Preservation of agri-food byproducts by acidification and fermentation in black soldier fly larvae bioconversion.

Maintaining a consistent supply of feedstock for efficient bioconversion of black soldier fly larvae (BSFL) presents challenges due to the fluctuating availability of biowastes and agri-food products. To address the challenge of consistent feedstock supply for BSFL, this study investigated the influence of three preservation methods: wild fermentation, inoculated fermentation, and acidification on agri-food by-products applied over three storage durations (1, 7, and 14 days), evaluating their impact on BSFL bioconversion, and feedstock nutrient and microbiota composition. The preserved feedstocks were characterized for gross nutrient, sugar, fermentation metabolite, and bacterial community analyses. All feedstock preservation methods and storage durations had a high bioconversion rate (21-25 % dry mass) and wet larval mass (170-196 mg). Notably, 7-and-14-day acidified feedstock had a significantly higher bioconversion rate compared to fermented feedstock. Acidification preserved feedstock nutrients best with only a 10 % difference compared to initial nutrient values. Fermentation produced typical lactic acid fermentation metabolites with reducing sugar contents; however, adding a lactic acid bacterial inoculum (7 log10 CFU kg feedstock-1) had no benefit, presumably due to the high nutrient content and existing richness in lactic acid bacteria. Preservations had little influence on Enterobacteriaceae (6.2-7.5 log10 CFU g-1) in freshly harvested larvae. Future research should assess the acidification and fermentation of different BSFL feedstocks and investigate the roles of feedstock pH, organic acids, and fermentation metabolites in more detail. Therefore, this study advances toward reliable and efficient insect-based nutrient recovery from agri-food by-products within the food system.

Physical pretreatment of three biowastes to improve black soldier fly larvae bioconversion efficiency.

Black soldier fly larvae (BSFL, Hermetia illucens (L.)) are recognized for efficient biowaste reduction while yielding valuable proteins and fats for animals. However, lignocellulosic fibers in biowastes are difficult to digest by biowaste and larval digestive tract microorganisms as well as the larvae themselves. This study investigated two biowaste physical pretreatments (thermal, mechanical) for improving BSFL processing of fibrous biowastes. Cow manure, spent grain, and grass clippings were thermally pretreated at 90 °C for three durations (0.5, 1 and 4 h). Contrary to expectations, thermal pretreatment resulted in either no improvement or decreased larval performance on all substrates, regardless of treatment duration. In contrast, mechanical pretreatment of spent grain and grass clippings, involving milling with three screen sizes (0.5, 1 and 2 mm) showed promising results. Specifically, bioconversion rates on 0.5 mm-milled spent grain and grass clippings increased by 0-53 % and 25-44 % dry mass, respectively compared to untreated. Additionally, larval protein conversion increased by 41 % and 23 % on spent grain and grass clippings, respectively. However, mechanical pretreatment did not affect fiber degradation by larval conversion, as hemicellulose decreased by 25 % and 75 % for spent grain and grass clippings, respectively, regardless of particle size. Particle size reduction influenced substrate microbial respiration (CO2 mg/min), with 0.5-mm milled grass clippings exhibiting higher respiration compared to untreated, although this effect was not observed for spent grain. This study highlights mechanical pretreatment's potential in enhancing BSFL bioconversion of fibrous biowastes and the importance of understanding substrate physical properties influencing substrate microorganisms and BSFL.

Evaluation of ammonia pretreatment of four fibrous biowastes and its effect on black soldier fly larvae rearing performance.

