Synergistic Interactions Between Black Soldier Fly Larvae and Thiobacillus thioparus Beijerinck 1904 for Ammonia Odor Control in Food Waste Bioconversion

22 23 Black Soldier Fly larvae (BSFL, Hermetia illucens) are highly effective for the 24 bioconversion of food waste. However, their rearing process often produces 25 substantial ammonia emissions, which are malodorous and environmentally 26 concerning. We investigated the co-cultivation of BSFL with the sulfur-oxidizing 27 bacterium Thiobacillus thioparus as a strategy to mitigate ammonia release. 28 Importantly, under conditions where ammonia emissions were significantly reduced, 29 neither larval growth nor bacterial viability was negatively affected. Furthermore, 30 even when the initial bacterial inoculum was reduced to 3.3*105 CFU/g-food wastes, 31 the bacterium rapidly recovered to functional levels and effectively controlled 32 ammonia emissions. This indicates the absence of harmful interaction or nutrient 33 competition between BSFL and T. thioparus. These findings suggest an efficient 34

for controlling ammonia in large-scale BSFL waste treatment. By reducing 35 the required bacterial inoculum, this approach enables scalable microbial co-culturing 36 with environmental and production benefits. 37 38 39 40 41 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 2 1. Introduction 42 Organic waste management has emerged as a global environmental challenge due to 43 the rapid increase in its generation. The accelerating growth of the world population, 44 projected to reach 9.7 billion by 2050 (Danny Dorling 2021) . Factors such as rapid 45 urbanization, accelerated economic growth, and population expansion have placed 46 severe pressure on waste management systems, often rendering conventional treatment 47

insufficient. In particular, the treatment of household food waste has become 48 extremely challenging with the progress of urbanization. Traditional approaches, 49 including landfilling, incineration, and open dumping, can be effective as short -term 50 solutions but impose substantial environmental burdens in the long term and cannot be 51 regarded as sustainable waste management measures (Hefa Cheng, Yuanan Hu 2010; L. 52 Giusti 2009; Daniela Porta et al.2009). 53 Black soldier fly (Hermetia illucens L., Diptera: Stratiomyidae), has attracted 54 considerable attention as a sustainable solution to organic waste management 55 challenges (D. Purkayastha1, S. Sarkar 2022; Qihang Zhang et al.2025). Black soldier 56 fly larvae (BSFL) can digest a wide range of organic waste and convert it into valuable 57 resources through their metabolic processes, enabling the sustainable recycling of food 58 waste and livestock manure through bioconversion (Lorenzo Mazza et al. 2020; 59 Junhua Ma et al. 2018; Trinh T. X. Nguyen et al. 2015). However, the large -scale 60 application of BSFL in food waste treatment faces critical challenges, particularly 61 regarding odor emissions. Ammonia is recognized as one of the most significant 62 contributors to offensive odors and gaseous emissions during waste treatment 63 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 3 (Alejandro Parodi et al. 2020 ; Shuguang Wang, Yang Zeng 2018 ). Beyond odor 64 concerns, nitrogen loss through ammonia volatilization during composting reduces the 65 overall recycling efficiency of organic waste (Dennis Beesigamukama et al. 2020; Hong 66 Giang Hoang et al. 2022 ), representing both an environmental and resource 67 conservation issue. Addressing ammonia emissions is therefore crucial for improving 68 the sustainability and feasibility of BSFL-based food waste treatment systems. 69 Understanding the role of microbial communities in BSFL rearing systems may 70 provide insights into mitigating these challenges (Jeroen De Smet 2018). Recent studies 71 on BSFL -mediated food waste bioconversion have revealed that the dominant 72 microbial communities in BSFL -rearing environments are highly variable rather than 73 fixed. Distinct microbial assemblages have been reported across different studies, with 74 compositions largely dependent on the regional source and characteristics of the food 75 waste used. In some cases, these variations have even led to contradictory outcomes 76 regarding system performance (Cheng-Liang Jiang et al. 2019; Moritz Gold et al. 2020). 77 These findings suggest that BSFL possess limited intrinsic capacity to regulate or 78 stabilize their surrounding microbiota and that their development does not rely on 79 obligatory symbiotic microorganisms ( Xin-Yu Li et al. 2019; Shu-Wei Lin, Matan 80 Shelomi 2024). This ecological flexibility presents a unique opportunity: the microbial 81 community within BSFL rearing environments can potentially be deliberately 82 manipulated to achieve desired functional outcomes, such as mitigating ammonia 83 emissions during the treatment of food waste (Lusheng Li et al. 2023). 