Production and Incorporation of Black Soldier Fly Larvae Frass in Retaining Nitrogen From Urea

European
Journal of Applied Sciences – Vol. 10, No. 4

Publication Date: August 25, 2022

DOI:10.14738/aivp.104.12220.

Visvini, L., Latifah, O., Ahmed, O. H., Kurk, W. J., & Cassandra, D. S. (2022). Production and Incorporation of Black Soldier Fly Larvae

Frass in Retaining Nitrogen from Urea. European Journal of Applied Sciences, 10(4). 395-412.
Production and Incorporation of Black Soldier Fly Larvae Frass in

Retaining Nitrogen from Urea

Lohanathan Visvini
Department of Crop Science, Faculty of Agricultural and
Forestry Sciences, Universiti Putra Malaysia
Bintulu Sarawak Campus, 97008 Bintulu, Sarawak, Malaysia

Omar Latifah
Institute of Ecosystem Science Borneo, Universiti Putra Malaysia
Bintulu, Sarawak, Malaysia

Osumanu Haruna Ahmed
Faculty of Agriculture,
University Sultan Sharif Ali Brunei, Kampus Sinaut,
Km 33, Jalan Tutong, Kampung Sinaut, Tutong TB1741,
Brunei Darussalam

Wei Jie Kurk

David Sarah Cassandra

ABSTRACT
Nitrogen (N) from urea application is not available in soils due to the fast release
and loss through ammonia volatilization or nitrate leaching. Nitrogen could be
retained in soil using organic amendment such as frass. The objectives of this study
were to: (i) produce frass by rearing black soldier fly larvae (BSLF) with decanter
cake (DC), (ii) determine the physico-chemical properties of frass produced from
BSFL fed on DC, and (iii) determine urea-N retention in soil following BSFL frass
application. The frass production was carried out by rearing BSFL using DC with the
feeding rate of 200 mg larvae-1 day-1. The maturity of the BSFL frass was determined
through physical, biological (phytotoxicity test), and chemical tests. A leaching
study was carried out for 30 days by incorporating different rates of BSFL frass and
urea. At 45 days of rearing, BSFL frass was produced whereby the insect larvae turn
into pupae. The BSFL frass has good agronomic qualities which appropriate to be
Services for Science and Education – United Kingdom

European Journal of Applied Sciences (EJAS) Vol. 10, Issue 4, August-2022

used as organic fertilizer. The BSFL frass were not toxic to white radish germination
as reflected by the germination index ranged from 85% to 97%. Incorporation of
BSFL frass with urea improved retention of N in the form of exchangeable NH4+ and
available NO3- because of high TOC and TOM which related to the organic N
decomposition into inorganic N. Combined application of BSFL frass and urea
particularly reduced by half of recommended urea suggests that urea-N application
can be properly improved if it is amended with BSFL frass.

Keywords: Ammonium; Insect larvae; Frass; Decomposition; Insect larvae; Nitrogen
retention

INTRODUCTION
Nitrogen (N) availability in soils derived from two major sources which are organic N and
inorganic N. The prominent plant-available N in the soil is the inorganic N form (ammonium
and nitrate) that mineralized from organic N. The insufficient N for crops uptake is due to
ammonium (NH4+) loss via ammonia (NH3) volatilization particularly without proper retention
whereas nitrate (NO3-) loss through leaching and did not retain in the soil due to its nature as
negatively charged ions [1]. Chemical and synthetic N fertilizers could be applied to soil to
overcome N deficiency, but they are not necessarily effective in recovering all the N needed by
the crops due to their fast release properties. Urea for example, is the common type of N
fertilizer that have been widely used in agriculture because it provides the most sources of N to
the crops, low production cost, readily available N (fast release N fertilizer), and widely used
for any type of crops. However, the excessive use of urea fertilizer did not only lead to high
production cost, but it is also polluting the environment due to loss via NH3 volatilization [2,3].
In traditional farming, organic amendments such as composts and frass have been the main
source of plant nutrients, especially N.

In this study, an attempt was made on producing frass using decanter cake (DC) as feeding
substrate for rearing black soldier fly larvae (BSFL). The black soldier fly (BSF) scientifically
known as Hermetia illucens L. considered as the bio-waste recyclers within the insect category
which able to transform various types of organic waste into biomass that could be used as bio
fertilizer due to high content of plant nutrients [4,5]. Frass is the excreta of BSFL, indigestible
material, metabolic products, and other parts from the other BSF stages of development [6].
The plant nutrients in the BSFL frass such as N, phosphorus (P), and potassium (K) at levels of
approximating 5-2-2, respectively could be used as organic fertilizer [6]. In addition, BSFL frass
also have properties that suppressed plant pathogens [7,8]. Our approach on frass production
is one of the ways to manage wastes generated from the processing of palm oil such as DC.

