Insights on Lipid Biodegradation in Domestic Biodegradable Waste at a Full-scale Black Soldier Fly Larvae (Hermetia illucens L.) Bioconversion

Insights on Lipid Biodegradation in Domestic Biodegradable Waste at a Full-scale Black Soldier Fly Larvae (Hermetia illucens L.) 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Bioconversion Shilin Fan, Jingjin Ma, Shuoyun Jiang, Faw Khan, FA Xiang, zhang Zhijian This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4007947/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract The lipids in the domestic biodegradable waste (DBW) pose a challenge to resource regeneration, and few studies have examined the evolution of lipid profiles during the process of black soldier fly larvae ( Hermetia illucens L. , BSFL) bioconversion. This study aimed to explore the dynamic features of lipid fraction and their associated responses of microbial community succession in residue during a full-scale BSFL bioconversion. Data showed that the lipid content decreased by95%, while the seed germination index increased by 20% through the synergistic effects of BSFL and microbiota. The results of spectral and Gas chromatography-mass spectrometry showed that free fatty acids and medium-chain fatty acids were given first priority in degrading in larval and microbial coexistence systems, resulting in the relative accumulation of sterols. The lipid content (71.1%, P = 0.002) was the prime environmental factor that promoted the succession of the bacterial community. The diversity and structure of the bacterial community varied at different stages of the bioprocess, where BSFL induced Corynebacterium, Marinobacter, and Brevibacterium . EC: 4.2.1.17 (Enoyl-CoA hydratase) and EC: 1.1.1.35 (3-hydroxyacyl-CoA dehydrogenase) were the key lipid metabolic enzymes, promoting the degradation and transformation of materials and lipids. The synergistic effect of BSFL and microbiota promotes lipid metabolisms in DBW, which is conducive to the sustainable utilization of BSFL biotechnology to convert wastes into high-value resources. Insect Bioconversion Microbiota Gut Bioproducts Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Highlights The lipid content was reduced by 95% by a combination of larvae and microbiota. MCFAs were more rapidly degraded than LCFAs and sterols during BSFL bioconversion. Lipid content is the key driver of microbial community during BSFL bioconversion. BSFL enhance fatty acid β oxidation and biosynthesis of microbiota in the DBW. Corynebacterium , Marinobacter and Brevibacterium are key taxa in lipid degradation. Introduction Domestic biodegradable waste (DBW) including food waste, kitchen waste, and fruit and vegetable waste has become a major issue for waste management [1]. High lipid content, ranging from 14–40% [2], in DBW poses a challenge for common composting methods, including windrow composting and aerated static pile composting. This is because in most cases, it covers the surface of the raw material particles, making it difficult to maintain aerobic conditions [3, 4], leading to incomplete degradation, odor issues, and even greenhouse gas emissions [5]. Fortunately, bioconversion using black soldier fly larvae (BSFL, Hermetia illucens L., Diptera: Stratiomyidae) offers a rapid and efficient biodegradation solution for DBW management [1, 6, 7]. Our previous studies showed that, without the use of three-phase separation technology (a common technology to separate lipid, water and solid waste in food waste treatment), BSFL bioconversion achieved a remarkable 71% total biomass reduction rate for DBW with an average 18.6% (m/m, dry basis) lipid content [8] at a full-scale plant. Furthermore, bioconversion of DBW using BSFL can yield eco-friendly and high value-added bioproducts such as biofuels and biofertilizers [9], with substantial potential to contribute to a circular economy [10]. Despite the widespread application of this method for high-lipid DBW management, there is limited information concerning the evolution of lipid profiles in the DBW during BSFL bioconversion. On the one hand, lipid content and fatty acid composition directly affect the respiration, growth, and metabolism of BSFL [6], further affecting larval quality as aquatic feed and biodiesel raw materials [11]. On the other hand, excessive lipids remaining in BSFL vermicompost (i.e., the product of the bioconversion process using BSFL) will reduce the efficiency of downstream secondary composting, leading to incomplete humification and low seed germination index (GI) [12]. Chinese organic fertilizer standard “NY/T 525–2021” (referred to http://www.moa.gov.cn/govpublic/ncpzlaq/202107/t20210714_6371843.htm ) already require raw materials to be degreased when needed to avoid secondary toxicity. Therefore, for the technical operation of BSFL bioconversion and utilization of associated bio-products, lipids play a ‘double-edged sword’ role in DBW management and thus require careful consideration and handling. Lipids are more difficult to be degraded by microorganisms than other macronutrients (e.g., carbohydrates and proteins) in the natural environment [3, 13]. After the introduction of BSFL, the microbial diversity and composition in DBW will be significantly different from that under natural conditions [14, 15]. Existing evidence suggests a synergistic relationship between BSFL and microorganisms in organic waste bioconversion [16]. The gut microbiota of BSFL accelerates the bioconversion of organic waste, enhancing the efficient conversion of nutrients from this resource [17–19]. Additionally, the addition of specific microorganisms into the organic waste can improve the bioconversion efficiency of BSFL [20, 21]. Therefore, we hypothesize that microbiota from both BSFL gut and the left-over residues (i.e., the mixture of frass and substrate) contribute significantly to lipid degradation during bioconversion. While research has extensively explored the microbial roles in carbohydrate (including lignocellulose) and protein degradation, the relationship between microbiota and lipid degradation remains relatively understudied. Elucidating this key relationship is crucial to understanding the evolution of lipid profiles in DBW during BSFL bioconversion. In this study, we conducted a 17-day controlled experiment on a full-scale DBW-based BSFL bioconversion plant, comparing DBW exposed to the natural environment (CK) with DBW inoculated with BSFL (BR). The aims of the current study were to 1) explore the dynamic properties in lipid structure, fatty acid composition, and functional groups in DBW using FTIR, GC-MS, and 13 C-NMR; 2) characterize the microbial community in the larval gut and residues, and predict functional enzymes involved in lipid metabolism using high-throughput sequencing; 3) investigate the functional role of gut microbiota in DBW lipid metabolism and its association with the observed dynamic lipid profiles. This study will provide a theoretical basis for optimizing bioconversion processes and improving bioproduct quality for BSFL bioconversion. Materials and methods Materials, BSFL composting process, and sample collection The experiment was conducted in a full-scale DBW-based BSFL bioconversion plant (Hangzhou GuSheng Biotechnology Co. Ltd) in Hangzhou, China (30°24′ 10.8′′ N, 120°09′ 43.3′′ E). The treatment facility has the capacity to treat 15 tons of domestic biodegradable waste (DBW, wet weight, WW) per day [8, 22], with a larval density of 35g eggs/ton DBW. The waste treatment plant receives raw DBW from households, restaurants, and cafeterias. After a series of pre-treatments, DBW slurry was completely mixed with rice hull powder [7] and a self-prepared strain (patented) to adjust the moisture content to ~75%, followed by a fermentation break of 24 hours. The composition of DBW used in this study is presented in Table 1. Fermented DBW was then subjected to two different treatments: 1) exposure to natural conditions with no BSFL incubation is denoted as ‘CK’; and 2) bioconversion with BSFL. Residues collected from BSFL bioconversion is denoted as ‘BR’, while samples of the extracted larval gut during the different stage of the BSFL process is denoted as ‘LG’. Both treatments were repeated thrice. BSFL bioconversion took place in ditches having a total area of 1200 m 2 . Each ditch measured 28 × 2 × 0.3 m (length × width × depth), whereas CK groups took place in plastic boxes (70 × 40 × 20 cm – length × width × height), which were placed alongside the ditch. The experiment was conducted for 17 days beginning June 1, 2020. The process was terminated when one-third of the prepupa emerged. Firstly, 4-day-old larvae were obtained from a fly breeding room and were added to 15%-25% of the total DBW. The fermented DBW was added at 10:00 am on days 1, 5, 7, 8, 9, 10, 11, 12, 13, 14 and 15 at a feeding rate of 15-30 kg/(m 2 ·d). Sampling was done before adding DBW on days 1, 3, 5, 7, 9, 11, 13, 15, and 17. The collected samples were stored at -80 ℃ [8, 22]. On day 17, the larvae were separated from the residue through a screen by passage. Physicochemical analysis The physicochemical characteristics of both group samples, including temperature, moisture content, pH, ammonium nitrogen (NH 4 + -N), total nitrogen (TN), and total carbon (TC) were measured as per standard protocol [22]. Briefly, the moisture content of samples was determined by assessing weight loss with a vacuum freeze drier. The pH of a 1:10 ( w/v ) deionized water extract was measured with a pH meter (PHB-4, China). NH 4 + -N concentration was measured with a continuous flow analyzer (SAN++, SKALAR, Netherlands). TN and TC were obtained with an elemental analyzer (Vario EL Cube, Elementar, German). The GI level of residue was tested as previously described [23]. Lipid extraction and characterization Extraction of lipids in residue samples was sequentially performed using a Soxhlet extraction method [24]. Briefly, 5g of freeze-dried compost in a Soxhlet apparatus was extracted with 200 mL organic solvent mixture (a 3/2 v/v mixture of n-hexane and isopropanol) for 12 h at 95 ℃. Each of the extracts was moved to a 10 mL tube and homogenized in 80 ℃ boiling water using a pressure-blowing concentrator (MTN-2800W, Auto Science, Tianjin) to remove most of the solvent and achieve a constant weight. The lipid content was calculated using the following formula: FTIR spectra of lipid mixed with potassium bromide (KBr) were measured in the wavenumber range of 4000-400 cm −1 using a spectrometer (NicoLET iS50FTIR, Thermo Scientific, USA) with a resolution of 4 cm −1 . 