84 Among microbial candidates for addressing this issue, Thiobacillus thioparus has 85 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 4 drawn considerable attention due to its unique sulfur -oxidizing capability (Luc 86 Malhautier et al. 2003) . T. thioparus is a motile rod -shaped bacterium, with optimal 87 growth occurring at 25 –30°C and pH 6.0–8.0 conditions similar to those preferred by 88 BSFL (Wancheng Pang et al. 2020) . Owing to its ability to oxidize organic and 89 inorganic sulfur compounds, T. thioparus has been widely applied in biofilters to 90 suppress hydrogen sulfide emissions (Patricio Oyarzu´n et al. 2003; Heesung Kim et al. 91 2002). In peat-based biofilters, it has been demonstrated that T. thioparus can remove 92 ammonia through chemical reactions between NH₃ and H₂SO₄ (Wenjie Gu et al. 2018; 93 Michael J. Gibson et al. 2006). Furthermore, research reported that the addition of only 94 0.25% sulfur significantly enhanced the ability of T. thioparus to inhibit ammonia 95 emissions (Yusheng Lu et al. 2018). In our preliminary studies, inoculation with BSFL 96 was shown to suppress the formation of volatile sulfur compounds (VSCs) during the 97 composting of food waste, which consequently led to an increase in sulfur concentration 98 in the substrate (Rena Michishita et al. 2023). 99 This study aimed to evaluate the synergistic effects of BSFL and T. thioparus on 100 ammonia reduction during food waste conversion. Specifically, we examined the effect 101 of sulfide emission suppression by BSFL —resulting in elevated sulfide levels in the 102 substrate—on the ammonia inhibition capacity of T. thioparus. The growth of both 103 BSFL and bacteria was monitored, and ammonia emissions were measured throughout 104 the conversion process. Our findings provide new insights into the integration of insect-105 based bioconversion with microbial interventions as a sustainable strategy for 106 mitigating nitrogen loss and odor emissions in organic waste management. 107 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 5 2. Materials and methods 108 2.1. Strain 109 Thiobacillus thioparus Beijerinck 1904 was from National Institute of Technology 110 and Evaluation (NITE) Japan, and the colony was maintained at the Laboratory of 111 Applied Entomology, The University of Tokyo. For long -term preservation, cultures 112 were mixed with 10% glycerol and stored at −80°C (Rich Boden et al. 2019). 113 Experimental BSFL used in this study were originally collected in Kagoshima, Japan, 114 in 2020, and the colony was maintained at the Laboratory of Applied Entomology, The 115 University of Tokyo, following established protocols. Hatched larvae were reared in 116 plastic cups (inner diameter 130 mm × height 100 mm, 860MB, Mineron Kasei Co., 117 Osaka, Japan) and fed a diet of breadcrumbs and rice bran. Prepupae were collected at 118 the prepupal stage and transferred to new plastic containers filled with wood chips for 119 pupation. The containers were placed in insect-proof cages and maintained in a biotron 120 under a 16L:8D light cycle at 32.5 ± 2.5°C and 60 ± 10% relative humidity and were 121 provided with water (Hiroto Ohki et al. 2025). 122 2.2. Media formulations 123 A solution of KH₂PO₄ 1.8 g, Na₂HPO₄ 1.2 g, (NH₄)₂SO₄ 0.1 g, MgSO₄·7H₂O 0.1 g, 124 FeCl₃·6H₂O 30 mg, MnSO₄·H₂O 30 mg, and CaCl₂·2H₂O 40 mg in 900 mL distilled 125 water was adjusted to pH 7 with sodium bicarbonate, autoclaved at 121 °C for 20 min, 126 and supplemented under aseptic conditions with 100 mL of sterile-filtered 10%Na₂S₂O₃ 127 solution. To enhance solubility and minimize precipitation, the conventional S6 128 medium was modified by substituting some compounds with their hydrated forms. (M. 129 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 6 Hutchinson et al. 1965) . For agar medium, 15 g of agar was added to the same base 130 solution before autoclaving (Kim D. Jones et al. 2012). The prepared liquid and solid 131 media were stored at 4 °C. Media stored for more than one month exhibited a markedly 132 decreased cultivation efficiency. 133 For research, 50 mL of the liquid medium was added to a 300 mL Erlenmeyer flask. 134 A frozen T. thioparus stock (150 µL, stored at −80 °C) was thawed and inoculated into 135 the liquid medium. The flasks were sealed with cotton plugs and incubated at 30 °C 136 under aerated conditions with a 16 L:8 D light cycle (Rich Boden et al. 2019 ). The 137 optical density (OD) of the culture was measured every 12 h to construct a growth curve. 