Waste Management 130 (2021) 155–166

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Waste Management
journal homepage: www.elsevier.com/locate/wasman

Applications of food waste-derived black soldier fly larval frass as

incorporated compost, side-dress fertilizer and frass-tea drench for
soilless cultivation of leafy vegetables in biochar-based growing media
Jonathan Koon Ngee Tan a, Jonathan Tian En Lee b, Zhongyu Chiam a,⇑, Shuang Song a, Srishti Arora b,
Yen Wah Tong c, Hugh Tiang Wah Tan a
a
Department of Biological Sciences, National University of Singapore, 16 Science Drive 4, 117558, Singapore
b
Environmental Research Institute, National University of Singapore, 1 Create Way, 138602, Singapore
c
Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4, 117585, Singapore

a r t i c l e i n f o a b s t r a c t

Article history: Black soldier fly (BSF) larval bioconversion can recycle nutrients in organic wastes into larval biomass and
Received 25 August 2020 frass. While the frass has been commonly marketed as a soil amendment, its usefulness in soilless culti-
Revised 16 March 2021 vation remains largely unexplored. Growth experiments were conducted to investigate the effectiveness
Accepted 20 May 2021
of surplus food-derived and okara-derived BSF larval frass as an incorporated compost, side-dress fertil-
izer and frass-tea drench for the cultivation of pak choi and lettuce in waste-wood derived biochar grow-
ing media. Pak choi yields from treatments with surplus food-derived frass and biochar at a 10:90 (v/v)
Keywords:
ratio and inorganic fertilizer were comparable to those of the control which consisted of soil, peat-based
Waste valorization
Food waste
compost and inorganic fertilizer. However, yields decreased with increasing frass incorporation rates
Black soldier fly owing to high salinity and potentially low oxygen conditions in the growing media. When used as a fer-
Sustainable agriculture tilizer on biochar-coir growing media, the direct application of frass as a side-dress fertilizer was 1.6–6.8
Soilless cultivation times more effective in promoting lettuce growth than the application as a frass-tea drench. Frass fertil-
izers derived from surplus food outperformed those derived from okara by 1.3–5.3 times. Lettuce yields
were not significantly different between the treatment with surplus food-derived frass applied as a side-
dress fertilizer and the control of liquid inorganic fertilizer. Variations in fertilizing potential were attrib-
uted to nutrient availability and the presence of plant growth promoting microbes in the growing media.
BSF larval frass derived from food waste shows promise in partially replacing unsustainable agricultural
inputs for leafy vegetable cultivation, including soil and inorganic fertilizers.
Ó 2021 Elsevier Ltd. All rights reserved.

1. Introduction et al., 2016). BSF larvae is known to be valuable as an animal feed

One way of managing wet organic wastes, including food waste,
human excreta and decaying organic materials of plant or animal
origins, is bioconversion using the larvae of Hermetia illucens L.,
commonly known as black soldier fly (BSF) (Diener et al., 2011;
Nguyen et al., 2015). The key advantages of BSF bioconversion
are its ability to accept diverse types of organic wastes as inputs
and recycle nutrients from the wastes into larval biomass and frass
(da Silva and Hesselberg, 2020; Palma et al., 2019). This valorises
the wastes and diverts them from incineration and land disposal
practices which could result in air and soil pollution (Kiyasudeen
⇑ Corresponding author.
E-mail addresses: dbstknj@nus.edu.sg (J.K.N. Tan), eriltej@nus.edu.sg (J.T.E. Lee),
chiamzy@u.nus.edu (Z. Chiam), e0223230@u.nus.edu (S. Song), erisa@nus.edu.sg
(S. Arora), chetyw@nus.edu.sg (Y.W. Tong), hughtan@nus.edu.sg (H.T.W. Tan).
supplement (Barragan-Fonseca et al., 2017; Wang and Shelomi,
2017) while the frass of the larvae, which is a by-product of the
bioconversion process, is marketed and commercially sold as a soil
amendment—a compost or fertilizer (Quilliam et al., 2020; Xiao
et al., 2018). However, results from the literature regarding the
effectiveness of the larval frass as a soil amendment are mixed.
In terms of promoting plant growth, some studies reported better
or comparable performance to conventional soil amendments
including commercial chemical fertilizers and chicken manure
(Choi et al., 2009; Quilliam et al., 2020; Setti et al., 2019) whereas
others showed worse performance (Alattar et al., 2016; Rosmiati
et al., 2017). This variability amongst the studies could be owing
to differences in feedstock of the larvae, rearing process, frass
application and experiment designs (Bosch et al., 2019; Schmitt
and de Vries, 2020). As we seek to upscale certain BSF

https://doi.org/10.1016/j.wasman.2021.05.025
0956-053X/Ó 2021 Elsevier Ltd. All rights reserved.
J.K.N. Tan, J.T.E. Lee, Z. Chiam et al. Waste Management 130 (2021) 155–166