32 scans were collected for each sample [25]. GC-MS analysis were operated as previously study [26]. The methyl-esterification reaction of 100 mg lipid with 1% sulfuric acid-methanol was performed. The targeted compounds (fatty acid methyl ester) were analyzed using an Agilent Technologies gas chromatograph model 7890 A coupled to an Agilent 7000 C mass spectrometer (Agilent Technologies, USA) and equipment with an HP-5MS capillary column (30 m × 0.25 mm, 0.25 μm, Agilent Technologies, USA). The NIST17.L library was used for the chemical identification. The area normalization method was used to obtain the relative content of each detected sample in compost. The specific operation procedure and parameters see supplementary material. A 13 C-NMR spectrometer (JNM-ECAR, JEOL, Japan) operating at 500 MHz was used to collect additional information about 100 mg lipid (dissolved in CDCl 3 ) [27]. Signal areas at 0-50 ppm, 50-110 ppm, 110-160 ppm, and 160-200 ppm were integrated and normalized according to the total integral areas (see supplementary material). DNA extraction and sequencing analysis DNA of samples collected from CK and BR was extracted using a DNeasy ® PowerSoil Pro Kit (QIAGEN, Dusseldorf, Germany) according to the manufacturer’s instructions. The DNA of LG was extracted using a QIAamp ® PowerFecal Pro DNA kit (QIAGEN, Dusseldorf, Germany). All samples were tested with an ultra-micro DNA detector (NanoDrop 2000, Thermo). After the quality and concentration were determined, 16S rRNA high-throughput sequencing analysis was performed. The V3-V4 regions of the bacterial 16S rRNA gene were amplified using 341F (5′−CCTAYGGGRBGCASCAG−3′) and 806R (5′−GGACTACHVGGGTWTCTAA−3′) primers. The PCR products were resolved with 2% agarose gel electrophoresis. The Illumina Novaseq PE250 platform was selected for double-end sequencing with an average of 30,000 raw reads. Pandaseq software was used to merge paired reads. Clean reads were obtained after quality filtering. Usearch software was used to determine data quality as well as identify and remove chimeric sequences. Operational taxonomic units (OTUs), obtained according to a 97% similarity threshold, were compared to the Greengenes database to obtain the microbial community structure and composition information for the samples. Raw data for high-throughput sequencing are available at Mendeley Data (https://doi.org/10.17632/rxdgmvx4x9.1). Statistical analysis One-way analysis of variance (ANOVA) was performed using SPSS 22.0 and a criterion of P < 0.05 was used. The creation of graphs werer performed with Origin 2017, Excel and R version 4.1.2. High-throughput data were analyzed by Quantitative Insights into Microbial Ecology (QIIME) [28]. Both α- and β-diversity indices were calculated at the OTU level to evaluate how larvae affected microbial dynamics. Alpha indexes (Chao1, Shannon, Simpson, and Observed Species) were calculated using Quantitative Insights Into Microbial Ecology (QIIME). Heat maps, principal coordinate analysis (PCoA), and a UPGMA tree based on the weighted Unifrac distance were used to visualize the OTU data. Linear discriminant analysis (LDA) effect size (LEfSe) [29] based on Kruskal-Wallis sum-rank testing was performed to identify significantly different species (biomarkers) of microbial taxa among groups. PICRUSt2 was applied to predict metabolically relevant functions based on the KEGG Database [30]. Functional enzymes involved in the lipid metabolism of the microbial communities were predicted after OTU standardization. In addition, Spearman correlation analysis of microbial species and functional enzymes involved in lipid metabolism was conducted. Redundancy analysis (RDA) using Canoco 5 software was carried out to evaluate physicochemical properties that influence microbial communities [31]. Results and discussion Variation in physicochemical properties The temperature of DBW without BSFL treatment (CK) followed the ambient temperature. In contrast, BR rapidly increased to 40 ℃ on day 9, 43 ℃ on day 15, and then decreased to 35 ℃ (close to ambient temperature) by day 17 (Fig. 1a). The temperature trend is essentially consistent with our previous research [14]. The moisture content of BR exhibited a reduction from 73% (day 1) to 56% (day 17) owing to the raised temperature, while that of CK decreased slightly and stabilized at 70% (Fig. 1b). The matrix pH of CK stabilized at 5 with slight fluctuation, while that of BR increased from 4.7 to 8.0, and then gradually rose to 9.0 (Fig. 1c). This increase in pH could be ascribed to high ammonia levels commonly occurring during BSFL bioconversion. In contrast to CK, the total nitrogen (TN) content of BR decreased from day 1 to day 5 but then rebounded under the relatively high feeding rate (Fig. 1d). The TN concentration remained relatively stable in BR after day 7, which is beneficial to the fertility of BSFL compost. The TC content of BR decreased from 463 g/kg to 280 g/kg, whereas TC in CK had no substantial changes over time (Fig. 1e). It is worth noting that the lipid content in BR decreased from 238 g/kg to 25 g/kg within 11 days (Fig. 1f), and continued to gradually decrease to approximately 11 g/kg (over 95% of total lipid was degraded), accompanied by an increase in seed GI to 20%. Based on our previous study's 15% fresh larvae yield and 71% biomass reduction rate [8], the 17-day BSFL bioconversion resulted in crude lipid reduction rates of 13.6±1.7 g/(kgDBW.d), equivalent to 25.1±3.3 g/(kg fresh larvae per day) (Table 2). The increased GI might be attributed to the utilization of lipids by the larvae and their associated microbiota, leading to the reduction of lipid phytotoxicity [12]. Evolution of lipid fraction in DBW Fourier-transformed infrared spectroscopy The FTIR spectra depicting the functional group composition in the extracted lipid samples at various time points for both CK and BR are shown in Fig. 2a and 2b. All samples of CK and BR exhibited peaks with high intensity between 3000 cm -1 and 2800 cm -1 . These peaks could be assigned to the stretching of groups in fatty acid [32, 33], indicating that fatty acids are the most abundant components in the extracted lipid samples. This aligns with the properties of fats and oils, which are rich in triglycerides [34]. During BSFL bioconversion, the intensity of the broadband near 3381 cm −1 assigned to –OH [35, 36] exhibited an obvious increasing trend over time. This increase of –OH abundance might be attributed to the larval and microbial preference for free fatty acids, resulting in the relative accumulation of other hydrolysates of triglyceride (e.g., monoglyceride, diglyceride, and glycerol) and sterols. In contrast, DBW without larval treatment remained in the hydrolysis process of triglycerides, while the oxidation process of fatty acid was weak. This view could be fully confirmed by the decline in the intensity of peaks at 1712 cm −1 and the increase in the intensity of peaks at 1059 cm −1 , corresponding to the stretching of C=O associated with free fatty acids [37] and –C–O bending out-of-plane [33] associated with sterols in BR. Gas chromatography-mass spectrometry The GC-MS analysis identified 868 chemical substances in the extracted lipid samples of CK and BR, with fatty acid methyl esters accounting for over 90% of the identified compounds (Fig. 2c). From these chemical substances, we focused on 111 fatty acid methyl esters and 12 sterols for further analysis. The chemical formulas of the selected substances, along with the corresponding carbon atom numbers of the fatty acid methyl esters in fatty acid form, are summarized in Table S1 and S2. The composition of fatty acids in the lipids varied across different treatments, but those with chain lengths of C16 and C18 predominated in both CK and BR (Fig. 2c). These fatty acids were reported to be prevalent in animal fats and plant oils [38]. The sterols content increased (Fig. 2d) significantly in BR. Additionally, long-chain fatty acids (i.e., ≥C12:0; LCFAs) were enriched in the residues after BSFL bioconversion, whereas medium-chain fatty acids (i.e., ≥C6:0, <C12:0; MCFAs) were found to be less abundant (Fig. 2e). Compared with LCFAs and sterols, MCFAs are more easily absorbed and utilized, due to their simple molecular structure. Therefore, these observations could be attributed to the larval and microbial rapid digestion of MCFAs, and/or the biosynthesis of LCFAs via MCFAs by Actinobacteria and some fungi [39, 40]. Another probable reason is the release of lipid molecules trapped in part of the lignocellulose complex [41]. 13 C-nuclear magnetic resonance 13 C-NMR spectra of the extracted lipids were divided into four main regions (i.e., alkyl-C, O-alkyl/N-alkyl-C, aromatic-C, carboxylic-C). Alkyl-C (0-50 ppm), which can be ascribed to the carbon chain of fatty acids in lipids [27], accounted for the largest proportion in both groups (Fig. 2f, Fig. S1, Table S3), meaning that triglycerides accounted for the largest proportion of lipids. This observation aligns with the GC-MS results on the predominance of fatty acid methyl esters. With the relative decrease in the intensity of alkyl-C, the signals for O-alkyl/N-alkyl-C (50-110 ppm) showed a parallel upward trend compared with day 1 in both groups, suggesting that fatty acids were gradually substituted by oxygen/nitrogen [27]. Meanwhile, the decrease in the signals for carboxylic-C (160-200 ppm) in BR might be due to a higher rate of fatty acid β oxidation relative to triglyceride hydrolysis, which is consistent with the decrease of C=O as evidenced by FTIR spectroscopy. Overall, the dynamic properties of lipids in DBW indicate significant changes in lipid metabolism following BSFL inoculation. DBW without larval treatment remained in the hydrolysis process of triglycerides, the dominant lipid component, suggesting a stalled metabolic shift towards fatty acid oxidation. In contrast, BSFL bioconversion altered the abundance of functional groups of lipid fraction. The presence of BSFL promoted the degradation of triglycerides and exhibited a preference for easily degradable components in the DBW lipids, resulting in the relative accumulation of hardily degradable components such as sterols. Few studies have quantified the amount of lipids in this reactor that are converted into larval biomass. Thus, future research should focus on determining how each mechanism promotes overall lipid degradation and exploring maximum lipid degradation rates of larval ingestion to avoid excessive lipids without compromising larval growth and bioconversion efficiencies. Dynamics of the microbial community and its main drivers Microbial community diversity and structure No significant differences (p>0.05) in alpha-diversity (Chao1 and species richness) were observed between microbial communities in CK and BR. However, LG exhibited significantly lower (p<0.001) alpha-diversity compared with CK and BR (Fig. 3a). These results are consistent with previous evidence [14], suggesting that specific taxa might be enriched in the larval gut. Shannon and Simpson indices exhibited some discrepancies but supported these results to a certain extent. Principal coordinate analysis (PCoA) (Fig. 3b) showed that CK, BR, and LG samples were well distinguished on the first axis (PCoA1, 52.0% variance explained), which reveals significant differences in their microbial community structures. This finding was fully supported by the heat map and UPGMA tree (Fig. 3c), which shows distinct clusters of samples from the same group. Since CK and BR were derived from the same raw material, they still exhibited the similarity of the microbial communities on day 3 (Fig. 3c). However, as BSFL bioconversion progressed, the taxonomical difference between CK and BR increased significantly (ANOSIM, R = 0.897, p = 0.001) from day 5 (Fig. S2). Microbial community structures during composting are dependent on environmental factors such as nutrients, temperature, and moisture content [2]. In this study, inoculation of BSFL into the DBW induced a dramatic change in the physicochemical environment (Fig. 1), which potentially reshaped the microbial community structure in the residues. Drivers of the microbial community Redundancy analysis (RDA) was performed to evaluate the relationship between physicochemical environmental factors and the microbial community structures in CK and BR (Fig. 4a). RDA1 explained most of the variance (79.0%) between the samples. BR samples were primarily positioned in the right quadrant, except for BR1 and BR3 felling within the left quadrant, whereas CK samples were positioned in the left quadrant. Among the environmental factors examined, lipid content had the most significant impact on microbial communities, and explained up to 71.1% of the total variation ( P = 0.002), followed by TN (5.0%, P = 0.046) and temperature (4.7%, P = 0.032). A previous study reported that BSFL could alter the microbial community of compost, and then promote the degradation of complex substrates such as lipids, carbohydrates, and amino compounds [14]. Given the fact that lipid content is the key driver in reshaping microbial community structure during