TYPE Original Research PUBLISHED 26 September 2022 DOI 10.3389/fmicb.2022.994091 Residues from black soldier fly OPEN ACCESS (Hermetia illucens) larvae rearing EDITED BY Kim Yrjälä, influence the plant-associated Zhejiang Agriculture and Forestry University, China soil microbiome in the short REVIEWED BY Blaz Stres, University of Ljubljana, term Slovenia Andrei Alyokhin, University of Maine, Adrian Fuhrmann 1,2, Benjamin Wilde 1, Rafaela Feola Conz 1, United States Speciose Kantengwa 3, Matieyedou Konlambigue 3, *CORRESPONDENCE Martin Hartmann Barthazar Masengesho 4, Kokou Kintche 3, Kinfe Kassa 5, martin.hartmann@usys.ethz.ch William Musazura 6, Leonhard Späth 1,7, Moritz Gold 8,9, SPECIALTY SECTION 8 1 This article was submitted to Alexander Mathys , Johan Six and Martin Hartmann 1* Terrestrial Microbiology, 1 a section of the journal Sustainable Agroecosystems Group, Institute of Agricultural Sciences, Department of Frontiers in Microbiology Environmental Systems Science, ETH Zürich, Zürich, Switzerland, 2 Singapore-ETH Centre, Singapore, Singapore, 3 International Institute of Tropical Agriculture, Kigali, Rwanda, 4 Maggot Farm RECEIVED 14 July 2022 Production Ltd., Kamonyi, Kamonyi, Rwanda, 5 Faculty of Water Supply and Environmental ACCEPTED 06 September 2022 Engineering, Arba Minch University, Arba Minch, Ethiopia, 6 School of Agricultural, Earth and PUBLISHED 26 September 2022 Environmental Sciences, University of Kwazulu-Natal, Pietermaritzburg, South Africa, 7 Transdisciplinary Lab, Department of Environmental Systems Science, ETH Zürich, Zürich, CITATION 8 Fuhrmann A, Wilde B, Conz RF, Switzerland, Sustainable Food Processing Laboratory, Institute of Food, Nutrition and Health, Kantengwa S, Konlambigue M, Department of Health Science and Technology, ETH Zürich, Zürich, Switzerland, 9 Department of Masengesho B, Kintche K, Kassa K, Sanitation, Water and Solid Waste for Development (Sandec), Swiss Federal Institute of Aquatic Musazura W, Späth L, Gold M, Mathys A, Science and Technology, Dübendorf, Switzerland Six J and Hartmann M (2022) Residues from black soldier fly (Hermetia illucens) larvae rearing influence the plant- The larvae of the black soldier fly (BSFL, Hermetia illucens) efficiently close associated soil microbiome in the short resource cycles. Next to the nutrient-rich insect biomass used as animal term. Front. Microbiol. 13:994091. feed, the residues from the process are promising plant fertilizers. Besides a doi: 10.3389/fmicb.2022.994091 high nutrient content, the residues contain a diverse microbial community COPYRIGHT and application to soil can potentially promote soil fertility and agricultural © 2022 Fuhrmann, Wilde, Conz, production through the introduction of beneficial microbes. This research Kantengwa, Konlambigue, Masengesho, Kintche, Kassa, Musazura, Späth, Gold, assessed the application of the residues on plant-associated bacterial and Mathys, Six and Hartmann. This is an open- fungal communities in the rhizosphere of a grass-clover mix in a 42-day access article distributed under the terms greenhouse pot study. Potted soil was amended with BSFL residues (BR+) of the Creative Commons Attribution License (CC BY). The use, distribution or or conventional compost (CC+) produced by Rwandan waste management reproduction in other forums is permitted, companies in parallel to residues and compost sterilized (BR-, CC-) by high- provided the original author(s) and the copyright owner(s) are credited and that energy electron beam (HEEB) as abiotic controls. The fertilizers were applied the original publication in this journal is at a rate of 150   kg  N   ha−1. Soil bacterial and fungal communities in both cited, in accordance with accepted fertilizer and soil were assessed by high-throughput sequencing of ribosomal academic practice. No use, distribution or reproduction is permitted which does not markers at different times after fertilizer application. Additionally, indicators comply with these terms. 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, Frontiers in Microbiology 01 frontiersin.org Fuhrmann et al. 10.3389/fmicb.2022.994091 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. KEYWORDS Hermentia illucens, soil microbiome, organic fertilizers, frass, plant growth promotion, circular economy, black soldier fly larvae Introduction human consumption, invertebrates are suitable to become a replacement for products associated with comparably negative The intensification of ecological imbalances combined with a environmental externalities like soy or fish based protein feed growing world population poses an enormous challenge for the (Smetana et al., 2019). Especially the larva of the black soldier fly global society of the 21st century. The demand for food is steadily (BSFL, Hermetia illucens) is considered to have great potential for increasing, and sustainable approaches are needed to solve the circular economy (Varelas, 2019), being able to metabolize a wide resulting challenges in agricultural production (McKenzie and range of organic substances including human excreta (Banks et al., Williams, 2015). The soil microbiome is essential to the viability 2014) and to reduce dry weight of waste material by 27–72% of global ecosystems, since it is a major driver of soil structure (Gold et  al., 2018). Since BSFL reproduce more efficiently in formation, organic matter breakdown, nutrient provisioning, tropical regions, the technology offers an opportunity for plant growth promotion, and stress and disease control (Barrios, innovation, particularly in low-income countries (Barragan- 2007). Its functional capacity to influence soil fertility and thus Fonseca et al., 2017). In addition to the sustainability potential of crop performance (Van Der Heijden et al., 2016; Saleem et al., the larva, the extensively accruing residues from insect rearing, 2019) has gained increasing attention in sustainable food i.e., excreta, undigested substrates and shed exoskeletons, bear the production. Intentionally steering the soil microbiome by adapting chance of returning versatile services to the agri-food chain via agricultural management practices is considered a promising their provision of nutrients, bioactive molecules, and potentially approach to increasing the resilience of a global agriculture facing beneficial microbes when amended to the soil–plant system environmental challenges (Wang and Haney, 2020). For example, (Poveda, 2021; Barragán-Fonseca et al., 2022). Hartmann et  al. (2015) showed that the type of agricultural The fertilizer effect of BSFL residues (in some studies also practice and especially the type of applied fertilizer can play a referred to as “frass”) on plants has been subjected to several crucial role in shaping long-term soil bacterial and fungal studies. Outcomes from greenhouse and field trials were mostly communities in agroecosystems. Sun et  al. (2016) further comparable or superior to mineral fertilization (Kebli and Sinaj, indicated that exogenous microorganisms associated with organic 2017; Klammsteiner et al., 2020a; Beesigamukama et al., 2020b; fertilizers such as animal manure can establish and alter the soil Anyega et  al., 2021) with exceptions (Gärttling et  al., 2020; microbiome. Compost fertilizer is a promising source of Gebremikael et al., 2022). Chirere et al. (2021) pointed out that the exogenous microorganisms with nutrient-cycling and disease- generally higher nutrient contents of BSFL residues compared to suppressive capacities that can improve soil health (Lutz et al., other organic soil amendments make the residues an agronomically 2020). Although organic sources of fertilizer such as compost have practical alternative. It should be noted that, depending on the demonstrated positive impacts on the soil microbiome, providing substrates fed to BSFL, physicochemical properties of the residues sufficient quantities of compost is a major impediment to may strongly differ (Schmitt and de Vries, 2020; Klammsteiner sustainable agricultural production. et  al., 2020a), which results in distinct effects on soil fertility The utilization of insects in the agri-food-chain is seen as a (Gebremikael et al., 2022). Furthermore, postprocessing of BSFL promising way to generate valuable protein feed and fertilizer in a residues, especially composting, with potential influence on the more sustainable way. Van Huis and Oonincx (2017) pointed out composition of the residues, is not conducted consistently across that insects emit less greenhouse gases, require less land, convert studies. This variability in post-processing potentially also inputs more efficiently than conventional livestock, and can feed influences attributed toxic effects on the soil–plant system (Zorrilla on organic side-streams. Besides rearing insects directly for and Hussain, 2020), whereas high concentrations of phenols in Frontiers in Microbiology 02 frontiersin.org Fuhrmann et al. 10.3389/fmicb.2022.994091 uncomposted residues were suggested to be  one reason for a amendments using high-energy electron beam (HEEB) radiation reduced germination rate of Pak Choi plants (Song et al., 2021). prior to their application, we sought to delineate the impact of The question of composting insect residues for agricultural use is biotic and abiotic components. We examined a possible transfer crucial for the insect industry because the requisite infrastructure of species stemming from BSFL residues to the soil. To shed light bears significant economic and logistic implications. In fact, BSFL on the consequences for soil fertility, we assessed changes in the residues can reduce plant growth