In palm oil production, approximately 4 to 5 tons of DC has been generated from every 100 tons
of palm fresh fruit bunch processed. It is important to adopt proper and safe methods to manage
the DC and turning them into valuable product that could be reusable. Most of animal and plant-
based wastes are managed through composting and used with chemical fertilizer in food crop
production. Owing to the high carbon and N contents in organic wastes, additives or
decomposer agents are introduced in feedstock (wastes either from animal or plant sources) to
fasten the decomposition and to ascertain the maturity and stability of the composts [1]. The
additives used in the composting are include molasses, zeolites, chicken feed, liming materials,
Services for Science and Education – United Kingdom 396

Visvini, L., Latifah, O., Ahmed, O. H., Kurk, W. J., & Cassandra, D. S. (2022). Production and Incorporation of Black Soldier Fly Larvae Frass in Retaining
Nitrogen from Urea. European Journal of Applied Sciences, 10(4). 395-412.

or animal manures. The common decomposer agents are wide range of microbes such as
effective microorganisms including insect such as worms, grubs and BSFL [1].

The BSFL frass has high degradation efficiency, rich in macronutrients and micronutrients, and
also organic matter (OM) that improved soil fertility and food crops productivity [9,10]. The
addition of BSFL frass to the soil provide beneficial effects on the plant growth and development
[11]. However, the quality of BSFL frass depends on the nature of the organic wastes consumed
by the BSFL and also the stabilization during the process of degradation which involved
biological, physical, and chemical factors. It is stated that low nutritional quality of organic
wastes could lead to low content of plant nutrients in frass [12,13]. The study of insect frass for
reducing the N loss from urea have been reported [9] which confirmed the retention of N due
to high levels of organic carbon which bound organically with NH4+-N.

It is hypothesized that managing DC using BSFL as decomposer agent could produce high
quality frass that could be incorporated with urea to improve N retention in soil. However, the
use of BSFL frass incorporated with urea for better N retention in soil is scarcely explored.
Research questions that need to be explored in this study are: (i) what is the physico-chemical
properties of frass produced by BSFL after being fed on DC? (ii) does frass produced from
rearing BSFL with DC safe to be used and have no toxic effects on plant? and (iii) does BSFL
frass has ability to retain N from urea application?

Owing to urea and BSFL frass are both sources of N, the adjustment in the rates of urea and
BSFL frass to complement each other were tested for the optimum rates and compared against
recommended urea that mostly used in crop production for example in vegetable production.
This study was carried out to determine: (i) selected physico-chemical properties of the frass
produced from BSFL after being fed on DC, (ii) the effects of different rates of BSFL frass in
retaining N in the form of NH4+ from different rates of urea and (ii) optimum rate of urea
amended with BSFL frass for NH4+ retention. The implications of this study could provide an
alternative approach of managing agro-industrial wastes such as DC using BSFL as decomposer
agent, develop organic fertilizer (frass), and confirm the benefits of BSFL frass in improving N
retention from urea application.

MATERIALS AND METHODS
Rearing black soldier fly larvae for frass production
The eggs of the BSF and the main rearing substrates which is DC were obtained from the BSF
Farming and Trading Bintulu, Sarawak, Malaysia. A 13 gram of BSF eggs were laid in the plastic
tray size of 65 cm length x 37 cm width x 15 cm depth and hatched after four days of the
incubation. Black soldier fly juvenile larvae were fed on the starter diet consisted of sago starch,
rice, bread crumble, and fruits such as banana and apple (leftover food). After the BSFL fed on
starter diet for two weeks, 3.1 kg of DC were placed in another plastic tray and the rearing of
the BSFL continued to feed on DC for another 31 days. During the rearing progress, the feeding
substrate (DC) was sprinkled with 20 mL of water to maintain moisture content and to
ascertain the humidity of the substrate. On the day 45, the DC was fully digested by the BSFL
whereby the substrate showed crumble in texture and the BSFL turn into pre-pupa and some
of them turn into pupa. The separation of the pupa from the frass was carried out using 2 mm
sieve and further analyzed for physico-chemical properties and phytotoxicity test. pH, electrical

URL: http://dx.doi.org/10.14738/aivp.104.12220 397


conductivity (EC), ammonium/nitrate(NH4+/NO3-) ratio, total organic matter (TOM), total
organic carbon (TOC) N, and C/N ratio were characterized using the standard procedures as
described by [14].

Selected physico-chemical analyses of black soldier fly larvae frass
pH of BSFL frass was extracted using 1 M potassium chloride (KCl) and distilled water at ratio
of 1:2.5 of sample and the extractant. A 10 g of air-dried frass was weighed in a plastic vial and
25 mL of distilled water was added to determine soil pH in water whereas 25 mL of 1 M KCl
was added to determine pH in KCl. Thereafter, the suspension was shaken using an orbital
shaker at 180 rpm for 15 minutes. The pH of mixture with distilled water was determined after
24 hours of equilibration whereas soil pH of mixture with KCl was determined directly after
shaken. The pH was determined using a digital pH meter.

