Suitability of Black Soldier Fly Frass as Soil Amendment and Implication for Organic Waste Hygienization

Abstract

Because of its nutritious properties, the black soldier fly has emerged as one of the most popular species in advancing circular economy through the re-valorization of anthropogenic organic wastes to insect biomass. Black soldier fly frass accumulates as a major by-product in artificial rearing setups and harbors great potential to complement or replace commercial fertilizers. We applied frass from larvae raised on different diets in nitrogen-equivalent amounts as soil amendment, comparing it to NH 4 NO 3 fertilizer as a control. While the soil properties did not reveal any difference between mineral fertilizer and frass, principal component analysis showed significant differences that are mainly attributed to nitrate and dissolved nitrogen contents. We did not find significant differences in the growth of perennial ryegrass between the treatments, indicating that frass serves as a rapidly acting fertilizer comparable to NH 4 NO 3. While the abundance of coliform bacteria increased during frass maturation, after application to the soil, they were outcompeted by gram-negatives. We thus conclude that frass may serve as a valuable fertilizer and does not impair the hygienic properties of soils.

Full text

agronomy
Communication
Suitability of Black Soldier Fly Frass as Soil
Amendment and Implication for Organic
Waste Hygienization
Thomas Klammsteiner 1, * , Veysel Turan 2, Marina Fernández-Delgado Juárez 1,
Simon Oberegger 1and Heribert Insam 1
1Department of Microbiology, University of Innsbruck, Technikerstraße 25d, 6020 Innsbruck, Austria;
marinafdj84@gmail.com (M.F.-D.J.); s.oberegger@student.uibk.ac.at (S.O.); heribert.insam@uibk.ac.at (H.I.)
2
Department of Soil Science and Plant Nutrition, Bingöl University, Selahaddin-I Eyyubi, Üniversite Caddesi,
12000 Bingöl, Turkey; vturan@bingol.edu.tr
*Correspondence: thomas.klammsteiner@uibk.ac.at; Tel.: +43-512-507-51322
Received: 20 September 2020; Accepted: 14 October 2020; Published: 15 October 2020


Abstract:
Because of its nutritious properties, the black soldier fly has emerged as one of the most
popular species in advancing circular economy through the re-valorization of anthropogenic organic
wastes to insect biomass. Black soldier fly frass accumulates as a major by-product in artificial rearing
set-ups and harbors great potential to complement or replace commercial fertilizers. We applied frass
from larvae raised on different diets in nitrogen-equivalent amounts as soil amendment, comparing it
to NH
4
NO
3
fertilizer as a control. While the soil properties did not reveal any difference between
mineral fertilizer and frass, principal component analysis showed significant differences that are
mainly attributed to nitrate and dissolved nitrogen contents. We did not find significant differences
in the growth of perennial ryegrass between the treatments, indicating that frass serves as a rapidly
acting fertilizer comparable to NH
4
NO
3
. While the abundance of coliform bacteria increased during
frass maturation, after application to the soil, they were outcompeted by gram-negatives. We thus
conclude that frass may serve as a valuable fertilizer and does not impair the hygienic properties
of soils.
Keywords: animal feedstuff; circular economy; fertilizer; greenhouse; insect larva; organic waste
1. Introduction
In recent years, the use of saprobic insect larvae from the mealworm beetle (Tenebrio molitor),
the black soldier fly (Hermetia illucens; BSF), or the house fly (Musca domestica) has attracted interest in
the face of rising prices of animal feedstuffand accumulating amounts of waste [
1
,
2
]. In the European
Union, green waste and food waste largely contribute to an annual amount of 118 to 138 million tons
of organic wastes [
3
]. Especially BSF larvae (BSFL) have been shown to efficiently convert organic
wastes into high quality fat and protein [
4
]. The economic potential and meaningful reintroduction of
otherwise wasted nutrients into the biosphere via a circular economy enticed researchers, investors,
and the public to contribute to a more efficient recycling of organic wastes by exploiting the potential of
insect larvae on a large scale [
5
,
6
]. BSFL could also play a valuable role for smaller decentralized waste
management systems operated by e.g., hobbyists or farmers in areas where the fly occurs naturally [
7
–
9
].
Additionally, the exploitation of BSF and its by-products could create an affordable opportunity for
revenue generation by entrepreneurs and smallholder farmers in low-income countries [
9
–
11
]. The main
by-product in the bioconversion of wastes into high quality protein for animal feedstuffis summarized
as ‘frass’. Frass in general describes insect excretions, but in a commercial context it often refers
Agronomy 2020,10, 1578; doi:10.3390/agronomy10101578 www.mdpi.com/journal/agronomy