138 2.3. Artificial food waste 139 In the laboratory, an artificial food waste was formulated to reflect the composition 140 of a typical Japanese household, including five food groups: vegetables, fruits, 141 carbohydrates, meat, and fish (Supplementary Table 1; Food Waste Suitable for 142 Treatment Using Black Soldier Fly Larvae). Prior to introducing the larvae, freshly 143 prepared waste was left to partially decompose for 3 days (Rena Michishita et al. 2023). 144 2.4. Experimental design 145 Four treatments were established in this study: (1) food waste only (T1), (2) food 146 waste with larvae (T2), (3) food waste with T. thioparus suspension (T3), and (4) food 147 waste with both larvae and T. thioparus suspension (T4). Each treatment contained 100 148 g of food waste, 30 black soldier fly larvae, and 2% (v/w) T. thioparus suspension, 149 which were added when applicable. The T. thioparus suspension was evenly sprayed 150 onto the food waste and thoroughly mixed. Newly hatched larvae were fed an artificial 151 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 7 diet containing glucose, molasses, yeast, p-hydroxybenzoic acid methyl 152 ester, propionic acid, agar, distilled water and cornmeal (Supplementary Table 2; 153 artificial diet Suitable for Black Soldier Fly Larvae) (C. -M. Liu et al. 2025) for seven 154 days prior to the experiment to ensure a relatively uniform body weight before 155

and a high survival rate after being added to the food waste. The prepared 156 mixtures were incubated in plastic containers (130 mm diameter × 100 mm height, 157 860MB) at 30°C under a 16L:8D light cycle with ventilation for seven days. The lids 158 of the plastic containers were perforated with 50 holes (2 mm in diameter) using a 159 conical needle to allow aeration . Each day, the containers were removed from the 160 incubator and placed in a well-ventilated area for 5 min to minimize mutual interference 161 among samples before measurement. Ammonia concentrations were then measured 162 using a GX-6000 gas detector (Riken Keiki Co., Ltd., Japan) at a height of 2 cm above 163 each sample for 5 min. The highest value recorded during this period was defined as 164 the ammonia concentration associated with odor intensity. On each day, five samples 165 (0.2g each) were collected from five different locations within the container and 166 homogenized for analysis , all samples were stored at −20 °C until further use (Rena 167 Michishita et al. 2023; Wenjie Gu et al. 2011). In treatments containing larvae, fifteen 168 larvae were randomly collected daily to measure the mean body weight for constructing 169 growth curves. On the final day, all 30 larvae were collected and weighed to evaluate 170 the effect of T. thioparus on BSFL growth, and the frass was also collected and sent to 171 Shimadzu Corporation (Kawasaki, Japan) for metabolite analysis using a GCMS-8040 172 Triple Quadrupole Gas Chromatograph Mass Spectrometer (TQ). 173 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 8 2.5. DNA extraction 174 Total DNA was extracted from the samples using the ISOFECAL DNA Extraction 175 Kit (Nippon Gene Co., Ltd., Japan) following the manufacturer’s instructions. Briefly, 176 0.1 g of sample was placed in a 1.5 mL microcentrifuge tube and 0.5 mL of Lysis 177 Solution F was added. Samples were resuspended, vortexed for 1 min, and incubated at 178 65 °C for 1 h. After centrifugation at 12000rpm for 5 min at room temperature, 300 μL 179 of supernatant was transferred to a new tube and mixed with 200 μL of Purification 180 Solution, followed by the addition of 300 μL of chloroform. The mixture was vortexed 181 briefly and centrifuged at 12000rpm for 15 min. The aqueous phase (400 μL) was 182 carefully transferred to a new tube, avoiding the interphase, and combined with 400 μL 183 of precipitation solution. This mixture was then centrifuged at 4 °C. The pellet was 184 washed with 0.5 mL of Wash Solution, centrifuged at 12000rpm for 10 min at 4 °C, and 185 subsequently treated with 0.5 mL of 70% ethanol and 2 μL Ethachinmate. After 186 centrifugation at 12000rpm for 5 min at 4 °C, the supernatant was removed, and the 187 pellet was air -dried and resuspended in 50 μL TE buffer (pH 8.0). All DNA samples 188 were stored at −20 °C until further use. 189 2.6. Primer design and QPCR analysis 190 Degenerate primers targeting T. thioparus were designed based on the 16S rRNA 191 sequence of T. thioparus ATCC 815816S (GenBank accession no. M79426) and other 