bioconversion systems and make them fully circular, it is impera- a nutrient-poor growing media comprising of biochar and coir.
tive that we valorise the BSF larval frass generated by discovering To investigate this, we conducted a lettuce (Lactuca sativa L.) bioas-
novel applications. say and chemical and microbial characterizations of the soilless
Researchers have studied the application of BSF larval frass to growing media.
soil (Alattar et al., 2016; Quilliam et al., 2020; Rosmiati et al.,
2017). However, given that existing arable land suitable for soil-
based agriculture is increasingly scarce (Cameron et al., 2015;
2. Materials and methods
United Nations, 2019) and advances in soilless cultivation could
enable more efficient use of water, fertilizer and space as well as
2.1. Production of food waste derived BSF larval frass and frass teas
control of soil-borne diseases (Raviv and Lieth, 2008), there are
environmental and economic incentives to study the applications
2.1.1. BSF larval frass
of BSF larval frass to soilless growing media. The commonly used
BSF individuals were obtained from the Evolutionary Biology
media components in soilless cultivation include peat, vermiculite
Laboratory of the National University of Singapore and came from
and perlite but rising concerns regarding the sustainability of these
a population comprising of individuals from the wild (Kent Ridge,
materials have led to the search for more environmentally friendly
Singapore) and from commercial cultures (Johor Bahru, Malaysia).
alternatives (Barrett et al., 2016). Numerous studies have sug-
The surplus food-derived frass was produced by feeding third
gested various types of biochar produced from green waste as
instar BSF larvae (7–10 days old) surplus food from residential din-
renewable alternatives to peat moss and vermiculite (Nieto et al.,
ing halls in the National University of Singapore which were desig-
2016; Ronga et al., 2019; Vaughn et al., 2015). Biochar is the solid
nated for disposal. Comprising primarily rice and noodles with
carbon-rich material produced from the pyrolysis of organic mate-
lesser amounts of leafy vegetables and meat, the surplus food
rials under anaerobic conditions. In certain formulations of soilless
was processed and homogenized using an industrial hammer mill
growing media, biochar was shown to improve nutrient availabil-
and/or a handheld cement mixer. A total of 7–8 kg of surplus food
ity, water holding capacity and stability of the media (Headlee
were fed to batches of approximately 10,000 BSF larvae over the
et al., 2014; Kaudal et al., 2016; Méndez et al., 2015). A mixture
duration of 14 days (50–57 mg/larva/day). At the end of the feeding
of BSF larval frass and commercial peat moss was shown to be a
period, the larvae were mechanically separated from the frass
viable soilless growing medium for baby leaf lettuce, basil and
using a vibrating rotary sifter with a 2 mm sieve screen. The sur-
tomato (Bortolini et al., 2020; Setti et al., 2019). This lends cre-
plus food-derived frass used in experiments 1 and 2 were produced
dence to further experiments incorporating BSF larval frass in soil-
in separate batches. Owing to heterogeneity of the surplus food
less growing media. We hypothesized that a growing medium
from the dining halls, the nutrient contents of the batches of frass
comprising of food waste-derived BSF larval frass and wood bio-
were not identical (Tables 1 and 2). The okara-derived frass was
char would have a favourable balance of nutrients from the former
produced by feeding five-day-old BSF larvae fresh okara collected
and nutrient retention, porosity and structure from the latter.
from local stores selling soybean milk and soybean curd (N&B
Compost teas are the solutions obtained from infusing com-
Snacks, Singapore). The feeding was carried out over 12 days, at
posted materials in water for a period of time (Hargreaves et al.,
a rate of 30 mg (dry weight)/larva/day. At the end of the feeding
2009). Taking inspiration from vermicompost tea, we conjectured
period, the frass was separated by sieving the larvae-frass mixture
that in addition to the direct applications of BSF larval frass as an
through a 2 mm sieve followed by manual removal of BSF larvae
incorporated compost or side-dress fertilizer, the aqueous extract
which had passed through the sieve. These forms of frass were
of the frass (frass tea) could be used as a liquid fertilizer drench.
stored in air-conditioned (23 °C) and dry conditions for two to
Vermicompost tea application to peat-perlite media has been
six weeks till they were used. For each specific feedstock, the dif-
shown to increase crop productivity, total carotenoids and glucosi-
ferent batches of frass were thoroughly mixed before they were
nolates in plant tissues as well as the biological activity in the soil-
used in the experiments.
less growing media which is associated with more efficient
mineralization of nutrients (Roy et al., 2006). These positive effects
could be contributed by the presence of mineral nitrogen, gib-
berellin and microbial by-products in the tea (Roy et al., 2006). 2.1.2. BSF larval frass teas
The liquid tea allows growers to achieve plant nutritional and For experiment 2, the frass teas were freshly made one day prior
health outcomes without changing the physical properties of the to each application. We added deionized water to the surplus food-
soilless growing media and at a lower cost than inorganic fertiliz- derived and okara-derived BSF larval frass at a ratio of 200 ml of
ers (Elekhtyar et al., 2017). However, current knowledge on the water for every 0.18 g of total nitrogen in the respective frass.
plant growth enhancing potential of BSF larval frass teas and their The quantification of total nitrogen in the frass is described in Sec-
mineral nutrients and microorganisms are lacking. tion 2.4. To these compost-water mixtures, we added molasses
We present in this paper the results of two nursery cultivation (Redman, Phoon Huat Pte. Ltd., Singapore) at a rate of 0.5% (v/v)
experiments which investigated whether BSF larval frass can be as a nutrient supplement to stimulate microbial growth
used together with biochar as novel growing medium and fertilizer (Shrestha et al., 2011). The mixtures were then brewed in batches