Agronomy 2020,10, 1578 2 of 12
to a mixture of mainly insect feces, substrate residues, and shed exoskeletons. It is an inevitable
side-stream during the mass-rearing of insects that can add up to 75% of the fed substrate [
12
] and is
often merchandised as a fertilizing product. In recent years, an increasing number of studies started
focusing on meaningful applications of insect frass [
9
,
13
–
15
], and the first large-scale field studies
provided promising perspectives for its application in agriculture, especially in terms of plant nutrient
availability [10,11,16].
The substrate used to grow insects affects the properties of the frass, since undegradable residues
remain unused, while the digested fraction is modified by the gut microbiota when passing through
the gastrointestinal tract [
17
,
18
]. Wang et al. [
19
] used frass from T. molitor for subsequent rearing of
BSFL to exploit leftover nutrients that T. molitor could not take up or digest. In substrates carrying
a high bioburden like human feces and manure, BSFL have shown to reduce pathogenic bacteria
such as Salmonella enterica [
20
,
21
] and Escherichia coli [
20
,
22
], which is attributed to their production of
antimicrobial peptides [
23
]. In the wild, frass from various insects can help to increase the chances of
survival and reproduction by either deterring [
24
,
25
] or attracting [
26
,
27
] conspecifics. Frass can act as
a vector for phytopathogenic microorganisms [
28
,
29
] and as a source of probiotic yeasts [
30
]. Its effect
on the insects’ environment can be observed in forests, where frass deposition goes hand in hand with
insect canopy herbivory. It has been shown that frass has an impact on C and N dynamics, and has
beneficial effects for tree growth by increasing soil total C, N, and NH
4+
, as well as microbial soil
respiration [
31
,
32
]. In industrial environments, frass pyrolyzed to biochar has been successfully tested
as a bioadsorbent for wastewater detoxification [
33
]. According to recent studies, frass’ agriculturally
and economically most meaningful potential could lie in its application as fertilizer [9,34].
In this study, we assessed the fertilizing potential of process residues (frass) from three generic diets
degraded by BSFL. Two of the diets represent major streams of organic waste, namely grass-cuttings
(GC) and fruit/vegetable (FV) mix, while the chicken feed (CF) control diet is a commonly used insect
breeding substrate. We hypothesized that (I) microbial colonization increases with frass maturation
and (II) frass may serve as a valuable alternative to mineral fertilizer by inducing beneficial effects on
plant growth.
2. Materials and Methods
2.1. Black Soldier Fly Frass Collection
The frass was collected from a preparatory feeding experiment conducted at 27
◦
C, 60% relative
humidity (Figures S1 and S2, Table 1). The chicken feed (CF; Grünes Legekorn Premium,
Unser Lagerhaus, Klagenfurt, Austria) was processed with a Fidibus flour mill (Komo Mills, Hopfgarten,
Austria) and mixed with water in a 40:60 ratio.
Table 1.
Feeding experiment termination summary. The feeding experiment was terminated after a
total of 23 days when more than 90% larvae from one treatment group transitioned to prepupal stage.
Different lower-case letters indicate differences between treatments (p
≤
0.05) according to the Tukey’s
HSD test. (n=4; average
±
standard deviation; CF =Chicken feed diet, GC =Grass-cuttings diet,
FW =Fruit/Vegetables diet).
CF GC FV
Pupation rate [%] 55.2 ±5.3 b 98.7 ±2.0 c 22.4 ±2.0 a
Prepupae fresh weight [mg] 198 ±10 167 ±11 165 ±31
Prepupae dry weight [%] 32.6 ±1.9 29.6 ±1.9 32.2 ±9.3
Prepupae water content [%] 67.4 ±3.7 70.4 ±5.6 67.8 ±9.9
Prepupae organic content [%] 27.6 ±1.6 26.5 ±1.8 31.4 ±8.4
Prepupae inorganic content [%] 5.1 ±0.3 c 3.1 ±0.2 b 0.9 ±0.9 a
Frass residues [%] 43.7 ±1.0 c 46.0 ±1.5 b 28.5 ±0.5 a