when applied in high amounts (≥ soil microbial community and its responding microbial taxa as 15–20 t ha−1), whereas the generally high ammonium and salt well as changes in plant yield and soil respiration. As a reference, content has been discussed as a potential reason (Temple et al., we compared BSFL residues with a conventional compost as an 2013; Alattar et  al., 2016; Chiam et  al., 2021). Chitin and its established organic fertilizer. derivates stemming from molting BSFL are associated with plant performance promotion through inhibition of pathogenic and support of beneficial soil microbes as well as through a direct Materials and methods stimulation of plant growth (Sharp, 2013). The BSFL residues are likely to contain further bioactive compounds of importance for Organic fertilizers the soil microbiome, since BSFL genes encoding for several antimicrobial peptides and their diet-dependent expression were The influence of the BSFL residues (BR) on the soil identified (Elhag et al., 2017; Vogel et al., 2018). microbiome was assessed in a pot experiment by comparing a Poveda (2021) emphasized the structural and functional sterile and a non-sterile form (BR-, BR+) to conventional compost similarities between plant root and insect gut, suggesting not just (CC) in a sterile and a non-sterile form (CC-, CC+). BR was an overlap of microbial communities between both habitats but provided by Maggot Farms Ltd. (Kamonyi, Rwanda), specialized also possible synergistic benefits when combined. As demonstrated in the bioconversion of organic waste-streams into animal feed in former studies, BSFL can substantially influence microbial with BSFL. Five day old larvae were added to a brewery waste- community composition in its residues presumably via fecal stream (spent malted barley grain from SKOL Brewery Ltd., transmission, leaving behind a characteristically imprinted residue Kigali, Rwanda). After around 14 days of feeding, first prepupae (Gold et al., 2020b). When the organic amendment is introduced appeared and insects were separated from the residues. During the to the rhizosphere soil, the insect gut derived flora may merge feeding process the waste material was reduced by 50–60% on a with its plant-related analogue. For example, the microbiome of wet weight basis. BR was stored for 12 days in closed plastic bags mealworm (Tenebrio molitor) excreta was reported to contain at ambient temperature before shipping to Switzerland. CC was plant growth promoting bacterial taxa and to have a positive provided by COPED Ltd., a waste management company based in influence on the performance of chard plants when applied as Kigali, Rwanda. The compost was produced with municipal waste fertilizer under greenhouse conditions (Poveda et  al., 2019). (mainly organics: food waste, green waste; inorganic material that Findings from Chiam et al. (2021) suggested that BSFL residues was not sorted out) and turned once during the composting time applied at high rates to soil (≥ 10% v/v BSFL residue) can change of around 6 months. The companies that provided the fertilizers the soil bacterial community structure. Gebremikael et al. (2020) are taking part in the RUNRES1 project which aims to improve found in their soil incubation experiment that fertilization with circular food systems in city regions across Africa. BSFL residues produced from food waste (110 kg N ha−1) changed After sampling in Rwanda, fertilizers were stored in resealable the microbial community significantly after 42 days based on an polyethylene bags and either transported on ice or stored in the assessment with bacterial and fungal biomarkers. The authors fridge at 5°C. After shipping to Switzerland, a fraction of both suggested both microbes and chitin inherent to BSFL residues to fertilizers was irradiated by HEEB radiation (Leoni Studer AG, be major drivers of soil microbial diversity. Däniken, Switzerland) with a 10 MeV electron beam (Rhodotron Recently, Barragán-Fonseca et al. (2022) urged the clarification TT300, IBA Corp., Louvain-la-Neuve, Belgium) at a dose of of the impact of insect residues on plant growth promoting >32 kGy in accordance with the ISO 11137-3:2017 standard rhizobacteria, crop growth and of potential underlying (International Organization for Standardization, 2017) as mechanisms. Several authors studying the application of insect described before by Gold et  al. (2020a). Sterilization was residues in agricultural contexts suggest an indirect effect on the confirmed by conventional plate counting on tryptic soy agar crop through an impact on the soil microbiome (Setti et al., 2019; (Sigma-Aldrich, Burlington, United States) at 25°C for 36 h. In Schmitt and de Vries, 2020; Beesigamukama et  al., 2020a; technical triplicates, 2.5 g of each fertilizer was added to 22.5 ml of Torgerson et al., 2021; Gebremikael et al., 2022; Tanga et al., 2022). Milli-Q-water, vortexed, and 1 ml suspension was spread on an The present pot study addressed the depicted knowledge gap. 8.8 cm diameter agar plate. Prior to application to the pots, We sequenced the bacterial, archaeal and fungal community of the fertilizers were gently crushed and homogenized by hand without BSFL residues, and the rhizosphere fertilized with BSFL residues. opening the sealed polyethylene bags. The aim was to investigate the potential of the novel organic fertilizer to impact the plant-associated soil microbiome and mechanisms behind observed changes. By sterilizing soil 1 https://www.runres.ethz.ch Frontiers in Microbiology 03 frontiersin.org Fuhrmann et al. 10.3389/fmicb.2022.994091 TABLE 1 Characteristics of the experimental soil. corresponded to 52.5 kg seeds ha−1. Temperatures in the Parameter 1 Content 2 greenhouse ranged from 14°C to 26°C, with an average of EC [μS cm−1] 450 17°C. Nine days after sowing, artificial light (400-W metal halide pH 7.1 lamps, Hugentobler Spezialleuchten AG, Weinfelden, Switzerland) N [g kg−1] 2.8 ± 0.1 with a photoperiod of 14/10 h was imposed in addition to the C [g kg−1] 45.4 ± 1.7 natural light source in the greenhouse. Throughout the C:N ratio 16.3 ± 1.0 experiment, pots were watered three times a week with the same P [g kg−1] 0.6 ± 0.0 amount of purified water (reverse osmosis) per pot. Watering K [g kg−1] 15.4 ± 0.1 targeted a water filled pore space of 60% (Haney and Haney, 2010). Weeds were removed manually on a weekly basis. 1EC (electrical conductivity), pH (in H2O), N (total nitrogen), C (total carbon), C:N Soil samples were collected on day 0, day 24 and day 42 ratio (ratio between C and N), P (total phosphorus), K (total potassium). 2Values are based on dry weight (average ± standard deviation if n = 3). following equilibration. Soil samples were collected over the whole soil profile (0–17 cm) with a steel core soil sampler (diameter of 1.3 cm), collecting at least three soil cores from each pot. Coring Experimental soil locations were evenly distributed over the pot surface. Soil samples were immediately stored in resealable polyethylene bags and The experimental soil was collected at a non-managed stored on ice until further processing and manual removal of plant grassland in Winterthur, Switzerland. The soil had a silt loam roots. Sampling cores were re-filled with autoclaved quartz sand texture (Soil Science Division Staff, 2017) containing 53 g clay, 524 (0.4–1.2 mm). After 42 days, plant shoot biomass was harvested g silt and 424 g sand kg−1 soil. The neutral-to-slightly alkaline soil for each pot, weighed, dried at 60°C for 96 h, and weighed again (pH 7.1) had an electrical conductivity (EC) of 450 μS cm−1 (see to calculate wet and dry weight yield (DWY) respectively. After Table 1 for further characteristics). After collection, the soil was 42 days, roots thoroughly permeated the entire pot such that all passed through a 4 mm sieve and stored at ambient temperature soil can be considered as rhizosphere (i.e., directly influenced by (≈15°C). the roots). Throughout the experiment, none of the plants showed symptoms of nutrient deficiency, drought stress, or disease. Pot experiment Physicochemical analysis of fertilizers The six-week long pot experiment was conducted in a climate- and soil controlled greenhouse at the ETH Zurich research station (Lindau, Switzerland, 47°44′92.5”N 8°68′20.7″E) from January 8th to Fertilizers and soil samples were analyzed for water and DW February 19th, 2021. BR+, BR-, CC+, and CC- were well mixed content, EC, pH, nitrate (NO − 3 ), and ammonium (NH + 4 ). with the experimental soil and placed into plastic pots (27 cm Elemental composition was determined for fertilizers and diameter x 22.5 cm height) with five repetitions per soil-fertilizer untreated experimental soil only. To assess gravimetric water mixture (sBR+, sBR-, sCC+ and sCC-). Similarly, a no-fertilizer content (GWC) and DW, 5 g of fertilizer or soil were dried at treatment (sN0) was prepared as a control without any 105°C for 72 h. EC and pH in soil samples were determined in a amendment, amounting to a total of 25 experimental units (pots). suspension of 10 g of dried soil (40°C, 72 h) and Milli-Q-water The four different fertilizers (BR+, BR-, CR+, CR-) were applied (1:2.5) using a multi-parameter meter (pHenomonal® MU6100L, to the experimental soil at a rate of 75 mg nitrogen (N) kg−1 dry VWR, Radnor, United States) after shaking for 24 h. EC and pH weight (DW) of soil. That corresponds to a rate 150 kg N ha−1 given in fertilizer samples was determined in a suspension with a ratio a bulk density of 1 g cm−3 and a 20 cm mixing zone of the upper of 1:5 in Milli-Q-water, because of strong water absorption, and soil layer in the