for sustainable agriculture (Fig. 1). The detailed research objectives of 2.5 L for 24 h and with aeration at a rate of 9 L/min before use.
are as follows. Experiment 1 was conducted to test whether sur-
plus food-derived BSF larval frass can be used effectively as a com-
post when incorporated into nutrient-poor, wood-derived biochar 2.2. Production of wood-derived biochar
at different rates, with and without inorganic fertilizer applied.
This experiment involved a pak choi (Brassica rapa L. Cultivar The biochar used was obtained commercially (GreenBack Pte.
Group Pak Choi Green-Petioled Form) bioassay, which compared Ltd., Singapore). They were produced from natural wood waste of
the frass-biochar media with the conventional soil medium, and parks, gardens and timber sawmills subjected to pyrolysis at
chemical characterizations of the experimental growing media. 650 °C for 10 to 12 h followed by grinding to sizes of 6 mm or less.
Experiment 2 aimed to evaluate whether BSF larval frass derived The ash content of the biochar was 4.3% (w/w), the volatile carbon
from surplus food or from okara are effective as an organic fertil- content was 25.6% and the fixed carbon content was 70.1% (Song
izer when applied as a side-dress fertilizer or frass-tea drench to et al., 2021).
156
J.K.N. Tan, J.T.E. Lee, Z. Chiam et al.
Fig. 1. Graphical representation of the two nursery cultivation experiments.
2.3. Experiment design
2.3.1. Germination phase for experiments 1 and 2
Two cultivation experiments were conducted at the nursery of
the National University of Singapore. For experiment 1, seeds of
pak choi (F1 Hybrid Special Pak Choy, Ban Lee Huat Seed Pte Ltd,
Singapore) were sown in in peat-based compost (Go Bio4, Jiffy
International AS, Kristiansand, Norway) and left to germinate and
grow for 14 days. On Day 14, healthy seedlings of similar sizes
were transplanted into plastic pots containing 2 L of the experi-
mental growing media (see Section 2.3.2). For experiment 2, let-
tuce seeds (Lettuce 211, Ban Lee Huat Seed Pte Ltd, Singapore)
were sowed in peat-based compost (Florafleur, Jiffy International
AS, Kristiansand, Norway) and allowed to grow for 21 days. On
Day 21, the seedlings were individually transplanted into pots
filled with 2 L of growing media comprising of biochar and coir
(Hua Hng Trading Co. Pte. Ltd., Singapore) at a 3:2 (v/v) ratio.
The pots were randomly arranged on the nursery benches.
Throughout the experiments, all the crops were exposed to natural
sunlight and watered equally by automated sprinklers three times
a day, at 9 am, 12 pm and 3 pm, for three minutes each time (ap-
proximately 5 mm of water per watering session). The average
recorded ambient temperature in the nursery was 28.6 °C and
the average daily light integral was 17.9 mol/m2/day. As the nurs-
ery was not enclosed, the crops were subjected to fluctuations in
temperature, humidity and light.
2.3.2. Experiment 1 — Application of surplus food-derived BSF larval
frass as media-incorporated compost
The study design was a 4  2 factorial experiment where four
BSF larval frass application rates in biochar and two inorganic fer-
tilizer application regimes were tested. Surplus food-derived frass
was incorporated to biochar at the ratios of 10:90, 20:80, 30:70 and
40:60 (v/v) to make up the four experimental growing media. NPK
15-15-15 fertilizer (Mc-Queen, Hua Hng Trading Co Pte Ltd, Singa-
pore) was either applied throughout the experiment or not at all.
The positive control medium simulating the commercial practice
was peat-based compost (Go Bio4, Jiffy International AS, Kris-
tiansand, Norway) and loamy top soil (Hua Hng Trading Co Pte
Ltd, Singapore) mixed at a ratio of 30:70 (v/v), while the negative
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Waste Management 130 (2021) 155–166
control medium was pure biochar. Both controls had inorganic fer-
tilizer applied. Ten replicates of pak choi were cultivated for each
treatment and the controls. After transplanting to the experimental
growing media, the pak choi seedlings were grown over a 20-day
period during which the controls and treatment groups with fertil-
izer applied had side-dressing of 1.2 g given on Days 16 and 26. On
Day 34, the crops were harvested and their fresh aboveground bio-
mass was measured. After oven-drying at 60 °C for a week, their
dry aboveground biomass was measured.
2.3.3. Experiment 2 — Applications of surplus food-derived and okara-
derived BSF larval frass as side-dress organic fertilizer and frass-tea
drench
In the second experiment, we investigated the growth
responses of lettuce to five fertilizer treatments and a control with
no fertilizer (CONT). The fertilizers applied included surplus food-
derived BSF larval frass (FOOD.F), frass tea made using the surplus
food-derived frass (FOOD.T), okara-derived BSF larval frass
(OKARA.F), frass tea made using the okara-derived frass (OKARA.
T) and NPK 15-15-15 fertilizer solution (COMM) which represents
the commercial practice. Five replicates of lettuces were cultivated
for each treatment and the control. FOOD.F and OKARA.F were
applied as side-dressings on the growing media, in a circle around
the individual crops. We standardized the total nitrogen content of
the frass added to each pot to be 0.18 g per application which is
equivalent to a nitrogen application rate of 95 kg/ha. Immediately
after each side-dressing application, the crop and the growing
media were drenched with 200 ml of deionized water which had
been aerated for 24 h. For FOOD.T, OKARA.T and COMM, each fer-
tilizer application involved drenching of both the crop and the
growing media with 200 ml of the respective liquid fertilizer. To
produce COMM, the same brewing procedure for the frass teas
detailed in Section 2.1.2, except the addition of molasses, was car-
ried out with pelleted NPK 15-15-15 fertilizer (Mc-queen Com-
pound, Hua Hng Trading Co. Pte. Ltd., Singapore). The
experimental fertilizers were applied three days after transplant-
ing, i.e., Day 24, and on a weekly interval thereafter, i.e., Days 31,
38 and 45. On Day 52, we harvested the lettuces by loosening
the growing media by hand, carefully scooping up the root masses
and gently washing them under running water to remove any
J.K.N. Tan, J.T.E. Lee, Z. Chiam et al. Waste Management 130 (2021) 155–166