BSFL bioconversion, it is necessary to investigate the dominant microbial taxa in the larval gut and residue, tracking their succession to identify the key taxa in lipid metabolism. Key taxa during BSFL bioconversion Firmicutes , Proteobacteria , Actinobacteria , and Bacteroidetes were the dominant phyla in the larval gut (above 86% abundance) (Fig. 4b). The phylum Fusobacteria also had a high abundance (0.25%-28.2%), which is consistent with findings for food waste reduction via BSFL [42]. Parabacteroides (0.54%-30.86%) and Dysgonomonas (0.21%-16.19%), which were commonly present in the gut of housefly ( Musca domestica ) [43], were the dominant microbial genus in LG (Fig. 4c). Parabacteroides can utilize polysaccharides to produce volatile fatty acids, acetate, and succinate [44], and Dysgonomonas can convert hexose into propionic acid and acetic acid to facilitate carbohydrate degradation [45]. Firmicutes , Proteobacteria , Actinobacteria , and Bacteroidetes were also the dominant phyla in the residues, accounting for over 97% of the overall microbial abundance (Fig. 4b). These phyla were commonly observed on residues after BSFL bioconversion [46]. Lactobacillus was initially the dominant genus (60%, day 3) in BR due to the application of a self-prepared microbial agent (predominantly Lactobacillus ) during pre-treatment for DBW fermentation. Corynebacterium (50.1%, day 5), Marinobacter (27.9%, day 15), and Brevibacterium (13.3%, day 7) gradually colonized in the residues and replaced the dominant position of genus Lactobacillus from day 5. Many species in Corynebacterium genus (e.g. Corynebacterium macginleyi ) are lipophilic, and their growth can be enhanced by the addition of LCFAs [47]. These features make it easier for Corynebacterium genus to colonize DBW with high lipid content, and serve as a potential key taxa for the degradation of LCFAs. The linear discriminant analysis effect size (LEfSe) also shows that the genus Corynebacterium affiliated with the phylum Actinobacteria was the biomarker (microbial taxa with significantly different abundances) in the residue microbiota (Fig. 4d). Additionally, Brevibacterium -related and Marinobacter -related bacteria were reported to play an important role in lipid metabolism: 1) some Brevibacterium strains could synthesize fatty acids having C16 and C18 chain lengths like oleic acid, stearic acid, and palmitic acid [48]; 2) some Marinobacter strains could degrade a very large spectrum of lipids belonging to triglyceride, fatty acid, fatty alcohol, and wax ester classes through the formation of oleolytic biofilms [49, 50] . Taken together, during the BSFL bioconversion, Parabacteroides, and Dysgonomonas in the larval gut, along with Corynebacterium , Marinobacter , and Brevibacterium in the residues might be the key taxa that play an important role in lipid metabolism (discussed below). Functional enzymes involved in lipid metabolism Identifying changes in microbial functional enzymes during BSFL bioconversion is critical for understanding the role of microbial communities in lipid metabolism. To elucidate the underlying mechanisms of the degradation of lipids in DBW, we analyzed the relative abundance of functional enzymes involved in acylglycerol degradation, fatty acid β oxidation, and fatty acid biosynthesis modules. Functional enzymes related to fatty acid β oxidation and fatty acid biosynthesis (type II FAS) were categorized into two circular reactions (Fig. 5a). Fatty acid β oxidation is the prime pathway for fatty acid degradation in eukaryotes and prokaryotes [51]. Considering acylglycerol degradation, it was worth noting that the abundance of triglyceride lipase (EC:3.1.1.3) was 4.9-fold higher in CK samples compared to BR samples on day 7, indicating that the bacteria community in CK was more efficient at triglyceride hydrolysis (Fig. 5b). This result provides additional molecular evidence for our hypothesis that DBW without BSFL treatment remained in the hydrolysis process of triglycerides. Our previous study reported that BSFL recruit functional microbiota into the gut to promote lignocellulosic degradation [15]. However, in the present study, BSFL gut microbiota did not exhibit strong lipid degradation including acylglycerol degradation and fatty acid β oxidation (Fig. 5b). The genome determines that BSFL lacks the ability to produce lignocellulosic degrading enzymes [6, 52], but BSFL could secrete high-activity lipases in the gut [53]. This suggests that BSFL may rely on their own lipase to hydrolyze lipids rather than recruiting functional microbiota into the gut. Free fatty acids with other hydrolysates are then absorbed by larval gut cells or excreted into the DBW, explaining why the functional enzymes of acylglycerol degradation were so weak in both the residue and the larval gut. The gut microbiota exhibited a strong fatty acid synthesis process, and the dominant genus Parabacteroides had a high relative abundance of functional enzymes that were related to fatty acid synthesis in LG (Fig. 5c). Gut microbiota could secret metabolites including some short-chain fatty acids to regulate lipid digestion, absorption, intestinal immune function, and inflammatory response of the host [54]. A previous study also reported that gut microbiota could increase the activity of proteinases to enhance the BSFL protein degradation ability [19]. Therefore, though our findings suggested that the key taxa in the gut microbiota of BSFL did not play a direct role in lipid degradation, and they might still affect their host digestion, resulting in the accumulation of larval biomass and increases in larval fat. In BR samples, the relative abundance of enzymes involved in fatty acid β oxidation increased rapidly over time and reached significantly higher than those in CK samples (Fig. 5b). Notably, the key genera Corynebacterium , Marinobacter , and Brevibacterium in BR residues were positively correlated with functional enzymes related to fatty acid β oxidation, especially the functional enzymes EC: 4.2.1.17 and EC: 1.1.1.35 (Fig. 5c). Considering fatty acid biosynthesis, the abundance of the enzymes in BR was also significantly higher than that in CK group, possibly one of the reasons for increased levels of LCFAs in the BR samples (Fig. 2c). Type II FAS is the only essential biosynthesis pathway that produces saturated and unsaturated fatty acids for bacteria cell assembly and cellular metabolism [55]. Although Corynebacterium glutamicum was reported to grow on propionate and produce LCFAs (e.g., C15:0, C17:0, C17:1) via type I FAS [56], we found no functional enzymes associated with type I FAS in this study. Instead, Corynebacterium exhibited the strongest correlation with functional enzymes associated with Type II FAS, such as EC:6.4.1.2, EC:2.3.1.41, and EC:2.3.1.179. Combined with the analysis of the remodeling of microbial communities and metabolic functions, we have found that the presence of BSFL enhanced the fatty acid β oxidation and synthesis in the DBW, and the colonization of these key taxa with various functions was the fundamental reason for promoting lipid metabolism. In addition to the BSFL and its intestinal microbiota, we believe that a symbiotic relationship may also exist between the BSFL and the residue microbiota. “Holobiont” is an assemblage of a host and the many other species living in or around it, which together form a discrete ecological unit through symbiosis [57]. Viewing the BSFL and the residue microbiota as a holobiont, they synergistically function as a bioreactor, effectively biodegrading lipids in the DBW. Therefore, we can conclude that BSFL coupled with microbiota in the residue boosted lipid biodegradation in DBW, gradually making the features of BSFL compost closer to the standards of organic fertilizer. Future studies could focus on 1) creating a germ-free BSFL model to investigate the respective contributions of the BSFL larvae and its microbiota in lipid degradation; 2) using the microbiome-metabolome integration approach to investigate the molecular mechanisms of host-microbiota interactions [57] between the BSFL and the residue microbiota; 3) constructing synthetic communities (SynComs) [3, 4] from the key taxa as a microbial agent that can used in BSFL bioconversion to improve the lipid reduction efficiency and upper limit of lipid tolerance. Conclusions In this study, the lipid components of DBW exhibited unique features of biodegradation and synthesis during BSFL bioconversion and promoted the biochemical stability of residues. Coupling BSFL with bacteria resulted in enhanced lipid biodegradation by 95%, along with a 20% increase in seed GI in this full-scale DBW bioconversion plant. In a system containing both larvae and microbes, larvae preferentially utilized free fatty acids and medium-chain fatty acids, resulting in the relative accumulation of non-degradable sterols. BSFL inoculation induced the presence of Corynebacterium , Marinobacter , and Brevibacterium , as well as EC: 4.2.1.17 and EC: 1.1.1.35, to promote the metabolism of lipids in DBW, beneficial for the production of BSFL compost that meets organic fertilizer standards. Exploring the evolutionary characteristics and microbial metabolism mechanism of lipids in DBW under the full-scale BSFL bioconversion is conducive to further improving the BSFL bioconversion efficiency and the value of regenerated bioproducts for DBW treatment. Declarations Author contributions ShiLin Fan : Data analysis, Writing. JingJin Ma: Lab testing, Writing – original draft. ShuoYun Jiang: Data analysis, Writing. Fawad Zafar Ahmad Khan: Review & editing. FangMing Xiang: Visualization, Writing. ZhiJian Zhang: Conceptualization, Design, Writing – review & editing. Funding information This work was supported by the National Natural Science Foundation of China (32171466, 41673081) and Science and Technology Innovation Programs of ZheJiang Province (2021C03024). Statements and Declarations The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Supporting information The following are the Supplementary data to this article: Supplementary Information. References Tao, X. H.; Xiang, F. M.; Khan, F. Z. A.; Yan, Y. L.; Ma, J. J.; Xu, B. X.; Zhang, Z. J.: Decomposition and humification process of domestic biodegradable waste by black soldier fly ( Hermetia illucens L.) larvae from the perspective of dissolved organic matter. Chemosphere (2023). https://doi.org/10.1016/j.chemosphere.2023.137861 Palaniveloo, K.; Amran, M. A.; Norhashim, N. 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J.; Sahm, H.; Eggeling, L.: Two functional FAS-I type fatty acid synthases in Corynebacterium glutamicum . Microbiol. (2005). https://doi.org/10.1099/mic.0.28012-0 Marasco, R.; Fusi, M.; Callegari, M.; Jucker, C.; Mapelli, F.; Borin, S.; Savoldelli, S.; Daffonchio, D.; Crotti, E.: Destabilization of the bacterial interactome identifies nutrient restriction-induced dysbiosis in insect guts. Microbiol. Spectrum (2022). https://doi.org/10.1128/spectrum.01580-21 Tables Table 1 . Key physicochemical parameters of raw bioconversion materials. Parameter pH Moisture content (%) Lipid content (g/kg) a Total nitrogen (g/kg) a Ammonium nitrogen (g/kg) a Total carbon (g/kg) a C/N DBW slurry 4.78 ± 0.01 72.9 ± 0.15 241 ± 24 27.1 ± 0.7 1.12 ± 0.03 451 ± 0.8 16.7 ± 0.5 a Parameters were measured based on the dry mass (DM). Table 2 . The dry mass reduction rates of DBW after 17 days BSFL bioconversion. Reduction rate g/(kgDBW•d) a g/(kg fresh larvae•d) b Total weight 30.8±2.2 55.5±4.4 TC 10.1±0.1 33.7±1.2 TN 0.21±0.04 1.69±0.10 Crude lipid 13.6±1.7 25.1±3.3 a Refers to the reduced dry mass weight per amount of dry DBW per day during BSFL bioconversion b Refers to the reduced dry mass weight per amount of fresh larvae per day during BSFL bioconversion Supplementary Files floatimage1.jpg Graphical abstract StatementofNovelty20240303.docx SupplementaryData2024224.xlsx SupplementaryInformation20240221.