field, following the approach of Klammsteiner shaken for just 1 hour to avoid fermentation. et al. (2020a). Thus 15.9 g (13.9 g DW) BR, or 39.6 g (25.1 g DW) Inorganic N was determined colorimetrically (NO − 3 : 540 nm, CC were thoroughly mixed with 7.5 kg (5.9 kg DW) of soil for each NH + 4 : 650 nm, V-1200, VWR) following (Forster, 1995) and experimental unit separately and filled into the pots. The unsealed Doane and Horwáth (2003) after extraction by the addition of pots were lined at the bottom with synthetic felt inside to avoid 50 ml KCl (2 M) to 10 g of wet fertilizer or soil sample, 1 h of loss of substrate. All pots were arranged in the greenhouse in a shaking and filtration through ashless filters (Grade 42, Whatman, randomized complete block design containing five blocks with a Little Chalfont). Total mineral N was estimated as the sum of size of 1 m × 1 m each (Supplementary Figure 1). NO − + 3 -N and NH4 -N. Total C and N were determined using After a three-day equilibration phase, red clover (Trifolium 200 mg dried (40°C, 72 h) and milled (MM200, Retsch GmbH, pratense var. Taifun) and Italian ryegrass (Loilium multiflorum var. Haan, Germany) sample using a C/N analyzer (CN628, LECO Oryx) seeds were sown at a ratio of 0.75 (g seed g−1 seed) Corporation, St. Joseph, United States). respectively as suggested for two-year-lasting grass-clover- Phosphorus (P), potassium (K), calcium (Ca), magnesium mixtures (Lehmann et  al., 2000). The total seeding rate (Mg), sodium (Na), iron (Fe), zinc (Zn), manganese (Mn), Frontiers in Microbiology 04 frontiersin.org Fuhrmann et al. 10.3389/fmicb.2022.994091 aluminum (Al), copper (Cu) and cadmium (Cd) were determined were kept on ice for direct processing or stored frozen with ICP-OES (5,100 SVDV, Agilent Technologies, Santa Clara, at – 20°C. All samples were exposed to not more than one freezing United States) after digestion of 200 mg dried (40°C, 72 h) and and thawing cycle before the DNA extraction from 250 mg of wet milled (MM200, Retsch GmbH) fertilizer or 1,000 mg of dried soil or fertilizer (DNeasy® PowerSoil® Pro Kit, QIAGEN, Hilden, (40°C, 72 h) and milled (MM200, Retsch GmbH) experimental Germany). Processing followed the manufacturer’s instructions soil sample. Fertilizer samples were digested with 15 ml HNO3 and included a bead beating step (FastPrep-24™ 5G, MP (65%) for 90 min at 120°C, and 3 ml H2O2 (30%) of 90 min at Biomedical, Santa Ana, United States) running for two cycles of 120°C (DigiPREP MS, Baie-D’Urfé, Canada). The experimental 60 s in total. Additional PCR-grade-water (VWR) was added to soil was mixed with 2 ml of Milli-Q-water and digested with 2 ml the three samples drawn from each fertilizer before bead beating, HNO3 (70%) and 6 ml HCl (37%) at 120°C for 90 min (DigiPREP because of strong absorption of the lysis buffer from the DNeasy® MS, Baie-D’Urfé, Canada). Digests were filtered through ashless PowerSoil® Pro Kit. Purity and quantity of extracted nucleic acids filters (Grade 41, Whatman) before ICP-OES analysis. For were analyzed by Uv–Vis spectrophotometry (Qiaxpert, quantification of effective cation exchange capacity (CECeff) and QIAGEN). DNA concentration of each sample was normalized to base saturation, based on Hendershot and Duquette (1986), 25 ml 10 ng μL−1 using a QIAgility system (QIAGEN). of BaCl2 (0.1 M) were added to 3 g of dried (40°C, 72 h) and milled PCR amplification of the bacterial and archaeal (V3-V4 (MM200, Retsch GmbH) soil and shaken for 2 h. After filtering region of the 16S rRNA gene) and fungal (ITS2 region of the rrn through ashless filters (Grade 41, Whatman) exchangeable cations operon) markers were conducted using primers 341F (5′– (Ca, Mg, Na, K, Al, Mn, and Fe) were analyzed with ICP-OES CCTAYGGGDBGCWSCAG-3′) and 806R (5′-GGACTACN (5,100 SVDV, Agilent Technologies). VGGGTHTCTAAT-3′) for the bacterial/archaeal marker (Frey et al., 2016) and ITS3ngs (5′-CANCGATGAAGAACGYRG-3′) and ITS4ngs (5′-CCTSCSCTTANTDATATGC-3′) for the fungal Basal soil respiration markers (Tedersoo and Lindahl, 2016), respectively. PCR amplification was performed in a mixture containing 40 ng of At the end of the pot experiment (day 42) soil samples were template DNA, 0.4 μM of each primer (Microsynth, Balgach, analyzed for basal respiration. 30 g of soil were incubated at room Switzerland), 1x GoTaq® Colorless Master Mix (Promega, temperature (≈22°C, 12 h) in valved, air-proof jars (7.4 cm Madison, WI, United States), and 0.5 mM MgCl2 (Promega) in a diameter × 10 cm height). Concentrations of CO2 emitted to the total reaction volume of 25 μl. Thermocycling consisted of an headspace of jars via soil basal respiration were determined using initial denaturation step (95°C, 2 min) followed by 30 (bacteria/ a LI-840A infrared gas analyzer (Li-Cor Inc., Lincoln, archaea) or 35 (fungi) cycles of denaturation (95°C, 40 s), United States). It was integrated into a continuous flow system annealing (58°C for bacteria/archaea and 55°C for fungi, 40 s), built according to Carbone et al. (2019) including the Flux Puppy and elongation (72°C for 1 min), and a final elongation step at app (v1.0.0). After flushing with ambient air, jars were successively 72°C for 10 min (C1000 Touch Thermal Cycler, BioRad connected to the system for each sampling (logging period: 60 s, Laboratories, Hercules, United States). Markers were amplified in logging interval: 1 s) within four consecutive sampling series three technical replicates, checked with gel electrophoreses (series interval: 4 h). Calculation of CO2 production by soil (QIAxcel Advanced, Qiagen), and pooled prior to sequencing. microbial respiration was performed based on Curiel Yuste et al. PCR products were sent to the Functional Genomics Center (2007). In brief, first means of CO2 concentrations (ppm) over the Zurich (FGCZ, Zurich, Switzerland) for indexing PCR. Indexed logging period were calculated for each timepoint. These PCR products were purified, quantified, and pooled in equal concentrations were corrected for temperature changes over the concentrations before preliminary sequencing on the Illumina duration of the experiment. Further, a correction for an inevitable MiniSeq platform (Illumina Inc., San Diego, United States) to dilution with ambient air present in the system (tubes, internal inform library re-pooling for optimal equimolarity across optical cell) upon each measurement was conducted. CO2 efflux samples. Final sequencing was conducted using the v3 chemistry (μmol g−1 h−1) was estimated from slopes of regression models (PE300) on the Illumina MiSeq platform (Illumina Inc.). applied to the generated CO2 concentration time series on each experimental unit taking into account jar volume and DW content of soil. Bioinformatics Data obtained from sequencing were processed using a Microbial community structure in customized pipeline based on VSEARCH v.2.17.0 (Rognes et al., 2016) fertilizers and soil as described previously (Longepierre et al., 2021, 2022). Briefly, PhiX contaminants were removed by aligning reads to the PhiX genome Bacterial, archaeal, and fungal community structure in (accession NC_001422.1) using Bowtie2 v.2.4.2 (Langmead and fertilizers and soil was determined using a metabarcoding Salzberg, 2012). PCR primers were trimmed with CUTADAPT v.3.4 approach targeting ribosomal markers. For this purpose, samples (Martin, 2011) allowing one mismatch. Paired-end reads were merged Frontiers in Microbiology 05 frontiersin.org Fuhrmann et al. 10.3389/fmicb.2022.994091 using the fastq_mergepairs function in VSEARCH, followed by filtering 100-fold subsampled ASV-matrices using the function vegdist in low quality reads using the fastq_filter function in VSEARCH allowing vegan and followed by a step-wise approach laid out by Anderson and a maximum expected error of one (Edgar and Flyvbjerg, 2015). Willis (Anderson and Willis, 2003). Briefly, first major variance Dereplication was performed using the derep_fulllength function in components were identified by a principal coordinate analysis (PCoA, VSEARCH and followed by delineation of amplicon sequence variants Gower, 2015) by the function cmdscale. Relationships between soil (ASVs; Callahan et  al., 2017) using the UNOISE algorithm physicochemical properties and microbial beta-diversity were implemented in VSEARCH with the cluster_unoise function (Edgar, assessed by creating join-biplot PCoAs using the function envfit in 2016b) using an alpha of two and a minsize of eight. Identification and vegan. Treatment effects were assessed using a multivariate removal of potentially chimeric ASV sequences was performed using permutational analysis of variance (PERMANOVA, Anderson, 2001) the UCHIME2 algorithm via the uchime3_denovo function (Edgar implemented as the adonis2 function in vegan. Since significant et  al., 2011) in VSEARCH. For the remaining sequences, target differences detected by PERMANOVA can not only arise from verification was conducted using Metaxa2 v.2.2.0 (Bengtsson-Palme differences in means but also from differences in dispersion et al., 2015) for the 16S rRNA gene (bacteria/archaea) and ITSx v1.1.3 (analogous to heteroscedasticity in a regular ANOVA), homogeneity (Bengtsson-Palme et al., 2013) for the ITS region (fungi), whereas of variance was checked using permutational analysis of multivariate unverified sequences were discarded. The quality filtered reads were dispersion (PERMDISP, Anderson, 2006) implemented as the run against the verified ASV sequences using the usearch_global betadisper function in vegan. p-values of pairwise PERMANOVA algorithm with settings maxrejects at 32, maxaccepts at 0, maxhits at 1, tests