Total organic matter (OM) and total organic carbon (TOC) were determined using loss on
ignition method [17]. Air-dried samples were placed in an oven and dried for 24 hours at 60°C.
Afterwards, the samples were left to cool in a desiccator. The initial weight of empty porcelain
dish (crucible) was taken. Then, 5 g of sample was added into the crucible. The sample was
placed in a muffle furnace and initially ashed at 300°C for 1 hour. The temperature was raised
to 550°C and the ashing was continued for 8 hours. The weight of sample and crucible were
taken after ashing. The OM and TOC were calculated using formula below:
!"#$#%& ()#*+$ ,- .%/0&) (*)34#"%& ()#*+$ ,- .%/0&) (*)
OM= !"#$#%& ()#*+$ ,- .%/0&) (*)
X 100% (1)
!"#$#%& ()#*+$ ,- .%/0&) (*)34#"%& ()#*+$ ,- .%/0&) (*)
TOC= !"#$#%& ()#*+$ ,- .%/0&) (*)
X 100% × 0.58 (2)

The inorganic N (NH4+ and NO3-) was determined following the method of Keeney and Nelson
[18]. Exchangeable NH4+ and available NO3- were extracted by shaking the sample with 2M of
potassium chloride (KCl) at a ratio of 1:10 for 1 hour using a reciprocal shaker. The NH4+ ion
was determined using 20 mL aliquot and 0.2 g of magnesium oxide (MgO) after which
distillation was run for 5 minutes and the NH4+ was collected in 10ml of 2% boric acid- indicator
solution. Afterwards, 0.2 g of Devarda’s alloy was added to the solution in distillation apparatus
after which distillation was run for another 5 minutes to determine NO3-. The NO3- was also
collected in 10ml of 2% boric acid-indicator solution after which the NH4+ and NO3- trapped in
the boric acid were estimated by titrating with 0.01M HCl. The concentration of NH4+ and NO3-
were calculated using the equation below: 17
(5 6 7 6 89.;8)
% NH4+ or NO3- = (5< 6 8;;;) X 100 (3)

Kjeldhal method was used to determine the total N by digestion and distillation [16]. A 0.5 g of
sample (sieved to pass 0.5mm) was weighed into 50ml Kjeldhal digestion tube. The sample was
wet with five drops of water and 10 mL concentrated H2SO4 added. A tablet of Kjeldhal catalyst
was added and the sample were shaken to equilibrate for 30 minutes. The samples heated in a
digestion block for 1 hour (180°C) and then increased to 320°C for 4 to 5 hours until the
samples turn colourless. The samples were cooled down, then 30 mL of distilled water was
added. A 10 mL of the samples were pipetted into distillation flask and added with 10 mL of
40% NaOH. The distillate is then distilled and collected in 10 mL of 2% boric acid-indicator
solution when the colour changed from purple to green. The distillate titrated against 0.01M


HCl or 0.01M H2SO4 until the colour changes from green to purple. The percentage of N in
sample was calculated using the formula given:
(53=)6 7 6 > 6 89.;8)
% N= ?$ 6 8;;;)
X 100 (4)

Phytotoxicity test
The plant bioassays method was used for phytotoxicity test which was determined under
controlled conditions [19,20]. The BSFL frass extracts were prepared by diluting 5 g of frass
with 50 mL of distilled water (1:10, w/v) then the mixtures were shaken using orbital shaker
for 1 hour at 180 rpm. The samples were then filtered through Whatman filter paper no. 2 and
diluted according to dilution factor of 1, 10, 100 and 1000, respectively. A 5 mL of each dilution
factor (1x, 10x, 100x, 1000x) sample was extracted and placed in petri dish. The Raphanus
sativus, white radish seeds were used and placed on filter papers which moistened with 5 mL
frass extracts in petri dishes for 72 hours at 25℃. Then the similar procedure was repeated for
the other frass extracts. The germination of white radish was carried out in distilled water
which serves as the positive control that used for the comparison against BSLF frass
extractants. After 3 days, the germinated seeds were recorded, and their radicle or root lengths
were determined. The germination index (GI) calculated using the equation below.
>@A% >>A%)
GI = 8;;
(5)

The RSG (%) is the relative seed germination whereas RRG (%) is the relative root growth. Both
can be calculated as formula below.
"C/D)E ,- .))<. *)E/#"%$)< #" -E%.. )6$E%F$.
RSG = "C/D)E ,- .))<. *)E/#"%$)< #" F,"$E,& X 100 (6)
/)%" E,,$ &)"*$+ #" -E%.. )6$E%F$
RRG = /)%" E,,$ &)"*$+ #" F,"$E,&
X 100 (7)

After the final GI values calculated, if seeds germinated in the frass extracts below than 50%, it
is considered as highly phytotoxic, germination under 50% and 80% are moderately
phytotoxic, and if the GI value more than 80%, the frass considered as not toxic to plant [21,22].