192 related 16S sequences. The primer sequences were: [QYF: TGA GGG GGA AAG TGG 193 GGG AT; QYR: GTA GGC CAT TAC CCC ACC AAC] Primer specificity was 194 confirmed using BLAST against the NBCI bacteria (taxid:2) database (V .L. Barbosa et 195 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 9 al. 2006; Michael J. Gibson et al. 2006 ). Quantitative real -time PCR (qPCR) was 196 performed on a QuantStudio 3 instrument (Thermo Fisher Scientific, USA) using 197 THUNDERBIRD Next Probe qPCR Mix (TOYOBO, Japan). The qPCR cycling 198 conditions followed the manufacturer’s universal protocol: an initial denaturation at 199 95 °C for 20 s, followed by 40 cycles of 95 °C for 1 s and 60 °C for 1 min. 200 2.7. Construction of the standard curve analysis for real‐time PCR 201 The population of T. thioparus was determined using the serial dilution and spread 202 plate method (Janeta Starosvetsky et al. 2013 ). For qPCR analysis, the amplification 203 efficiency of T. thioparus DNA was determined by performing regression analysis 204 between the log₁₀ -transformed cell equivalents or DNA concentrations of a series of 205 diluted DNA samples and their corresponding Cₜ values. The detection sensitivity of the 206 T. thioparus qPCR assay was further estimated from the regression line of the dilution 207 series (Nichole E. Brinkman et al. 2003; Richard A. Haugland et al. 2005). 208 2.8. Measurement of the degradation of T. thioparus 16S rDNA 209 T. thioparus suspension was sterilized by autoclaving at 121 °C for 20 min. Then, 70 210 µL of the sterilized bacterial suspension, 3 g of food waste, and one larva were placed 211 in a 50 mL centrifuge tube and incubated at 30 °C. At 0 min, 10 min, 20 min, 40 min, 1 212 h, 2 h, 3 h, 4 h, and 6 h, one centrifuge tube was removed, and 30 mL of distilled water 213 was added. After vigorous vortexing, 0.2 mL of the suspension was collected for DNA 214 extraction. The extracted DNA was analyzed by qPCR to determine the degradation of 215 T. thioparus 16S rDNA. 216 2.9. Confirmation of T. thioparus survival in the gut of BSFL 217 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 10 BSFL reared on food waste(T2) and food waste with T. thioparus(T4) were collected 218 on the final day. The larvae were starved for 24 h to minimize the influence of gut 219 contents, then washed three times with 70% ethanol and three times with sterile water. 220 Under sterile conditions, the guts were dissected, and total DNA was extracted. The 221 presence of T. thioparus was confirmed by amplifying its 16S rDNA using the primers 222 [LQYF: GGG TGA GTA ATG CGT CGG AA; LQYR: GTT CAA AAT GCC ATT CCC 223 AGG T] Primer specificity was confirmed using BLAST against the NBCI bacteria 224 (taxid:2) database. PCR amplification was performed under the following conditions: 225 35 cycles of denaturation at 94 °C for 30 s, annealing at 5 5 °C for 30 s, and extension 226 at 72 °C for 1 min. The PCR products were examined by 2% agarose gel electrophoresis 227 to verify the expected band size (Cheng-Liang Jiang et al. 2019; Xin-Yu Li et al. 2019). 228 3. Results and discussion 229 3.1. Growth curve of T. thioparus in medium 230 As shown in Figure 1, due to initial adaptation to the new environment, T. thioparus 231 exhibited a prolonged lag phase of approximately 48h. Consequently, the biosynthesis 232 of the required inducible enzymes and cytoplasmic components also required additional 233 time. The exponential growth phase began around 60h, during which enzyme activity 234 was high and metabolism accelerated, resulting in the maximum growth rate of T. 235 thioparus. As nutrients were depleted and metabolic by -products accumulated and 236 degraded in the environment, the growth rate slowed by 192h. This growth pattern is 237 consistent with the observations reported by Gu et al. For subsequent co -culture 238 experiments, T. thioparus cultures aged 72-192h will be used to ensure optimal growth 239 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 11 efficiency and viable cell numbers (Wenjie Gu et al. 2011). 240 3.2. Effect of BSFL and T. thioparus on ammonia-associated odor emissions from food 241 waste 242 As shown in Figure 2A, the addition of BSFL in treatments T2 and T4 accelerated 243 NH₃ emissions, whereas in treatments without BSFL (T1 and T3), ammonia release was 244 not detected until the second day. This may be because microbial composting requires 245 time to initiate, while the digestive enzymes (Wontae Kim et al. 2011). secreted from 246 the salivary glands and gut of the larvae during feeding promote nitrogen mineralization, 247 thereby increasing the concentration of ammonium (NH₄⁺) in the residual food waste 248 (Terrence R. Green, Radu Popa 2012).From the second day onward, consistent with 249 previous studies (Wancheng Pang et al. 2020) , the T3 group, which contained only 250 BSFL, exhibited suppression of ammonia emissions compared with T1 (food waste 251 only) (P < 0.05). This may also be partly due to substrate reduction caused by larval 252 feeding. 