Agronomy 2020,10, 1578 3 of 12
The fruit/vegetable mix (FV; cucumber, tomato, apple, orange, in ratio 0.5:1:1:1) and fresh
grass-cutting diet (GC) were shredded and homogenized using a Total Nutrition Center blender
(Vitamix, Olmsted Township, United States). Feeding was done in organic content-equivalents (100,
250, and 370 mg larvae
−1
day
−1
for CF, GC, and FV). After termination of the feeding experiment,
the black soldier fly frass (BSFF) from each treatment was collected in plastic bags and stored at room
temperature until further use.2.2. Soil Preparation and Greenhouse Set Up
A greenhouse trial using soil collected from an agricultural site (47
◦
15
0
54” N, 11
◦
20
0
20” E; Table 2)
was set up to evaluate the fertilizing effect of the BSFF on the soil. The neutral-to-slightly basic
soil (pH 7.3
±
0.4) had an electrical conductivity of 78.0
±
2.7
µ
s cm
−1
and a volatile solids content
of
78.0 ±26.6 g kg−1
. In addition to a P
total
content of 823
±
190 mg kg
−1
(P
bioavailable
proportion
6.88 ±1.28 mg kg−1
), elemental analysis determined a C/N ratio of 24 (40 g C
total
kg
−1
, 1.7 g N
total
kg
−1
).
The soil classified as a calcaric Fluvisol (IUSS Working Group WRB, 2015) was sieved (Ø <4 mm) and
homogenously mixed with a vermiculite/sand blend (1:1; v:v) at a ratio 2:1 w:w(soil:blend).
Table 2.
Characterization of the soil used for the greenhouse trial. Values expressed on a dry mass basis
for n=3 (average
±
standard deviation). pH (pH CaCl
2
), EC (Electrical conductivity), VS (Volatile solids),
Ctot (Total carbon), Ntot (Total nitrogen), Ptot (Total phosphorous), Pav (Plant available P).
Parameter Value
pH 7.3 ±0.4
EC [µs cm-1]78.1 ±2.7
VS [g kg-1]78.5 ±26.6
Ctot [g kg-1]40
Ntot [g kg-1]1.7
Ptot [mg kg-1]823 ±190
Pav [mg kg-1]6.88 ±1.28
The four experimental treatments were performed in 500 mL pots: soil was mixed with (1) mineral
fertilizer (which served as control); (2) GC BSFF; (3) FV BSFF; (4) and CF BSFF. The mineral fertilizer
(NH
4
NO
3
) and the different types of BSFF (Table 3) were added in an amount of 40 mg N kg
−1
soil,
which is equivalent to 80 kg N ha
−1
, considering the soil bulk density of 1 g cm
−3
and a plough depth
of 20 cm as described by Goberna et al. [
35
]. Thereby, all treatments received the same dose of total N.
Table 3.
Main properties of the three different black soldier fly frass fractions (CF-F: Chicken feed frass;
GC-F: Grass-cuttings frass; FV-F: Fruit/Vegetables frass). Values expressed on a dry mass basis for n=3
(average
±
standard deviation). Different lower-case letters indicate differences between treatments
(p
≤
0.05) according to the Tukey
´
s HSD test. Different capital letters indicate significant differences
between treatments (p
≤
0.05) according to the Mann–Whitney test. EC (Electrical conductivity),
VS (Volatile solids), Ctot (Total carbon content), Ntot (Total nitrogen content).
CF-F GC-F FV-F
pH 6.22 ±0.14 C 5.40 ±0.03 A 5.58 ±0.01 B
EC [mS cm-1]5.67 ±0.27 c 3.06 ±0.03 b 2.36 ±0.11 a
Dry matter [%] 90.9 ±0.0 89.9 ±0.0 90.4 ±0.0
Ctot [g kg-1]479 ±8 B 443 ±6 A 488 ±4 B
Ntot [g kg-1]25.9 ±0.9 b 24.4 ±0.2 b 18.3 ±1.2 a
C:N ratio 18.5 ±0.3 a 18.2 ±0.4 a 26.6 ±1.7 b
VS [g kg-1]910 ±7 c 825 ±9 a 873 ±4 b
After an equilibration period of 16 h at 4
◦
C, pots were randomly placed in a greenhouse.
Ryegrass (Lolium perenne; seed amount based on 30 kg seeds ha
−1
) was sown and left to develop.
During the incubation period of 28 days, at an average temperature of 20
◦
C with a light/darkness cycle
of 10/14 h, the soil moisture was kept at field capacity (moisture of the soil after drainage by gravity).