Biowaste treatment with black soldier fly larvae (BSFL, Hermetia illucens L.) can promote a more sustainable food system by reusing nutrients that would otherwise be wasted. However, many agri-food wastes and byproducts are typically high in lignocellulosic fibers (i.e., cellulose, hemicellulose, and lignin), making it resistant to efficient larval and/or microbial degradation. Ammonia pretreatment could be used to partially degrade lignocellulose, making the biowaste more easily degradable by the larvae and/or microorganisms. This study evaluated ammonia pretreatment for lignocellulose degradation and its effect on BSFL performance on four fibrous biowastes: brewers spent grain, cow manure, oat pulp, and grass clippings. First, the optimal ammonia dose (1 % or 5 % dry mass) and pretreatment time (three or seven days) were assessed by measuring fibers after treatment and further examined using Fourier transform infrared spectroscopy (FTIR) spectra and scanning electron microscopy (SEM) images. Second, BSFL rearing performance on ammonia-pretreated substrates was assessed with a 9-day feeding experiment. Three-day pretreatment with 5 % ammonia was chosen as it decreased the total fiber content by 8-23 % for all substrates except cow manure. Contrary to expectations, ammonia pretreatment with all substates decreased BSFL rearing performance metrics by more than half compared to the untreated control. Follow-up experiments suggested that ammonia pretreatment had a dose-dependent toxicity to BSFL. Interestingly, three-day fermentation of cow manure and oat pulp increased bioconversion rate by 25-31 %. This study shows that ammonia pretreatment is not suitable before BSFL rearing. Ammonia toxicity to BSFL and other pretreatments, such as fermentation, should be further studied.

Residues from black soldier fly (Hermetia illucens) larvae rearing influence the plant-associated soil microbiome in the short term.

The larvae of the black soldier fly (BSFL, Hermetia illucens) efficiently close resource cycles. Next to the nutrient-rich insect biomass used as animal feed, the residues from the process are promising plant fertilizers. Besides a high nutrient content, the residues contain a diverse microbial community and application to soil can potentially promote soil fertility and agricultural production through the introduction of beneficial microbes. This research assessed the application of the residues on plant-associated bacterial and fungal communities in the rhizosphere of a grass-clover mix in a 42-day greenhouse pot study. Potted soil was amended with BSFL residues (BR+) or conventional compost (CC+) produced by Rwandan waste management companies in parallel to residues and compost sterilized (BR-, CC-) by high-energy electron beam (HEEB) as abiotic controls. The fertilizers were applied at a rate of 150 kg N ha-1. Soil bacterial and fungal communities in both fertilizer and soil were assessed by high-throughput sequencing of ribosomal markers at different times after fertilizer application. Additionally, indicators for soil fertility such as basal respiration, plant yield and soil physicochemical properties were analyzed. Results showed that the application of BSFL residues influenced the soil microbial communities, and especially fungi, stronger than CC fertilizers. These effects on the microbial community structure could partly be attributed to a potential introduction of microbes to the soil by BSFL residues (e.g., members of genus Bacillus) since untreated and sterilized BSFL residues promoted different microbial communities. With respect to the abiotic effects, we emphasize a potential driving role of particular classes of organic matter like fiber and chitin. Indeed, especially taxa associated with decomposition of organic matter (e.g., members of the fungal genus Mortierella) were promoted by the application of BSFL residues. Soil fertility with respect to plant yield (+17% increase compared to unamended control) and basal respiration (+16% increase compared to unamended control) tended to be improved with the addition of BSFL residues. Findings underline the versatile opportunities for soil fertility arising from the application of BSFL residues in plant production and point to further research on quantification of the described effects.

Transfer of Lauric and Myristic Acid from Black Soldier Fly Larval Lipids to Egg Yolk Lipids of Hens Is Low.

Implementing insects, such as the black soldier fly larvae (BSFL), as animal feed commonly includes the previous removal of substantial amounts of fat. This fat may represent an as yet underutilized energy source for livestock. However, transfer of lauric and myristic acid, prevalent in BSFL fat and undesired in human nutrition, into animal-source foods like eggs may limit its implementation. To quantify this, a laying hen experiment was performed comprising five different diets (10 hens/diet). These were a control diet with soybean oil and meal and a second diet with soybean oil but with partially defatted BSFL meal as protein source. The other three diets were based on different combinations of partially defatted BSFL meal and fat obtained by two different production methods. Lauric acid made up half of the BSFL fat from both origins. Both BSFL fats also contained substantial amounts of myristic and palmitic acid. However, in the insect-based diets, the net transfer from diet to egg yolk was less than 1% for lauric acid, whereas the net transfer for myristic and palmitic acid was about 30% and 100%, respectively. The net transfer did not vary between BSFL originating from production on different larval feeding substrates. The results illustrate that hens are able to metabolize or elongate very large proportions of ingested lauric acid and myristic acid, which are predominant in the BSFL lipids (together accounting for as much as 37 mol%), such that they collectively account for less than 3.5 mol% of egg yolk fatty acids.