253 Regarding T. thioparus, we observed that in the absence of BSFL, the addition of T. 254 thioparus (T3) slightly inhibited ammonia production compared with T1. However, 255 when combined with BSFL, the T4 treatment significantly reduced the ammonia 256 emission rate (P < 0.05). These results are consistent with the findings of Gu et al. 257 (Wenjie Gu et al. 2018) , showing that while the addition of T. thioparus alone can 258 slightly reduce ammonia emission rates, the combined addition of BSFL and T. 259 thioparus can effectively suppress ammonia release. This observation partially 260 confirms our hypothesis that during the early stage of composting, proteins in meat and 261 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 12 fish within the food waste degrade first, releasing sulfur compounds (D. Dave A.E., 262 Ghaly 2011; Ruo He et al. 201 8). The sulfur content in the meat exceeds the 0.25% 263 sulfur addition used in previous studies. BSFL feeding suppresses the volatilization of 264 these sulfur compounds, providing an opportunity for T. thioparus to oxidize organic 265 and inorganic sulfur compounds into SO₄²⁻, which can combine with NH₄⁺ to form more 266 stable compounds, thereby reducing ammonia emissions. 267 As shown in Figure 2B, ammonia concentrations were converted into odor intensity 268 based on the Weber–Fechner law (JOANNA KOŚMIDER, BARTOSZ WYSZYŃSKI 269 2002) and Japanese governmental guidelines reported online (Environment Bureau of 270 Osaka City Govt 2025) (I = 1.67 log C + 2.38, where I represents odor intensity and C 271 represents ammonia concentration). Regardless of BSFL addition, the odor intensity far 272 exceeded the comfort threshold of 3 for humans (Supplementary Table 3). However, 273 co-cultivation of BSFL and T. thioparus not only markedly reduced ammonia 274 concentrations but also suppressed the odor intensity caused by ammonia to below 4, 275 making ammonia-related odor during BSFL production more acceptable. 276 3.3. Effect of T. thioparus on the growth of BSFL 277 Figure 3 shows the growth performance of BSFL co -cultured with T. thioparus. 278 During the experimental period, the larval body weights in both treatments (T2 and T4) 279 increased continuously, displaying typical sigmoidal growth curves (Fig. 3A). No 280 significant difference (p > 0.05) was observed between the two treatments, indicating 281 that the presence of T. thioparus did not significantly affect larval growth. During the 282 growth process, half of the larvae were randomly sampled each day for body weight 283 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 13 measurement, and their average values were used for analysis. To minimize the effects 284 of individual size variation since — larger individuals are more likely to be found and 285 collected in the substrate, all larvae were collected and weighed at the end of the 286 experiment. The average individual body weight was 0.16 ± 0.06 g (n = 90) in the T2 287 group and 0.17 ± 0.05 g (n = 89) in the T4 group (Fig. 3B). Only one larva died in the 288 T4 group, while all others survived, suggesting that the co -culture condition was 289 suitable for larval development. Although the larvae in the co -culture group were 290 slightly heavier, statistical analysis showed no significant difference (p > 0.05) 291 These results indicate that under the tested conditions, co-culturing with T. thioparus 292 did not enhance larval growth; however, the metabolic activity of T. thioparus did not 293 harm the larvae either. The similar weight gain patterns and high survival rates in both 294 treatments suggest that nutrient availability in the food waste substrate was the main 295 factor determining larval growth performance, and that T. thioparus did not compete 296 for nutrients with the larvae. Although T. thioparus effectively suppressed ammonia 297 emissions in the co -culture system, its activity did not affect larval productivity. This 298 finding suggests that inoculating T. thioparus to mitigate odor emissions does not 299 negatively impact the growth of BSFL. Consistent with the hypothesis, BSFL exhibit 300 high environmental adaptability, and the introduction of suitable exogenous 301 microorganisms—even those altering the rearing environment to some extent —does 302 not adversely affect larval performance. This finding supports the potential application 303 of BSFL–microbe co-culture in integrated and sustainable food waste bioconversion 304 systems. 