Materials
Pig manure (21.1 ± 1.7 % total solids) was collected on a pig farm in Uppsala municipality, Sweden. Dog food (Puppy Original, Pro Plan, Purina) mixed with water (21.9 ± 0.2 % total solids) was used as the model substrate for organic waste (Vinnerås et al. 2003). Human feces were collected fresh in plastic bags and stored at −20 °C immediately upon collection. A mixture of pig manure, dog food, and human feces was prepared (4:4:2; 28.7 ± 1.2 % total solids), divided into feeding portions, and kept at −20 °C until use.
Ascaris suum, which infects pigs, is often used as model for Ascaris lumbricoides, which infects humans (Johnson et al. 1998). A. suum extracted from the feces of infected pigs were purchased from Excelsior Entinel, Inc. and received in aqueous solution at a concentration of 100,000 eggs mL−1.
The bacterial inoculate solutions consisted of 108 colony forming units (CFU) mL−1
Enterococcus faecalis (ATCC 29212) and 109 CFU mL−1
Salmonella enterica subspecies ~ 1 serovar Typhimurium phage type 178 (isolated from sewage sludge (Sahlström et al. 2004), grown in unselective bacterial nutrient medium (NB, Oxoid AB, Sweden).
Propagation of bacteriophage фX174 to a concentration of 109 plaque forming units (PFU) mL−1 was performed in unselective bacterial nutrient medium (NB, Oxoid AB, Sweden) using the host strain Escherichia coli (ATCC 13706). The phage was collected by centrifuging the solution at 2,000 × g for 10 min followed by sterile filtration and kept at 4–6 °C until use.
The virus inoculate comprised the following: Reovirus type 3 (ATCC VR-232); strain Abney (human); genus Orthoreovirus; Canine Adenovirus I, strain vacc-98, genus Mastadenovirus; and Porcine enterovirus I, genus Teschovirus.
Black soldier fly eggs were received twice a week from the Research Institute of Organic Agriculture, Frick, Switzerland.
Experimental set-up and sampling
Three fly larvae reactors consistent of white 37 L Sortera bins (37 × 55 × 28 cm), with a lid and slanting wall for prepupal migration, that were placed inside 45 L black Samla bins (39 × 57 × 28 cm) with a lid, both from IKEA (Uppsala, Sweden). The Samla bins were used to keep light out of the reactor and as collection bins for migrating prepupae. The reactors were kept at 25 °C throughout the experiment.
Black soldier fly egg packages were received in cardboard strips. Individual egg packages were divided into small (around 150–250 larvae), medium (around 400–500 larvae), and large (around 800–1,000 larvae) egg packages and hatched individually. Twice a week, a more or less equal number (based on the number and size of egg packages) of 5–10-day-old larvae were added to each reactor. The eggs were hatched in the same feed mixture used in the reactors. The total number of larvae at steady-state was estimated to be 7,200.
The reactors were fed three times a week. The pre-prepared feed mixture was thawed overnight and added in the mornings. The feed portions were adjusted to the estimated number of larvae present in the reactors, to a feeding rate of 100 mg per larvae per day (Diener et al. 2009); at steady-state each unit was fed 5.4 kg per week.
The reactors were operated as plug-flow: old material in the back of the reactor was pushed 5 cm to the front prior to fresh feed mixture being added. All samples were collected from the front 10 cm of the reactor.
On each feeding occasion, the feed mixture was inoculated with Salmonella typhimurium (1 % w/w), E. faecalis (1 % w/w), and фX174 (1 % w/w).
On the first feeding occasion in week 4, the feed mixture was inoculated with 1 mL A. suum of concentration 100,000 egg mL−1.
On the first feeding occasion in week 7, the feed mixture was inoculated with Reovirus type 3, Canine Adenovirus I, and Porcine enterovirus I.
The reactors were weighed weekly throughout the course of the experiment. All migrating prepupae were counted/weighed. Once a week, triplicate samples of 5 g were collected in each reactor and analyzed separately to determine the concentration of bacteria and bacteriophages in the influent and effluent material. The concentration and viability of A. suum eggs was determined on days 7, 14, and 28 after inoculation. The concentration of viruses in the material was determined 2, 4, 7, 9, 11, and 14 days after inoculation. Samples for physicochemical analysis were collected at the start and end of the experiment.
Physicochemical analyses
The material was dried at 105 °C for 14 h to determine total solids and at 550 °C for 6 h to determine volatile solids, i.e., the organic content.