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 18 Mar, 2024 Reviewers invited by journal 18 Mar, 2024 Editor invited by journal 16 Mar, 2024 Editor assigned by journal 05 Mar, 2024 First submitted to journal 02 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4007947","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":280899791,"identity":"fd418ba8-a094-431a-aee2-854ea1790e62","order_by":0,"name":"Shilin Fan","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Shilin","middleName":"","lastName":"Fan","suffix":""},{"id":280899792,"identity":"c30d943c-d459-4b78-80b3-81fc0e321894","order_by":1,"name":"Jingjin Ma","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Jingjin","middleName":"","lastName":"Ma","suffix":""},{"id":280899793,"identity":"e785c46f-3d17-43a7-a632-faa9a8fa0369","order_by":2,"name":"Shuoyun Jiang","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Shuoyun","middleName":"","lastName":"Jiang","suffix":""},{"id":280899794,"identity":"3193141b-20c9-4e77-b950-d353c7e16d97","order_by":3,"name":"Faw Khan","email":"","orcid":"","institution":"MNS-University of Agriculture: Muhammad Nawaz Shareef University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Faw","middleName":"","lastName":"Khan","suffix":""},{"id":280899795,"identity":"a2440675-31a3-40ff-bdcd-36ad05c1fbea","order_by":4,"name":"FA Xiang","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"FA","middleName":"","lastName":"Xiang","suffix":""},{"id":280899796,"identity":"bad4ce3d-2fb3-478f-9d54-f4bd204ef271","order_by":5,"name":"zhang Zhijian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYDACCTB5gIexGUh9YGAD8QyI18I4gxQtYJKZByKGX4v87OZnD7/8uSPD3M787LHNH77EBvbmbRIMNXdwamGcc8zcWIbnGdBhbObGOTxsiQ08x8okGI49w6mFWSLBTFpC4jDIL2bSORJALRI5ZhKMDYdxamGTSP8mLWEA0sL+TdrCAKhF/g1+LTxAMyU/JIC08JhJMySAbOHBr0VCIqdMmuEAWEuZZM8BNuM2nrRii4RjuLXIz0jfJvnjz2F7w/7j2yR+/Dkm289+eOONDzW4tYCDABQdhg1g9jFIZCbg1QAM6B8g6yDsGgJqR8EoGAWjYCQCAFRvS/ozqCikAAAAAElFTkSuQmCC","orcid":"","institution":"Zhejiang University","correspondingAuthor":true,"prefix":"","firstName":"zhang","middleName":"","lastName":"Zhijian","suffix":""}],"badges":[],"createdAt":"2024-03-03 08:10:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4007947/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4007947/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53081625,"identity":"607daf66-454b-4123-b62c-d96eba1ad913","added_by":"auto","created_at":"2024-03-20 10:51:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":385206,"visible":true,"origin":"","legend":"\u003cp\u003eDynamic variations of physicochemical parameters during composting: (a) Temperature, (b) moisture content, (c) pH, (d) total nitrogen (TN) and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, (e) TC, (f) Lipid content and GI. CK: No BSFL incubation (CK); BR: BSFL bioconversion. All data are the mean of three replicates and error bars indicate standard deviations.\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4007947/v1/358a3519049034b64a9467d7.jpg"},{"id":53081628,"identity":"4e0090c4-7b46-4871-8ed4-b5e5b353c281","added_by":"auto","created_at":"2024-03-20 10:51:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":323948,"visible":true,"origin":"","legend":"\u003cp\u003e(a) FTIR spectra of lipid in CK; (b) FTIR spectra of lipid in BR. (c) Results of GC-MS for relative abundance of fatty acids and sterols derived from lipids in CK and BR; (d) Results of GC-MS for percentage of sterols derived from lipids in CK and BR; (e) Results of GC-MS for changes in fatty acid and sterols abundance over time; and (f) \u003csup\u003e13\u003c/sup\u003eC NMR spectra of abundance values of various C-containing groups of lipids. The chemical shift regions were assigned as follows: 0-50 ppm (alkyl-C), 50-110 ppm (O-alkyl/N-alkyl-C), 110-160 ppm (aromatic-C), and 160-200 ppm (carboxylic-C).\u0026nbsp; CK: No BSFL incubation; BR: BSFL bioconversion.\u003c/p\u003e","description":"","filename":"floatimage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4007947/v1/8bc092e42f2842e55fc1e78a.jpg"},{"id":53081630,"identity":"6cb31d79-c7ff-4262-b4a0-13704dec20e7","added_by":"auto","created_at":"2024-03-20 10:51:05","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":558560,"visible":true,"origin":"","legend":"\u003cp\u003eThe diversity and similarity of bacterial communities in CK, BR, and LG during BSFL conversion. (a) alpha indices (Chao1, Observed Species, Shannon, and Simpson); (b) Heat map and UPGMA tree; and (c) PCoA analysis.\u003c/p\u003e","description":"","filename":"floatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4007947/v1/05066fc03fb8b40eca85222d.jpg"},{"id":53081629,"identity":"18f340fa-038d-444b-b808-2625ce248871","added_by":"auto","created_at":"2024-03-20 10:51:05","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":319006,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Redundancy analysis of bacterial community (genus level) and environmental factors. Relative abundance of bacterial communities in CK, BR, and LG during BSFL bioconversion: \u0026nbsp;(b) Phylum level; (c) Genus level; and (d) Lefse (LDA score \u0026gt;5.0).\u003c/p\u003e","description":"","filename":"floatimage5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4007947/v1/6997371285805759d1261ed2.jpg"},{"id":53081631,"identity":"8fda3110-eedb-4164-b7a4-95370d20f522","added_by":"auto","created_at":"2024-03-20 10:51:06","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":442251,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Potential lipid metabolism pathways and functional enzymes; (b) Abundance of corresponding lipid metabolism functional enzymes; and (c) Spearman correlation between functional enzymes and genus (top 21 genus in relative abundance) across all groups.\u003c/p\u003e","description":"","filename":"floatimage6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4007947/v1/64db450cfd50d5547f4eb5f8.jpg"},{"id":53082271,"identity":"c4c8e6b5-a3e2-43fc-8788-6c236f00423c","added_by":"auto","created_at":"2024-03-20 10:59:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1168607,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4007947/v1/ea88f86c-87ed-4d11-95b7-30e4ff9008d8.pdf"},{"id":53081627,"identity":"b59467a4-c6e2-4298-a105-642691888c72","added_by":"auto","created_at":"2024-03-20 10:51:05","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":228458,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"floatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4007947/v1/4ca0860471d4b9904ab24cf0.jpg"},{"id":53081626,"identity":"6cb60808-467e-4cf1-a2fc-412e4b02559c","added_by":"auto","created_at":"2024-03-20 10:51:05","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12799,"visible":true,"origin":"","legend":"","description":"","filename":"StatementofNovelty20240303.docx","url":"https://assets-eu.researchsquare.com/files/rs-4007947/v1/29407b7f8fa4aa7e4b1358a1.docx"},{"id":53081634,"identity":"e95ebb42-97d6-4fe3-a585-ed2890f4e26e","added_by":"auto","created_at":"2024-03-20 10:51:06","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":226885,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData2024224.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4007947/v1/9ef2c9d638784c3800b2dfd5.xlsx"},{"id":53081632,"identity":"0def582f-7db7-4029-96ed-4a8e571765f2","added_by":"auto","created_at":"2024-03-20 10:51:06","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":447621,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation20240221.docx","url":"https://assets-eu.researchsquare.com/files/rs-4007947/v1/5ea756566823c74c83e0ab5d.docx"}],"financialInterests":"","formattedTitle":"Insights on Lipid Biodegradation in Domestic Biodegradable Waste at a Full-scale Black Soldier Fly Larvae (Hermetia illucens L.) Bioconversion","fulltext":[{"header":"Highlights","content":"\u003cul start=\"12\"\u003e\n \u003cli\u003eThe lipid content was reduced by 95% by a combination of larvae and microbiota.\u003c/li\u003e\n \u003cli\u003eMCFAs were more rapidly degraded than LCFAs and sterols during BSFL bioconversion.\u003c/li\u003e\n \u003cli\u003eLipid content is the key driver of microbial community during BSFL bioconversion.\u003c/li\u003e\n \u003cli\u003eBSFL enhance fatty acid \u0026beta; oxidation and biosynthesis of microbiota in the DBW.\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eCorynebacterium\u003c/em\u003e, \u003cem\u003eMarinobacter\u003c/em\u003e and \u003cem\u003eBrevibacterium\u003c/em\u003e are key taxa in lipid degradation.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eDomestic biodegradable waste (DBW) including food waste, kitchen waste, and fruit and vegetable waste has become a major issue for waste management [1]. High lipid content, ranging from 14\u0026ndash;40% [2], in DBW poses a challenge for common composting methods, including windrow composting and aerated static pile composting. This is because in most cases, it covers the surface of the raw material particles, making it difficult to maintain aerobic conditions [3, 4], leading to incomplete degradation, odor issues, and even greenhouse gas emissions [5].\u003c/p\u003e \u003cp\u003eFortunately, bioconversion using black soldier fly larvae (BSFL, \u003cem\u003eHermetia illucens\u003c/em\u003e L., Diptera: Stratiomyidae) offers a rapid and efficient biodegradation solution for DBW management [1, 6, 7]. Our previous studies showed that, without the use of three-phase separation technology (a common technology to separate lipid, water and solid waste in food waste treatment), BSFL bioconversion achieved a remarkable 71% total biomass reduction rate for DBW with an average 18.6% (m/m, dry basis) lipid content [8] at a full-scale plant. Furthermore, bioconversion of DBW using BSFL can yield eco-friendly and high value-added bioproducts such as biofuels and biofertilizers [9], with substantial potential to contribute to a circular economy [10]. Despite the widespread application of this method for high-lipid DBW management, there is limited information concerning the evolution of lipid profiles in the DBW during BSFL bioconversion. On the one hand, lipid content and fatty acid composition directly affect the respiration, growth, and metabolism of BSFL [6], further affecting larval quality as aquatic feed and biodiesel raw materials [11]. On the other hand, excessive lipids remaining in BSFL vermicompost (i.e., the product of the bioconversion process using BSFL) will reduce the efficiency of downstream secondary composting, leading to incomplete humification and low seed germination index (GI) [12]. Chinese organic fertilizer standard \u0026ldquo;NY/T 525\u0026ndash;2021\u0026rdquo; (referred to \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.moa.gov.cn/govpublic/ncpzlaq/202107/t20210714_6371843.htm\u003c/span\u003e\u003cspan address=\"http://www.moa.gov.cn/govpublic/ncpzlaq/202107/t20210714_6371843.htm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) already require raw materials to be degreased when needed to avoid secondary toxicity. Therefore, for the technical operation of BSFL bioconversion and utilization of associated bio-products, lipids play a \u0026lsquo;double-edged sword\u0026rsquo; role in DBW management and thus require careful consideration and handling.