305 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 14 3.4. Quantitative PCR Detection and PCR Detection Performance Evaluation 306 Figure 4A shows the standard curve generated from tenfold serial dilutions of DNA 307 extracted from food waste inoculated with T. thioparus (3.33 × 10⁹ CFU/mL), along 308 with the corresponding regression equation and coefficient of determination 309 (R²=0.9833). Based on the first standard dilution that failed to amplify in at least one 310 replicate (10⁻⁵ dilution in the figure), the Cq cutoff value was set at 36.5. In this study, 311 for the limiting dilution of 10⁻⁵, two out of three replicates were negative. The limit of 312 detection (LOD) was calculated from the obtained Cq values and was determined to be 313 <3.33 × 10 6 CFU per gram of sample (Bojan Papić et al. 2017; Sungwoo Bae, Stefan 314 Wuertz 2009). 315 To evaluate whether dead cells could interfere with the daily growth curve 316 measurements, we investigated the degradation of T. thioparus 16S rDNA in a complex 317 food waste environment following autoclaving (Geoffrey Young et al. 2007). Samples 318 were collected before autoclaving (BF), immediately after autoclaving (AF), and at 319 various time points within 360 min post -treatment. DNA was extracted from each 320 sample, and the relative abundance of T. thioparus 16S rDNA was quantified by qPCR. 321 As shown in Figure 4B, autoclaving did not destroy the 16S rDNA of T. thioparus. 322 During the first 10 min after autoclaving, the DNA concentration remained relatively 323 stable, comparable to the BF and AF controls. However, between 20 and 40 min, the 324 DNA level decreased sharply from approximately 1.0 to less than 0.05 of the initial 325 amounts. From 40 to 120 min, the DNA concentration remained negligible (relative 326 abundance <0.1), and by 180 min it was completely degraded below the limit of 327 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 15 detection (LOD). The rapid degradation kinetics observed here effectively eliminate 328 concerns about DNA accumulation from dead cells, confirming that qPCR -based 329 quantification can reliably track changes in viable bacterial populations over time. The 330 rapid degradation may be attributed to several factors, including high microbial activity 331 in the food waste (Nur Syahidah Zulkefli et al. 2019), the inherent instability of DNA 332 once released from cellular protection (Tomas Lindahl 1993), and potential physical 333 degradation in the complex food waste matrix (Rizal F. Hariadi et al. 2015; Sara Hope 334 Sirois, Daniel H. Buckley 2019). 335 3.5. T. thioparus growth in food waste with black soldier fly larvae 336 To assess the growth and persistence of T. thioparus, we co -cultured T. thioparus 337 with BSFL in food waste and monitored the population dynamics over seven days, with 338 the primary goal of confirming the bacterial activity throughout one week. Although 339 various reports indicate that DNA can persist for extended periods in vitro (Taner Çevik, 340 Nazife Çevik 2025; Kaare M. NIELSEN et al. 200 7), fortunately, the portion of T. 341 thioparus 16S rDNA amplified by the qPCR primers degrades rapidly after cell death, 342 dropping to less than 5% of the initial amount within 20 –40 minutes (Fig. 4B). Since 343 the growth curve was measured at 24 -hour intervals, residual DNA from dead cells is 344 unlikely to significantly affect daily measurements. At the start of the experiment, T. 345 thioparus cultures of varying initial cell densities were inoculated into food waste. 346 During the first two days, nutrients present in the bacterial inoculum promoted rapid 347 exponential growth, resulting in a 2–3 log increase in cell numbers and reaching a peak 348 on days 1–2. Furthermore, even when the initial bacterial inoculum was reduced to 3.3 349 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 16 × 10⁵ CFU g⁻¹ food waste, the bacterium rapidly recovered to functional levels and 350 effectively controlled ammonia emissions, demonstrating strong resilience at low 351 starting densities. This initial burst confirms that the introduced bacteria were active 352 and metabolically competent. However, this peak was short -lived. After day 2, we 353 observed a decline in viable cell numbers, likely due to nutrient depletion in the initial 354 inoculum and subsequent decline may be due to the consumption of available substrates 