Table 1
Nutrient contents (n = 3) of the inorganic fertilizer, surplus food-derived frass and commercial peat-based compost used in experiment 1, and the fertilizers, quantified on a single
application event basis, used in experiment 2. For experiment 1, nutrients were quantified from aqueous extracts of solid samples. For experiment 2, each application was
standardised such that the frass directly applied or the pelleted inorganic fertilizer or frass used to make the liquid fertilizers contained 0.18 g of nitrogen in total. Nutrients of BSF
larval frass were quantified from aqueous extracts of the frass. ND = not detected.

Experiment 1
Water extractable ion/element (ppm) NPK fertilizer Surplus food-derived frass Peat-based compost
(batch 1)
NO3- 26,100 8.26 33.97
PO3- 4 15,410 566 5.00
SO2- 4 5224 656 33.63
-
Cl 11,950 3399 22.11
P 5231 393 4.58
K 12,340 1334 101
Ca 143 5.51 30.39
Mg 232 9.73 19.92
B 1.16 0.93 0.12
Cu 0.02 0.37 <0.01
Fe 0.29 4.71 0.02
Mn 0.46 0.11 ND
Ni 0.06 0.08 ND
Mo ND 0.08 <0.01
Zn 4.75 1.25 ND
As ND ND ND
Cd ND ND ND
Pb ND ND ND
Cr ND 0.01 ND
Experiment 2
Water extractable ion/element (mg per NPK fertilizer Surplus food-derived frass Surplus food-derived Okara-derived Okara-derived frass
side-dress or drench application) solution (COMM) (batch 2, FOOD.F) frass tea (FOOD.T) frass (OKARA.F) tea (OKARA.T)
NO3- 357 0.4 <0.1 <0.1 <0.1
PO3- 4 149 20 48 20 44
SO2- 4 74 29 50 13 32
-
Cl 146 146 195 0.4 15
P 68 17.17 22.13 10.35 23.60
K 145 58 93 68 121
Ca 2.81 0.24 9.82 0.59 7.79
Mg 3.15 0.43 4.84 0.37 8.32
B 0.03 0.04 0.07 0.17 0.24
Cu <0.01 0.02 0.02 0.03 0.03
Fe 0.02 0.21 0.45 0.13 0.23
Mn 0.01 <0.01 0.03 <0.01 0.03
Ni <0.01 <0.01 <0.01 <0.01 <0.01
Mo ND <0.01 <0.01 <0.01 <0.01
Zn ND 0.05 0.07 0.06 0.06
As ND ND ND ND ND
Cd ND ND ND ND ND
Pb ND ND ND ND ND
Cr <0.01 <0.01 <0.01 <0.01 ND

Table 2 char as well as the peat-based compost and soil used in experiment
Mean (±SE) pH and EC of biochar-coir growing media in experiment 2 under different 1 were determined using a FlashSmartTM CHNS Elemental Analyser
fertilization post-harvest. Values in a column that do not share a letter are
(ThermoFisher Scientific, Massachusetts, United States) and a mod-
significantly different (p < 0.05).
ified DUMAS method. The concentrations of selected elements and
Fertilization pH EC (bulk dS/m) ions in the aqueous extracts of these materials were also quantified
No fertilizer 7.65 ± 0.10 a 32.00 ± 3.34b to estimate the amounts of nutrients from them which were
NPK solution 7.08 ± 0.28 a 34.00 ± 4.68b directly available to the crops (Tables 1 and 2). The same suite of
Surplus food-derived frass 7.14 ± 0.13 a 45.00 ± 9.33 ab
nutrients was quantified for the BSF larval frass teas and the NPK
Surplus food-derived frass tea 7.38 ± 0.10 a 58.25 ± 5.81 ab
Okara-derived frass 7.24 ± 0.13 a 56.40 ± 10.07 ab 15-15-15 fertilizer solution used in experiment 2. The anions in
Okara-derived frass tea 7.67 ± 0.08 a 71.20 ± 10.66 a the liquid samples were analysed by ion chromatography (820 IC
Separation Center with 732 IC Detector, Metrohm AG, Herisau,
Switzerland) while the elements were analysed by inductively cou-
growing media that were stuck. Subsequently, the fresh above- pled plasma-optical emission spectroscopy (Avio 500 ICP Optical
ground and belowground biomass of the harvested lettuces were Emission Spectrometer, PerkinElmer, Massachusetts, United
measured. The lettuces were oven-dried at 60 °C for a week before States).
their dry aboveground and belowground biomass were measured. The pH and electrical conductivity (EC) of the growing media
from each treatment and the controls in experiment 1 were mea-
2.4. Chemical characteristics of growing media and fertilizers sured before any inorganic fertilizer was added, on Day 14, and
after harvesting, on Day 34, to investigate the effects of the increas-
The total nitrogen contents (% w/w) of the two batches of sur- ing rates of incorporation of BSF larval frass on the chemical char-
plus food-derived BSF larval frass, the okara-derived frass, the bio- acteristics of the media. The same measurements were conducted