Identification of Bacteria in Two Food Waste Black Soldier Fly Larvae Rearing Residues.

Significant economic, environmental, and social impacts are associated with the avoidable disposal of foods worldwide. Mass-rearing of black soldier fly (Hermetia illucens) larvae using organic wastes and food- and agro-industry side products is promising for recycling resources within the food system. One current challenge of this approach is ensuring a reliable and high conversion performance of larvae with inherently variable substrates. Research has been devoted to increasing rearing performance by optimizing substrate nutrient contents and ratios, while the potential of the substrate and larval gut microbiota to increase rearing performance remains untapped. Since previous research has focused on gut microbiota, here, we describe bacterial dynamics in the residue (i.e., the mixture of frass and substrate) of black soldier fly larvae reared on two food wastes (i.e., canteen and household waste). To identify members of the substrate and residue microbiota, potentially associated with rearing performance, bacterial dynamics were also studied in the canteen waste without larvae, and after inactivation by irradiation of the initial microbiota in canteen waste. The food waste substrates had similar microbiota; both were dominated by common lactic acid bacteria. Inactivation of the canteen waste microbiota, which was dominated by Leuconostoc, Bacillus, and Staphylococcus, decreased the levels of all rearing performance indicators by 31-46% relative to canteen waste with the native microbiota. In both food waste substrates, larval rearing decreased the bacterial richness and changed the physicochemical residue properties and composition over the rearing period of 12 days, and typical members of the larval intestinal microbiota (i.e., Providencia, Dysgonomonas, Morganella, and Proteus) became more abundant, suggesting their transfer into the residue through excretions. Future studies should isolate members of these taxa and elucidate their true potential to influence black soldier fly mass-rearing performance.

Novel Experimental Methods for the Investigation of Hermetia illucens (Diptera: Stratiomyidae) Larvae.

Large-scale insect rearing for food and feed production can be improved by understanding diet digestion and host-microbe interactions. To examine these processes in black soldier fly (Hermetia illucens L.; Diptera: Stratiomyidae) larvae, two protocols were developed. Protocol 1 describes a method to produce viable, sterile black soldier fly larvae and a gentle method for diet sterilization. Sterile black soldier fly larvae can be used to study the diverse role of microbes in larval development. Nutrient requirements of sterile black soldier fly larvae are met only through diet. Viable sterile black soldier fly larvae were consistently generated using a four-step treatment with alternating immersions of eggs for 2 min each in ethanol (70%) and sodium hypochlorite (0.6%), over two cycles. A nonthermal method of diet sterilization, namely high-energy electron beam (HEEB) treatment, was introduced. Subsequently, growth of sterile black soldier fly larvae was observed on the HEEB-treated diets (40, 60, and 40% of replicates with poultry feed, liver pie, and an artificial diet, respectively) but not on autoclaved diets. In Protocol 2, we propose a novel method to collect frass from individual larvae. We then measured the metabolites in frass, using high-pressure liquid chromatography. Results on metabolites confirmed the influence of digestion. For instance, succinate increased from 1 to 2 and 7 µmol/g sample from diet to gut homogenate and frass, respectively. The collection method is a promising tool to estimate the diet and nutrient requirements of black soldier fly larvae, thus increasing the performance and reliability of black soldier fly larvae rearing. We discuss in detail the possible applications and limitations of our methods in black soldier fly larvae research.

Estimating black soldier fly larvae biowaste conversion performance by simulation of midgut digestion.

Black soldier fly larvae treatment is an emerging technology for the conversion of biowaste into potentially more sustainable and marketable high-value products, according to circular economy principles. Unknown or variable performance for different biowastes is currently one challenge that prohibits the global technology up-scaling. This study describes simulated midgut digestion for black soldier fly larvae to estimate biowaste conversion performance. Before simulation, the unknown biowaste residence time in the three midgut regions was determined on three diets varying in protein and non-fiber carbohydrate content. For the static in vitro model, diet residence times of 15 min, 45 min, and 90 min were used for the anterior, middle, and posterior midgut region, respectively. The model was validated by comparing the ranking of diets based on in vitro digestion products to the ranking found in in vivo feeding experiments. Four artificial diets and five biowastes were digested using the model, and diet digestibility and supernatant nutrient contents were determined. This approach was able to distinguish broadly the worst and best performing rearing diets. However, for some of the diets, the performance estimated based on in vitro results did not match with the results of the feeding experiments. Future studies should try to establish a stronger correlation by considering fly larvae nutrient requirements, hemicellulose digestion, and the diet/gut microbiota. In vitro digestion models could be a powerful tool for academia and industry to increase conversion performance of biowastes with black soldier fly larvae.