355 as BSFL gained weight, reducing the resources accessible to T. thioparus. This direct 356 competition created a significant nutritional bottleneck, leading to a gradual reduction 357 in bacterial abundance as the food waste was assimilated by BSFL. 358 Interestingly, while bacterial numbers generally declined steadily across most 359 replicates, in a few instances where an unusually sharp drop occurred, a slight rebound 360 in cell numbers was observed afterward. This “rebound” phenomenon is particularly 361 noteworthy, as it strongly indicates that the decline in T. thioparus abundance was 362 primarily due to nutrient limitation rather than inhibition by BSFL -derived 363 antimicrobial compounds, such as previously reported antimicrobial peptides (Osama 364 Elhag et al. 2022). This suggests that T. thioparus and BSFL are capable of coexisting. 365 The results confirm that throughout the one-week period required for BSFL to process 366 food waste, T. thioparus remained above the detection limit and retained high activity. 367 In practical applications, the amount of food waste is typically much greater than that 368 consumed by BSFL, so nutrient limitation-induced declines in bacterial abundance are 369 unlikely to be a major concern. But for future studies, supplementation with additional 370 materials, such as specific nutrient sources for T. thioparus, could be considered to 371 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 17 further enhance ammonia suppression in the synergistic system. 372 3.6. Effect of T. thioparus and BSFL on Nitrogen Metabolism 373 The nitrogen content in frass is a critical determinant of its value as an organic 374 fertilizer. In this study, the urea content in the frass of the T2 and T4 groups increased 375 by 207% and 199.1% (Figure 6B), compared with the T1 control, indicating that the 376 activity of BSFL substantially enhanced nitrogen retention in the frass. This is 377 consistent with previous studies showing that BSFL accelerate nitrogen mineralization 378 in food waste, with the nitrogen content in the residues nearly doubling that of the raw 379 waste (Shwe S. Win et al. 20 18). Interestingly, although the total urea content was 380 similar between T2 and T4, the concentrations of urea precursors —ornithine and 381 glutamic acid—were significantly higher in T4, increasing by 61.6% and 84.1% (Figure 382 6C, Figure 6D), respectively. This suggests that the addition of T. thioparus not only 383 helps preserve bioavailable nitrogen but may also stimulate nitrogen assimilation 384 pathways. 385 The observed increase in precursor amino acids can be explained by coupled chemical 386 and biological processes. BSFL activity enhances protein degradation and 387 ammonification, leading to elevated ammonium levels in the substrate. Under such 388 conditions, the reversible amination of α-ketoglutarate shifts toward glutamic acid 389 formation, increasing the glutamic acid . Subsequently, sulfuric acid produced by T. 390 thioparus reacts with ammonium to form ammonium sulfate, effectively reducing free 391 ammonia volatilization while maintaining nitrogen in a biologically available form. The 392 resulting decrease in free NH₃ concentration likely promotes assimilatory nitrogen 393 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 18 metabolism, favoring pathways such as GS –GOGAT that incorporate ammonium into 394 amino acids. This mechanism explains the significantly higher glutamic acid levels 395 observed in T4 (Figure 6D) (Lin Zhu et al. 2024; ROBERT B. HELLING 1998; Jie 396 Yuan et al. 200 9). In addition, the acidification caused by H₂SO₄ accumulation can 397 inhibit ornithine decarboxylase activity, limiting the conversion of ornithine to 398 putrescine. Consistent with this, putrescine concentrations in T4 were markedly lower 399 than in T2 (Figure 6E), indicating reduced ornithine catabolism and consequent 400 ornithine accumulation (Sara Bover Cid et al. 2008). 401 These results indicate that synergistic interactions between BSFL and T. thioparus 402 may be an effective strategy for enhancing nitrogen retention in frass. From an applied 403 perspective, the increased urea and amino acid contents imply that frass produced under 404 synergistic conditions may have higher fertilizer value, contributing to sustainable 405 waste management and nutrient recycling. 