.67 m²) with a 7-cm layer (80% usable area) required 32.5 tons of manure per batch, yielding a theoretical larval protein output of 590.6 kg per batch. Considering the 6-day larval growth cycle, allowing about 50 production cycles per year, the annual protein yield per mu could reach 29,530 kg. This output is approximately 206.5 times higher than that of soybean protein per unit land area.

In summary, housefly larvae demonstrated high manure bioconversion efficiency, nutrient enrichment capacity, and protein production potential. The protein yield per area greatly surpassed that of soybean, while maintaining a favorable amino acid profile and low heavy metal concentrations, thereby supporting sustainable manure recycling and novel protein supply strategies. Nevertheless, practical challenges such as the costs of larval production, biosecurity risks, and scalability under farm conditions should be considered, and future studies need to evaluate the technoeconomic feasibility of large-scale implementation.

3.2. Physicochemical Variations and Nitrogen Transformation

The temperature profiles of the three composting treatments significantly differed, as shown in Figure 1a–c (F = 27.22, p < 0.05). Under low ambient conditions (~10 °C), all piles experienced delayed warming. On the basis of temperature dynamics, the process can be divided into mesophilic, heating, thermophilic, and maturation phases. In HL, the mesophilic phase lasted 10 days at ~22.5 °C—well above ambient—demonstrating the improved physical properties and insulating effects of housefly larval pretreatment. The HL pile reached 50 °C by day 42, was maintained at ≥50 °C for 16 days (≥60 °C for 3 days), and stabilized at ~30 °C after day 99. In contrast, CK1 remained in the mesophilic phase for 41 days (~10 °C) due to increased moisture and bulk density, which limited aeration and delayed microbial activity [29]. After day 42, it reached the thermophilic phase by day 77 (≥50 °C for 15 days; ≥60 °C for the last 2 days) and stabilized at ~28 °C after day 93. In CK2, the temperature remained low during the first 6 days before sawdust addition, after which heating accelerated, sustaining ≥50 °C for 40 days and ≥60 °C for 8 days, stabilizing at 24–28 °C by day 104. Sawdust amendment significantly increased porosity and prolonged the thermophilic phase, which is consistent with previous findings [30]. No significant temperature differences were found between HL and CK2 (p = 0.884), indicating that both biological and physical amendments improved porosity, aeration, and cold resistance, thereby supporting efficient organic matter degradation.

The changes in moisture, bulk density, and pH (Figure 1d–f) also reflected structural improvements. On day 0, HL had lower moisture (66.3%) and bulk density (0.71 g·cm⁻³) but higher pH (9.60) than did CK1 and CK2 (p < 0.05), because of larval-driven dehydration and ammonia release [31,32]. During the mesophilic phase, the moisture content remained similar in HL and CK2 (p > 0.05), whereas that in CK1 slightly decreased. Bulk density decreased in all the treatments, with CK2 showing the largest decrease after sawdust addition on day 6, confirming improved porosity and heating. The pH remained stable in HL and CK2 but increased to 8.60 in CK1. During the heating phase, elevated temperatures enhanced evaporation and convection, reducing the moisture content by 5.6 (HL), 8.31 (CK1), and 10.41 (CK2) percentage points (p < 0.05). The bulk density significantly decreased in all the treatments: 0.21 g/cm³ (HL), 0.15 g/cm³ (CK1), and 0.53 g/cm³ (CK2). The pH decreased in HL and CK1 (to 8.27 and 8.17, respectively) but peaked in CK2 (8.77), showing the buffering effect of sawdust [33]. After the thermophilic phase, further decreases in bulk density and moisture reflected organic matter degradation and structural collapse. By the maturation phase, these parameters had stabilized. These results confirm that both HL and CK2 improved porosity and stability, promoting maturity.