158
J.K.N. Tan, J.T.E. Lee, Z. Chiam et al.
for experiment 2 after harvesting, on Day 52, to study the effects of
the applications of BSF larval frass as side-dress fertilizer or frass-
tea drench. These measurements were done directly in the growing
media for experiment 1 using a pH meter (HI99121, Hanna Instru-
ments Inc., Rhode Island, United States) and a GS3 multipurpose
sensor (METER Group Inc., Washington, United States). For exper-
iment 2, the measurements were carried out in a 1:10 (w/v) mix-
ture of growing medium and deionized water using the same
instruments.
2.5. Microbial characteristics of growing media under different
fertilizer treatments
At the end (Day 52) of experiment 2, growing media samples
were collected from three random pots from each treatment and
the control and pooled. These samples were stored at 20 °C
before the genomic DNA were extracted using a soil DNA isolation
kit (DNeasyÒ PowerSoil Ò Kit, QIAGEN, Hilden, Germany). Amplifi-
cation of the V3-V4 region of the 16S rRNA gene was carried out
with the use of specific forward (CCTAYGGGRBGCASCAG) and
reverse (GGACTACNNGGGTATCTAAT) primers. All PCR reactions
were carried out with PhusionÒ High-Fidelity PCR Master Mix
(New England Biolabs) with the mixed PCR products purified with
Qiagen Gel Extraction Kit (Qiagen, Germany). The libraries were
generated with NEBNextÒ UltraTM DNA Library Prep Kit for Illu-
mina and quantified via Qubit and Q-PCR. Sequences for each sam-
ple were sorted according to the unique barcodes and paired-end
reads were merged using FLASH. Quality filtering on the raw tags
were performed under specific filtering conditions to obtain the
high-quality clean tags utilising the QIIME software. The tags were
then compared against the Gold database using the UCHIME algo-
rithm to detect chimera sequences which were removed.
Sequences analyses were performed by Uparse software and
sequences with 97% similarity were assigned to the same OTUs.
The representative sequence for each OTU was screened for further
annotation. Mothur software was used for each representative
sequence to compare against the SSUrRNA database of SILVA Data-
base for species annotation at each taxonomic rank. OTUs abun-
dance information were then normalized using a standard of
sequence number corresponding to the sample with the least
sequences. Subsequent analysis of alpha diversity and beta diver-
sity were all performed basing on this output normalized data on
QIIME and R softwares.
2.6. Statistical analyses
For experiment 1, two-sample t-tests were conducted to test for
differences between the dry aboveground biomass of the crops
from the most productive frass-biochar medium and that from
the conventional soil medium and pure biochar. The effects of
the amount of biochar incorporated to the growing media and
the application of inorganic fertilizer on the dry aboveground bio-
mass were modelled using analysis of covariance (ANCOVA). Linear
regressions were calculated to predict the trends of the pH and EC
of the biochar growing media with increasing incorporation of
frass. As heteroscedasticity was detected with the EC values, gen-
eralised least squares estimators were used in its model.
For experiment 2, one-way analysis of variance (ANOVA) was
applied to the log-transformed plant growth parameters, which
include dry aboveground biomass (DAB) and dry belowground bio-
mass (DBB), and the pH and EC of the growing media post-harvest
to evaluate the effects of the fertilizer treatments on these vari-
ables. The plant growth parameters were log transformed to satisfy
the assumption of normality of the ANOVA test. Tukey’s (HD) pair-
wise comparison was performed to separate the differences
between the means and compare the effects caused by the differ-
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Waste Management 130 (2021) 155–166
ent fertilizer treatments. Statistical significance was tested at a
95% confidence level. All analyses were conducted in the R version
3.6.1 software environment (R Core Team, 2019).
3. Results
3.1. Pak choi yields in biochar growing media with surplus food-
derived BSF larval frass incorporated at different rates
In experiment 1, the best performing experimental growing
media comprised of surplus food-derived BSF larval frass and bio-
char at a ratio of 10:90, with inorganic fertilizer applied (Fig. 2).
Pak choi grown in this proposed medium had dry aboveground
biomass that was not significantly different from those of the soil
and peat-based compost mixture with fertilizer (t(13) = 0.88,
p = 0.39) but were significantly higher than the yields from pure
biochar plus fertilizer (t(16) = 3.38, p < 0.01). The ANCOVA model
predicted that the dry aboveground biomass would decrease by
0.09 g per 10 percentage points increase (in terms of total volume
of the growing media) in the amount of BSF frass incorporated to
the biochar growing media (F(1,68) = 21.72, p < 0.01). The covariate
of inorganic fertilization of the experimental growing media was
estimated to increase the dry aboveground biomass by 0.11 g
which amounted to an increase of 19–48% as compared to the
yields obtained from the same growing media without fertilizer
applied (F(1,68) = 5.75, p = 0.02).
3.2. Lettuce yields in biochar-coir growing media with surplus food-
derived and okara-derived BSF larval frass applied as side-dress
organic fertilizer or frass-tea drench
In experiment 2, lettuces grown under COMM treatment gener-
ally had higher dry aboveground biomass than those grown under
the BSF larval frass fertilizers treatments. However, the difference
between COMM and FOOD.F was not statistically significant
(Fig. 3). For dry belowground biomass, there was no significant dif-
ferences between COMM, FOOD.F, FOOD.T and OKARA.F. Generally,
lettuce yields with side-dress fertilizer application were higher
compared to their respective frass-tea application. This was espe-
cially noticeable for okara-derived BSF larval frass where dry
aboveground and belowground biomass was significantly higher
under OKARA.F than OKARA.T treatment (6.8 times higher for
aboveground biomass). Comparing the frass teas, lettuce dry bio-
mass was higher under FOOD.T than OKARA.T treatment (5.3 times
higher for aboveground biomass). Relative to CONT (no fertilizer),
the dry biomass was significantly increased by the addition of sur-
plus food-derived or okara-derived BSF larval frass, either as a side-
dress organic fertilizer or frass-tea drench.
3.3. Nutrient contents of BSF larval frass growing media component
and fertilizers
Chemical characterization showed that the surplus food-
derived BSF larval frass used in experiment 1 had more than six
times the total nitrogen content (3.15% w/w) as compared to the
commercial peat-based compost (<0.50% w/w). However, the bulk
of this nitrogen was likely in the organic form as indicated by the
low nitrate concentration (8.26 ppm) from the frass aqueous
extract. The frass had higher phosphate and potassium contents
than the peat-based compost, the differences being more than
100 times and close to 20 times, respectively. Based on its high
availability of macronutrients and numerous of the essential
micronutrients, the frass can be a nutrient source for crops.
Our results indicated that relative to COMM, the different BSF
larval frass fertilizers used in experiment 2 had lower nitrate, phos-
J.K.N. Tan, J.T.E. Lee, Z. Chiam et al. Waste Management 130 (2021) 155–166