406 4. Conclusions 407 This study demonstrates that the synergistic interaction between Thiobacillus 408 thioparus and black soldier fly larvae (BSFL) effectively suppresses ammonia 409 emissions from food waste, thereby mitigating odor during rearing without negatively 410 affecting the growth or survival of either organism. T. thioparus maintained high 411 metabolic activity throughout the seven -day bioconversion period, and even when 412 applied at low initial inoculation levels, it rapidly proliferated to functional populations 413 capable of controlling ammonia release, highlighting its suitability for large -scale 414 industrial applications. BSFL activity accelerated nitrogen mineralization and 415 significantly increased urea accumulation in the frass, while T. thioparus stabilized NH₃ 416 through acid -driven conversion to NH₄⁺, further enhancing nitrogen retention. In 417 addition, the increased ammonium availability promoted glutamate synthesis, and the 418 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 19 lowered environmental pH inhibited ornithine decarboxylation, resulting in greater 419 preservation and accumulation of amino acids in the frass, which contributes to 420 improving its nutritional value when applied as an organic fertilizer. 421 422 Overall, this cooperative system reduces odor emissions, improves the fertilizer quality 423 of the resulting frass, and maintains larval productivity. 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It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 31 707 708 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 32 709 710 711 712 713 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint 33 714 715 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint Fig. 1 Growth of Thiobacillus thioparus in m density (OD) of T. thioparus in the culture med medium. Changes in optical edium over time. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint A B Fig. 2 Effect of different treatments on ammoni (A) Temporal changes in ammonia concentration u (B) Odor intensity calculated by converting ammo using the equation I = 1.67 log C + 2.38, where I r onia emission from food waste substrate. n under different treatments during the experimental period. monia concentrations at the corresponding time points for each treatment I represents odor intensity and C represents ammonia concentration. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint A B Fig. 3 Effect of T. thioparus on the growth of black so (A) Daily changes in larval body weight under treatmen collected from each replicate, and the mean body weigh significant differences were detected between treatmen the experiment. Each dot represents an individual larva minimum–maximum values. No significant difference w soldier fly larvae. ents T2 and T4 over the experimental period. For each time point, 15 larvae were ight was calculated. Data are presented as mean ± SD from three independent repli ents at any time point (p > 0.05).(B) Body weight distribution of all larvae on the f va (T2: n = 90; T4: n = 89). Box plots indicate the median, interquartile range, and ce was observed between treatments (p > 0.05) .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint A B Fig. 4 Quantification and degradation analysis of Thiobacil (A) qPCR standard curve showing the relationship between Ct coefficient (R²) are indicated. (B) Measurement of the degradation of T. thioparus 16S rDNA (n = 9). BF and AF represent samples collected before and afte each box represents the median, and the boxes and whiskers in cillus thioparus 16S rDNA. Ct values and log-transformed cell numbers (log CFU). The regression equation an NA over time, quantified by qPCR. Box plots represent the distribution of relative D fter autoclaving, respectively. The “+” symbol indicates the mean value, the horizo indicate the interquartile range and minimum–maximum values, respectively. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint Fig. 5 Growth of Thiobacillus thio Temporal changes in T. thioparus 1 the experimental period. Each line replicate (n = 9), with different col bacterial concentrations. hioparus in food waste. 16S rDNA abundance in T4 over ne represents an independent olors indicating different initial .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint ig. 6 Effect of T. thioparus and black soldier fly larvae on m icrobial nitrogen metabolism pathways involved in food waste –E) Relative abundances of nitrogen-related metabolites meas rea, (C) ornithine, (D) glutamic acid, and (E) putrescine. Data a BC D E microbial nitrogen metabolism. (A) Schematic overview of ste degradation, key enzymes and metabolic steps are indicated easured as GC–MS peak area (a.u.) under different treatments: ta are presented as mean ± SD (n = 3). .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 6, 2026. ; https://doi.org/10.64898/2026.05.04.722119doi: bioRxiv preprint