A radiometer electrode was used to measure pH. All analyses were conducted at room temperature on 10 g of sample diluted with 50 mL deionized water and were left to settle for 1 h prior to analysis.
In a closed container, 1 g of sample was mixed with 20 mL deionized water. The material was thoroughly mixed and diluted 1:10 in deionized water. Spectroquant© test kit number 114544 was used for total ammonium nitrogen (NH4-N) analyses and kit number 114764 for nitrate (NO3-N) analyses.
In a 50-mL volumetric flask, 0.5–0.6 g samples were dissolved into 10 mL concentrated H2SO4 and brought to “rolling boil” on a hotplate. When the sample had visually dissolved, an additional 5 mL H2SO4 was added. The sample was allowed to cool and was thereby diluted—1:100 for total nitrogen analysis and 1:50 for total phosphorous analysis—in deionized water. Spectroquant© test kit number 1.00613.0001 was used for total nitrogen analysis and number 1.00673.0001 was used for total phosphorous analysis.
Microbiological analyses
Bacterial and bacteriophage analyses
The buffer used for all experiments was buffered NaCl peptone water with Tween 80 at pH 7, hereafter referred to as Tween buffer. Sample (5 g) was extracted and further diluted in the Tween buffer.
A 100 µL volume of selected dilution was spread on Slanetz–Bartley agar (Oxoid AB, Sweden) and incubated at 44 °C for 48 h for Ent. spp. enumeration and on xylose lysine desoxycholate agar (XLD) (Oxoid AB, Sweden) containing 0.15 % sodium-novobiocin and incubated at 37 °C for 12 h for Salmonella spp. enumeration. The plates were enumerated with a detection limit of 100 CFU mL−1.
To lower the detection limit of Salmonella spp. concentration, 200 μL per plate of sample of dilution 10−1 was spread on five plates per sample and enumerated with a detection limit of 10 CFU mL−1. To lower the detection limit further, the sample was enriched as follows: 5 g per unit was immersed in 45 mL buffered peptone water and incubated at 37 °C for 24 h, upon which 100 μL was immersed into Rappaport–Vassiliadis salmonella enrichment broth (RVB) and incubated at 41.5 °C for 24 h. Positive results were evaluated by spreading 10 μL on a XLD plate and incubated at 37 °C for 12 h, lowering the detection limit to 1 CFU g−1.
Total thermotolerant coliforms were enumerated in double layer agar using violet red bile agar (VRB) (Oxoid AB, Sweden); 1 mL of sample was mixed with 7–8 mL of agar, and upon solidification of the first layer, an additional 7–8 mL agar was added. The plates were incubated at 44 °C for 24 h and counted with a detection limit of 10 CFU g−1.
For sampling of coliphage фX174, the host E. coli (ATCC 13706) was cultured in unselective microbial medium (NB, Oxoid AB, Sweden) at 37 °C for up to 4 h. A 1 mL sample volume of suitable dilution was mixed with 2 mL soft agar and 1 mL host solution, poured onto blood agar base (BAB) plates (Oxoid AB, Sweden) and incubated at 37 °C for 16 ± 2 h.
A. suum ova extraction
A. suum ova extraction was conducted according to the procedure of the United States Environmental Protection Agency (EPA/625/R-92/013), using 30 g of material. Prior to incubation, 15–20 of the extracted ascaris eggs were verified. The tubes were incubated at 28 °C for 30 days in 0.1 N H2SO4.
After approximately 30 days, the incubated material was analyzed under the microscope (~100 ova per sample). A 1 mL Sedgwick Rafter Counting Cell was used to count eggs; pre-larvae and larvae were counted as viable.
Virus analyses
Samples for virus analysis were diluted 1:5 in NaCl peptone water with Tween buffer, further diluted 1:2 in beef extract with glycine, and kept at −70 °C until analysis. After thawing, the samples were vortexed for 15 min at 60 rpm, centrifuged at 3,000 × g for 15 min at 5 °C, and the supernatant filtered through a 0.45 μm filter. The samples were then filtered through a sterile gel filter (PD10, GE Healthcare) to remove toxicity. The samples were analyzed by endpoint titration using the following cell cultures: MDCK (ATCC CCL-34) for adenovirus, BHK (ATCC CCL-10) for reovirus, and swine kidney cell line (SK-6) for enterovirus, all cells were prepared 24 h prior to infection. To assess the virus concentration in the ingoing material, the samples were diluted tenfold and eight replicates of 50 μL were assayed for each dilution, while 48 replicates of the lowest dilutions were used to lower the detection limit.