Agronomy 2020,10, 1578 4 of 12
All treatments were applied in four replicates, resulting in a total of 16 pots in this study. After the
incubation period, plants were removed from the pots, and soil samples were sieved (Ø <2 mm) and
immediately stored at +4◦C until analyses (Table 4).
Table 4.
Physicochemical and biological properties of the control (C-S: NH
4
NO
3
) and the frass amended
soils (CF-S: Chicken feed frass +soil; GC-S: Grass-cuttings frass +soil and FV-S: Fruit/Vegetables frass +soil).
Values expressed on a dry mass basis for
n=4
(average
±
standard deviation). Different lower-case letters
indicate differences between treatments (
p≤0.05
) according to the Tukey
´
s HSD test. Different capital
letters indicate significant differences between treatments (
p≤0.05
) according to the Mann–Whitney
test.
EC (Electrical conductivity)
,
VS (volatile solids)
,
Ctot (Total carbon
content),
Ntot (Total nitrogen
content),
NH4+(Ammonium content)
,
NO3−(Nitrate content)
,
DOC (Dissolved organic
carbon),
DC (Dissolved carbon)
,
DN (Dissolved nitrogen)
, P
av
(Plant available phosphorous content),
Ptot (Total phosphorous content), BR (Basal respiration), qCO2(Metabolic quotient).
C-S CF-S GC-S FV-S
pH CaCl27.53 ±0.02 a 7.57 ±0.01 ab 7.58 ±0.03 b 7.58 ±0.02 b
EC [µS cm-1]95.5 ±1.8 B 79.8 ±6.4 A 77.3 ±2.9 A 81.5 ±7.8 A
VS [g kg-1]37.3 ±1.1 35.4 ±1.3 38.4 ±2.0 37.8 ±2.1
Ctot [g kg-1]17.9 ±3.2 21.7 ±4. 22.5 ±6.1 20.6 ±5.2
Ntot [g kg-1]0.98 ±0.35 0.99 ±0.51 1.17 ±0.39 0.98 ±0.44
C:N ratio 20.3 ±8.1 26.2 ±11.7 21.0 ±9.8 22.1 ±9.0
NH4+[mg kg-1]0.57 ±0.13 0.58 ±0.06 0.58 ±0.08 0.61 ±0.14
NO3-[mg kg-1]45.2 ±4.1 b 15.4 ±3.3 a 17.0 ±3.5 a 12.1 ±4.4 a
DOC [mg kg-1]48.3 ±3.8 50.2 ±1.5 48.5 ±1.9 51.8 ±1.3
DC [mg kg-1]95.6 ±1.6 a 104.7 ±1.4 b 103.5 ±4.0 b 112.1 ±2.0 c
DN [mg kg-1]35.6 ±3.9 C 17.0 ±1.5 B 15.3 ±1.8 AB 15.0 ±0.6 A
Pav [mg kg-1]5.2 ±0.4 6.1 ±1.0 6.1 ±1.4 5.8 ±0.9
Ptot [mg kg-1]783 ±46 ab 866 ±35 b 757 ±33 a 721 ±45 a
P bioavailability [%] 67 ±8 70 ±12 81 ±23 80 ±12
BR [µg CO2g-1 dw h-1]5.6 ±0.15 4.6 ±1.5 6.7 ±0.7 5.6 ±0.5
Cmic [µg CO2g-1 dw soil] 416.1 ±103.5 276.4 ±38.7 279.0 ±22.7 336.2 ±16.1
qCO2[µg CO2-C h-1/µg-1 C mic] 14.7 ±4.7 16.4 ±4.3 24.5 ±4.5 16.6 ±1.4
Plant biomass [mg dw] 85 ±7 80 ±6 74 ±4 75 ±3
2.2. Frass and Soil Analyses
Frass and soil samples (10 g fresh weight) were placed into a glass Petri dish and oven-dried (105
◦
C)
for 24 h to determine the content of total solids. Volatile organic solid (VS) content was determined from
the weight loss following ignition in a muffle furnace (CWF 1000, Carbolite, Neuhausen, Germany)
at 550
◦
C for 5 h. Total C and N contents were analyzed in dried samples using a CN analyzer
(TruSpec CHN, LECO, St. Joseph, MI, USA). EC and pH were determined in distilled water and 0.01 M
CaCl2extracts (1:2.5, w/v), respectively.
Soil inorganic nitrogen (NH
4+
and NO
3−
) was determined in 0.0125 M CaCl
2
extracts as described
by Kandeler [
36
,
37
]. Soil total P (P
tot
) and plant available P (P
av
) were determined as described by
Illmer et al. [
38
]. To estimate dissolved organic carbon (DOC), dissolved carbon (DC), and dissolved
nitrogen (DN), 10 g of field-moist soil were shaken in 40 mL distilled water, filtered, and immediately
measured using a TOC-L analyzer (Shimadzu, Ky
¯
oto, Japan). Soil basal respiration (BR) and microbial
biomass (C
mic
) were measured according to Heinemeyer et al. [
39
]. The metabolic quotient (qCO
2
)
was calculated from BR and C
mic
according to Anderson and Domsch [
40
]. At the end of the trial,
aboveground plant biomass was determined by cutting plant shoots at the soil surface and drying
them at 60 ◦C for 48 h. Samples were then re-weighted to determine the dry biomass.
2.3. Preparation of Media
For the assessment of the total cultivable bacterial colony forming units (CFUs), we used standard
methods agar (0.5% peptone, 0.25% yeast extract, 0.1% glucose, 1.5% agar, pH adjusted to neutral).