Fig. 2. Dry aboveground biomass of Brassica rapa grown in various experimental growing media, with and without fertilizer applied, and in the positive control (soil and peat-
based compost) and negative control (pure biochar). Values are means ± 1 standard error. The ANCOVA model predictions for the yields with and without fertilizer applied are
denoted by the solid and dotted lines, respectively.

Fig. 3. Dry aboveground and belowground biomass of Lactuca sativa grown on biochar and coir growing media under different fertilizer treatments. Values are means ± 1
standard error. Statistically significant differences (p < 0.05) based on the log-transformed dry biomass are indicated with different letters above the bars. Biomass of the
different plant parts was analysed separately.

phate and potassium concentrations (Table 1). Relative to the solid
frass, the aerated brewing process increased the concentrations of
water extractable phosphate (surplus food: 2.4 times; okara: 2.2
times), phosphorous (surplus food: 1.3 times; okara: 2.3 times),
potassium (surplus food: 1.6 times; okara: 1.8 times), calcium (sur-
plus food: 40.8 times; okara: 13.2 times), magnesium (surplus
food: 11.4 times; okara: 22.4 times), sulphate, chlorine, boron, iron,
manganese and zinc for both types of frass. Heavy metals, namely
arsenic, cadmium, lead and chromium, were not detected or
present in negligible quantities in the BSF larval frass and
frass teas.
160
3.4. pH and electrical conductance of growing media under various
BSF larval frass application
In experiment 1, the pH of the various experimental growing
media was within the tolerable range of pak choi, i.e., 4.3–7.5
(Roy et al., 2006). The initial (Day 34) pH decreased by 0.15 for
every 10% increase (in terms of total volume of the growing media)
in the amount of BSF frass incorporated to the biochar growing
media (F(1,67) = 24.56, p < 0.01) while the initial EC increased sig-
nificantly with higher frass incorporation rates (F(1,68) = 96.53,
p < 0.01) (Fig. 4).
J.K.N. Tan, J.T.E. Lee, Z. Chiam et al.
Fig. 4. Predicted trends of the pH (A) and EC (B) of the biochar-based growing media in experiment 1 with increasing incorporation of surplus food-derived BSF larval frass
(percentage based on total volume of the growing media) on Day 14 post-transplanting and Day 34 post-harvesting. Recorded values are represented by points.
In experiment 2, the pH values of the biochar-coir growing
media under different fertilization ranged between 7.08 and 7.65,
which were above the optimal media pH range for lettuce, i.e.,
6.0–7.0 (Roy et al., 2006). The EC values of the growing media
increased with all fertilization and were higher with frass-tea
application as compared to the corresponding organic fertilizer
application (Table 2). Notably, the EC values of OKARA.T were sig-
nificantly higher relative to COMM and CONT.
3.5. Bacterial communities of biochar-coir growing media under
various BSF larval frass application
The rarefaction curves of the samples after Illumina sequencing
(Fig. S1) suggested that the sequencing depth for bacteria was not
enough to cover the entire diversity, although the Good’s coverage
of 99% indicated that it is sufficient for inferring patterns of beta
diversity and overall taxon relative abundances of dominant lin-
eages (Bates et al., 2011). Species richness derived from the num-
ber of OTUs and Chao 1 (Fig. S2) reveal that commercial fertiliser
resulted in a lower diversity as compared to non-amended control
media, with food-fed frass tea displaying the least diverse micro-
bial community. On the other hand, okara-derived frass resulted
in the most diversity regardless of the fertilisation method. The
Shannon function, which additionally measures species evenness,
is mostly in agreement with these results with smaller variability,
whilst the Simpson index, which skews towards more dominant
species, displays the least variance between the samples with the
exception of FOOD.T. The PCoA plot based on weighted Unifrac dis-
tances (Fig. 5) show that in terms of the bacterial community, the
best performing group in terms of dry biomass productivity (Group
R consisting of the positive control and FOOD.F) were a little more
similar to each other compared to the other samples. Meanwhile,
the community resulting from surplus food-derived frass-tea
application was vastly different from all the others. Assuming that
microbial community evolution is deterministic, the only nutrient
variable that fits the trend would be the chloride anion (Table 1).
In terms of the taxonomic data, at the Phylum level (Fig. 6),
there are no Synergistes detected in any of the samples, and very
low amounts of Verrucomicrobia. Proteobacteria and Bacteroidetes
are the most abundant phyla, with Acidobacteria, Actinobacteria
Cyanobacteria and Firmicutes making up the remainder. From the
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Waste Management 130 (2021) 155–166
heatmap (Fig. 6a), it can be seen that the best performing condi-
tions of the commercial fertiliser and FOOD.F owe their results to
different communities of putative syntrophic bacteria. COMM has
high amounts of Sediminibacterium, Lavobacterium, Lacibacter, and
Flavisolibacter belonging to Bacteroidetes and Bdellovibrio, Methy-
lobacillus, Methylophilus, and Acidibacter that are Proteobacteria. In
contrast, FOOD.F has a community rich in the Proteobacteria Lacto-
bacillus, Ruminiclostridium, Clostridium sensu stricto, and
Ruminococcaceae and Bacteroidetes genera Flavitalea and Cloacibac-
terium. In the compounded heatmap (Fig. 6b), it is also noteworthy
that Terrimonas is common to the best performing samples of
COMM and FOOD.F. Of note also is the genera of Proteobacteria:
Hirschia, Ramlibacter, Rhodoplanes, Devosia and Microvirga which
are found in abundance in the poorest performing samples (Group
Q), in lower numbers in the middle performance Group S, and
Group R having a dearth of these microbes.
In addition, analyses of the bacterial genus of the different
media samples were unable to detect common foodborne patho-
gens like Campylobacter, Listeria, Salmonella and Yersinia.
4. Discussion
4.1. Pak choi yields in biochar growing media with surplus food-
derived BSF larval frass incorporated at different rates
Our results showed that a growing medium made entirely of
upcycled food and wood waste materials could produce yields of
a leafy vegetable comparable to that from the conventional med-
ium of soil and peat-based compost and therefore, be a plausible
alternative to the latter. This highlights a potential application of
BSF larval bioconversion for sustainable agriculture where food
production is carried out utilising organic wastes instead of soil
as the growing medium. When food is produced in such a way,
organic wastes are managed as they are upcycled and soil as well
as peat, which are finite natural resources, remain in the environ-
ment where they support ecosystems (Jónsson and Davídsdóttir,
2016; Osland et al., 2020). Moreover, as the nutrients in the BSF
larval frass are expected to be completely utilized in a few growth
cycles, there will be a constant demand for the frass by agricultural
systems, justifying the sustained bioconversion of food wastes.
J.K.N. Tan, J.T.E. Lee, Z. Chiam et al. Waste Management 130 (2021) 155–166