The carbon and nitrogen dynamics (Figure 1g–i) revealed further differences. On day 0, the TC, TN, and C/N ratios were 25.36%, 3.21%, and 7.90, respectively, in HL; 34.66%, 5.07%, and 6.83, respectively, in CK1; and 29.07%, 3.91%, and 7.44, respectively, in CK2 (all p < 0.05), reflecting the impact of substrate structure and pretreatment. In the mesophilic phase, the TC content and C/N ratio remained stable in HL and CK2 but fluctuated in CK1 due to its compact structure and high moisture. The TN content decreased by 0.20 and 1.33 percentage points in the HL and CK1 treatments, respectively, whereas that in the CK2 treatment did not significantly change. During the heating phase, the TC content peaked in all the groups (HL: 36.34%, CK1: 41.53%, CK2: 48.99%; p < 0.05) and then decreased to 17.80% in the HL treatment group and 31.30% in the CK2 treatment group. TN decreased to 2.70% in HL and 2.95% in CK2; in CK1, TN first increased to 4.68% and then decreased to 3.93%. The C/N ratio exhibited a similar trend, peaking at 12.06 (HL), 10.33 (CK1), and 15.06 (CK2) (p < 0.001). In the thermophilic phase, the TC content in HL decreased to 19.56% and then increased to 25%; in CK1, it decreased from 40.57% to 35.74%; and in CK2, it increased from 31.30% to 41.93% before decreasing to 29.17%. The TN content in the HL treatment increased to 3.33%, that in the CK1 treatment decreased to 3.58%, and that in the CK2 treatment increased to 4.49% and then decreased to 3.57%. These patterns reflect different balances between carbon degradation and nitrogen retention, with HL resulting in greater nutrient conservation.

The evolution of nitrogen forms during composting highlights the regulatory mechanisms of nitrogen cycling and compost maturity (Figure 1j–l). Across all three treatments, nitrate nitrogen (NO₃⁻-N) remained relatively stable, with only a small peak in the HL treatment (1.65% on day 90), reflecting slow nitrate accumulation under weak nitrification [34]. The NH₄⁺/NO₃⁻ ratio closely followed that of NH₄⁺-N, confirming that ammonium was the primary driver. Initially, NH₄⁺-N in HL (0.63%) and CK2 (0.60%) was significantly lower than that in CK1 (0.91%, p < 0.05), indicating better structural conditions. During the mesophilic phase, NH₄⁺-N in HL steadily declined to 0.57%. In CK1, NH₄⁺-N decreased to 0.68%, rebounded to 0.93% with ongoing mineralization, and eventually decreased to 0.64%. In CK2, limited initial improvement led to NH₄⁺-N accumulation, reaching 0.91%. These results highlight the interplay between organic N mineralization and suppressed nitrification under low temperatures, with CK1 experiencing larger NH₄⁺-N fluctuations due to poor aeration [34]. During the heating phase, the addition of sawdust to CK2 improved the porosity and accelerated nitrification, causing NH₄⁺-N to decrease rapidly. In the HL treatment, NH₄⁺-N decreased to 0.37%, briefly increased to 0.48%, and then decreased to 0.16%. In CK1, NH₄⁺-N fluctuated between 0.70% and 0.80%, eventually stabilizing at approximately 0.80%. These trends illustrate the combined impacts of mineralization, ammonia volatilization, and enhanced nitrification, making treatment effects more pronounced [35,36]. In the thermophilic and maturation phases, NH₄⁺-N stabilized at low levels: HL (0.084–0.14%), CK1 (0.24–0.26%), and CK2 (0.29–0.36%). HL consistently presented the lowest concentrations, indicating the most effective ammonia removal and nitrogen mineralization. CK1 remained intermediate, while CK2 maintained the highest levels, suggesting that physical conditioning alone was less effective at mitigating ammonium build-up than was biological pretreatment.

To further assess compost maturity and end-product quality, key physicochemical parameters before and after composting are presented in

Table 4. Physicochemical properties of the compost before and after treatment.