Biowaste treatment with black soldier fly larvae: Increasing performance through the formulation of biowastes based on protein and carbohydrates.

A key challenge for black soldier fly larvae (BSFL) treatment is its variable reliability and efficiency when applied to different biowastes. Similar to other biowaste treatment technologies, co-conversion could compensate for variability in the composition of biowastes. Using detailed nutrient analyses, this study assessed whether mixing biowastes to similar protein and non-fibre carbohydrate (NFC) contents increased the performance and reduced the variability of BSFL treatment in comparison to the treatment of individual wastes. The biowastes examined were mill by-products, human faeces, poultry slaughterhouse waste, cow manure, and canteen waste. Biowaste formulations had a protein-to-NFC ratio of 1:1, a protein content of 14-19%, and a NFC content of 13-15% (dry mass). Performance parameters that were assessed included survival and bioconversion rate, waste reduction, and waste conversion and protein conversion efficiency. In comparison to poultry feed (benchmark), vegetable canteen waste showed the best performance and cow manure performed worst. Formulations showed significantly improved performance and lower variability in comparison to the individual wastes. However, variability in performance was higher than expected for the formulations. One reason for this variability could be different fibre and lipid contents, which correlated with the performance results of the formulations. Overall, this research provides baseline knowledge and guidance on how BSFL treatment facilities may systematically operate using biowastes of varying types and compositions.

Influence of Lactobacillus buchneri on soybean curd residue co-conversion by black soldier fly larvae (Hermetia illucens) for food and feedstock production.

Black soldier fly larvae (BSFL), Hermetia illucens (Diptera: Stratiomyidae) can reduce environmental pollution and convert organic wastes into biomass that is rich in protein and fat. The influence of the nutritional characteristics of organic waste on BSFL characteristics relevant for food and feed safety remains poorly understood. To evaluate the conversion of soybean curd residues (SCR) into high-quality animal-derived proteins and fats for human and livestock consumption, this study assessed the co-conversion efficacy, nutrient composition, safety, and anti-nutritional factor concentrations in BSFL after the development on SCR with Lactobacillus buchneri (L3-9). SCR was pretreated with L. buchneri (108 cfu/ml), and then BSFL was employed for conversion. BSFL fed with SCR and L. buchneri had a significantly higher dry mass reduction (55.7 ±â€¯0.9%), bioconversion rate (6.9 ±â€¯0.3%), crude protein content (55.3 ±â€¯0.6%), and fat content (30.0 ±â€¯0.6%) than SCR (49.0 ±â€¯0.7%, 5.0 ±â€¯0.3%, 52.8 ±â€¯0.3%, and 26.1 ±â€¯0.8%, respectively) and artificial feed (43.9 ±â€¯0.8%, 3.9 ±â€¯0.1%, 50.3 ±â€¯0.4%, and 24.3 ±â€¯0.4%, respectively). However, the feed conversion ratio (8.0 ±â€¯0.3), of BSFL fed with SCR and L. buchneri was lower than that of the BSFL fed with SCR (9.8 ±â€¯0.1) and artificial feed (11.1 ±â€¯0.5). In addition, BSFL had satisfactory concentrations of all essential amino acids and fatty acids required for human consumption as recommended by WHO/FAO/UNU. The heavy metals and anti-nutritional factor concentrations were within the safety intake levels for food and feedstock. Therefore, the addition of L. buchneri with BSFL on SCR did not only increase co-conversion performance but also enhanced the nutritional value of BSFL.

Decomposition of biowaste macronutrients, microbes, and chemicals in black soldier fly larval treatment: A review.