Fig. 5. PCoA Plot (Group Q consists of poorly performing samples CONT and OKARA.T. Group R consists of most productive samples of COMM and FOOD.F, and Group S of
intermediate performance samples of FOOD.T and OKARA.F).

Fig. 6. Heatmap of soil bacterial phyla and genera by samples (a) and by group (b).

162
J.K.N. Tan, J.T.E. Lee, Z. Chiam et al.
The negative correlation between the concentration of surplus
food-derived frass in the growing media and the yield of pak choi
could imply that the frass had some phytotoxic properties which
outweighed the expected positive effects of its relatively high
nutrient contents at high application rates. One possibility is that
high salinity of the frass, as indicated by high Cl concentration
(Table 1) and EC values (Fig. 4), stunted the crops’ growth
(Ghoulam et al., 2002; Romero-Aranda et al., 2001). The immediate
effect of high media salinity is an interference with the plants’ abil-
ity to take up water from their roots and maintain their structures
through turgor pressure (Sohan et al., 1999). When Cl concentra-
tion is high in the growing medium, the uptake of Cl could com-
pete with the uptake of nutrients such as NO3 (Bañuls et al., 1990;
Fisarakis et al., 2001). In addition to the adverse effects of high
salinity, we hypothesized that at higher incorporation rates, the
tendency of the frass to act like sticky clay in the macropores
between the biochar, especially when wet, could have reduced
oxygen supply to the plant roots and hindered crop growth. Appli-
cation of composted organic waste to soilless growing media was
shown to reduce aeration (Bugbee, 2002; Graceson et al., 2014).
In low-oxygen growing media with organic matter incorporated,
reduced compounds and by-products of microbial metabolism
such as volatile fatty acids, phenolic acids and hydrocarbons could
also accumulate to harmful levels and result in plant damage.
Despite the surplus food-derived frass having a higher nutrient
availability than the commercial peat-based compost, the signifi-
cant positive effect of inorganic fertiliser on the aboveground dry
biomass of pak choi indicates that the frass alone was unable to
supply nutrients at the amount and rate required by the crop to
achieve its full potential growth. This contrasts with the results
of (Choi et al., 2009) which reported that food waste-derived BSF
larval frass alone was as effective as conventional inorganic fer-
tiliser for the cultivation of Chinese cabbage (Brassica rapa subsp.
pekinensis). This discrepancy is likely owed to the variability in
the feedstocks, which affect the residue quality (Kagata and
Ohgushi, 2012), as well as the different application rates amongst
the studies.
4.2. Lettuce yields in biochar-coir growing media with surplus food-
derived and okara-derived BSF larval frass applied as side-dress
organic fertilizer or frass-tea drench
In the absence of diseases and pests and grown under the same
environmental conditions, the variations in the biomass of the let-
tuces which experienced different fertilization could be attributed
to nutrient availability in the growing media. FOOD.F was the only
experimental fertilization with lettuce yields that were not signif-
icantly different than those from COMM, the positive control. The
comparable productivity of FOOD.F to COMM despite the lower
concentrations of water extractable nutrients in FOOD.F implies
that nutrients in the frass were more efficiently utilized by the
plants. This is likely because the nutrients were supplied to the
plants over a longer duration and at a more suitable rate. The lower
EC values of the growing media after COMM treatment as com-
pared to after FOOD.F and other BSF frass treatments support the
hypothesis that the nutrients in COMM, which were mostly avail-
able for instantaneous absorption by plants, were present in excess
quantities and leached out of the pots at greater rates. Moreover,
similar to vermicompost, FOOD.F likely contained microbes, phyto-
hormones and other substances absent in COMM which increased
nutrient availability, stimulated plant growth and conferred to
crops, protection against abiotic stress relating to water, salinity
and temperature (Amara et al., 2015; Wong et al., 2020).
Generally, direct application of frass as a side-dress fertilizer
was more effective than application of frass teas in promoting
the growth of lettuces. The difference in yield between FOOD.F
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Waste Management 130 (2021) 155–166
and FOOD.T, though not significant, could be partially explained
by the higher nitrate content of the frass as compared to the frass
tea. However, OKARA.F did not exhibit significantly higher nitrate
content than OKARA.T. Moreover, the frass teas typically had
higher quantities of water extractable nutrients compared to the
respective frass. The better performance of the solid frass could
be attributed to the bulk of its nutrients being in organic forms
which were not quantified by our chemical analyses. These nutri-
ents were gradually converted into plant-available forms after
the frass was applied to the growing media. Therefore, the solid