Parameter	HL		CK1		CK2		Compost Quality Threshold
	Before (0 Day)	After (104 Day)	Before (0 Day)	After (104 Day)	Before (0 Day)	After (104 Day)
Moisture (%)	66.3 ± 1.3	33.3 ± 0.2	73.5 ± 0.5	21.5 ± 1.1	73.5 ± 0.4	36.7 ± 0.9	≤30 [22]
Bulk density (g·cm−3)	0.71	0.45	1.00	0.34	0.998	0.32
pH	9.60 ± 0.00	7.97 ± 0.14	7.40 ± 0.00	7.30 ± 0.08	7.43 ± 0.06	7.94 ± 0.06	5.5~8.5 [22]
Organic matter (%)	43.7 ± 0.88	50.5 ± 0.49	59.7 ± 0.88	62.7 ± 1.4	50.1 ± 1.0	56.3 ± 0.16	≥30 [22]
Total N (%)	3.21 ± 0.05	3.60 ± 0.01	5.07 ± 0.12	3.13 ± 0.15	3.91 ± 0.13	3.66 ± 0.15
C/N ratio	7.90 ± 0.58	8.14 ± 0.14	6.83 ± 0.17	11.61 ± 0.46	7.44 ± 0.27	8.92 ± 0.04

Note: HL = housefly larval pretreatment; CK1 = untreated control; CK2 = sawdust conditioning. Values are expressed as mean ± SD (n = 3). “Before” indicates the initial mixture at day 0; “After” indicates the matured compost at the end of composting. Compost quality thresholds are based on the Chinese standard for organic fertilizer (NY/T 525-2021).

. By the end of the process, CK1 achieved a final moisture content of 21.5%, fully meeting the national threshold of ≤30% [22]. In contrast, HL (33.3%) and CK2 (36.7%) fell slightly above the ≤30% threshold but still showed marked reductions from initial levels (>66%), remaining acceptable for storage and application. All treatments achieved organic matter levels above 50% and C/N ratios below 20, indicative of sufficient maturity. HL exhibited the highest nitrogen retention efficiency, with TN slightly increasing from 3.21% at the beginning to 3.60% at the end of composting. By contrast, CK1 experienced substantial nitrogen depletion, decreasing from 5.07% to 3.13%, while CK2 showed a moderate reduction from 3.91% to 3.66%. The pH of all treatments stabilized within the acceptable range (5.5–8.5), ensuring agronomic safety. These results confirm that housefly larval pretreatment not only improved composting dynamics but also enhanced the quality of the final compost product, yielding a nutrient-rich and stable organic fertilizer.

3.3. Microbial Enzyme Activity Dynamics and Regulatory Mechanisms

Figure 2 illustrates the dynamic changes in six key enzymes—protease, urease, catalase, luciferase, nitrate reductase (NR), and nitrite reductase (NiR)—across the four composting stages under different treatments. Heatmaps of Z score-standardized activities (Figure 2a–c) and temporal profiles (Figure 2d–i) highlight the synergistic regulation of enzymatic pathways by housefly larval pretreatment and sawdust conditioning. Protease and urease, as key enzymes for organic nitrogen degradation, reflect the rates of protein and organic nitrogen compound decomposition and transformation through their activity profiles. Both enzymes peaked during the mesophilic or heating phase In CK1 group, protease activity remained high throughout the maturation (Z = 1.73, with a peak of 40.72 mg TYR g⁻¹ 48 h⁻¹), suggesting that delayed aeration slowed but prolonged proteolysis. Urease activity peaked during the mesophilic and heating phases in all groups (e.g., CK1: Z = 1.50, 257.16 mg NH₄⁺-N g⁻¹ 3 h⁻¹) and then declined sharply, reflecting the early dominance of ammonification. In the HL group, protease activity increased in the maturation phase, whereas urease activity remained suppressed, indicating late-stage protein turnover with limited ammonium accumulation. In the CK2 group, the enzyme activities resembled those in the CK1 group during the first six days, but after sawdust addition, both the protease activity and urease activity decreased and remained low, reflecting substrate dilution and high C/N inhibition. These findings confirm that high-C/N substrates inhibit nitrogen-related enzyme activities and that sawdust conditioning relies primarily on structural and carbon buffering rather than promoting sustained nitrogen degradation. Enzyme dynamics are closely related to pile temperature and organic matter degradation, confirming that conditioning strategies optimize substrate structure and the microecological environment, thereby improving nitrogen cycling and compost quality [35,37].