\u003c/p\u003e \u003cp\u003eLipids are more difficult to be degraded by microorganisms than other macronutrients (e.g., carbohydrates and proteins) in the natural environment [3, 13]. After the introduction of BSFL, the microbial diversity and composition in DBW will be significantly different from that under natural conditions [14, 15]. Existing evidence suggests a synergistic relationship between BSFL and microorganisms in organic waste bioconversion [16]. The gut microbiota of BSFL accelerates the bioconversion of organic waste, enhancing the efficient conversion of nutrients from this resource [17\u0026ndash;19]. Additionally, the addition of specific microorganisms into the organic waste can improve the bioconversion efficiency of BSFL [20, 21]. Therefore, we hypothesize that microbiota from both BSFL gut and the left-over residues (i.e., the mixture of frass and substrate) contribute significantly to lipid degradation during bioconversion. While research has extensively explored the microbial roles in carbohydrate (including lignocellulose) and protein degradation, the relationship between microbiota and lipid degradation remains relatively understudied. Elucidating this key relationship is crucial to understanding the evolution of lipid profiles in DBW during BSFL bioconversion.\u003c/p\u003e \u003cp\u003eIn this study, we conducted a 17-day controlled experiment on a full-scale DBW-based BSFL bioconversion plant, comparing DBW exposed to the natural environment (CK) with DBW inoculated with BSFL (BR). The aims of the current study were to 1) explore the dynamic properties in lipid structure, fatty acid composition, and functional groups in DBW using FTIR, GC-MS, and \u003csup\u003e13\u003c/sup\u003eC-NMR; 2) characterize the microbial community in the larval gut and residues, and predict functional enzymes involved in lipid metabolism using high-throughput sequencing; 3) investigate the functional role of gut microbiota in DBW lipid metabolism and its association with the observed dynamic lipid profiles. This study will provide a theoretical basis for optimizing bioconversion processes and improving bioproduct quality for BSFL bioconversion.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials, BSFL composting process, and sample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiment was conducted in a full-scale DBW-based BSFL bioconversion plant (Hangzhou GuSheng Biotechnology Co. Ltd) in Hangzhou, China (30\u0026deg;24\u0026prime; 10.8\u0026prime;\u0026prime; N, 120\u0026deg;09\u0026prime; 43.3\u0026prime;\u0026prime; E). The treatment facility has the capacity to treat 15 tons of domestic biodegradable waste (DBW, wet weight, WW) per day [8, 22], with a larval density of 35g eggs/ton DBW. The waste treatment plant receives raw DBW from households, restaurants, and cafeterias. After a series of pre-treatments, DBW slurry was completely mixed with rice hull powder [7] and a self-prepared strain (patented) to adjust the moisture content to ~75%, followed by a fermentation break of 24 hours. The composition of DBW used in this study is presented in Table 1. Fermented DBW was then subjected to two different treatments: 1) exposure to natural conditions with no BSFL incubation is denoted as \u0026lsquo;CK\u0026rsquo;; and 2) bioconversion with BSFL. Residues collected from BSFL bioconversion is denoted as \u0026lsquo;BR\u0026rsquo;, while samples of the extracted larval gut during the different stage of the BSFL process is denoted as \u0026lsquo;LG\u0026rsquo;. Both treatments were repeated thrice. BSFL bioconversion took place in ditches having a total area of 1200 m\u003csup\u003e2\u003c/sup\u003e. Each ditch measured 28 \u0026times; 2 \u0026times; 0.3 m (length \u0026times; width \u0026times; depth), whereas CK groups took place in plastic boxes (70 \u0026times; 40 \u0026times; 20 cm \u0026ndash; length \u0026times; width \u0026times; height), which were placed alongside the ditch. The experiment was conducted for 17 days beginning June 1, 2020. The process was terminated when one-third of the prepupa emerged. \u003c/p\u003e\n\u003cp\u003eFirstly, 4-day-old larvae were obtained from a fly breeding room and were added to 15%-25% of the total DBW. The fermented DBW was added at 10:00 am on days 1, 5, 7, 8, 9, 10, 11, 12, 13, 14 and 15 at a feeding rate of 15-30 kg/(m\u003csup\u003e2\u003c/sup\u003e\u0026middot;d). Sampling was done before adding DBW on days 1, 3, 5, 7, 9, 11, 13, 15, and 17. The collected samples were stored at -80 ℃ [8, 22]. On day 17, the larvae were separated from the residue through a screen by passage. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysicochemical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe physicochemical characteristics of both group samples, including temperature, moisture content, pH, ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N), total nitrogen (TN), and total carbon (TC) were measured as per standard protocol [22]. Briefly, the moisture content of samples was determined by assessing weight loss with a vacuum freeze drier. The pH of a 1:10 (\u003cem\u003ew/v\u003c/em\u003e) deionized water extract was measured with a pH meter (PHB-4, China). NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N concentration was measured with a continuous flow analyzer (SAN++, SKALAR, Netherlands). TN and TC were obtained with an elemental analyzer (Vario EL Cube, Elementar, German). The GI level of residue was tested as previously described [23].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLipid extraction and characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExtraction of lipids in residue samples was sequentially performed using a Soxhlet extraction method [24]. Briefly, 5g of freeze-dried compost in a Soxhlet apparatus was extracted with 200 mL organic solvent mixture (a 3/2 v/v mixture of n-hexane and isopropanol) for 12 h at 95 ℃. Each of the extracts was moved to a 10 mL tube and homogenized in 80 ℃ boiling water using a pressure-blowing concentrator (MTN-2800W, Auto Science, Tianjin) to remove most of the solvent and achieve a constant weight. The lipid content was calculated using the following formula: \u003c/p\u003e\n\u003cp\u003eFTIR spectra of lipid mixed with potassium bromide (KBr) were measured in the wavenumber range of 4000-400 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e using a spectrometer (NicoLET iS50FTIR, Thermo Scientific, USA) with a resolution of 4 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e. 32 scans were collected for each sample [25]. \u003c/p\u003e\n\u003cp\u003eGC-MS analysis were operated as previously study [26]. The methyl-esterification reaction of 100 mg lipid with 1% sulfuric acid-methanol was performed. The targeted compounds (fatty acid methyl ester) were analyzed using an Agilent Technologies gas chromatograph model 7890 A coupled to an Agilent 7000 C mass spectrometer (Agilent Technologies, USA) and equipment with an HP-5MS capillary column (30 m \u0026times; 0.25 mm, 0.25 \u0026mu;m, Agilent Technologies, USA). The NIST17.L library was used for the chemical identification. The area normalization method was used to obtain the relative content of each detected sample in compost. The specific operation procedure and parameters see supplementary material.\u003c/p\u003e\n\u003cp\u003eA \u003csup\u003e13\u003c/sup\u003eC-NMR spectrometer (JNM-ECAR, JEOL, Japan) operating at 500 MHz was used to collect additional information about 100 mg lipid (dissolved in CDCl\u003csub\u003e3\u003c/sub\u003e) [27]. Signal areas at 0-50 ppm, 50-110 ppm, 110-160 ppm, and 160-200 ppm were integrated and normalized according to the total integral areas (see supplementary material).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA extraction and sequencing analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA of samples collected from CK and BR was extracted using a DNeasy\u003csup\u003e\u0026reg;\u003c/sup\u003e PowerSoil Pro Kit (QIAGEN, Dusseldorf, Germany) according to the manufacturer\u0026rsquo;s instructions. The DNA of LG was extracted using a QIAamp\u003csup\u003e\u0026reg;\u003c/sup\u003e PowerFecal Pro DNA kit (QIAGEN, Dusseldorf, Germany). All samples were tested with an ultra-micro DNA detector (NanoDrop 2000, Thermo). After the quality and concentration were determined, 16S rRNA high-throughput sequencing analysis was performed. The V3-V4 regions of the bacterial 16S rRNA gene were amplified using 341F (5\u0026prime;\u0026minus;CCTAYGGGRBGCASCAG\u0026minus;3\u0026prime;) and 806R (5\u0026prime;\u0026minus;GGACTACHVGGGTWTCTAA\u0026minus;3\u0026prime;) primers. The PCR products were resolved with 2% agarose gel electrophoresis. The Illumina Novaseq PE250 platform was selected for double-end sequencing with an average of 30,000 raw reads. Pandaseq software was used to merge paired reads. Clean reads were obtained after quality filtering. Usearch software was used to determine data quality as well as identify and remove chimeric sequences. Operational taxonomic units (OTUs), obtained according to a 97% similarity threshold, were compared to the Greengenes database to obtain the microbial community structure and composition information for the samples. Raw data for high-throughput sequencing are available at Mendeley Data (https://doi.org/10.17632/rxdgmvx4x9.1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne-way analysis of variance (ANOVA) was performed using SPSS 22.0 and a criterion of \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was used. The creation of graphs werer performed with Origin 2017, Excel and R version 4.1.2. High-throughput data were analyzed by Quantitative Insights into Microbial Ecology (QIIME) [28]. Both \u0026alpha;- and \u0026beta;-diversity indices were calculated at the OTU level to evaluate how larvae affected microbial dynamics. Alpha indexes (Chao1, Shannon, Simpson, and Observed Species) were calculated using Quantitative Insights Into Microbial Ecology (QIIME). Heat maps, principal coordinate analysis (PCoA), and a UPGMA tree based on the weighted Unifrac distance were used to visualize the OTU data. Linear discriminant analysis (LDA) effect size (LEfSe) [29] based on Kruskal-Wallis sum-rank testing was performed to identify significantly different species (biomarkers) of microbial taxa among groups. PICRUSt2 was applied to predict metabolically relevant functions based on the KEGG Database [30]. Functional enzymes involved in the lipid metabolism of the microbial communities were predicted after OTU standardization. In addition, Spearman correlation analysis of microbial species and functional enzymes involved in lipid metabolism was conducted. Redundancy analysis (RDA) using Canoco 5 software was carried out to evaluate physicochemical properties that influence microbial communities [31].\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cstrong\u003eVariation in physicochemical properties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe temperature of DBW without BSFL treatment (CK) followed the ambient temperature. In contrast, BR rapidly increased to 40 ℃ on day 9, 43 ℃ on day 15, and then decreased to 35 ℃ (close to ambient temperature) by day 17 (Fig. 1a). The temperature trend is essentially consistent with our previous research [14]. The moisture content of BR exhibited a reduction from 73% (day 1) to 56% (day 17) owing to the raised temperature, while that of CK decreased slightly and stabilized at 70% (Fig. 1b). The matrix pH of CK stabilized at 5 with slight fluctuation, while that of BR increased from 4.7 to 8.0, and then gradually rose to 9.0 (Fig. 1c). This increase in pH could be ascribed to high ammonia levels commonly occurring during BSFL bioconversion. In contrast to CK, the total nitrogen (TN) content of BR decreased from day 1 to day 5 but then rebounded under the relatively high feeding rate (Fig. 1d). The TN concentration remained relatively stable in BR after day 7, which is beneficial to the fertility of BSFL compost. The TC content of BR decreased from 463 g/kg to 280 g/kg, whereas TC in CK had no substantial changes over time (Fig. 1e). It is worth noting that the lipid content in BR decreased from 238 g/kg to 25 g/kg within 11 days (Fig. 1f), and continued to gradually decrease to approximately 11 g/kg (over 95% of total lipid was degraded), accompanied by an increase in seed GI to 20%. Based on our previous study\u0026apos;s 15% fresh larvae yield and 71% biomass reduction rate [8], the 17-day BSFL bioconversion resulted in crude lipid reduction rates of 13.6\u0026plusmn;1.7 g/(kgDBW.d), equivalent to 25.1\u0026plusmn;3.3 g/(kg fresh larvae per day) (Table 2). The increased GI might be attributed to the utilization of lipids by the larvae and their associated microbiota, leading to the reduction of lipid phytotoxicity [12]. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvolution of lipid \u003c/strong\u003e\u003cstrong\u003efraction in DBW\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFourier-transformed infrared spectroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe FTIR spectra depicting the functional group composition in the extracted lipid samples at various time points for both CK and BR are shown in Fig. 2a and 2b. All samples of CK and BR exhibited peaks with high intensity between 3000 cm\u003csup\u003e-1\u003c/sup\u003e and 2800 cm\u003csup\u003e-1\u003c/sup\u003e. These peaks could be assigned to the stretching of groups in fatty acid [32, 33], indicating that fatty acids are the most abundant components in the extracted lipid samples. This aligns with the properties of fats and oils, which are rich in triglycerides [34]. During BSFL bioconversion, the intensity of the broadband near 3381 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e assigned to \u0026ndash;OH [35, 36] exhibited an obvious increasing trend over time. This increase of \u0026ndash;OH abundance might be attributed to the larval and microbial preference for free fatty acids, resulting in the relative accumulation of other hydrolysates of triglyceride (e.g., monoglyceride, diglyceride, and glycerol) and sterols. In contrast, DBW without larval treatment remained in the hydrolysis process of triglycerides, while the oxidation process of fatty acid was weak. This view could be fully confirmed by the decline in the intensity of peaks at 1712 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e and the increase in the intensity of peaks at 1059 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e, corresponding to the stretching of C=O associated with free fatty acids [37] and \u0026ndash;C\u0026ndash;O bending out-of-plane [33] associated with sterols in BR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGas chromatography-mass spectrometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe GC-MS analysis identified 868 chemical substances in the extracted lipid samples of CK and BR, with fatty acid methyl esters accounting for over 90% of the identified compounds (Fig. 2c). From these chemical substances, we focused on 111 fatty acid methyl esters and 12 sterols for further analysis. The chemical formulas of the selected substances, along with the corresponding carbon atom numbers of the fatty acid methyl esters in fatty acid form, are summarized in Table S1 and S2. The composition of fatty acids in the lipids varied across different treatments, but those with chain lengths of C16 and C18 predominated in both CK and BR (Fig. 2c). These fatty acids were reported to be prevalent in animal fats and plant oils [38]. The sterols content increased (Fig. 2d) significantly in BR. Additionally, long-chain fatty acids (i.e., \u0026ge;C12:0; LCFAs) were enriched in the residues after BSFL bioconversion, whereas medium-chain fatty acids (i.e., \u0026ge;C6:0, \u0026lt;C12:0; MCFAs) were found to be less abundant (Fig. 2e). Compared with LCFAs and sterols, MCFAs are more easily absorbed and utilized, due to their simple molecular structure. Therefore, these observations could be attributed to the larval and microbial rapid digestion of MCFAs, and/or the biosynthesis of LCFAs via MCFAs by \u003cem\u003eActinobacteria\u003c/em\u003e and some fungi [39, 40]. Another probable reason is the release of lipid molecules trapped in part of the lignocellulose complex [41]. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csup\u003e13\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003eC-nuclear magnetic resonance \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e13\u003c/sup\u003eC-NMR spectra of the extracted lipids were divided into four main regions (i.e., alkyl-C, O-alkyl/N-alkyl-C, aromatic-C, carboxylic-C). Alkyl-C (0-50 ppm), which can be ascribed to the carbon chain of fatty acids in lipids [27], accounted for the largest proportion in both groups (Fig. 2f, Fig. S1, Table S3), meaning that triglycerides accounted for the largest proportion of lipids. This observation aligns with the GC-MS results on the predominance of fatty acid methyl esters. With the relative decrease in the intensity of alkyl-C, the signals for O-alkyl/N-alkyl-C (50-110 ppm) showed a parallel upward trend compared with day 1 in both groups, suggesting that fatty acids were gradually substituted by oxygen/nitrogen [27]. Meanwhile, the decrease in the signals for carboxylic-C (160-200 ppm) in BR might be due to a higher rate of fatty acid \u0026beta; oxidation relative to triglyceride hydrolysis, which is consistent with the decrease of C=O as evidenced by FTIR spectroscopy. \u003c/p\u003e\n\u003cp\u003eOverall, the dynamic properties of lipids in DBW indicate significant changes in lipid metabolism following BSFL inoculation. DBW without larval treatment remained in the hydrolysis process of triglycerides, the dominant lipid component, suggesting a stalled metabolic shift towards fatty acid oxidation. In contrast, BSFL bioconversion altered the abundance of functional groups of lipid fraction. The presence of BSFL promoted the degradation of triglycerides and exhibited a preference for easily degradable components in the DBW lipids, resulting in the relative accumulation of hardily degradable components such as sterols. Few studies have quantified the amount of lipids in this reactor that are converted into larval biomass. Thus, future research should focus on determining how each mechanism promotes overall lipid degradation and exploring maximum lipid degradation rates of larval ingestion to avoid excessive lipids without compromising larval growth and bioconversion efficiencies. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDynamics of the microbial community and its main drivers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicrobial community\u003c/strong\u003e\u003cstrong\u003e diversity and structure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo significant differences (p\u0026gt;0.05) in alpha-diversity (Chao1 and species richness) were observed between microbial communities in CK and BR. However, LG exhibited significantly lower (p\u0026lt;0.001) alpha-diversity compared with CK and BR (Fig. 3a). These results are consistent with previous evidence [14], suggesting that specific taxa might be enriched in the larval gut. Shannon and Simpson indices exhibited some discrepancies but supported these results to a certain extent.\u003c/p\u003e\n\u003cp\u003ePrincipal coordinate analysis (PCoA) (Fig. 3b) showed that CK, BR, and LG samples were well distinguished on the first axis (PCoA1, 52.0% variance explained), which reveals significant differences in their microbial community structures. This finding was fully supported by the heat map and UPGMA tree (Fig. 3c), which shows distinct clusters of samples from the same group. Since CK and BR were derived from the same raw material, they still exhibited the similarity of the microbial communities on day 3 (Fig. 3c). However, as BSFL bioconversion progressed, the taxonomical difference between CK and BR increased significantly (ANOSIM, R = 0.897, p = 0.001) from day 5 (Fig. S2). Microbial community structures during composting are dependent on environmental factors such as nutrients, temperature, and moisture content [2]. In this study, \u003cstrong\u003einoculation of BSFL into the\u003c/strong\u003e DBW induced a dramatic change in the physicochemical environment (Fig. 1), which potentially reshaped the microbial community structure in the residues.\u003c/p\u003e\n\u003ch3\u003eDrivers of the microbial community\u003c/h3\u003e\n\u003cp\u003eRedundancy analysis (RDA) was performed to evaluate the relationship between physicochemical environmental factors and the microbial community structures in CK and BR (Fig. 4a). RDA1 explained most of the variance (79.0%) between the samples. BR samples were primarily positioned in the right quadrant, except for BR1 and BR3 felling within the left quadrant, whereas CK samples were positioned in the left quadrant. Among the environmental factors examined, lipid content had the most significant impact on microbial communities, and explained up to 71.1% of the total variation (\u003cem\u003eP\u003c/em\u003e = 0.002), followed by TN (5.0%, \u003cem\u003eP\u003c/em\u003e = 0.046) and temperature (4.7%, \u003cem\u003eP\u003c/em\u003e = 0.032). A previous study reported that BSFL could alter the microbial community of compost, and then promote the degradation of complex substrates such as lipids, carbohydrates, and amino compounds [14]. Given the fact that lipid content is the key driver in reshaping microbial community structure during BSFL bioconversion, it is necessary to investigate the dominant microbial taxa in the larval gut and residue, tracking their succession to identify the key taxa in lipid metabolism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKey taxa during BSFL bioconversion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eActinobacteria\u003c/em\u003e, and \u003cem\u003eBacteroidetes\u003c/em\u003e were the dominant phyla in the larval gut (above 86% abundance) (Fig. 4b). The phylum \u003cem\u003eFusobacteria\u003c/em\u003e also had a high abundance (0.25%-28.2%), which is consistent with findings for food waste reduction via BSFL [42]. \u003cem\u003eParabacteroides \u003c/em\u003e(0.54%-30.86%) and \u003cem\u003eDysgonomonas \u003c/em\u003e(0.21%-16.19%), which were commonly present in the gut of housefly (\u003cem\u003eMusca domestica\u003c/em\u003e)\u003cem\u003e \u003c/em\u003e[43], were the dominant microbial genus in LG (Fig. 4c). \u003cem\u003eParabacteroides\u003c/em\u003e can utilize polysaccharides to produce volatile fatty acids, acetate, and succinate [44], and \u003cem\u003eDysgonomonas\u003c/em\u003e can convert hexose into propionic acid and acetic acid to facilitate carbohydrate degradation [45]. \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eActinobacteria\u003c/em\u003e, and \u003cem\u003eBacteroidetes\u003c/em\u003e were also the dominant phyla in the residues, accounting for over 97% of the overall microbial abundance (Fig. 4b). These phyla were commonly observed on residues after BSFL bioconversion [46]. \u003cem\u003eLactobacillus\u003c/em\u003e was initially the dominant genus (60%, day 3) in BR due to the application of a self-prepared microbial agent (predominantly \u003cem\u003eLactobacillus\u003c/em\u003e) during pre-treatment for DBW fermentation. \u003cem\u003eCorynebacterium \u003c/em\u003e(50.1%, day 5), \u003cem\u003eMarinobacter \u003c/em\u003e(27.9%, day 15), and \u003cem\u003eBrevibacterium\u003c/em\u003e\u003cem\u003e \u003c/em\u003e(13.3%, day 7) gradually colonized in the residues\u003cem\u003e \u003c/em\u003eand replaced the dominant position of genus \u003cem\u003eLactobacillus\u003c/em\u003e from day 5. Many species in \u003cem\u003eCorynebacterium\u003c/em\u003e genus (e.g. \u003cem\u003eCorynebacterium macginleyi\u003c/em\u003e) are lipophilic, and their growth can be enhanced by the addition of LCFAs [47]. These features make it easier for \u003cem\u003eCorynebacterium\u003c/em\u003e genus to colonize DBW with high lipid content, and serve as a potential key taxa for the degradation of LCFAs. The linear discriminant analysis effect size (LEfSe) also shows that the genus\u003cem\u003e Corynebacterium\u003c/em\u003e affiliated with the phylum\u003cem\u003e Actinobacteria\u003c/em\u003e was the biomarker (microbial taxa with significantly different abundances) in the residue microbiota (Fig. 4d). Additionally, \u003cem\u003eBrevibacterium\u003c/em\u003e-related and \u003cem\u003eMarinobacter\u003c/em\u003e-related\u003cem\u003e \u003c/em\u003ebacteria were reported to play an important role in lipid metabolism: 1) some \u003cem\u003eBrevibacterium\u003c/em\u003e strains could synthesize fatty acids having C16 and C18 chain lengths like oleic acid, stearic acid, and palmitic acid [48]; 2) some \u003cem\u003eMarinobacter \u003c/em\u003estrains\u003cem\u003e \u003c/em\u003ecould degrade a very large spectrum of lipids belonging to triglyceride, fatty acid, fatty alcohol, and wax ester classes through the formation of oleolytic biofilms [49, 50] . Taken together, during the BSFL bioconversion, \u003cem\u003eParabacteroides,\u003c/em\u003e and \u003cem\u003eDysgonomonas \u003c/em\u003ein the larval gut, along with\u003cem\u003e Corynebacterium\u003c/em\u003e, \u003cem\u003eMarinobacter\u003c/em\u003e, and \u003cem\u003eBrevibacterium \u003c/em\u003ein the residues might be the key taxa that play an important role in lipid metabolism (discussed below).