Agronomy 2020,10, 1578 5 of 12
To determine the abundance of Salmonella sp., E. coli, coliforms, and other gram-negative bacteria,
XLT-4 and ChromoCult
®
coliform agar (Merck, Darmstadt, Germany) were prepared according to the
enclosed recipe.
2.4. Pathogen Quantification/Assessment of Microbial Colonization in Frass and Soil
An amount of 2 g frass or soil sample was added to 18 mL sterile saline solution (0.95% NaCl)
and placed on a rotation shaker at 200 rpm for 15 min. Samples were diluted to 10
−2
and 10
−3
for soil,
and 10
−5
and 10
−6
for frass using sterile 0.95% NaCl. From each dilution, 50
µ
L was plated using the
spread plate technique. Plates were then incubated at 37 ◦C for 24 h, and the CFUs were counted.
2.5. Statistical Analyses
The effect of the BSFF application on soil parameters was tested with a one-way analysis of
variance (ANOVA). In case of significant F-values, a Tukey’s HSD (honestly significant difference)
post hoc test (p<0.05) was performed. Prior to analysis, the homogeneity of the variances was
tested (Levene’s test), and data were also tested for normality. Non-normal data were subjected to
non-parametric tests for several independent samples (Kruskal–Wallis test), and pairwise comparisons
between treatments were performed using the Mann–Whitney U test (p<0.05). Statistical analyses
were performed using the SPSS v. 23.0 Software (IBM, Armonk, NY, USA). Principal component
analysis was performed in R [
41
] using the vegan package [
42
]. Analysis of similarity (ANOSIM) on the
physicochemical data (999 permutations) was also conducted with vegan. All graphical representations
of data were created with ggplot2 [43].
3. Results and Discussion
3.1. Assessment of Microbial Load in Frass and Frass-Amended Soils
The high moisture content of substrates and air, as well as the pleasantly warm temperature
common in insect breeding, favor microbial growth. While the type of diet is known to directly
influence the BSFL gut microbiome [
17
,
44
], the excrements in turn may influence the microbiome in the
frass. It is likely that by agitating and mixing their surrounding substrate with feces and their inherent
microorganisms, the larvae have an impact on their habitat. Similar effects are known from the widely
used earthworms (Eisenia fetida), which can stabilize organic wastes and introduce ammonia-oxidizing
microorganisms, thereby boosting nitrification and increasing nitrate concentrations in the resulting
vermicompost [
45
]. Other insect species inoculate the soil with excreted microorganisms and provide
beneficial effects for its quality both in wild and artificial settings [46–48].
Before and after applying frass as soil amendment, the number of cultivable E. coli, coliform,
and other gram-negative bacteria were assessed (Figure 1). While frass counted up to 10
9
CFU g
−1
,
the count in soil was down to 10
3
–10
5
g
−1
. With the nutrient media used in this study, untreated soil
contained no cultivable E. coli or coliforms, and only low abundances of cultivable gram-negatives
with 10
2
CFUs g
−1
. In particular, frass from the CF treatment acted as a reservoir for coliforms with
a CFU count of 1.9
×
10
9
, thereby exceeding CFU counts recorded on larval surfaces (Figure S2).
Gram-negative bacteria predominated the cultivable microbiota in frass-amended soil with highest
CFU counts of up to 10
5
in soil treated with frass from a FV diet. High microbial load and dominance
of coliforms in frass shifted to lower CFU numbers and predominantly gram-negative bacteria in
the frass-soil mix, indicating that the autochthonous soil microbiota outcompeted allochthonous
microorganisms introduced with frass [49–51].

