Bioconversion of Cattle Manure by Hermetia Illucens Larvae: Mineral Content Changes in Manure and Larval Biomass.

Bioconversion by Hermetia illucens larvae is a novel technology for organic waste treatment and valorization. However, since the possible uses of products from this process are in agriculture and livestock, the bioconversion must guarantee the mineral quality of both the Hermetia illucens larvae frass and larval biomass. Therefore, this study aimed to assess the mineral content changes for both the larval biomass and larvae frass of Hermetia illucens after the manure bioconversion to determine their suitability as animal feed and organic fertilizer, respectively. Hermetia illucens larvae were put into a plastic box containing fresh cattle manure, and the control treatment with the same conditions without larvae was established. After the rst pre-pupae were detected, frass and larvae were collected, and their mineral content was analyzed. At the end of the experiment, the larvae showed increases in some micro and macronutrients, especially calcium and manganese, increasing up to 2.6 and 22.6 times the initial concentration, respectively. The toxic elements concentration was increased in larval biomass, but these levels met the international legislation for animal feed. As a result, the mineral content in larval biomass revealed that Hermetia illucens could be potentially used as animal feed, which could be comparable with sh meal, and is probably better than soybean meal. However, the larvae frass could only be used as organic fertilizer in a Canadian context, with further treatment for decreasing the chromium content being necessary.


Introduction
Animal manure is a type of organic waste generated by livestock. With the increase of intensive production systems in response to the growing worldwide demand for animal protein, high amounts of manure must be managed (Sungur et al. 2016;Feng et al. 2018). Due to manure`s nutrient and mineral content, the most common management method used for it is its direct application to soils with or without prior storage (IPCC 2006) to improve soil quality and crop yield (Fengsong et al. 2011). However, the presence of pathogens, antibiotics, veterinary drugs, and some toxic elements could limit its use on agricultural soils and carry a risk to the environment and human health (Sahito et  . For example, Cu, Zn, As, and Cr promote animal growth, disease mitigation, and feed use e ciency. Pig feed often contains higher concentrations of Cu and Zn, and the animal's gut absorbs only 10-20% of these metals in feed. Thus the remaining amount of metals is excreted, which is why pig manure often shows higher levels of these metals than chicken and cattle manure (Nicholson et al. 1999;Fengsong et al. 2011;Ji et al. 2012;Wang et al. 2014;Ding et al. 2017;Feng et al. 2018).
The concern here is that toxic elements from manure entering the ecosystem may lead to accumulation, bioaccumulation, and biomagni cation in the food chain (Zhao et al. 2014), through crop leaching and surface run-off to freshwater after their application to soils (Shi et al. 2018(Shi et al. , 2019. Current studies reveal that land fertilizing with animal manure is the highest source of toxic elements in agricultural soils in countries with low industrial activities and the second source of toxic elements in soils, after atmospheric deposition, in industrialized countries (Shi et al. 2018(Shi et al. , 2019. Annually, the land fertilizing with animal manure contributes, at a global level, approximately 2.9 kt of toxic elements to the environment (Leclerc and Laurent 2017).
In addition to spreading manure on soils, the main methods of manure management used are anaerobic digestion and composting. However, toxic elements could be concentrated by these process (Hu et al. 2017;Zubair et al. 2020). Therefore, better methods of manure management must be developed which allow for the reduction and recycling of the toxic, macro and micronutrients of this waste.
A novel method for waste management that is gaining more attention due to the possibility of obtaining products of high value from this type of residue is bioconversion using y larvae (Čičková et al. 2015; Huis 2019). Hermetia illucens is the most proposed species for the treatment and valorization of animal manure. Using this species in the process can produce larval biomass and larvae frass that could be used as alternative feed and biofertilizer, respectively (Liu et al. 2019). However, when the y larvae are fed with manure from livestock activities, the generated larval biomass as feed is not recommended. Since 2017, the processed protein from seven edible insect species was approved by the European Union (EU) to use in aquaculture feeding. However, manure use is not allowed as an insect breeding substrate (European Commission 2017). Recently, the use of processed protein from these insects in poultry and swine feeds was authorized (European Commission 2021). Considering, that globally there are about 45.6 million tonnes per day of manure potentially available for processing (FAO; Chávez-Fuentes et al. 2017), and the potential for growing y larvae on animal manure to recycle protein and manage organic waste from livestock production (Nordentoft et al. 2017), more research may be needed to con rm the suitability of the use of animal manure (Huis 2019).
Larval biomass can accumulate high levels of some toxic elements (up to 9 fold the cadmium concentration in the substrate) (Tschirner and Simon 2015; Purschke et al. 2017). Charlton et al. (2015) found that y larvae fed with the swine manure of four different companies contained cadmium concentrations higher than the EU allowed limits for animal feed. Diverse studies agree with this nding and have highlighted the ability of Hermetia illucens to accumulate cadmium from spiked substrates Despite this concern, Hermetia illucens can also accumulate essential minerals for animal nutrition, such as Cu, Zn, and Ca Similarly, after co-products bioconversion, larvae frass have shown to be an interesting alternative for chemical fertilizer, increasing the development of crops such as tomato and leaf lettuce (Setti et al. 2019). However, when the substrates are contaminated with toxic elements, some elements can become concentrated, and others reduced. For example, Hermetia illucens larvae can concentrate Pb in spiked substrates (Diener et  demonstrated that Hermetia illucens could reduce animal manure's mineral content depending on the system scale. Accordingly, Hermetia illucens larvae could be a potential agent for treating toxic elements in contaminated wastes. Thus, in the context of the need for manure treatment and valorization, besides the potential of Hermetia illucens for feed and biofertilizer production from decayed materials, the aims of this study were: 1) to assess the suitability of Hermetia illucens larvae and frass from animal manure bioconversion as animal feed and biofertilizer by taking into consideration its mineral content and 2) to analyze changes in mineral concentrations, from larvae to pre-pupae, on larvae biomass and cattle manure after the bioconversion.