Calculation
Material conversion
The waste-to-biomass conversion rate (BCR) on a total solids basis was calculated by:
$$ \mathrm{BCR}=\frac{\mathrm{Mig}.{\mathrm{PP}}_{\mathrm{Tot}.\mathrm{TS}}}{{\mathrm{Waste}}_{\mathrm{Tot}.\mathrm{TS}}}\times 100 $$
(1)
where Mig.PPTot.TS and WasteTot.TS were the total solids in the migrating prepupae and waste, respectively.
The calculations for the nutrient flow balance and material degradation were based on the assumption that:
$$ {\mathrm{Waste}}_{\mathrm{Tot}.\mathrm{Ash}}={\mathrm{Residue}}_{\mathrm{Tot}.\mathrm{Ash}}+\mathrm{Mig}.{\mathrm{PP}}_{\mathrm{Tot}.\mathrm{Ash}} $$
(2)
where WasteTot.Ash, ResidueTot.Ash, and Mig.PPTot.Ash were the total amount of ash in the inflow waste, the residue, and the migrated prepupae, respectively. The amount of ash in the residue was estimated based on the measured values in the inflow material and migrated prepupae.
The material reduction (MatTS.Red) on a total solids basis was calculated as:
$$ {\mathrm{Mat}}_{\mathrm{TS}.\mathrm{Red}}=\left(1-\frac{{\mathrm{Redsidue}}_{\mathrm{Tot}.\mathrm{TS}}}{{\mathrm{Waste}}_{\mathrm{Tot}.\mathrm{TS}}}\right)\times 100 $$
(3)
where ResidueTot.TS and WasteTot.TS were the total amount of total solids in the treatment residue and the inflow waste, respectively.
Reduction of microorganisms
The log10 reduction of evaluated microorganisms that occurred during 1 week in the treatment (∆ log10 Red) was calculated as:
$$ \varDelta { \log}_{10}\mathrm{Red}={ \log}_{10}\left(\frac{C_{{\mathrm{Mat}}_i}}{C_{\mathrm{Mat}.{\mathrm{out}}_{i+1}}}\right) $$
(4)
where \( {C}_{{\mathrm{Mat}}_i} \) was the estimated concentration of the material in the treatment unit after substrate inoculation week i and \( {C}_{\mathrm{Mat}.{\mathrm{out}}_{i+1}} \) the theoretical concentration of the material in the treatment unit prior to substrate inoculation 1 week later (i + 1).
The theoretical concentration of the material in the treatment unit week i (\( {C}_{{\mathrm{Mat}}_i} \)) was calculated as:
$$ {C}_{{\mathrm{Mat}}_i}=\left(\frac{C_{\mathrm{Sub}.{\mathrm{in}}_i}\times {m}_{\mathrm{Sub}.{\mathrm{in}}_i}+{C}_{\mathrm{Mat}.{\mathrm{out}}_i}\times {m}_{{\mathrm{Mat}}_i}}{m_{\mathrm{Sub}.{\mathrm{in}}_i}+{m}_{{\mathrm{Mat}}_i}}\right) $$
(5)
where \( {C}_{\mathrm{Sub}.{\mathrm{in}}_i} \) and \( {m}_{\mathrm{Sub}.{\mathrm{in}}_i} \) were the concentration and mass, respectively, of the inflow substrate week i and \( {C}_{{\mathrm{Mat}}_i} \) and \( {m}_{{\mathrm{Mat}}_i} \) the concentration and the mass, respectively, of the treatment unit week i.
Mass balance
The material in the fly larvae composts was assumed to be mainly polysaccharides (cellulose and starch); thus, the chemical formula C6H10O5 was used when calculating the amount of oxygen needed for the respiration. For each polysaccharide, six oxygen molecules were required for full degradation into six carbon dioxide and five water molecules. For the mass balance, it was assumed that the total mass of material reduced on organic content basis had been completely respired into carbon dioxide and water.
The nutrient mass balance was conducted by multiplying the measured concentration of the evaluated nutrients with the mass of the total solids, for each fraction.
Statistical analyses
Two-tailed, paired Student’s t test (95 % confidence interval) was used to evaluate whether the reduction of microorganisms was significant and if a statistically significant difference occurred between the total solids, organic content and the concentrations of nutrients in the inflow and outflow materials. Linear regression with t test was used to evaluate if the pH and the reduction of microorganisms changed over time. All analyses and graphical presentations were conducted in R (R Core Team 2012).