Soil sampling, preparation, and initial physico-chemical characterization
The soil used for the leaching experiment was sampled at depth of 0 – 20 cm from an
uncultivated area in Universiti Putra Malaysia Bintulu Sarawak Campus (UPMKB), Sarawak,
Malaysia. The soil was air-dried for seven days, crushed using a soil mortar and a pestle, after
which sieved to pass a 2 mm sieve for a laboratory analysis and the leaching study. Selected
physical and chemical characteristics of soil such as pH, electrical conductivity (EC), total OM,
OC, N, exchangeable NH4+, available NO3- soil cation exchange capacity (CEC), available P and
exchangeable cations were analyzed using standard procedure as described by Tan [14].

Leaching study
Leaching experiments was carried out for 30 days in the Soil Science Laboratory of Universiti
Putra Malaysia Bintulu, Sarawak Campus, Malaysia. The treatments evaluated in this
experiment were: (i) T1 (300 g soil only), (ii) T2 (300 g soil + 4 g urea), (iii) T3 (300 g soil + 40
g BSFL frass), (iv) T4 (300 g soil + 3 g urea + 30 g BSFL frass), (v) T5 (300 g soil + 2 g urea + 20
g BSFL frass). The rates of the urea and BSFL frass used in this study were based on standard



fertilizers’ recommendation for white radish cultivation. However, the amount of urea applied
in T4 and T5 were reduced by 25 and 50% of the standard recommendation for white radish,
whereby complemented by the addition of 30 g and 40 g BSFL frass based on standard
recommendation of 10 and 5 t ha-1 of BSFL frass, respectively. The mixture of treatment was
filled in leaching tubes and leached with distilled water after which the leachates were collected
at three days interval based on a five-year rainfall. The leaching experiment was carried out for
30 days at 26 °C. Each treatment had 3 replications and at 30 days the treatment samples were
air-dried and analyzed for pH, total N, NH4+ and NO3- content.

Experimental design and statistical analysis
Leaching experiment was arranged in a completely randomized design (CRD) with three
replications. All of the analyses in this study were carried out in triplicates for each analysis to
obtain accurate and valid data. The significant means were separated using Tukey’s test at p<
0.05. The Statistical Analysis Software (SAS version 9.2) was used for all of the statistical
analysis.

RESULTS
Rearing black soldier fly larvae for frass production
The average of ambient temperature of the rearing room for BSFL frass production was 26 ℃
whereas the average of daily temperature of the rearing substrate (DC) was 30 °C throughout
45 days (Figure 1). Both of the temperatures are within the optimum temperature that facilitate
the efficiency of the BSFL to feed on the DC and excreted them as frass from day 35 to 45 of
rearing days (Figure 1).

Ambient temperature Substrate temperature
35
30
Temperature °C
25
20
15
10
5
0
1 5 10 15 20 25 30 35 40 45
Days of rearing black soldier fly larvae

Figure 1. Temperature profiles of substrate during rearing black soldier fly larvae

Selected physico-chemical analysis of black soldier fly larvae frass
The BSF egg hatched into juvenile larvae after four days of incubation and the first instar of the
BSFL were fed with starter diet. At 14 days, second instar of the BSFL were fed with the main
diet which was DC. The DC was moistened with water as the growth of the BSFL observed on
day 15 to 35 of rearing day. Selected physico-chemical properties of the BSFL frass presented
in Table 1 showed a good pH of the BSFL frass for agronomic use. Electrical conductivity, total
OM, OC, N, NH4+, NO3-, available P, exchangeable K, NH4+/NO3- ratio, and C/N ratio of the BSFL


frass are in the range of a good quality of organic amendment that commonly use as growing
media (Table 1).

Table 1. Selected physico-chemical properties of black soldier fly larvae frass
Property Value obtained
pH 6.85
Electrical conductivity µS cm-1 95.70
Total organic matter (%) 45.10
Total carbon (%) 26.10
Total nitrogen 1.64
Ammonium (ppm) 1.92
Nitrate (ppm) 1.76
Ammonium/nitrate ratio 1
Available phosphorus (%) 0.52
Exchangeable potassium (%) 2.97
C/N ratio
16
Note. All analysis is based on dry weight

Phytotoxicity test
Germination index (GI) of the white radish in distilled water (T0: control) and frass extractants
(T1, T2, T3, and T4) were not significantly different and all of those root length, relative root
growth, and relative shoot growth of white radish were greater than 80% suggested that the
BSFL frass had no toxic effects to white radish seeds germination (Table 2).