were corrected for multiple testing using the Benjamini- and a minimum identity threshold at 97%. Each verified ASV sequence Hochberg method (Benjamini and Hochberg, 1995) implemented in was taxonomically classified by running the Sintax algorithm (Edgar, the p.adjust function. Finally, beta-diversity was constrained by the 2016a) against the SILVA v.132 database (Pruesse et al., 2007) for the significant factors using canonical analysis of principal coordinates 16S rRNA gene sequences and the UNITE database v8.0 (Nilsson et al., (CAP, Anderson and Willis, 2003) implemented as the CAPdiscrim 2019) for the ITS region at a confidence cutoff of 0.8. ASVs not function in the R package BiodiversityR v2.14–2 (Kindt and Coe, assigned to bacteria, archaea, and fungi, or assigned to organelle 2005). PERMANOVA, PERMDISP, CAP were run with 999 structures (chloroplasts, mitochondria) were removed. permutations. In order to assess the dissimilarity between fertilizer and no-fertilizer treatments, we calculated the mean Bray-Curtis dissimilarities of one replicate of the fertilizer treatments to the five Statistics replicates of the no-fertilizer treatment at the same sampling day. Differences of individual ASVs across treatments was assessed Statistical analyses were conducted in R version 4.0.5 (R Core using univariate PERMANOVA and PERMDISP on the median of Team, 2021) and the R script is provided in the 100-fold subsampled ASV matrices. Type I error inflation due to Supplementary Material. Differences in physicochemical soil multiple testing was controlled by the false discovery rate correction characteristics, DWY, and basal respiration between treatments approach according to Storey (2002) using the package qvalue v.2.22.0 within timepoints (incorporating block as an additional factor) was (Storey et al., 2020). ASVs were classified as “sensitive” to treatment assessed using ANOVA. Subsequently, differences between factor if (I.) PERMANOVA results were significant and (II.) significance did levels were analyzed with Tukey’s HSD test. Normality of residuals not arise from significant dispersion (PERMDISP). The analysis of and homoscedasticity was confirmed with the Shapiro–Wilk test and sensitive ASVs was focused on the soil microbial data of sampling day standardized residuals plots, respectively. If these assumptions were 42 only to reduce the amount of confounding extracellular DNA not met, the Friedman Rank Sum Test was used analogously to assess potentially imported with fertilizers, which is further addressed in the the significance of factors, whereas the Conover post-hoc test from discussion section. Additionally, to reduce noise from rare ASVs (low the PMCMR package v.4.4 (Pohlert, 2014) was used as post-hoc test read counts across all samples) for the analysis of sensitive ASVs, for multiple comparisons between factor levels, adjusting p-values prefiltering was applied. ASVs with a minimum relative abundance with the Bonferroni method. Correlations among these variables were of 0.015% (rare) were removed. For all statistical tests, significance calculated using the squared Pearson’s correlation coefficient (R2). was accepted at p < 0.05 or q < 0.05, if applicable. The following depicted operations were largely based on functions from the vegan package v.2.5–7 (Oksanen et al., 2020) in R. An iterative (100 iterations) subsampling approach of the ASV Results matrices (Martiny et al., 2017; Hemkemeyer et al., 2019) was applied to remove systematic biases in read counts potentially arising from Physicochemical characteristics of the sequencing workflow (McKnight et al., 2019). Alpha-diversity was fertilizers and fertilizer-amended soils assessed by calculating observed richness, Shannon diversity index and Pielou’s evenness of ASVs (Chao et  al., 2014) based on the BR tended to be drier and contain more C, N, NH + 4 , and P median of 100-fold subsampled ASV matrices using the functions than CC (Table 2). BR also showed higher EC and was closer to a rarefy, specnumber, and diversity in vegan. Beta-diversity was assessed neutral pH compared to CC. CC in turn tended to show higher based on the median Bray–Curtis dissimilarity calculated from the contents of the remaining elements. Frontiers in Microbiology 06 frontiersin.org Fuhrmann et al. 10.3389/fmicb.2022.994091 TABLE 2 Physicochemical characteristics of the applied fertilizers. fungal reads delineated into 4,037 and 383 ASVs, respectively. For Parameter1 BR 2 CC fungi, one replicate of CC had to be excluded from the analysis for DM [%] 87.43 63.4 showing only 56 read counts. Fertilizers differed in bacterial/ NH +-N [mg kg−1] 4,667.3 34.8 archaeal taxonomic composition (Figures 2A,B). At phylum level, 4 NO − -N [mg kg−1] 250.2 181.4 Proteobacteria (31%), Actinobacteria (24%), Bacteroidetes (15%), 3 Firmicutes (15%), and Chloroflexi (11%) were the most abundant EC [μS cm−1] 1,236 772 groups, whereas Chloroflexi was proportionally more abundant in pH 6.9 7.5 CC (20%) than in BR (2%), at the expense of Bacteroidetes (5% in N [g kg−1] 31.6 ± 1.3 15.0 ± 1.1 CC, 25% in BR). At genus level, taxonomic composition differed C [g kg−1] 400.7 ± 9 173.5 ± 9.5 strongly, with only Bacillus (3%) and Streptomyces (2%) among the C:N ratio 12.7 ± 0.6 11.6 ± 0.3 predominant genera (>0.5%) shared between CC and BR P [g kg−1] 5.6 ± 0.3 2.4 ± 0.2 (Figure 2B). K [g kg−1] 2.7 ± 0.1 3.0 ± 0.2 Ascomycota and Basidiomycota were the predominant fungal Ca [g kg−1] 6.4 ± 0.4 23.7 ± 3.8 phyla in the fertilizers, with Basidiomycota showing much higher Mg [g kg−1] 2.2 ± 0.1 3.2 ± 0.5 relative abundance in BR (>64%) than CC (5%; Figure  2C). Na [g kg−1] 0.2 ± 0.0 0.5 ± 0.0 Interestingly, a large fraction (up to 28%) of the sequences in CC Fe [g kg−1] 4.1 ± 1.0 21.0 ± 6.4 could not be assigned at phylum level, whereas this fraction was Mn [g kg−1] 0.1 ± 0.0 0.9 ± 0.1 small (<1%) in BR. Again, only very few of the abundant genera −1 were shared between the fertilizers, e.g., Arthrographis (5%) and Zn [g kg ] 0.1 ± 0.0 0.1 ± 0.0 Aspergillus (4%, Figure 2D). Al [g kg−1] 3.6 ± 0.9 13.0 ± 0.5 For both domains but particularly for bacteria/archaea, CC Cu [mg kg−1] 12.8 ± 1.3 28.6 ± 5.2 tended to be richer in ASVs (bacteria: BR:1632 ± 7, CC: 2022 ± 78; Cd [mg kg−1] ND4 ND fungi: BR:171 ± 4, CC:184 ± 10). For bacteria, Pielou’s evenness 1Ammonium-N (NH + 4 -N), nitrate-N (NO − 3 -N), electrical conductivity (EC), pH (in tended to be higher in BR (BR:0.84 ± 0.00, CC:0.72 ± 0.00), while H2O), N (total nitrogen), C (total carbon), C:N ratio (ratio between C and N), P (total this trend did not apply to fungi (BR:0.52 ± 0.01, CC:0.6 ± 0.08). phosphorous), K (total potassium), Ca (total calcium), Mg (total magnesium), Na (total sodium), Fe (total iron), Mn (total manganese), Zn (total zinc), Al (total aluminum), Cu BR showed higher bacterial/archaeal (6.23 ± 0.03) and lower (total copper), CD (total cadmium). fungal Shannon diversity (2.66 ± 0.07) when compared to CC 2Values are based on dry weight (average ± standard deviation if measured in technical (5.49 ± 0.03 and 3.14 ± 0.44, respectively). replicates, n = 3). 3The first five properties could not have been measured in triplicates since they require larger quantities of dried material and only small quantities of the fertilizers were received. 4ND, below detection limit. Impact of fertilizers on soil microbial community Overall, application of fertilizers had a minor influence on soil physicochemical parameters (Supplementary Tables 1, 2). Soil microbial diversity However, BR fertilizers significantly (p < 0.05) increased NO − 3 -N The bacterial/archaeal community in the initial unamended content in soil over all sampling dates compared to sN0 (Figure 1). soil (sN0 at day 0, Supplementary Figure 2) was dominated by On day 24 post application, BR fertilizers significantly (p < 0.05) Proteobacteria (26%), Actinobacteria (23%) and Acidobacteria increased NO − 3 -N also compared to CC fertilizers. Furthermore, (16%) and the most abundant genera included Candidatus NO − 3 -N in soil generally dropped by more than half from day 24 Xiphinematobacter (2%), Bacillus (2%), Gaiella (2%), and to 42. Soil NH + 4 -N contents did not show this pattern and Candidatus Udaeobacter (2%). All taxa assigned to the domain accounted for only around 2% (1.12 ± 0.89 mg NH + kg−1 4 soil) of archaea (2%) belonged to the family Nitrososphaeraceae (2%) and the total mineral N. EC values strongly correlated with NO − 3 -N were not further classified. The fungal community was dominated contents (R2 = 0.85, p < 0.001) and showed a similar significance by Ascomycota (68%) followed by Mortierellomycota (14%), and pattern across the treatments (Supplementary Table 2). GWC Basidiomycota (9%) and the most abundant genera included varied among sampling timepoints only. Significant differences in Mortierella (10%), Fusarium (3%), Staphylotrichum (3%), pH, as well as C and N contents among treatments and over time Lecythophora (3%). occurred only erratically and lacked trends. The addition of fertilizer was a significant (PERMANOVA, p < 0.001) driver of soil microbial community structure (Table  3). The impact on soil fungi was more pronounced Microbial community composition in compared to the impact on soil bacteria/archaea (Figure  3; fertilizers Supplementary Figure  3). Fertilization explained bacterial/ archaeal and fungal variation in soil community structures by Sequencing yielded 326,647 (54,441 ± 4,680 per replicate) on average 7 and 21%, respectively. Application of BR+ led to bacterial/archaeal and 176,527 (35,305 ± 7,100 per replicate) soil microbial communities significantly (PERMANOVA, Frontiers in Microbiology 07 frontiersin.org Fuhrmann et al. 10.3389/fmicb.2022.994091 FIGURE 1 Nitrate-N (NO − 3 -N) content in the different fertilizer-amended soils and in the no-fertilizer treatment. Distinct lower-case letters indicate significant (p  < 0.05, n = 5) differences among treatments for the same sampling date. sBR+, soil fertilized with BSFL residue; sBR-, soil fertilized with sterile BSFL residue; sCC+, soil fertilized with conventional compost; sCC-, soil fertilized with sterile conventional compost; sN0, no-fertilizer treatment. p ≤ 0.002) distinct from soil amended with BR- and unfertilized of their variation, respectively. Bacterial/archaeal soil. However, significant differences between bacterial/archaeal communities were significantly (p < 0.001) changing between communities among fertilizer treatments were not only arising each sampling day. In contrast, only the fungal community at from dissimilarity but also from dispersion. day 0 was significantly distinct from the later sampling days BR+ appeared to introduce stronger effects to the microbial 24 and 42 (p = 0.028 and p = 0.006, respectively). The community than CC+ (Figure  3), but notably for bacteria/ interaction between sampling day and treatment had a archaea both pairwise comparisons with sN0 were significant significant (p ≤ 0.001) impact on bacterial/archaeal and (p < 0.05). For bacteria/archaea differences between the fungal communities and accounted for on average 12 and influence of BR+ and CC+ became most apparent at sampling 13% of the variation, respectively. days 0 and 42 based on the dissimilarity to sN0 Comparing among treatments, physicochemical soil (Supplementary Figure 4). In general, BR fertilizers tended to properties C, N, C:N and pH minorly affected soil microbial have a more clear-cut effect on the soil fungal community than communities. NO − 3 -N content and EC were the only soil the composts. properties significantly correlated with bacterial/archaeal Time had a significant (PERMANOVA, p < 0.001) impact (p = 0.004 and p = 0.004, respectively) and fungal (p = 0.014 on bacterial/archaeal and fungal communities in soil (Table 3; and p = 0.025, respectively) beta-diversity patterns. Along the Figure 3). The sampling day explained on average 4 and 5% gradient of NO − 3 -N bacterial/archaeal communities were Frontiers in Microbiology 08 frontiersin.org Fuhrmann et al. 10.3389/fmicb.2022.994091 A B C D FIGURE 2 Most abundant bacterial and fungal phyla (A and C, respectively) and genera (B and D, respectively) in the two organic fertilizers. BR: BSFL residue, CC: conventional compost. TABLE 3 Effects of fertilizer treatment on microbial beta-diversity determined by PERMANOVA. Significant values (p < 0.05) are indicated in bold. Bacteria & Archaea Fungi Test1 F P R2 F P R2 Treatment 1.5 0.001SD 0.07 5.1 0.001 0.21 Sampling day 1.7 0.001 0.04 2.4 0.001 0.05 Treatment x 1.2 0.001SD 0.12 1.6 0.001 0.13 Sampling day Treatments2 sBR+ sBR− sCC+ sCC− sBR+ sBR− sCC+ sCC− sBR- 0.002 0.001 sCC+ 0.001 0.001 0.001 0.001 sCC- 0.001 0.002 0.028 0.001 0.001 0.221 sN0 0.001 0.001 0.001 0.001 0.001 0.001 0.552 0.075 1Effects of factors and their interaction were analyzed by permutational analysis of variance (PERMANOVA). Factors are fertilizer treatment (degrees of freedom = 4, sBR+, soil fertilized with BSFL residue; sBR-, soil fertilized with sterile BSFL residue; sCC+, soil fertilized with conventional compost; sCC-, soil fertilized with sterile conventional compost; sN0, no-fertilizer treatment soil), day of sampling (degrees of freedom = 2, day 0, 24, 42), and their interaction. Values represent the pseudo-F ratio (F) and the level of significance (P). Values at p < 0.05 are shown in bold. SD indicates significant dispersion. 2P-values of pairwise comparisons between fertilizer treatments with P-values adjusted for multiple comparisons using the Benjamini–Hochberg method. Values at p < 0.05 are shown in bold. Frontiers in Microbiology 09 frontiersin.org Fuhrmann et al. 10.3389/fmicb.2022.994091 A B FIGURE 3 Constrained analysis of principal coordinates (CAP) ordinations elucidating a priori differences in bacterial/archaeal (A) and fungal (B) community structure upon fertilizer amendment at days 0, 24, 42 of the experiment. Percent between group variation represented by each canonical axis are provided in parentheses next to the axis headers. CAP reclassification success rates for bacteria/archaea at day 0: sBR+, 100%; sBR-, 100%; sCC+, 100%; sCC-, 60%; sN0, 80%; day 24: sBR+, 80%; sBR-, 80%; sCC+, 100%; sCC-, 40%; sN0, 80%; day 42: sBR+, 100%; sBR-, 100%; sCC+, 40%; sCC-, 60%; sN0, 80%. CAP reclassification success rates for fungi at day 0: sBR+, 80%; sBR-, 100%; sCC+, 0%; sCC-, 20%; sN0, 60%; day 24: sBR+, 100%; sBR-, 100%; sCC+, 0%; sCC-, 60%; sN0, 80%; day 42: sBR+, 100%; sBR-, 100%; sCC+, 40%; sCC-, 40%; sN0, 0%. sBR+, soil fertilized with BSFL residue; sBR-, soil fertilized with sterile BSFL residue; sCC+, soil fertilized with conventional compost; sCC-, soil fertilized with sterile conventional compost; sN0, no-fertilizer treatment. lining up by timepoints whereas fungal communities were Impact of fertilizers on basal respiration rather lining up by fertilizer types (Supplementary Figure 5). Regression models fitted well to the development of CO2 Sensitive taxa concentrations over time in the headspace of incubated soil The application of BSFL residues significantly (q < 0.05) samples with an R2 of 0.97 ± 0.02 over all experimental units. At impacted the soil microbiome on individual ASV levels. A total the end of the experiment, soil fertilized with BR+ emitted of 42 fungal and 16 bacterial/archaeal ASVs were identified as significantly (p < 0.02) more CO2 than soil treated with CC sensitive to treatments (Supplementary Tables 5, 6) at day 42. A fertilizers whereas BR- showed values in between (Figure  4A; comparable number of fungal and bacterial/archaeal ASVs (11 Supplementary Table 2). BR+ amended soil emitted around 20% and 12 respectively) were enriched under BR+ treatment. (≈0.01 μmol g−1  h−1) more CO2 than CC+ and CC- amended Nevertheless, the total relative abundance of sensitive ASVs treatments (Supplementary Table 2). Even though BR+ treated assigned to fungi and enriched under BR+ was around five soils showed the highest respiration values, these were not times higher than for bacteria. ASVs thriving under BR statistically different to BR-amended or unamended soils (sN0). fertilizers in general (BR+ and BR-) were among others assigned The positive correlation between basal respiration (corrected for to the fungal genus Mortierella and the family Lasiosphaeriaceae. block effect) and NO − 3 -N at day 42 was significant but weak However, under BR+ treatment only ASVs assigned to the (R2 = 0.22, p = 0.018). genera Mucor, Cephaliophora and a not further classified fungus were identified as enriched. With respect to bacteria /archaea just one ASV assigned to bacterial family Bacillaceae was thriving under BR fertilizers in general. Bacterial /archaeal Impact of fertilizers on yield ASVs thriving under BR+ only were among others assigned to the bacterial genus Bacillus, the family Chitinophagaceae as well At the end of the pot experiment, plants growing on soil as to the archaeal family Nitrososphaeraceae fertilized with BR+ had around 17% more (p < 0.05) biomass than (Supplementary Tables 5, 6). Few fungal and bacterial/archaeal the no-fertilizer treatment (Figure 4B; Supplementary Table 2). ASVs that were promoted in soil by BR+ application were also However, even though sBR+ showed the highest mean values, no identified in BR itself hinting to potential introduction. These significant difference was found compared to the other fertilization ASVs were assigned to the fungal genera Mucor, Botryotrichum, treatments. Only on sampling day 24 there was a significant but Cephaliophora, and Mortierella and to the bacterial genus weak correlation between DWY and NO − 3 -N observable Bacillus, family Bacillaceae and order RBG-13-54-9. (R2 = 0.14, p = 0.036). Frontiers in Microbiology 10 frontiersin.org Fuhrmann et al. 10.3389/fmicb.2022.994091 A B FIGURE 4 Basal respiration (A) and grass-clover dry weight yield (B) at the end of the experiment (day 42) from the different fertilizer amended soils. Distinct lower-case letters indicate significant (p < 0.05), n = 5 differences among treatments as determined by Tukey’s HSD test, values were corrected for the significant block effect as determined by ANOVA. sBR+, soil fertilized with BSFL residue; sBR-, soil fertilized with sterile BSFL residue; sCC+, soil fertilized with conventional compost; sCC-, soil fertilized with sterile conventional compost; sN0, no-fertilizer treatment. Discussion enriched in BR. These genera were reported to be characteristic for the gut of the larva (Klammsteiner et al., 2020b). Other genera Microbial taxa associated with BSFL guts recurringly associated with the gut and residues of BSFL like and residues Dysgonomonas, Morganella, Providencia, and Proteus (Gold et al., 2020b) were not present in BR or only in low abundances (<1%). The recurrence of certain microbial taxa in BSFL residues is The BSFL residues used in this study had a high DM content especially interesting for the assessment of their reliability in (87.4%) compared to average reported values for BSFL residues of introducing BSFL- and soil fertility-related microbes to the soil. around 69% (values ranging between 40 and 90%; Gärttling and Therefore, taxa found in BR are contrasted with those that are Schulz, 2021). Thus, BR might have resembled less the conditions commonly