Materials
Hermetia illucens larvae and cattle manure collecting Larvae of Hermetia illucens were obtained from eggs of wild ies found on the composting project of the State University of Santa Cruz (Ilhéus-Bahia-Brazil). The cattle manure was collected from an experimental farm from the university mentioned above. The Hermetia illucens larvae rearing process during the rst six days and cattle manure collecting are described in the recently published paper of Sanchez Matos and colleagues (2021). Table 1 shows the mineral content of chicken feed and cattle manure used for the rearing of Hermetia illucens larvae and the experiments, respectively.

Mineralization and elemental analysis
The larvae and manure were dried at 60˚C for 48 hours and ground in a ceramic mortar. Dry samples of 0.3 g of manure and larvae were weighed and digested with 4 mL HNO3, 0.5 mL HCl and 2.5 mL H2O2 into per uoroalkoxy (PFA) vessels using a multimode microwave apparatus (CEM Mars Xpress). The samples were digested according to the following program: ramp in 2 min to 120 ºC, 8 min at 120 ºC, 5 min from 120 ºC to 180 ºC, and 15 min at 180 ºC. After digestion, the contents in the PFA vessels were transferred to 50 mL conical centrifuge tubes, then lled to 15 mL with deionized water. For digested samples of larvae, aliquots of these were diluted 20-fold with ultrapure water. These solutions were analyzed by an inductively coupled plasma optical emission spectrometry (ICP OES), model 710-ES Varian (Mulgrave, Australia), with axial con guration. This instrument was equipped with a MEINHARD® concentric nebulizer (Santa Clara, USA), coupled to a cyclonic nebulization chamber -single pass Varian The bioconversion of organic materials was able to cause changes in the mineral content of the substrates. In this study, as shown in Table 2, Cu, Mn, and Na content were signi cantly increased, and Ca and K content showed a signi cant reduction in Hermetia illucens frass after cattle manure bioconversion. However, Fe, Mg, P, S, Zn, and the content of the toxic metals showed no marked changes.
This nding is similar to the effect of Musca domestica larvae only in Cu, Fe, Na, P, and S content of cattle manure reported by Hussein et al. (2017). These differences could be linked to two factors: 1) the difference between the bioaccumulation factors of Musca domestica and Hermetia illucens, and 2) the higher water and dry matter reduction by Musca domestica in the cited study (37%) than for Hermetia illucens in this study (16.8%). Considering the potential use of larvae frass as biofertilizers, great attention should be given to high rates of reduction of organic matter of waste and low levels of element uptake, which could lead to an accumulation of undesired elements.  The mineral content of Hermetia illucens larvae frass obtained in this study was compared with European, Canadian, American, and Brazilian maximum limits of contaminants in organic fertilizers to assess its suitability for agricultural use. As shown in Tables 2 and 3, Cu, Zn, Cd, and Pb content met the maximum limits established by cited legislations; however, Cr content only met the Canadian maximum limits. A reason for this nding could be linked to Cr supplementation in cattle feeds and subsequent transfer to excreta. Usually, diverse forms of Cr are used in feed to treat mental, physical, or metabolic stress in cattle They sometimes can exceed the maximum limits for animal feed by six fold, as reported by Li et al.(2019). Therefore, in this case, it would be recommendable that a subsequent treatment process can be used to improve the suitability of larvae frass, for example, for co-composting with other residual materials that have low Cr content. Ca (