Table 2. Plant bioassay test for different extractants of a frass as compared with distilled water
Treatment Media Root Length RRG (%) RSG (%) GI (%)
(cm)
T0 Distilled water 5.95 ± 0.2 100 100 100
T1 Frass extractant (1x) 5.50 ± 0.67 91.97 96.33 40.42± 27.19
T2 Frass extractant (10x 5.70 ± 0.86 95.89 100 95.89 ± 2.05

T3 Frass extractant 5.78 ± 0.87 97.18 100 97.18 ± 6.97
(100x)
T4 Frass extractant 5.70 ± 0.09 95.90 88.89 85.25 ± 3.51
(1000x)

Leaching study
Exchangeable ammonium in leachate over thirty days of leaching
There was no NH4+ leached in T1 throughout the leaching period of 30 days, whereas T2 which
consist of urea only showed the highest amount of NH4+ leached particularly at day 6 of leaching
study compared with those of soil incorporated with BSFL frass (T3, T4, and T5) (Figure 2). Use
of BSFL frass alone in T3 showed gradual leached of NH4+ from day 3 to day 15, then the amount
of NH4+ leached was not detectable at day 24 to day 30 (Figure 2). The reduction of urea by 25
and 50% in T4 and T5, respectively showed lower NH4+ leached compared with urea without
incorporation with BSFL frass (Figure 2).



1600.00
T1 T2 T3 T4 T5
Exchangeable NH4+ Loss (mg kg-1)

1400.00
1200.00
1000.00
800.00
600.00
400.00
200.00
0.00
3 6 9 12 15 18 21 24 27 30
Days of leaching

Figure 2 Amount of ammonium leached at three days interval over thirty days of leaching. T1
(300g soil only), T2 (300g soil + 4g urea), T3 (300g soil + 40g BSFL frass), T4 (300g soil + 3g
urea + 30g BSFL frass) and T5 (300g soil + 2g urea + 20g BSFL frass)

Cumulative losses of exchangeable NH4+ over 30 days of leaching presented in Figure 3 showed
the highest amount of NH4+ leached in urea only without any addition of BSFL frass compared
with soil incorporated with BSFL frass.

3500.00 a
Exchangeable NH4+ Loss (mg kg-1)
3000.00 b
2500.00 c
2000.00
1500.00
1000.00
d
500.00
e
0.00
T1 T2 T3 T4 T5
Treatment

Figure 3 Cumulative amount of exchangeable ammonium loss at thirty days of leaching. T1
(300g soil only), T2 (300g soil + 4g urea), T3 (300g soil + 40g BSFL frass), T4 (300g soil + 3g
urea + 30g BSFL frass) and T5 (300g soil + 2g urea + 20g BSFL frass). Means between columns
with different letter(s) indicate significant difference between treatments by Tukey’s test at
p<0.05.

Available nitrate in leachate over thirty days of leaching
In accordance with NH4+, the amount of NO3- leached in urea alone (T2) at three days interval
and cumulative loss of NO3- for 30 days of leaching was statistically higher than those of NO3-
leached in soil incorporated with the BSFL frass (T3, T4, and T5) (Figures 4 and 5). Both NH4+


and NO3- leached at the peak of day 6 to day 9 after which decreased in their amount at day 12
to day 30 (Figures 3 and 4).

1600.00
T1 T2 T3 T4 T5
1400.00
1200.00
Available NO3- (mg kg-1)
1000.00
800.00
600.00
400.00
200.00
0.00
3 6 9 12 15 18 21 24 27 30
Days of leaching

Figure 4 Amount of nitrate leached at three days interval over thirty days of leaching. T1 (300g
soil only), T2 (300g soil + 4g urea), T3 (300g soil + 40g BSFL frass), T4 (300g soil + 3g urea +
30g BSFL frass) and T5 (300g soil + 2g urea + 20g BSFL frass)

3500.00
a
3000.00
b
2500.00
2000.00 c
1500.00
1000.00
d
500.00
e
0.00
T1 T2 T3 T4 T5
Treatment

Figure 5 Cumulative amounts of available nitrate at thirty days of leaching. T1 (300g soil only),
T2 (300g soil + 4g urea), T3 (300g soil + 40g BSFL frass), T4 (300g soil + 3g urea + 30g BSFL
frass) and T5 (300g soil + 2g urea + 20g BSFL frass). Means between columns with different
letter(s) indicate significant difference between treatments by Tukey’s test at p<0.05.



pH of Leachate over Thirty Days of Leaching
At day 3 to day 6, all leachates from 5 treatments have high pH except for soil alone (T1) (Table
3). pH of leachate in urea alone (T2) was decrease from alkaline (pH 9) at day 3 and 6 to acidic
(pH 5 and below 5) from day 15 to day 30 of leaching study (Table 3). Throughout the leaching
study, pH of leachate in urea incorporated with BSFL frass were remained in alkaline nature
(Table 3).