found to be associated with BSFL residues or BSFL of insect guts compared to moister residues, which are based on themselves in literature. distinct substrates or rearing conditions. That could have limited the maintenance of gut-associated taxa in BR. Bacteria/Archaea Sphingobacterium, Pseudomonas and Bacillus were predominant Firmicutes, Proteobacteria, and Bacteroidetes were identified bacterial genera in BR, that were also previously identified in BSFL as characteristic for guts and residues of BSFL (Gold et al., 2020b). residues from brewery waste and fruits summarized by Gold et al. Those were three of the four most abundant bacterial phyla (2020b) from Wynants et al. (2019). These three genera were also we  have found in BR. Especially Bacteroidetes were strongly identified in mealworm residues and are known plant-associated taxa enriched in BR compared to CC (Figure  2). Members of the potentially involved in plant growth promotion (Poveda et al., 2019). bacterial phylum Bacteroidetes are important animal symbionts Members of Sphingobacterium were reported to play a major role in and decomposers of high molecular weight organic matter the decomposition of complex organic matter, whereas Pseudomonas (Thomas et  al., 2011). Members of the proteobacterial genus and Bacillus were associated with the control of plant pathogens (Lutz Pseudomonas and the actinobacterial genus Actinomyces were also et al., 2020; Scotti et al., 2020). Frontiers in Microbiology 11 frontiersin.org Fuhrmann et al. 10.3389/fmicb.2022.994091 Fungi extent than composts. Results from Chiam et al. (2021) suggest Compared to bacteria little data is available on fungi associated the same finding for bacteria only and using much higher with BSFL. One study suggests that the fungal community found fertilization rates (30% v/v BSFL residue or compost +70% v/v in BSFL guts is mainly shaped by the diet (Boccazzi et al., 2017), soil). By sterilizing fertilizers prior to their application, we sought which lowers the probability of a core mycobiome to be passed on to disentangle the impact of biotic and abiotic drivers of BR on the from BSFL to residues. The study by Tanga et al. (2021) proposed soil microbial community. Both biotic and abiotic components Pichia as a prevalent genus in BSFL guts across different diets appeared to influence the soil microbial community structure in including brewery waste. Furthermore, Kuznetsova et al. (2021) general. However, on the soil bacterial/archaeal community, biotic reported that Pichia and Diutina dominated (99.9%) the residues factors appeared to have a stronger effect, since the unsterilized after rearing BSFL for 5 days on food waste. We found Pichia to BR+ introduced stronger changes than the sterilized counterpart be present in BR, but only in low abundances (<0.1%). The high BR- (Figure 3; Supplementary Figure 4). Furthermore, differences relative abundance of Basidiomycota in BR compared to CC can in the bacterial/archaeal community structure were not as almost exclusively be attributed to members of the most abundant clear-cut as in the fungal community structure since dispersion genus Trichosporon, which has been identified previously in BSFL influenced the significance of our results (Table 3). guts (Tanga et al., 2021) and as the dominant basidiomycetous genus in substrates exposed to BSFL (Bernard et al., 2020). Indeed, Influence of the abiotic part of BSFL residues BSFL were suggested to associate with the genus to suppress We propose the addition of organic matter to the soil as a pathogenic bacteria in the rearing environment of the larvae major abiotic driver of the observed microbial patterns. Gärttling (Gorrens et al., 2021). Trichosporon has been reported to degrade and Schulz (2021) showed that BSFL residues comprise high aromatic compounds such as phenols (Middelhoven, 1993; DeRito contents of organic matter of around 86% and can thus be an and Madsen, 2009). In fact, the high abundance of Trichosporon in important energy source for soil microbes. However, in our BR might be an indicator of elevated concentrations of phenolic experiment, total soil C as a proxy for organic matter was not and thus aromatic substances, the chemical compounds of lignin. significantly influenced by the fertilization treatments. Interestingly, Song et al. (2021) related increased concentrations of Nevertheless, BR fertilizers had a stronger impact on soil microbial phenolic substances in uncomposted BSFL residues to hampered communities than the compost (Figure 3; Supplementary Figure 4). plant performance in comparison to their composted analogue. BR Thus, probably rather the type than the amount of organic matter did not undergo a composting process. However, effects of that was added was decisive. On one hand, the organic C in CC phytotoxicity were not observable in our experiment. Phytotoxicity was highly stabilized over the six-month composting process might anyway only become apparent upon direct comparison (Garcia et al., 1991) and was thereby less available to microbes. On between composted and uncomposted residues applied at the other hand, BSFL residues used in our experiment were higher rates. produced by BSFL feeding on brewer’s spent grain. Roth et al. (2019) report that brewer’s spent grain consists of up to 70% fiber (cellulose, hemicellulose and lignin), whereas the hardly Changes in soil microbial communities degradable lignin alone may be enriched by up to 28% in the upon fertilization with BSFL residues organic waste. Reduction of cellulose and hemicellulose by BSFL treatment is highly variable but may account for up to 50%, Extracellular DNA derived from dead cells is a well described whereas lignin reduction may account for up to 2% if any (Gold and potentially confounding factor in microbial community et  al., 2018). Thus, the residue used in our experiment was analysis (Carini et  al., 2016; Lennon et  al., 2018). Sirois and probably high in fiber and especially in lignin. As discussed above, Buckley (2019) reported >99% degradation of extracellular DNA the high abundance of the fungal genus Trichosporon in the (eDNA) in soils within 7 days irrespective of the moisture content, residue supports this assumption. These complex organic temperature and tillage of the soil. However, a small amount of compounds are known to be mainly degraded by fungi, whereas eDNA with little potential to bias beta-diversity analysis remained bacteria use the more labile C sources (Insam and De Bertoldi, determinable throughout their trials (39–80 days). Consequently, 2007; Kirchman, 2018). The fact that also sterile BR fertilizers were an artifact arising from eDNA imported with sterile fertilizer strongly influencing soil fungal diversity might thus have been a especially at sampling day 0 cannot be ruled out. To minimize the result of fiber addition. This is supported by the fact that taxa from impact of eDNA on our individual ASV results, the analysis of the fungal species Mucor circinelloides, genus Mortierella and sensitive ASVs was conducted on the last sampling (day 42). family Lasiosphaeriaceae were enriched under BR fertilizer Furthermore, soil conditions were expected here to be closest to application in general. Members of the taxa have been reported an assumed “steady state” including thorough root permeation for decomposing cellulose (Saha, 2004), hemicellulose (Jackson, throughout the soil profile. 1965) and wood (Nordén and Paltto, 2001), a fiber-rich organic This study aimed to examine the impact of BSFL residues on matter, respectively. Our discussion emphasizes that the type of the plant-associated soil microbiome. We  showed that BSFL organic waste BSFL residues are originating from is likely residues can shape the soil microbial community and to a larger influencing the soil microbiome when applied as fertilizer. Frontiers in Microbiology 12 frontiersin.org Fuhrmann et al. 10.3389/fmicb.2022.994091 Whether our results are generalizable might therefore depend on only significant for soil NO − 3 -N content and EC. EC was highly a generally high fiber content in BSFL residues compared to other correlated to NO − 3 -N and thus probably mainly driven by this organic fertilizers, which to our knowledge has not yet been plant nutrient as observed before (Blume et al., 2010). Indeed, due assessed. The assumption is supported by recurrently highlighted to an already nutrient-rich experimental soil, the addition of stimulations of soil fungi after application of BSFL residues fertilizers had a limited effect on the physicochemical variables (Temple et al., 2013; Rummel et al., 2021; Watson et al., 2021). (Supplementary Table 2), except for NO − 3 -N (Figure 1). Long- The presence of chitin, a more labile form of organic matter, term mineral N addition may influence microbial diversity in soil in BSFL residues might have also contributed to a shift in the soil by favoring microbes with tolerance to high osmotic potential microbial community. Gebremikael et al. (2020) suggested chitin (Wang et al., 2018). However, the experimental period was short as a major abiotic driver of change in microbial communities and the increase in NO − 3 mediated by addition of BR fertilizers fertilized with BSFL residues. Due to BSFL shedding their was relatively moderate. BSFL residues are expected to have larger exoskeletons during rearing (Gold et  al., 2018) chitin and its effects on the soil microbial community when applied in nutrient derivates are an integral part of the residue. Crocker et al. (2019) poor agricultural systems. reported impacts on the soil microbial community structure when pure chitin