Processing of biowaste with larvae of the black soldier fly, Hermetia illucens L. (Diptera: Stratiomyidae), is an emerging waste treatment technology. Larvae grown on biowaste can be a relevant raw material for animal feed production and can therefore provide revenues for financially viable waste management systems. In addition, when produced on biowaste, insect-based feeds can be more sustainable than conventional feeds. Among others, the scalability of the technology will depend on the availability of large amounts of biowaste with a high process performance (e.g. bioconversion of organic matter to proteins and lipids) and microbial and chemical product safety. Currently, in contrast to other waste treatment technologies, such as composting or anaerobic digestion, the process performance is variable and the processes driving the decomposition of biowaste macronutrients, inactivation of microbes and fate of chemicals is poorly understood. This review presents the first summary of the most important processes involved in black soldier fly larvae (BSFL) treatment, based on the available knowledge concerning five well-studied fly species. This is a starting point to increase understanding regarding the processes of this technology, with the potential to increase its efficiency and uptake, and support the development of appropriate regulations. Based on this review, formulating different types of biowaste, e.g. to produce a diet with a similar protein content, a balanced amino acid profile and/or pre- and co-treatment of biowaste with beneficial microbes, has the potential to increase process performance. Following harvest, larvae require heat or other treatments for microbial inactivation and safety.

Cross-country analysis of faecal sludge dewatering.

Dewatering of faecal sludge (FS) is indispensable for adequate FS management. However, comprehensive knowledge is lacking on FS dewatering performance. This study compared the dewatering performance of FS from different countries and onsite sanitation technologies, to assess influential characteristics on dewatering, and to compare dewatering performance of FS with wastewater sludge. We collected 73 FS samples from septic tanks, lined pit latrines, unlined pit latrines and johkasou tanks in Uganda, Vietnam and Japan, and 18 samples of wastewater sludge in Switzerland. Capillary suction time (CST) and total solids (TS) of centrifuged sludge (%TSfinal) were determined as metrics of dewatering rate and dewaterability, respectively, together with relevant sludge characteristics. Data were analysed by bootstrapping comparison of median results of each sample category and by bootstrapping multiple regression analysis to quantify the relative importance of sludge characteristics on dewatering performance. Results showed that the dewatering rate was significantly different between FS from different technologies, whereas dewaterability was significantly different within the same technology. FS had a significantly lower dewatering rate than wastewater sludge. In contrast, FS dewaterability was greater than wastewater sludge. However, this could be attributed to higher concentrations of sand in FS. Electrochemical properties such as NH4-N and surface charge had the strongest correlation to dewatering rate, and solid properties such as sand content and total volatile solids to dewaterability. The results identify potential characteristics that could explain and predict the high variability of FS dewatering performance that is observed in the field.

Locally produced natural conditioners for dewatering of faecal sludge.

In urban areas of low-income countries, treatment of faecal sludge (FS) is insufficient or non-existent. This results in large amounts of FS being dumped into the environment. Existing treatment technologies for FS, such as settling-thickening tanks and drying beds, are land intensive which is limiting in urban areas. Enhanced settling and dewatering by conditioning was evaluated in order to reduce the treatment footprint (or increase treatment capacity). Conventional wastewater conditioners, such as commercially available lime and polymers, are expensive, and commonly rely on complex supply chains for use in low-income countries. Therefore, the treatment performance of five conditioners which could be produced locally was evaluated: Moringa oleifera seeds and press cake, Jatropha curcas seeds, Jatropha Calotropis leaves and chitosan. M. oleifera seeds and press cake, and chitosan improved settling and dewatering and had a similar performance compared to lime and polymers. Optimal dosages were 400-500 kg M. oleifera/t TS, 300-800 kg lime/t TS and 25-50 kg polymer solution/t TS. In comparison, chitosan required 1.5-3.75 kg/t TS. These dosages are comparable to those recommended for wastewater (sludge). The results indicate that conditioning of FS can reduce total suspended solids (TSS) in the effluent of settling-thickening tanks by 22-81% and reduce dewatering time with drying beds by 59-97%. This means that the area of drying beds could be reduced by 59-97% with end-use as soil conditioner, or 9-26% as solid fuel. Least expensive options and availability will depend on the local context. In Dakar, Senegal, chitosan produced from shrimp waste appears to be most promising.