Table 3. pH of Leachate over Thirty Days of Leaching
pH T1 T2 T3 T4 T5
D3 6.19b 9.18a 7.41b 9.16a 9.15a
D6 5.22c 9.06a 7.84b 8.98a 8.91ab
D9 4.71c 7.96b 8.75ab 8.87a 8.82a
D12 5.55b 6.25b 8.90a 8.77a 8.76a
D15 5.80b 5.01b 8.84a 8.63a 8.74a
D18 5.92b 4.9c 8.56a 8.78a 8.83a
D21 5.90c 4.76d 7.71b 8.94a 8.89a
D24 5.60c 4.94c 7.53b 8.92a 8.70a
D27 5.72c 5.12c 7.40b 8.99a 8.74a
D30 5.75c 5.22c 7.31b 8.90a 8.61ab
*Means with different letters within a row indicate significantly difference at p<0.05. T1 (300g
soil only), T2 (300g soil + 4g urea), T3 (300g soil + 40g BSFL frass), T4 (300g soil + 3g urea +
30g BSFL frass) and T5 (300g soil + 2g urea + 20g BSFL frass)

Selected Soil Physico-chemical Properties at Thirty Days of Leaching Study
Soil pH in water and potassium chloride
Soil pH after 30 days of leaching on the treatments amended with BSFL frass in T3, T4 and T5
were significantly higher than in the treatment without BSFL frass (T2) as shown in Figure 6.
Higher soil pH in soil after 30 days of leaching study is in line with the higher of pH in leachate
of all treatments with the inclusion of BSFL frass (Table 3 and Figure 6).



Water KCl
8 a
ab
7 c bc
a
6 b
b
d
5
c
Soil pH
4 d
0
T1 T2 T3 T4 T5
Treatment

Figure 6 Soil pH in water and potassium chloride at thirty days of leaching. T1 (300g soil only),
T2 (300g soil + 4g urea), T3 (300g soil + 40g BSFL frass), T4 (300g soil + 3g urea + 30g BSFL
frass) and T5 (300g soil + 2g urea + 20g BSFL frass). Means between columns with different
letter(s) indicate significant difference between treatments by Tukey’s test at p<0.05. Bars
represents the mean values ± SE

Total organic matter and total organic carbon in soil at thirty days of leaching
The inclusion of BSFL frass significantly affected total of carbon and organic matter in soil as
exhibited in T3, T4, and T5 compared with urea alone (Figures 7 and 8). As shown in Figure
10(a) and Figure 10(b), the soil organic matter and total organic carbon of treatments amended
with BSFL frass (T3, T4 and T5) were significantly higher compared with the treatment without
BSFL frass (T1 and T2).

9.00
8.00 a
7.00 b
Organic matter (%)
6.00 c
5.00
4.00 d d
3.00
2.00
1.00
0.00
T1 T2 T3 T4 T4
Treatment

Figure 7 Total organic matter of soil at thirty days of leaching. T1 (300g soil only), T2 (300g soil
+ 4g urea), T3 (300g soil + 40g BSFL frass), T4 (300g soil + 3g urea + 30g BSFL frass) and T5
(300g soil + 2g urea + 20g BSFL frass). Means between columns with different letter(s) indicate
significant difference between treatments by Tukey’s test at p<0.05. Bars represents the mean
values ± SE



5.00
a
4.50
Total Organic Carbon (%)

4.00 b
3.50
c
3.00
2.50
2.00 d d
1.50
1.00
0.50
0.00
T1 T2 T3 T4 T5
Treatment

Figure 8 Total organic carbon of soil at thirty days of leaching. T1 (300g soil only), T2 (300g soil
+ 4g urea), T3 (300g soil + 40g BSFL frass), T4 (300g soil + 3g urea + 30g BSFL frass) and T5
(300g soil + 2g urea + 20g BSFL frass). Means between columns with different letter(s) indicate
significant difference between treatments by Tukey’s test at p<0.05. Bars represents the mean
values ± SE

Soil exchangeable ammonium, available nitrate, and total nitrogen in soil at thirty days of
leaching
The amount of soil exchangeable NH4+, available NO3-, and total N retained in T4 was
significantly higher compared with other treatments (Figures 9 and 10).

250 a
Exchangeable NH4+ and
200 b
150
a
100 b
cd c
50 c c c
d
0
T1 T2 T3 T4 T5
Treatment
Exchangeable Ammonium Available Nitrate

Figure 9 Soil exchangeable ammonium and available nitrate from different treatment as T1
(300 g soil only), T2 (300 g soil + 4 g urea), T3 (300 g soil + 40 g BSFL frass), T4 (300 g soil + 3g
urea + 30 g BSFL frass) and T5 (300 g soil + 2g urea + 20 g BSFL frass) at 30 days of leaching.
Means between columns with different letter(s) indicate significant difference between
treatments by Tukey’s test at p<0.05. Bars represents the mean values ± SE