was added to the soil. Previous studies indicated effects Influence of the biotic part of BSFL residues of chitin contained in BSFL residues on the soil microbiome. In Gebremikael et  al. (2020) postulated that the microbiome fact, the content of chitin or its derivates in BSFL residues from inherent to insect residues can, similarly as suggested for other different origins is still to be assessed. However, Panov (2021) organic soil amendments (Sun et  al., 2016), impact the soil showed that chitinolytic organisms and chitinase activity were microbial community when used as a fertilizer. We could show that promoted when BSFL residues were added to soil even in low the biota inherent to BSFL residues can significantly influence the quantities (1.6% v/v). Gebremikael et al. (2022) found increased rhizosphere soil microbial community, since differences between chitinase activity in soil fertilized with BSFL residues produced on soil fertilized with untreated and sterile BSFL residues were distinct organic wastes (0.11 g N kg−1 DW soil) over the duration significant (Table 3). This effect might stem directly from microbes of their 103-day incubation experiment. Notably chitinase activity present in BR+ that were introduced to the soil. We showed that at was not only enhanced compared to the control soil but also to soil the end of our experiment few microbes significantly enriched in treated with a distinct organic fertilizer. In our experiment soil fertilized with BR+ were also identified in the fertilizer itself Mortierella, a fungal genus associated with strong chitinolytic (Supplementary Tables 5, 6) e.g. ASVs linked to the fungal species capabilities (Jackson, 1965; Okafor, 1966; Gray and Baxby, 1968), Cephaliophora tropica and Mucor circinelloides or the bacterial was enriched in soils treated with BR+ and BR-. That finding genus Bacillus. As mentioned, Bacillus has been identified in BSFL supports the assumption that chitin in the residues was impacting residues (Gold et al., 2020b) and insect residues in general and was the soil microbial community. What speaks against a strong effect linked to their positive effects on crops (Barragán-Fonseca et al., of chitin on soil microbes on community level is the limited 2022). Notably, also members of the beforementioned genus response of bacteria/archaea in soils fertilized with BR-. Since Trichosporon, that dominated BSFL residues, were only highly bacteria generally play a key role in chitin degradation (Beier and abundant in BR+ amended soil. However, those ASVs assigned to Bertilsson, 2013), a chitin-enriched BR- would probably have led Trichosporon did not qualify for being sensitive taxa due to to a more pronounced shift in their community composition. The significant inhomogeneity of variance (dispersion). Nevertheless, presence and bioavailability of chitin in BSFL residues should this taxon should further be observed as a potential candidate that undergo further scrutiny. The molecule and its derivates are is transferred from BSFL residues to the soil. Going one step considered promising substances for agricultural management further, results from our setting give little indication that BSFL (Sharp, 2013). Among others they can be used to control plant residues can act as a vector for microbes associated with the larvae pathogens like nematodes and pathogenic fungi. Stimulation of themselves to compete in the rhizosphere, as it has been implied for chitin degrading antagonists is one proposed underlying insect residue (Poveda, 2021). mechanism (Yang and Fukamizo, 2019). Former studies suggested We assume that microbes, that were potentially introduced via a link between the chitin inherent to BSFL residues and the BSFL residues, contributed also indirectly to the differences in suppression of soil-borne crop diseases (Gebremikael et al., 2020; microbial communities between soil fertilized with BR+ and BR-. Quilliam et al., 2020). However, the disease suppressiveness of In fact, especially bacterial/archaeal ASVs significantly enriched BSFL residues is not always given (Elissen et  al., 2019). And under BR+ alone were rarely identified in the fertilizer itself different ways of post-processing such as composting of the (Supplementary Table 5). It is possible that these taxa enriched residues might reduce the content of the easily degradable chitin under BR+ were promoted by metabolic products from introduced (Insam and De Bertoldi, 2007; Oberlintner et al., 2021). taxa. For instance, decomposers introduced with BR+ could have The physicochemical properties that were measured in the made nutrients and energy sources available to depending fertilizers and soils appeared to have little influence on differences bacteria/archaea (Beare et  al., 1997). Indeed, members of the in soil microbial community structure. Correlations of archaeal family Nitrososphaeraceae were enriched under BR+ physicochemical variables onto the community ordinations were fertilization only. They are known ammonia-oxidizing organisms Frontiers in Microbiology 13 frontiersin.org Fuhrmann et al. 10.3389/fmicb.2022.994091 that rely on the provision of NH + 4 and may use organic compounds development but they did not quantify their abundance. Also as carbon sources (Tourna et al., 2011; Kerou and Schleper, 2015). derivates of chitin were suggested to stimulate plant growth directly Both are principal sources that arise from microbial mineralization (Winkler et al., 2017; Xu and Mou, 2018) and are probably present of organic matter. Beyond that it is possible that organisms distinct in BSFL residues as discussed before. Screening and quantification from fungi or bacteria/archaea were introduced to the soil via of potential plant growth promoting substances across BSFL BSFL residues and affected the soil microbial community by their residues of distinct origin is certainly needed to advance the metabolites. Little is known about the meso-and microfauna of discussion. However, not only abiotic components of BSFL residues, BSFL residues, but it is probable that also here they are involved but also microbes enriched in soil via BSFL residues could have had in organic matter decomposition as much as they are generally in a positive influence on soil fertility. Similarly to the composts and soils (Blume et al., 2010; Steel and Bert, 2012) and beforementioned, Gebremikael et al. (2022) and Tan et al. (2021) would therefore also be  relevant to the biotic and abiotic reported that fertilization with BSFL residues led to a better plant environment of the soil they are introduced to. performance than expected from their supply with plant-available It has to be noted that the fertilizer sterilization could have nutrients, whereas they suggest an increased stimulation by plant contributed to a change in the soil microbial community. HEEB, growth promoting microbes under BSFL residue treatment. Poveda a non-thermal sterilization method, can be  considered as a et al. (2019) argued that plants can benefit from mealworm residue relatively gentle procedure in comparison to thermal sterilization. application as a fertilizer by the introduction of a variety of plant However, it cannot be ruled out that HEEB led to changes in the growth promoting rhizobacteria. The fungal taxon Mortierella bioavailability of nutrients in fertilizers. elongata that was thriving in our soils fertilized with BR+ and BR- (Supplementary Table 6) is associated with plant growth promotion via the stimulation of phytohormone production (Li et al., 2018). BSFL residues and soil fertility Members of the bacterial genus Bacillus, a bacterial group that harbors many plant growth promoting strains (Saxena et al., 2020), Our study sought to further identify whether BSFL residues was highly enriched under soil amended with BR+ and could have can contribute to soil fertility via an impact on the soil played a direct role in the significant yield increase under BR+ microbiome. Soil microbial diversity has previously been treatment. However, it is important to note that a comprehensive associated with soil fertility (Mäder et al., 2002), however a change number of metabolically diverse species belongs to this genus. in the microbial community composition does not automatically Again, the soil used in our experiment had a high nutritional status translate into a change in their performance due to functional and we expect microbes associated with BSFL residues to promote redundancy among microbes (Allison and Martiny, 2008). And crop yield more clearly under resource poor conditions, where although we identified changes in taxa for example associated with rhizosphere microbiomes tend to show an increasing impact on nutrient cycling to be enriched under BR fertilizers, we can only plant performance (Van Der Heijden et al., 2008). speculate about their actual role in our experiment. Thus, to Microbial heterotrophic activity is another potential indicator of complement our findings we  further analyzed plant yield and soil fertility and can be estimated by soil respiration (Robertson et al., basal respiration as a measure for soil activity. 