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional enzymes involved in lipid metabolism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIdentifying changes in microbial functional enzymes during BSFL bioconversion is critical for understanding the role of microbial communities in lipid metabolism. To elucidate the underlying mechanisms of the degradation of lipids in DBW, we analyzed the relative abundance of functional enzymes involved in acylglycerol degradation, fatty acid \u0026beta; oxidation, and fatty acid biosynthesis modules. Functional enzymes related to fatty acid \u0026beta; oxidation and fatty acid biosynthesis (type II FAS) were categorized into two circular reactions (Fig. 5a). Fatty acid \u0026beta; oxidation is the prime pathway for fatty acid degradation in eukaryotes and prokaryotes [51].\u003c/p\u003e\n\u003cp\u003eConsidering acylglycerol degradation, it was worth noting that the abundance of triglyceride lipase (EC:3.1.1.3) was 4.9-fold higher in CK samples compared to BR samples on day 7, indicating that the bacteria community in CK was more efficient at triglyceride hydrolysis (Fig. 5b). This result provides additional molecular evidence for our hypothesis that DBW without BSFL treatment remained in the hydrolysis process of triglycerides.\u003c/p\u003e\n\u003cp\u003eOur previous study reported that BSFL recruit functional microbiota into the gut to promote lignocellulosic degradation [15]. However, in the present study, BSFL gut microbiota did not exhibit strong lipid degradation including acylglycerol degradation and fatty acid \u0026beta; oxidation (Fig. 5b). The genome determines that BSFL lacks the ability to produce lignocellulosic degrading enzymes [6, 52], but BSFL could secrete high-activity lipases in the gut [53]. This suggests that BSFL may rely on their own lipase to hydrolyze lipids rather than recruiting functional microbiota into the gut. Free fatty acids with other hydrolysates are then absorbed by larval gut cells or excreted into the DBW, explaining why the functional enzymes of acylglycerol degradation were so weak in both the residue and the larval gut. The gut microbiota exhibited a strong fatty acid synthesis process, and the dominant genus \u003cem\u003eParabacteroides\u003c/em\u003e had a high relative abundance of functional enzymes that were related to fatty acid synthesis in LG (Fig. 5c). Gut microbiota could secret metabolites including some short-chain fatty acids to regulate lipid digestion, absorption, intestinal immune function, and inflammatory response of the host [54]. A previous study also reported that gut microbiota could increase the activity of proteinases to enhance the BSFL protein degradation ability [19]. Therefore, though our findings suggested that the key taxa in the gut microbiota of BSFL did not play a direct role in lipid degradation, and they might still affect their host digestion, resulting in the accumulation of larval biomass and increases in larval fat. \u003c/p\u003e\n\u003cp\u003eIn BR samples, the relative abundance of enzymes involved in fatty acid \u0026beta; oxidation increased rapidly over time and reached significantly higher than those in CK samples (Fig. 5b). Notably, the key genera \u003cem\u003eCorynebacterium\u003c/em\u003e, \u003cem\u003eMarinobacter\u003c/em\u003e, and \u003cem\u003eBrevibacterium\u003c/em\u003e in BR residues were positively correlated with functional enzymes related to fatty acid \u0026beta; oxidation, especially the functional enzymes EC: 4.2.1.17 and EC: 1.1.1.35 (Fig. 5c). Considering fatty acid biosynthesis, the abundance of the enzymes in BR was also significantly higher than that in CK group, possibly one of the reasons for increased levels of LCFAs in the BR samples (Fig. 2c). Type II FAS is the only essential biosynthesis pathway that produces saturated and unsaturated fatty acids for bacteria cell assembly and cellular metabolism [55]. Although \u003cem\u003eCorynebacterium glutamicum\u003c/em\u003e was reported to grow on propionate and produce LCFAs (e.g., C15:0, C17:0, C17:1) via type I FAS [56], we found no functional enzymes associated with type I FAS in this study. Instead,\u003cem\u003e Corynebacterium\u003c/em\u003e exhibited the strongest correlation with functional enzymes associated with Type II FAS, such as EC:6.4.1.2, EC:2.3.1.41, and EC:2.3.1.179. Combined with the analysis of the remodeling of microbial communities and metabolic functions, we have found that the presence of BSFL enhanced the fatty acid \u0026beta; oxidation and synthesis in the DBW, and the colonization of these key taxa with various functions was the fundamental reason for promoting lipid metabolism. In addition to the BSFL and its intestinal microbiota, we believe that a symbiotic relationship may also exist between the BSFL and the residue microbiota. \u0026ldquo;Holobiont\u0026rdquo; is an assemblage of a host and the many other species living in or around it, which together form a discrete ecological unit through symbiosis [57]. Viewing the BSFL and the residue microbiota as a holobiont, they synergistically function as a bioreactor, effectively biodegrading lipids in the DBW.\u003c/p\u003e\n\u003cp\u003eTherefore, we can conclude that BSFL coupled with microbiota in the residue boosted lipid biodegradation in DBW, gradually making the features of BSFL compost closer to the standards of organic fertilizer. Future studies could focus on 1) creating a germ-free BSFL model to investigate the respective contributions of the BSFL larvae and its microbiota in lipid degradation; 2) using the microbiome-metabolome integration approach to investigate the molecular mechanisms of host-microbiota interactions [57] between the BSFL and the residue microbiota; 3) constructing synthetic communities (SynComs) [3, 4] from the key taxa as a microbial agent that can used in BSFL bioconversion to improve the lipid reduction efficiency and upper limit of lipid tolerance.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, the lipid components of DBW exhibited unique features of biodegradation and synthesis during BSFL bioconversion and promoted the biochemical stability of residues. Coupling BSFL with bacteria resulted in enhanced lipid biodegradation by 95%, along with a 20% increase in seed GI in this full-scale DBW bioconversion plant. In a system containing both larvae and microbes, larvae preferentially utilized free fatty acids and medium-chain fatty acids, resulting in the relative accumulation of non-degradable sterols. BSFL inoculation induced the presence of \u003cem\u003eCorynebacterium\u003c/em\u003e, \u003cem\u003eMarinobacter\u003c/em\u003e, and \u003cem\u003eBrevibacterium\u003c/em\u003e, as well as EC: 4.2.1.17 and EC: 1.1.1.35, to promote the metabolism of lipids in DBW, beneficial for the production of BSFL compost that meets organic fertilizer standards. Exploring the evolutionary characteristics and microbial metabolism mechanism of lipids in DBW under the full-scale BSFL bioconversion is conducive to further improving the BSFL bioconversion efficiency and the value of regenerated bioproducts for DBW treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShiLin Fan\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Data analysis, Writing.\u0026nbsp;\u003cstrong\u003eJingJin Ma:\u003c/strong\u003e Lab testing, Writing \u0026ndash; original draft. \u003cstrong\u003eShuoYun Jiang:\u0026nbsp;\u003c/strong\u003eData analysis, Writing. \u003cstrong\u003eFawad Zafar Ahmad Khan:\u003c/strong\u003e Review \u0026amp; editing. \u003cstrong\u003eFangMing Xiang:\u003c/strong\u003e Visualization, Writing. \u003cstrong\u003e\u0026nbsp;ZhiJian Zhang:\u003c/strong\u003e Conceptualization, Design, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eFunding information\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32171466, 41673081) and Science and Technology Innovation Programs of ZheJiang Province (2021C03024).\u003c/p\u003e\n\u003cp\u003eStatements and Declarations\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003cp\u003eSupporting information\u003c/p\u003e\n\u003cp\u003eThe following are the Supplementary data to this article: Supplementary Information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTao, X. 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(2011). https://doi.org/10.1186/gb-2011-12-6-r60\u003c/li\u003e\n\u003cli\u003eDouglas, G. M.; Maffei, V. J.; Zaneveld, J. R.; Yurgel, S. N.; Brown, J. R.; Taylor, C. M.; Huttenhower, C.; Langille, M. G.: PICRUSt2 for prediction of metagenome functions. Nat. Biotechnol. (2020). https://doi.org/10.1038/s41587-020-0548-6\u003c/li\u003e\n\u003cli\u003e\u0026Scaron;milauer, P.; Lep\u0026scaron;, J.: Multivariate analysis of ecological data using CANOCO 5. Cambridge university press (2014)\u003c/li\u003e\n\u003cli\u003eDreissig, I.; Machill, S.; Salzer, R.; Krafft, C.: Quantification of brain lipids by FTIR spectroscopy and partial least squares regression. Spectrochim. Acta, Part A (2009). https://doi.org/10.1016/j.saa.2008.08.008\u003c/li\u003e\n\u003cli\u003eGao, F.; Han, L.; Yang, Z.; Xu, L.; Liu, X.: Characterization of lipid constitution in Fourier transform infrared spectra and spectroscopic discrimination of animal-derived feedstuffs from different species. J. Anim. Sci. (2017). https://doi.org/10.2527/jas.2016.1332\u003c/li\u003e\n\u003cli\u003eThomas, L. M.; Holub, B. J.: Nutritional aspects of fats and oils. Springer US Boston, MA. (1994)\u003c/li\u003e\n\u003cli\u003eDroussi, Z.; D\u0026rsquo;orazio, V.; Provenzano, M. R.; Hafidi, M.; Ouatmane, A.: Study of the biodegradation and transformation of olive-mill residues during composting using FTIR spectroscopy and differential scanning calorimetry. J. Hazard. Mater. (2009). https://doi.org/10.1016/j.jhazmat.2008.09.081\u003c/li\u003e\n\u003cli\u003eEl Fels, L.; Zamama, M.; El Asli, A.; Hafidi, M.: Assessment of biotransformation of organic matter during co-composting of sewage sludge-lignocelullosic waste by chemical, FTIR analyses, and phytotoxicity tests. Int. Biodeterior. Biodegrad. (2014). https://doi.org/10.1016/j.ibiod.2013.09.024\u003c/li\u003e\n\u003cli\u003eGuill\u0026eacute;n, M. D.; Cabo, N.: Characterization of edible oils and lard by fourier transform infrared spectroscopy. Relationships between composition and frequency of concrete bands in the fingerprint region. J. Am. Oil Chem. Soc. (1997). https://doi.org/10.1007/s11746-997-0058-4\u003c/li\u003e\n\u003cli\u003eSpaccini, R.; Piccolo, A.: Molecular characteristics of humic acids extracted from compost at increasing maturity stages. Soil Biol. Biochem. (2009). https://doi.org/10.1016/j.soilbio.2009.02.026\u003c/li\u003e\n\u003cli\u003eBhatia, S. K.; Gurav, R.; Choi, T., Rim; Han, Y. H.; Park, Y., Lim; Jung, H., Rim; Yang, S., Yeon; Song, H., Suk; Yang, Y., Hun. A clean and green approach for odd chain fatty acids production in \u003cem\u003eRhodococcus sp.