0.700
a
Total Nitrogen (%) 0.600
0.500
0.400
b
0.300 bc
c
0.200
0.100
d
0.000
T1 T2 T3 T4 T5
Treatment

Figure 10 Soil total N from different treatment as T1 (300g soil only), T2 (300g soil + 4g urea),
T3 (300g soil + 40g BSFL frass), T4 (300g soil + 3g urea + 30g BSFL frass) and T5 (300g soil + 2g
urea + 20g BSFL frass) at 30 days of leaching. Means between columns with different letter(s)
indicate significant difference between treatments by Tukey’s test at p<0.05. Bars represents
the mean value

DISCUSSIONS
Frass Production from Black Soldier Fly Larvae After Being Fed on Decanter Cake
The temperature of the DC as feeding substrate for frass production in rearing BSFL was 27 ℃
and it is the optimum temperature that favour the efficiency of the BSFL to digest DC [23]. The
slight fluctuation of the temperature as shown in Figure 1 is due to the difference of
temperature between day and night. Towards the end-of-life cycle in larvae phase, the
temperature of feeding substrate decreased and almost similar to ambient temperature (Figure
1). At day 39, the similar in temperature of ambient and frass indicated that the frass is
approaching a stable and mature state. The colour of the BSFL frass was darken from day 15 of
rearing BSFL to day 39 of the stabilization phase which was determined using Munsell color
chart. The dark colour of the BSFL frass after 39 suggested the appropriate amount of aeration
throughout the digestion of DC and the decomposition progress [24]. In addition, the frass
excreted by the BSFL at 39 days of digestion does not emits unpleasant odors which odourless
mixture is one of the maturity indicators [25]. Instead of unpleasant odor, the BSFL frass was
odourless whereby the odour is similar to earthy smells and inoffensive.

Selected Physico-chemical Characteristics of Black Soldier Fly Larvae Frass
The low pH of BSFL frass (6.85) after being fed on DC cake in Table 1 showed slightly acidic,
however the pH is in the range of suitable pH for most of plant growth. Total OM, TOC, and total
N content of the BSFL frass organic matter are common for organic amendment used as soil
conditioner [1]. The higher content of NH4+, NO3-, and exchangeable K, and available P are
suitable for growing media and improve soil fertility and crop productivity. High amount of
essential nutrient is important to improve low fertile soils such as Bekenu Series soil, thus the
addition of organic matter from BSFL frass could replenish nutrients in low fertile soil to favour
photosynthesis and other metabolic processes of plants because plant productivity will be



limited due to insufficient in essential elements [33]. Organic matter increases availability of
plant nutrients via CEC due to decomposition process, thus increasing the ability of soil to retain
Ca, K, Mg, and NH4+ [34].

Phytotoxicity test
Success germination of white radish (test crop) which reflected by insignificant differences in
root length, relative root growth (RRG) and relative shoot germination (RSG) in the BSFL frass
extractants (T1, T2, T3, and T4) and distilled water (T0: control) corroborated that no toxic
effects of the BSFL frass to white raddish germination regardless of dilution time (Table 2). The
GI values of distilled water and all BSFL frass extractants were above 80% and the findings are
in accordance with previous studies which justified that the GI values between 80% to 100%
proved as free from plant toxicity [35]. The T1, T2, T3, and T4 were safe to be used as growing
media or soil amendment because they undergone dilution series that lead to lower EC and
attributed to the successful radish seed germination which ranged from 85.25% to 97.18%
(Table 2).

Leaching of Exchangeable Ammonium and Available Nitrate in Leachate
The higher leaching of NH4+ and NO3- in T2 (urea only) compared with all soils added with the
BSFL frass (T3, T4, and T5) were because urea hydrolyzed on day 6 and then became lesser
until the end of leaching experiment (Figures 2-5). Lesser N in soil with urea only indicates that
the N from urea was not retain in the soil without the addition of BSFL frass when it was
hydrolyzed to released NH4+ and eventually to NO3- and those forms of N leached into the
leachate (Figures 2-5). The addition of BSFL frass in T3, T4, and T5 help to fix N in the form of
NH4+ because of the organic matter content of the BSFL frass provide negatively surface charge
for soil to retain NH4+. In addition, leaching of NH4+ can be reduced due to the affinity of
functional group such as phenolic and carboxyl group in wide range of organic amendment such
as frass and compost [31].