Mineral content changes in Hermetia illucens larval biomass
Fly larvae can degrade different types of substrates, assimilating the minerals therein for growth and development. Table 2 shows the mineral concentration of initial larvae (young larvae) and larvae ( rst prepupae appeared) of Hermetia illucens. Some macronutrients (K, Na, and P) did not have marked changes at the end of the bioconversion process. On the other hand, other micro and macronutrients, such as Ca, Cu, Fe, Mg, Mn, S, and Zn, presented signi cant increases. Proc et al.(2020b) also found increases in Ca, Cu, Fe, Mg, and Mn concentrations; however, K, Na, P, S, and Zn content decreased in larvae at the end of sh feed bioconversion. The reason for the differences between these studies could be linked to the nutritional composition of substrates. In this regard, Tschirner and Simon (2015) revealed that mineral content changes in biomass of Hermetia illucens larvae depend on the type of substrate. For example, the authors found that K, Na, P, and Mg content decreased in larvae fed with a mixture of middlings from a feed mill. When larvae were fed with dried distillers' grains with solubles made from barley, corn, wheat, and sugar syrups (protein group), the Cu, Fe, K, Mg, Mn, Na, P, and Zn content decreased. However, in larvae fed with dried sugar beet pulp, only Cu, Fe, P and Cu decreased, and the remaining elements increased.
Among all the evaluated elements, calcium was the most increased in larval biomass at the end of the experiment, with a 2.6 fold increase in the initial concentration. This result is consistent with the increase in the calcium content in Hermetia illucens larvae fed with sh feed, up to 2.3 times the initial concentration of young larvae, reported by Proc et al.(2020b), and it is also similar to the results obtained in larvae fed with co-products (dried sugar beet pulp), up to 2.7 times the initial calcium concentration, highlighted by Tschirner and Simon (2015). Previous studies with other y larvae (Musca autumnalis) have revealed that large amounts of Ca are ingested and stored in the Malpighian tubules during the larval stage, to subsequently be used during the pupariation process (Darlington et al. 1983;Grodowitz and Broce 1983). This calcium uptake by y larvae depends on the concentration of this element in the diet (Dube et al. 2000).
Regarding the toxic elements, the Cd, Cr, and Pb concentrations in larvae were signi cantly increased ( Table 2) On the other hand, a comparison of the mineral content in larval biomass was carried out in other studies to identify if the concentrations of minerals depend on the type of substrate (not spiked with mineral solutions) when it is feed co-product or waste. Table S1 (in supplementary material) shows a wide variation in the mineral content of larval biomass with apparently no pattern according to the type of substrate used for rearing. For instance, calcium content obtained in larvae was higher than the values obtained in larvae fed with feeds and co-products, and only similar with larvae reared in dried sugar beet pulp. However, this does not mean that all larvae reared in residues will have more calcium than those obtained from co-products or animal feed. For example, in the case of larvae fed with restaurant waste (Spranghers et al. 2017), kitchen waste and chicken manure (Shumo et al. 2019) had lower amounts of calcium than those from the other substrates. In this study, the Cu content in larvae was higher than that fed with feeds and in line with larvae fed with chicken manure and kitchen waste (Shumo et al. 2019) but lower than the Cu content in commercial larvae (Irungu et al. 2018). Furthermore, regarding K, Mg, and P content, these were similar to the content of larvae fed with chicken feed (Dierenfeld and King 2008) and Na content was in line with the content in larvae grown in poultry and pig manure (Newton et al. 2005). Zn and Fe content in the present study were higher in larvae fed with feeds or co-products and only lower than larvae reared in chicken manure (Shumo et  The micro and macronutrients in larvae were compared with the mineral content of sh and soybean meals, as shown in Table 2. The larvae's Ca, Fe, Mg, Mn, and S contents were higher than in sh and soybean meal. The Cu, Na, and P contents were higher in the sh meal than in the others; however, they were higher in larvae than in the soybean meal. The K content in larvae was lower than in soybean meal but higher than in sh meal. The Zn concentration was similar to that in larvae, sh meal, and soybean meal. Therefore, the mineral content of Hermetia illucens larvae is comparable with sh meal and possibly better than soybean meal. The mineral content in larvae was also compared with the maximum limits for feeds established by different countries. As shown in Tables 2 and 3, the toxic elements in larvae met the EU, USA (United States of America), and Canada concentration limits for Cd, Cr, and Pb. For micro and macronutrients, EU and Canadian legislation has not established maximum limits; therefore, the values of these elements in larvae were compared with the limits established by the USA. The Cu level in larval biomass met the maximum limit for pig, poultry, and sh feed. Furthermore, Fe and Zn content only met the maximum limit for pig feed. However, K, Mg, Mn, P, and S concentrations were slightly higher than the maximum limits for poultry, pig, and sh feed. These results are promising for two reasons: 1) the safe levels of toxic elements and 2) despite the slightly higher mineral levels compared with maximum levels for feed, the larval biomass could be used partially in feeds by adjusting the mineral content according to the animal's nutritional requirements.