1997). An increased basal respiration might arise from both an In our study the grass-clover mixture growing on soil amended increase in microbial activity and microbial biomass, which we, with BR fertilizers produced the highest plant yield (Figure 4B), however, did not disentangle here. Gebremikael et al. (2022) showed although only soil treatment with BR+ was statistically significant that application of BSFL residues produced on different organic compared to the no-fertilizer treatment. Not only abiotic but also wastes can increase soil microbial biomass more strongly than an biotic components of BSFL residues might have been the drivers, anaerobic digestate of food waste, which suggests that an increase in but soils treated with BR+ and BR-did not differ significantly in soil microbial biomass was likely contributing to basal respiration in yield, which would be  necessary to separate the effects. The our study. We could show that non-sterile BSFL residues can stimulate availability of plant nutrients had probably only a minor effect on the soil microbiome stronger than a conventional compost. The yield, since correlations between DWY and NO − 3 concentrations in application of BR+ increased basal respiration compared to the soil were weak. Differences in P and K amounts added with the application of CC fertilizers (Figure 4A). This is in line with Watson fertilizers were probably negligible considering the high nutritional et al. (2021), who observed even stronger differences in cumulated status of the soil. This corresponds to previous studies suggesting soil respiration between the addition of BSFL residues and an improvement of plant performance by BSFL residues that is not vermicompost in a 28-day soil incubation experiment. However, their mediated by a direct supply of plant available nutrients. Chirere fertilization rates were around 10 times higher compared to our study, et  al. (2021) and Klammsteiner et  al. (2020a) could not find and we measured microbial activity later after fertilizer application significant differences in yield of Swiss Chard and Perennial (45 days). Watson et al. (2021), suggested that the main driver of Ryegrass, respectively, when comparing BSFL residues to mineral differences in basal respiration among treated soils was labile C, a key fertilizer, however the latter significantly increased the mineral N fuel for microbial activity, and probably depleted in CC compared to content of the soil. Chirere et al. (2021) suggested the stimulatory BR fertilizers. However, in line with beforementioned assumptions capacity of humic acids in BSFL residues to contribute to plant yield we would propose that original labile C added to soil through BR Frontiers in Microbiology 14 frontiersin.org Fuhrmann et al. 10.3389/fmicb.2022.994091 fertilizers was likely to be majorly consumed after 45 days and thus community structure should be complemented with analyses on had probably a negligible effect on basal respiration. Thus, we would the functional capacity of the microbial community, for example by assume that rather the C sources released from the slower, fungi- measuring the genetic potential via quantification of key genes dominated decomposition of cellulose or lignin (Insam and De through quantitative PCR, metagenome sequencing to identify Bertoldi, 2007) probably inherent to BSFL residues were driving basal genes that are enriched under BSFL residue amendment, and respiration in our experiment. In fact, fertilization with BR fertilizers extracellular enzyme assays to directly measure shifts in specific led to an enrichment of members of fungal taxa that were previously enzymatic activities. The results of our study are specific to our associated with the decomposition of organic matter such as genus experimental setup since the composition of BSFL residues both in Mortierella (Jayasinghe and Parkinson, 2008) and family terms of biota and substrate composition differ largely according to Lasiosphaeriaceae (Nordén and Paltto, 2001). That might have led to their origin and processing. Soil type, crop type, and investigated a trickle-down effect with their metabolic products serving as a time scale are other important factors that need to be considered substrate for other microbial taxa resulting in an increased basal further, as these parameters were fixed in our experiment. Studies respiration. Again, it is not possible to clearly conclude that microbes on long-term effects of such amendments, especially under field introduced with BR+ contributed to the stimulation of soil microbes conditions, are also largely missing. All these investigations can since BR- and BR+ were not differing significantly in basal respiration. pave the way for using organic fertilizers derived from insect In this study we combined the sterilization of organic soil inputs rearing on waste products for a circular and sustainable agriculture. with physicochemical and microbial assessments. This method can serve as an example to test the validity of other soil amendments to which a positive influence of inherent microorganisms on soil fertility Data availability statement is attributed, also referred to as “biofertilizers” (Mahanty et al., 2017). The datasets presented in this study can be found in online repositories. The names of the repository/repositories and Conclusion accession number(s) can be found at: https://www.ebi.ac.uk/ena, PRJEB54639. Fertilization with BSFL residues altered the soil microbial community and tended to increase microbial activity and crop yield when compared to compost-amended or unamended soils, Author contributions respectively. By including a non-thermal soil sterilization treatment, we  could provide indication that these effects were partially AF, BW, MG, AM, and MH have conceived the study. AF mediated by the introduction of BSFL residue-derived microbes to conducted the greenhouse experiment. AF and RF performed the the soil. Among these, taxa commonly associated with organic laboratory analyses. BW, SK, MK, BM and LS organized sourcing matter decomposition and plant growth promotion were identified, and shipping of fertilizers. AF, BW and MH analysed the data. AF which could also have potential downstream effects on other wrote the initial draft of the manuscript. BW, MG and MH bacterial and archaeal taxa important for soil fertility. In contrast, provided substantial input. All authors contributed to the article the BSFL residues had limited influence on the measured soil and approved the submitted version. physicochemical properties, although we  assume that organic compounds characteristic for BSFL residues used in our experiment such as chitin and its derivates with promising applications in Funding agricultural production might have been enriched upon amendment. Thus, we can assume that both biotic effects via the The RUNRES project funded by the Swiss Agency for introduced organisms as well as abiotic effects via bioactive Development and Cooperation (project number 7F09521) provided substances can influence soil fertility and plant performance. us with the applied organic fertilizers through the two participating Therefore, BSFL residues with high plant-nutritional value and Rwandan bioconversion companies COPED and Maggot Farm microbial capabilities to promote plant growth have the potential Production Ltd. Open access funding provided by ETH Zurich. to improve sustainable agricultural production, especially in low-income countries, where BSFL rearing is meeting multiple needs as a promising, low-threshold technology in circular Acknowledgments economy. Notably the characteristics of relevant biotic and abiotic components in BSFL residues are likely to be impacted by their We want to express our gratefulness to the RUNRES project post-processing such as composting and should further be assessed funded by the Swiss Agency for Development and Cooperation to inform the debate with high practical significance. To corroborate that enabled our study by providing us with the applied organic ours and other findings, further research should focus on the fertilizers through the two participating Rwandan bioconversion quantification of chitin, labile carbon, fiber and compounds like companies COPED and Maggot Farm Production Ltd. as well as humic acids in BSFL residues. Results on changes in soil microbial the International Institute of Tropical Agriculture (IITA). We want Frontiers in Microbiology 15 frontiersin.org Fuhrmann et al. 10.3389/fmicb.2022.994091 to thank Horst Adelmann from the Laboratory of Food & Soft relationships that could be  construed as a potential conflict Materials, ETH Zurich for supporting the preparation and Conrad of interest. Günthard from LEONI Studer AG for the kind consultancy and execution of the HEEB sterilization of the fertilizers. We  are grateful for the support by Brigitta Herzog, Britta Jahn-Humphrey, Publisher’s note Elena Giuliano, Elena Kost and Tania Galindo Castaneda for their support with setting up the experiment and with physicochemical All claims expressed in this article are solely those of the analyses of the soil. We would like to thank Matti Barthel and authors and do not necessarily represent those of their affiliated Astrid Jäger for their guidance on the experimental set up and on organizations, or those of the publisher, the editors and the the soil respiration measurements. We thank Maria Domenica reviewers. Any product that may be evaluated in this article, or Moccia at the Functional Genomics Center Zurich (FGCZ) for claim that may be made by its manufacturer, is not guaranteed or providing the sequencing service on the Illumina MiSeq platform. endorsed by the publisher. Conflict of interest Supplementary material BM is employed by Maggot Farm. The Supplementary Material for this article can be found The remaining authors declare that the research was online at: https://www.frontiersin.org/articles/10.3389/fmicb. conducted in the absence of any commercial or financial 2022.994091/full#supplementary-material References Alattar, M. A., Alattar, F. N., and Popa, R. (2016). 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