Organic matter in BSFL frass affects the lower leaching of NH4+ and NO3- in soil incorporated
with BSFL frass because organic matter serves as storage for the stabilization of N. Therefore,
the availability of N in soil with BSFL frass was affected by the decomposition of organic N from
organic matter which gradually released inorganic N in the form of soil exchangeable NH4+ and
available NO3- [31]. This is true as exhibited by the inclusion of the BSFL frass in T3, T4 and T5.
Despite of the lower loss of exchangeable NH4+ and available NO3- in T3 (300g soil + 40g BSFL
frass) the availability of N in T3 was significantly lower compared with T4 and T5 indicated that
balance of N availability should incorporate urea and BSFL frass as source of N but at optimize
amount which is reduced by 25 and 50% from the 100% of urea applied in vegetable
production (Figures 2-5).

pH of Leachate at Three Days Interval over Thirty Days of Leaching Study
pH in T2 at day 6 was higher because urea has been hydrolyzed to release NH4+, OH- and CO32-
thus the concentration of these cations increased the pH in leachate. However, pH of T2 was
decreased after day 6 until day 30 due to the leaching of base cations from the soil. The lower
pH in urea alone towards the end of leaching study was related to the formation of dissolved
NH3 and the released of H+ from NH4+ that attribute to low pH of leachate [31].



Soil pH in Water and Potassium Chloride after Thirty Days of Leaching Study
Soil pH in treatment with BSFL frass (T3, T4 and T5) were higher than in T2 (urea only) because
of the ability of the frass to rise soil pH values from 8.0 to 8.5 [36]. The humic substances which
are naturally produced organic matter from the transformations of organic debris by microbial
and chemical activities in the soil during the decomposition of the organic matter also part of
the reasons for high in soil pH. The increased of soil pH in urea only on day 6 during leaching
study could also be attributed to the inherent content of N of the urea. Dissociation of NH4+ to
NH3 gases would increase in pH and shifting the equilibrium to NH3 which eventually
evaporates [37].

Organic Matter and Total Organic Carbon of Soil at Thirty Days of Leaching
Total OC and OM in soil with BSFL frass were higher than in urea alone were related to the
higher inherent contents of TOC and TOM in BSFL frass. Most of the organic amendments
including BSFL frass have high water holding capacity. Bekenu series soil used in the leaching
study have many spaces within the pores which could filled by the particles of BSFL frass and
hold the water in those spaces, hence, the water holding capacity also favour the decomposition
of organic and carbon which reflected by the higher amount of TOC and TOM in all soils with
incorporation of BSFL frass (Figures 7 and 8).

Soil Exchangeable Ammonium, Available Nitrate, and Total Nitrogen after Thirty Days of
Leaching
Higher concentration of soil exchangeable NH4+, available NO3-, and total N in urea with BSFL
frass particularly in T4 (reduction of urea by 50% and complemented by BSFL frass) among all
treatments at the end of leaching experiment suggested that the combined use of urea and BSFL
frass was optimized at the rate of urea reduced by half and BSFL frass applied at 10 t ha-1
(Figures 9 and 10). In addition, the decomposition of BSFL frass in T4 could affected the
concentration of NH4+ and NO3- in the soil because the decomposition is stimulated by high N
contents [42-44]. The higher content of NH4+ in T4 among all treatments in this study suggests
that the inclusion of BSFL frass improved NH4+ retention in soil as well as minimizing the
conversion of NH4+ to NO3-. In line with the higher retention of NH4+ and NO3- in all treatments
with BSFL frass, higher total N particularly in T4 suggested that the application of urea with
BSFL frass affect the availability of total N in soils.

CONCLUSIONS
The colour of the BSFL frass produced from BSFL fed on DC was dark and odorless with earthy
and inoffensive smell. The frass excreted by BSFL consists significant amount of N, available P,
exchangeable K, TOC, TOM, appropriate levels of pH, EC, NH4+/NO3- ratio, and C/N ratio which
are suitable to be used as growing media or soil organic amendment. Regardless of dilution
time, the BSFL frass extractants were not toxic as reflected by successful white radish
germination at GI ranged from 85% to 97%. The incorporation of BSFL frass able to improve
retention of N in the form of plant uptake N which is exchangeable NH4+ and available NO3-
because of high TOC and TOM which related to the decomposition of organic N into inorganic
N. Combined application of BSFL frass and urea particularly at reduction by half of
recommended urea suggest that application of urea in soil can be properly managed if it is
amended with BSFL frass to improve the N availability. However, pot and field trials are
essential to explore the ability of BSFL frass to retain N from urea.



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Production and Incorporation of Black Soldier Fly Larvae Frass in Retaining Nitrogen From Urea

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European

Journal of Applied Sciences – Vol. 10, No. 4

Production and Incorporation of Black Soldier Fly Larvae Frass in

Services for Science and Education – United Kingdom 396

Services for Science and Education – United Kingdom 398

Services for Science and Education – United Kingdom 400

T1 Frass extractant (1x) 5.50 ± 0.67 91.97 96.33 40.42± 27.19

T2 Frass extractant (10x 5.70 ± 0.86 95.89 100 95.89 ± 2.05

Exchangeable NH4+ Loss (mg kg-1)

Services for Science and Education – United Kingdom 402

Services for Science and Education – United Kingdom 404

Total Organic Carbon (%)

Services for Science and Education – United Kingdom 406

Services for Science and Education – United Kingdom 412

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