Agronomy 2020,10, 1578 6 of 12
Figure 1.
Colony forming units counted for gram-negative, coliform, and Escherichia coli from frass
samples after collection from the feeding experiment and soil samples after having mixed the soil with
frass (n=4). CF =Chicken feed, FV =Fruit/vegetable mix, GC =Grass-cuttings.
3.2. Black Soldier Fly Frass Properties, Soil Quality and Plant Performance
The physicochemical properties of frass were influenced by the larval diet (Table 3). Especially CF
frass was more alkaline, had a higher EC, and a higher content of VS. While total C contents were
similar in all types of frass, FV frass showed a C:N-ratio of 26.6, compared to 18.5 and 18.2 in CF frass
and GC frass, respectively. Similar C:N ratios as found in CF and GC frass have been reported by other
studies that used brewery spent grains as larval substrate [
11
,
16
]. A C:N-ratio >20 bears the risk of
soil N immobilization, which may favor plants with a more efficient N exploitation attributed to their
rhizobiome [
46
,
52
]. The addition of biochar to the larval waste conversion process might further improve
the frass’ N retention, while at the same time increasing larval biomass yield [
10
]. Moreover, larvae pass
through six instars continuously shedding their exoskeleton. Chitin, an N-acetylglucosamine-based
polymer (C
8
H
13
O
5
N)
n
, may influence not only the C:N ratio, but its degradation product chitosan
may also provide underrated benefits for plant health and pathogen resistance [
53
,
54
]. The C:N ratio
is one of the major parameters to consider when it comes to deciding whether frass should be used
as soil amendment or as co-substrate in anaerobic digestion or composting [
55
,
56
]. Chitin utilization
by insects is often associated with chitinolytic gut symbionts [
57
], which still needs to be further
investigated in the context of BSF larvae. Chitin-containing fertilizers have previously been found to
serve as splendid nitrogen sources [58].
Frass addition to the soil before planting Lolium perenne was adjusted on a basis of N-equivalence
(80 kg N ha
−1
; Tables 3and 4). Soil amended with CF frass exhibited a higher P
tot
content than the
other frass-amended soils; however, P
av
was not significantly different. Principal component analysis
(Figure 2) highlighted the parameters that influenced the properties of the soil-frass mix the most,
which was further confirmed by ANOSIM (R =0.5061, p<0.001). The three frass-amended soils
clustered closely together, with P
tot
and P
av
, pH, DC, N
tot
, C:N ratio, BR, and the qCO
2
being the most
influential parameters for their similarity.