Bioaccumulation of toxic elements, micro and macronutrients in Hermetia illucens larvae
The bioaccumulation factors (BAF) of micro and macronutrients and toxic elements in larval biomass increased from young to adult larvae (Fig. 1). The BAF of elements in larvae that were higher than 1 showed the following trend in this study: Ca > K > Mn > P > S > Mg > Na > Zn > Cd.
These results were compared with the BAF of different elements in Hermetia illucens larvae fed with arti cially uncontaminated substrates of previous studies (Tschirner and

Conclusion
In this study, the cattle manure bioconversion by Hermetia illucens larvae was studied by obtaining larval biomass and frass. The Ca, Cu, Fe, Mg, Mn, S, and Zn content in larvae increased through bioconversion.
The biomass larval showed promise for partial use as animal feed in countries where waste-fed insects would be allowed due to the high micro and macronutrients content and safe toxic metal levels. However, more studies are needed to assess the microbiological and chemical (antibiotics, veterinary medicines, and allergens) hazards of using animal manure as growing media for Hermetia illucens larvae. Furthermore, considering the mineral content, Hermetia illucens larvae frass was only suitable as organic fertilizer in a Canadian context due to its Cr concentration. So further treatment, such as co-composting with low chromium residual materials, would be necessary and more controls in chromium supplementation for livestock to improve the suitability of larvae frass obtained from the bioconversion of manure.

Declarations
Availability of data and materials The datasets supporting the conclusions of this manuscript are included within the article and the supplementary information. Not applicable.

Con icts of Interest/Competing Interests
The authors declare no con ict of interest. Figure 1 Bioaccumulation Factors (BAF) of different elements for Hermetia illucens larvae fed with cattle manure

Supplementary Files
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