Catalase, a key antioxidant enzyme, alleviates oxidative stress by degrading hydrogen peroxide. In HL, catalase activity increased rapidly during the heating phase and peaked on day 28 (Z = 0.93, 35.53 × 100 mL 2 mM KMnO₄ g⁻¹ 30 min⁻¹). CK1 reached its peak on day 40 (40.57 × 100 mL 2 mM KMnO₄ g⁻¹ 30 min⁻¹). Both groups declined sharply after the thermophilic phase. In CK2, catalase activity transiently increased after day 6 (peaked at 20.20 × 100 mL 2 mM KMnO₄ g⁻¹ 30 min⁻¹), followed by fluctuations and eventual decreases, suggesting that the sawdust stimulated but failed to sustain high antioxidant activity. Catalase activity is positively correlated with temperature and negatively correlated with the germination index and the E4/E6 ratio, underscoring its role in high-temperature and rapid degradation stages [13]. Luciferase activity, reflecting overall microbial metabolism, was significantly greater in HL (p < 0.05), peaked at 47.25 μg of fluorescein g⁻¹ 20 min⁻¹ on day 12 and stabilized at ~34.00 μg of fluorescein g⁻¹ 20 min⁻¹ during maturation. By contrast, CK1 sharply decreased under thermophilic conditions, and CK2 peaked much later (day 75), indicating slower microbial activation.

Nitrate reductase (NR) and nitrite reductase (NiR), which are pivotal for denitrification, exhibited treatment-specific dynamics. In the HL group, NR activity started low (8.07 μg NO₃⁻-N g⁻¹ 24 h⁻¹), peaked at 16.39 on day 40, stabilized at approximately 9.6 during the thermophilic phase, and increased to 17.06 by day 104, indicating early activation, mid-phase stabilization, and late-stage recovery. In CK1, NR activity reached its minimum (6.22) on day 28, increased to 17.44 by day 40, and then stabilized between 14.43 and 17.90, reflecting a steady but delayed transformation. In CK2, NR was initially high (15.24), decreased sharply to 3.93 by day 51, and remained unstable, suggesting weaker resilience. For NiR, all the treatments maintained high activity (>850 mg NO₂⁻-N g⁻¹ 24 h⁻¹) during the first 52 days, facilitating rapid NO₂⁻ reduction and suppressing N₂O emissions. The NiR activity subsequently decreased at different rates: the activity of CK1 and CK2 dropped quickly to ~410 by day 62 and remained low, whereas the activity of HL increased until day 75 (434.83), indicating prolonged pathway stability. This aligns with findings that nitrifier inoculation enhances denitrifying enzyme activities and nitrogen retention during composting [38]. The coupling of NR and NiR also varied by treatment. In HL, the synchronous NR and NiR peaks around day 40 supported efficient denitrification and full nitrogen conversion to N₂, with a late NR rebound further enhancing nitrate reduction. In CK1, NR and NiR were coordinated early (days 30–40) but diverged after day 62, risking NO₂⁻ accumulation and elevated N₂O emission. In CK2, both enzymes were active early but decoupled later (NR dropped on day 51, NiR on day 62), suggested instability and higher nitrogen loss risk.

In summary, the three conditioning strategies led to distinct enzymatic regulation patterns and nitrogen cycling outcomes. HL promoted early activation of protease, catalase, and luciferase, supporting rapid organic matter decomposition, microbial proliferation, while suppression excessive ammonification. Its coordinated regulation of NR and NiR ensured stable denitrification and reduced N₂O risk. In contrast, CK1 suffered from delayed enzymatic activation and asynchronous NR/NiR dynamics, whereas CK2 initially benefited from structural improvement but was constrained by high C/N ratios in later stages. Overall, larval pretreatment optimized both biochemical and structural conditions, enabling efficient nitrogen retention and high compost quality under cold-climate constraints.