Agronomy 2020,10, 1578 7 of 12
Figure 2.
Principal component analysis of samples from control soil and soil mixed with the three
different frass types. Data points represent replicates, and arrows show the most influential parameters
for the spread of the data. NO
3
=Nitrate, DN =Dissolved nitrogen, CN =Carbon/Nitrogen ratio,
P
bio
=Phosphorus bioavailability, BR =Soil basal respiration, qCO
2
=Metabolic quotient, C
mic
=
Microbial biomass, N
tot
=Total nitrogen, P
av
=Plant available phosphorus, DC =Dissolved carbon,
EC =Electric conductivity.
The qCO
2
describing the microbial soil respiration per unit C
mic
is known to be tightly connected
to the C:N ratio and increases when less N is available [
59
]. Higher qCO
2
can indicate stress or
disturbances within the soil because, although C sources are readily available, microbial metabolism
and substrate decomposition are limited by N [
60
]. NO
3
and DN, on the other hand, were the major
drivers for the deviation of the control group from the frass treatment groups, since they were both
significantly higher in control soil.
In our study, the frass treatments were compared with a control that received an equivalent of
80 kg ha−1
nitrogen in the form of NH
4
NO
3
. In a similar experiment, Ros et al. [
61
] found that such
an amount of mineral N increased the maize yield by 33% compared with an unfertilized control,
while N-equivalent additions of compost yielded only 15% increase. Recent observations at field-scale
by Beesigamukama et al. showed that even at lower application rates of 30 kg N ha
−1
, BSFF exceeded
the performance of mineral N fertilizer in terms of grain yield and nitrogen fertilizer replacement
values when applied at the same rates [
16
]. Compared with commercial fertilizers, nitrogen recovery
rates and nitrogen use efficiency of plants have been shown to be improved when amended with
BSFF [
11
]. Additionally, the higher P concentrations in the frass could facilitate N accumulation in
plants by improving N uptake, as P plays an important role in energy transfer [62,63].
Using BSFL instead of aerobic windrow composting has additionally been shown to reduce
the global warming potential of treating organic wastes by 50% [
64
]. The addition of frass did not
lead to significant differences in plant growth compared to the mineral fertilizer (Figure 3). In fact,

Agronomy 2020,10, 1578 8 of 12
the similar growth progress indicates that the nutrients from frass are readily available for uptake and
have no detrimental impact on plant growth. These results, however, do not support the findings of
Alattar et al. [13]
, who reported that the development of plant height and leaves in corn (Zea mays) was
inhibited by the addition of BSFL frass. In their study, they attributed the negative effects to the low
porosity of larval residues that may have created anaerobic conditions. The moisture content of the
frass harvested from our preliminary feeding experiment was only 10% (Table 3), thereby facilitating
aeration and miscibility in soil. Insufficient oxygen supply can occur when frass has a high moisture
content and is not subjected to adequate post-processing. In an environment specialized on insect
rearing, a multi-step treatment of frass could increase the efficiency of degradation. With additional
downstream composting or anaerobic digestion [
65
,
66
], the recovery as soil amendment represents the
economically most promising option.
Figure 3.
Plant biomass yield of Lolium perenne after application of black soldier fly frass (BSFF)
obtained from the degradation of various organic substrates. CF BSFF =Chicken feed frass, FV BSFF =
Fruit/vegetables frass, GC BSFF =Grass-cuttings frass (n=4).
4. Conclusions
The valorization of organic wastes by insect larvae generates frass as a side-product. From our
study we conclude that frass may serve as a soil nutrient source and does not impair soil hygiene.
In some cases, however, frass post-processing through anaerobic digestion or composting may be
advised to avoid soil nitrogen deficiencies or impairing soil gas permeability. In the light of the
increasing importance of insect rearing, the agricultural utilization of frass is demanding further
research, in particular, long-term studies.
Supplementary Materials:
The following are available online at http://www.mdpi.com/2073-4395/10/10/1578/s1,
Figure S1: Influence of three different diets on larval biomass increase, Figure S2: Microbial colonization of
larval surfaces.
Author Contributions:
Conceptualization, T.K. and M.F.-D.J.; formal analysis, V.T., T.K., and S.O.;
funding acquisition, H.I., V.T., and T.K.; investigation, S.O. and V.T.; methodology, V.T. and M.F.-D.J.; resources,
H.I.; supervision, T.K. and M.F.-D.J.; visualization, T.K.; writing—original draft, T.K. and M.F.-D.J. All authors
have read and agreed to the published version of the manuscript.
Funding:
This research was funded by the Austrian Science Fund (FWF; project number: P26444).
Thomas Klammsteiner was supported by a PhD grant from the Vizerektorat für Forschung of the Universität
Innsbruck (Doktoratsstipendium aus der Nachwuchsförderung). Veysel Turan was supported by a post-doctoral
fellowship from the Scientific and Technological Research Council of Turkey (TUBITAK, grant number,
1059B191601133).

Agronomy 2020,10, 1578 9 of 12
Acknowledgments:
The authors show their gratitude to Carina D. Heussler for providing the black soldier fly
larvae for this study. Open Access Funding by the Austrian Science Fund (FWF).
Conflicts of Interest: The authors declare no conflict of interest.
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