\u003c/em\u003e YHY01 by medium engineering. Bioresour. Technol. (2019). https://doi.org/10.1016/j.biortech.2019.121383\u003c/li\u003e\n\u003cli\u003eZhang, L. S.; Liang, S.; Zong, M. H.; Yang, J. G.; Lou, W. Y.: Microbial synthesis of functional odd-chain fatty acids: a review. World J. Microbiol. Biotechnol. (2020). https://doi.org/10.1007/s11274-020-02814-5\u003c/li\u003e\n\u003cli\u003eElouaqoudi, F. Z.; El Fels, L.; Amir, S.; Merlina, G.; Meddich, A.; Lemee, L.; Ambles, A.; Hafidi, M.: Lipid signature of the microbial community structure during composting of date palm waste alone or mixed with couch grass clippings. Int. Biodeterior. Biodegrad. (2015). https://doi.org/10.1016/j.ibiod.2014.08.016\u003c/li\u003e\n\u003cli\u003eJeon, H.; Park, S.; Choi, J.; Jeong, G.; Lee, S. B.; Choi, Y.; Lee, S. J.: The intestinal bacterial community in the food waste-reducing larvae of \u003cem\u003eHermetia illucens\u003c/em\u003e. Curr. Microbiol. (2011). https://doi.org/10.1007/s00284-011-9874-8\u003c/li\u003e\n\u003cli\u003eZhao, Y.; Wang, W.; Zhu, F.; Wang, X.; Wang, X.; Lei, C.: The gut microbiota in larvae of the housefly Musca domestica and their horizontal transfer through feeding. (2017). https://doi.org/10.1186/s13568-017-0445-7\u003c/li\u003e\n\u003cli\u003eTan, H. Q.; Li, T. T.; Zhu, C.; Zhang, X. Q.; Wu, M.; Zhu, X. F.: \u003cem\u003eParabacteroides chartae sp. nov\u003c/em\u003e., an obligately anaerobic species from wastewater of a paper mill. Int. J. Syst. Evol. Microbiol. (2012). https://doi.org/10.1099/ijs.0.038000-0\u003c/li\u003e\n\u003cli\u003eXiong, Z.; Hussain, A.; Lee, J.; Lee, H. S.: Food waste fermentation in a leach bed reactor: Reactor performance, and microbial ecology and dynamics. Bioresour. Technol. (2019). https://doi.org/10.1016/j.biortech.2018.11.066\u003c/li\u003e\n\u003cli\u003eLiu, T.; Klammsteiner, T.; Dregulo, A. M.; Kumar, V.; Zhou, Y. W.; Zhang, Z. Q.; Awasthi, M. K.: Black soldier fly larvae for organic manure recycling and its potential for a circular bioeconomy: A review. Sci. Total Environ. (2022). https://doi.org/10.1016/j.scitotenv.2022.155122\u003c/li\u003e\n\u003cli\u003eSmith, R. F.: Fatty acid requirements of human cutaneous lipophilic \u003cem\u003eCorynebacteria\u003c/em\u003e. J. Gen. Microbiol. (1970). https://doi.org/10.1099/00221287-60-2-259\u003c/li\u003e\n\u003cli\u003eStuible, H. P.; Meurer, G.; Schweizer, E.: Heterologous expression and biochemical characterization of two functionally different type I fatty acid synthases from \u003cem\u003eBrevibacterium ammoniagenes\u003c/em\u003e. Eur. J. Biochem. (1997). https://doi.org/10.1111/j.1432-1033.1997.00268.x\u003c/li\u003e\n\u003cli\u003eBonin, P.; Vieira, C.; Grimaud, R.; Militon, C.; Cuny, P.; Lima, O.; Guasco, S.; Brussaard, C. P. D.; Michotey, V.: Substrates specialization in lipid compounds and hydrocarbons of \u003cem\u003eMarinobacter\u003c/em\u003e genus. Environ. Sci. Pollut. Res. (2015). https://doi.org/10.1007/s11356-014-4009-y\u003c/li\u003e\n\u003cli\u003eMounier, J.; Camus, A.; Mitteau, I.; Vaysse, P.-J.; Goulas, P.; Grimaud, R.; Sivadon, P.: The marine bacterium \u003cem\u003eMarinobacter hydrocarbonoclasticusSP17\u003c/em\u003e degrades a wide range of lipids and hydrocarbons through the formation of oleolytic biofilms with distinct gene expression profiles. FEMS Microbiol. Ecol. (2014). https://doi.org/10.1111/1574-6941.12439\u003c/li\u003e\n\u003cli\u003eCintolesi, A.; Rodr\u0026iacute;guez Moy\u0026aacute;, M.; Gonzalez, R.: Fatty acid oxidation: systems analysis and applications. Wiley Interdiscip. Rev.: Syst. Biol. Med. (2013). https://doi.org/10.1002/wsbm.1226\u003c/li\u003e\n\u003cli\u003eZhan, S.; Fang, G. Q.; Cai, M. M.; Kou, Z. Q.; Xu, J.; Cao, Y. H.; Bai, L.; Zhang, Y. X.; Jiang, Y. M.; Luo, X. Y.; Xu, J.; Xu, X.; Zheng, L. Y.; Yu, Z. N.; Yang, H.; Zhang, Z. J.; Wang, S. B.; Tomberlin, J. K.; Zhang, J. B.; Huang, Y. P.: Genomic landscape and genetic manipulation of the black soldier fly \u003cem\u003eHermetia illucens\u003c/em\u003e, a natural waste recycler. Cell Res. (2020). https://doi.org/10.1038/s41422-019-0252-6\u003c/li\u003e\n\u003cli\u003eKim, W.; Bae, S.; Park, K.; Lee, S.; Choi, Y.; Han, S.; Koh, Y.: Biochemical characterization of digestive enzymes in the black soldier fly, \u003cem\u003eHermetia illucens \u003c/em\u003e(Diptera: Stratiomyidae). J. Asia-Pac. Entomol. (2011). https://doi.org/10.1016/j.aspen.2010.11.003\u003c/li\u003e\n\u003cli\u003eLiu, Y. L.: Fatty acids, inflammation and intestinal health in pigs. J. Anim. Sci. Biotechnol. (2015). https://doi.org/10.1186/s40104-015-0040-1\u003c/li\u003e\n\u003cli\u003eZhou, J.; Zhang, L.; Zhang, L.: Advances on mechanism and drug discovery of type-II fatty acid biosynthesis pathway. ACTA Chimica Sinica (2020). https://doi.org/10.6023/A20070299\u003c/li\u003e\n\u003cli\u003eRadmacher, E.; Alderwick, L. J.; Besra, G. S.; Brown, A. K.; Gibson, K. J.; Sahm, H.; Eggeling, L.: Two functional FAS-I type fatty acid synthases in \u003cem\u003eCorynebacterium glutamicum\u003c/em\u003e. Microbiol. (2005). https://doi.org/10.1099/mic.0.28012-0\u003c/li\u003e\n\u003cli\u003eMarasco, R.; Fusi, M.; Callegari, M.; Jucker, C.; Mapelli, F.; Borin, S.; Savoldelli, S.; Daffonchio, D.; Crotti, E.: Destabilization of the bacterial interactome identifies nutrient restriction-induced dysbiosis in insect guts. Microbiol. Spectrum (2022). https://doi.org/10.1128/spectrum.01580-21\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e. Key physicochemical parameters of raw bioconversion materials.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.370705244122966%\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.499095840867993%\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.924050632911392%\"\u003e\n \u003cp\u003eMoisture content (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.754068716094032%\"\u003e\n \u003cp\u003eLipid content (g/kg)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.296564195298373%\"\u003e\n \u003cp\u003eTotal nitrogen\u003c/p\u003e\n \u003cp\u003e(g/kg)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.009041591320072%\"\u003e\n \u003cp\u003eAmmonium nitrogen (g/kg)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.849909584086799%\"\u003e\n \u003cp\u003eTotal carbon (g/kg)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.296564195298373%\"\u003e\n \u003cp\u003eC/N\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.370705244122966%\"\u003e\n \u003cp\u003eDBW slurry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.499095840867993%\"\u003e\n \u003cp\u003e4.78\u0026nbsp;\u0026plusmn;\u0026nbsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.924050632911392%\"\u003e\n \u003cp\u003e72.9\u0026nbsp;\u0026plusmn;\u0026nbsp;0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.754068716094032%\"\u003e\n \u003cp\u003e241\u0026nbsp;\u0026plusmn;\u0026nbsp;24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.296564195298373%\"\u003e\n \u003cp\u003e27.1\u0026nbsp;\u0026plusmn;\u0026nbsp;0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.009041591320072%\"\u003e\n \u003cp\u003e1.12\u0026nbsp;\u0026plusmn;\u0026nbsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.849909584086799%\"\u003e\n \u003cp\u003e451\u0026nbsp;\u0026plusmn;\u0026nbsp;0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.296564195298373%\"\u003e\n \u003cp\u003e16.7\u0026nbsp;\u0026plusmn;\u0026nbsp;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u0026nbsp;\u003c/sup\u003eParameters were measured based on the dry mass (DM).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e. The dry mass reduction rates of DBW after 17 days BSFL bioconversion.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"81.02189781021897%\" colspan=\"2\"\u003e\n \u003cp\u003eReduction rate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"36.13138686131387%\"\u003e\n \u003cp\u003eg/(kgDBW\u0026bull;d)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"44.89051094890511%\"\u003e\n \u003cp\u003eg/(kg fresh larvae\u0026bull;d)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\"\u003e\n \u003cp\u003eTotal weight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.13138686131387%\"\u003e\n \u003cp\u003e30.8\u0026plusmn;2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"44.89051094890511%\"\u003e\n \u003cp\u003e55.5\u0026plusmn;4.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\"\u003e\n \u003cp\u003eTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.13138686131387%\"\u003e\n \u003cp\u003e10.1\u0026plusmn;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"44.89051094890511%\"\u003e\n \u003cp\u003e33.7\u0026plusmn;1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\"\u003e\n \u003cp\u003eTN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.13138686131387%\"\u003e\n \u003cp\u003e0.21\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"44.89051094890511%\"\u003e\n \u003cp\u003e1.69\u0026plusmn;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\"\u003e\n \u003cp\u003eCrude lipid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.13138686131387%\"\u003e\n \u003cp\u003e13.6\u0026plusmn;1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"44.89051094890511%\"\u003e\n \u003cp\u003e25.1\u0026plusmn;3.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u0026nbsp;\u003c/sup\u003eRefers to the reduced dry mass weight per amount of dry DBW per day during BSFL bioconversion\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u0026nbsp;\u003c/sup\u003eRefers to the reduced dry mass weight per amount of fresh larvae per day during BSFL bioconversion\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Insect, Bioconversion, Microbiota, Gut, Bioproducts","lastPublishedDoi":"10.21203/rs.3.rs-4007947/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4007947/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe lipids in the domestic biodegradable waste (DBW) pose a challenge to resource regeneration, and few studies have examined the evolution of lipid profiles during the process of black soldier fly larvae (\u003cem\u003eHermetia illucens L.\u003c/em\u003e, BSFL) bioconversion. This study aimed to explore the dynamic features of lipid fraction and their associated responses of microbial community succession in residue during a full-scale BSFL bioconversion. Data showed that the lipid content decreased by95%, while the seed germination index increased by 20% through the synergistic effects of BSFL and microbiota. The results of spectral and Gas chromatography-mass spectrometry showed that free fatty acids and medium-chain fatty acids were given first priority in degrading in larval and microbial coexistence systems, resulting in the relative accumulation of sterols. The lipid content (71.1%, \u003cem\u003eP\u003c/em\u003e = 0.002) was the prime environmental factor that promoted the succession of the bacterial community. The diversity and structure of the bacterial community varied at different stages of the bioprocess, where BSFL induced\u003cem\u003e Corynebacterium, Marinobacter, and Brevibacterium\u003c/em\u003e. EC: 4.2.1.17 (Enoyl-CoA hydratase) and EC: 1.1.1.35 (3-hydroxyacyl-CoA dehydrogenase) were the key lipid metabolic enzymes, promoting the degradation and transformation of materials and lipids. The synergistic effect of BSFL and microbiota promotes lipid metabolisms in DBW, which is conducive to the sustainable utilization of BSFL biotechnology to convert wastes into high-value resources.\u003c/p\u003e","manuscriptTitle":"Insights on Lipid Biodegradation in Domestic Biodegradable Waste at a Full-scale Black Soldier Fly Larvae (Hermetia illucens L.) 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