3.4. Evaluation of Compost Maturity and Quality

Figure 3 presents the dynamic changes in the germination index (GI), an indicator of compost detoxification and maturity. In HL, the GI increased rapidly during the heating phase, exceeding the safety threshold of 70% by day 51, peaking at 184.55% on day 75, and remaining above 120% through maturation (124.40% on day 104). These findings indicate that housefly larval pretreatment optimizes substrate structure and aeration, enhances microbial activity, and accelerates the removal of phytotoxic substances, thereby promoting early detoxification and faster compost stabilization, which is consistent with previous reports [39]. In the CK1 group, the GI remained below 70% for 75 days, increased sharply only at the end of the thermophilic phase and reached 235.6% by day 104, indicating delayed maturation and detoxification. In CK2, the GI was stable for the initial six days, increased after sawdust addition, reached the safety threshold by day 75, and stabilized at approximately 120% after day 90. These results indicate that physical conditioning can promote maturation and reduce toxicity, although it is less effective than larval pretreatment is, which is consistent with other studies on porous amendments [40]. Overall, all treatments achieved final GI values well above the 70% safety threshold, demonstrating that both biological pretreatment and physical conditioning improve compost safety and agronomic quality, with housefly larvae showing the greatest efficiency in accelerating maturity.

3.5. Correlation Analysis of the Compost Parameters and Enzymatic Activity

Figure 4 shows correlation heatmaps of key physicochemical, enzymatic, and maturity indicators across the four composting stages, highlighting treatment-specific regulatory mechanisms. During the mesophilic phase, in the HL group, duration was strongly negatively correlated with bulk density (r = −1.00), total nitrogen (r = −0.95), NH₄⁺–N (r = −0.94), and NiR (r = −0.94) but positively correlated with the NH₄⁺/NO₃⁻ ratio (r = 0.98) and catalase activity (r = 0.96). These results indicate improved aeration and oxygen supply, facilitating ammonium retention and antioxidant responses, while coordinated catalase–urease–NR activity suppressed excessive denitrification [41]. In contrast, CK1 exhibited rapid nitrogen and moisture loss, reflected by negative correlations with TN (r = −0.81) and moisture (r = −0.91), as well as increased pH (r = 0.81) and moderate urease/catalase activity, indicating limited enzymatic synergy. For CK2, duration was positively correlated with NH₄⁺–N (r = 0.99), NO₃⁻–N (r = 0.78), and the NH₄⁺/NO₃⁻ ratio (r = 0.91) but negatively correlated with pH (r = −0.89), bulk density (r = −0.80), and catalase (r = −0.89), suggesting that woodchip addition buffered pH and improved porosity but reduced antioxidant capacity [42].

During the heating phase, HL was positively correlated with temperature (r = 0.97) and GI (r = 0.81), but negatively correlated with NH₄⁺–N (r = −0.73), and catalase (r = −0.83). These findings suggest that reduced moisture and ammonium levels promote rapid organic matter decomposition, enhance nitrification, and accelerate compost maturation, whereas enzymatic activities decrease as compost maturity progresses [30]. In CK1, composting duration was positively correlated with GI (r = 0.67), and protease activity (r = 0.87) but negatively correlated with urease (r = −0.96), and catalase (r = −0.79). This aligns with the findings of Bohacz et al. [43], who reported that protein decomposition is a key driver of compost maturation and detoxification. However, CK1 also presented a rapid decline in nitrogen mineralization and antioxidant capacity, indicating limited enzymatic coordination and poor nitrogen retention. CK2 positively correlated with urease activity (r = 0.93), NiR (r = 0.85), and luciferase (r = 0.97), but negatively correlated with moisture (r = −0.84), bulk density (r = −1.00), total carbon (r = −0.98), and NH₄⁺–N (r = −1.00). These results indicate that increased porosity and alkalinity are accompanied by rapid depletion of organic matter and ammonium and intensified nitrogen cycling and microbial metabolism [44].

During the thermophilic phase, HL exhibited positively correlated with TN (r = 0.89), protease (r = 0.84), and luciferase activity (r = 1.00), and negatively correlated with NR (r = −0.97), and NiR (r = −1.00). This indicates sustained nitrogen retention with restricted denitrification, minimizing N loss. In the CK1 group, duration was strongly positively correlated with temperature (r = 0.88), the GI (r = 0.97), NO₃⁻–N (r = 0.92), urease activity (r = 0.96) and negatively correlated with moisture (r = −1.00), pH (r = −0.97), bulk density (r = −1.00), NH₄⁺–N (r = −0.99), pointing to substantial ammonium volatilization and incomplete stabilization. In the CK2 group, duration was positively correlated with pH (r = 0.74), GI (r = 0.81), but strong negative correlated with NiR reductase (r = −0.89), suggesting gradual maturation yet limited denitrification stability. These findings underscore the importance of carbon-based conditioners (e.g., biochar and zeolite) in improving compost stability and regulatory efficiency by modulating pH, structural porosity, and denitrification enzyme activity [45].

During the maturation phase, HL retained nutrients, as shown by strongly positively correlated with TC (r = 0.99), TN (r = 0.93), the C/N ratio (r = 0.98), NH₄⁺–N (r = 0.93), and nitrate reductase (r = 0.98), while negatively correlated with moisture (r = −0.64), pH (r = −0.59), bulk density (r = −1.00), NiR (r = −0.95), and protease activity (r = −0.90). These correlations suggest enhanced nutrient conservation, improved compost stability, and the suppression of further organic degradation and denitrification, indicating advanced maturity and minimal nitrogen loss. In the CK1 group, duration was positively correlated with the GI (r = 0.99), luciferase activity (r = 1.00), and NH₄⁺–N (r = 0.96) and negatively correlated with moisture (r = −0.98), pH (r = −0.56), bulk density (r = −1.00), and total nitrogen (r = −0.86), indicating maturity with nitrogen depletion. In the CK2 group, duration was positively correlated with bulk density (r = 0.92) and NR (r = 1.00), and negatively correlated with catalase activity (r = −0.98), and NiR (r = −0.88). These results suggest that although nutrient retention and denitrification were gradually enhanced by woodchip addition, compared with those in the HL group, compost stabilization and maturity progressed more slowly.

In summary, HL has achieved rapid maturity and high nitrogen retention through continuous optimization of physicochemical conditions and enzymatic activation. CK2 enhanced the stability and antioxidant balance via porosity and carbon buffering. In contrast, CK1 suffered from poor aeration and weak enzymatic coordination, resulting in slower maturation and greater nitrogen loss. These results highlight the importance of the integrative regulation of physical structure, enzymatic dynamics, and nitrogen pathways to increase compost performance and reduce nutrient loss.

4. Conclusions

Housefly larvae pretreated with swine manure at low ambient temperatures (~10 °C in the experimental greenhouse) yielded larval biomass equivalent to 12.71% of the fresh manure weight, retaining 10.69% of TC, 30.55% of TN, 8.54% of TP, and 11.53% of TK. The harvested larvae contained 53.35% crude protein, and the heavy metal concentrations were well below the regulatory safety limits for animal feed. The estimated annual protein yield per unit area reached 29,530 kg·mu⁻¹·year⁻¹, approximately 206.5 times greater than that of two soybean crops (143 kg·mu⁻¹·crop⁻¹) cultivated on the same land area. Compared with CK2, HL composting achieved faster temperature increases and maturation (GI exceeded 70% by day 51 versus day 75 in CK2) and maintained lower NH₄⁺-N levels, enhancing nitrogen retention. Housefly larval pretreatment reduced the bulk density, improved aeration, and accelerated organic matter decomposition, resulting in a faster temperature increase and sustained thermophilic conditions despite low ambient temperatures. These improvements, coupled with early peaks in protease and catalase activities, synergistically promoted organic matter degradation and reduced nitrogen loss via ammonia volatilization and denitrification.

In conclusion, HL composting enables efficient protein and nitrogen recovery in winter, achieving rapid manure stabilization and superior waste recycling. This study provides a climate-resilient and scalable strategy for manure management not only in eastern China but also in cold regions where composting efficiency is limited, as well as in intensive livestock systems worldwide. Nevertheless, practical challenges—such as the costs of larval production, biosecurity risks, and scalability under farm conditions—must be acknowledged, and future studies should focus on the multi-layered, mechanized, and enclosed transformation by housefly larvae from livestock and poultry manure. In the future, housefly larval pretreatment could become a key component of sustainable agricultural systems within the circular bioeconomy, simultaneously converting livestock manure into safe organic fertilizers and high-quality protein feed, thereby promoting nutrient recycling, reducing environmental emissions, and enhancing global food and feed security.