frass behaved more like slow-release fertilizer. In contrast, most
of the nutrients in the frass teas were likely present in plant-
available forms as we expect nutrient mineralisation and extrac-
tion of water-soluble nutrients from the frass to occur during the
brewing process. The higher yields obtained from the solid frass
treatments suggest that the gradual release action of the side-
dress fertilizer provided the lettuces with more nutrients over
the span of the experiment compared to the spikes of nutrients
supplied by the frass tea. To the author’s knowledge, few studies
compared the yields of crops under solid compost and compost
tea drench fertilizations and none of these studies compared the
nutrient contents of compost teas and the composts they were
derived from on a per application basis. Radin and Warman
(2011) found no significant differences in yields of tomato plants
fertilized with only municipal solid waste compost and only tea
made from the compost but provided no mechanistic explanations
for the observation. Similarly, Hargreaves et al. (2009) detected no
significant differences in yields of strawberry plants fertilized with
compost and compost tea derived from municipal solid waste. The
low yields across the treatments were attributed to insufficient
nitrogen provided by both the compost and the compost tea. Other
studies reported the nutrient contents of composts and the corre-
sponding compost teas but a comparison of fertilizing potentials
was not in their scopes (Shrestha et al., 2011; st. Martin et al.,
2012).
Comparing the yields in the treatments using different BSF lar-
val feedstock, FOOD.F had a higher dry aboveground biomass than
OKARA.F. This could be owing to the higher nitrate content in the
former. With regard to frass-tea application, FOOD.T had higher
dry aboveground and belowground biomass than OKARA.T. In con-
trast to FOOD.F and OKARA.F, the nutrient profiles of the frass teas
were similar, suggesting that microbial factors or the presence of
other compounds played a greater role in determining the fertiliz-
ing potential of frass teas.
Future works regarding the applications of larval frass in soil-
less growing media should expand on the scope of the agronomic
responses investigated, particularly the physiological responses
of plants to larval frass application, to understand the mechanisms
such as the effects on water use efficiency, nitrogen use efficiency
and leaf chlorophyll concentration, in addition to biomass
accumulation.
4.3. Bacterial communities of biochar-coir growing media under
various BSF larval frass application
Alpha diversity indices (Figs. S1 and S2) indicate that the addi-
tion of mineral fertilizers compared with non-amended growing
media reduces biodiversity, in congruence with literature (Zhong
et al., 2010). In contrast to findings of Enwall et al. (2007), the addi-
tion of organic fertilisers in this study did not have a significant
positive impact on the microbial community, with the exception
of FOOD.T. Whilst increased microbial diversity has been reported
to correlate with higher soil organic carbon content and fertility
(Wolińska, 2019), in this study the biomass yield of lettuce faces
a discrepancy. This is not likely to be due to the media, as a previ-
ous study has shown results which fit the hypothesis (Cheong
J.K.N. Tan, J.T.E. Lee, Z. Chiam et al.
et al., 2020). The PCoA plot (Fig. 5) also reveals that while the best
performing treatments of COMM and FOOD.F are similar in terms
of the community, biodiversity is not the sole determinant of per-
formance as the intermediate yield OKARA.F is clustered in the
middle of CONT and OKARA.T.
It has been proposed that changes in soil bacterial diversity may
not be correlated with functioning (Lupatini et al., 2013) and that
microbial function might be more important than simply the
microbial diversity (Gała˛zka and Furtak, 2019; Pan et al., 2014).
The heatmaps of the bacterial communities in Fig. 6 lends credence
to this hypothesis as it can be clearly observed that the best per-
forming COMM and FOOD.F have a high abundance of discrete gen-
era belonging mainly to Proteobacteria, and Bacteroidetes. In
agreement with literature, these two phyla make up the bulk of
the media bacterial community (Ishaq et al., 2017; Pershina
et al., 2015). Proteobacteria is a metabolically diverse taxonomical
group with several subphyla and Land et al. (2015) reported that
about half of all the sequences deposited in the soil genome
libraries belonged to this phylum. Constituents of this group have
been reported to display functions such as nitrogen fixing, degra-
dation of toxic compounds, and methane oxidation (Aislabie
et al., 2013), and contain genera often present in arable soils
(Janssen, 2006). Bacteroidetes on the other hand, are generally
involved in the aerobic degradation of cellulose, chitin and proteins
(Aislabie et al., 2013), and positively correlated with net carbon
mineralisation (Fierer et al., 2007). Nemergut et al. (2008) reported
that fertilisation of soil increased the abundance of this phylum
and with the exception of the FOOD.T sample, the results of this
study are in agreement with that finding. Thus, it is conjectured
that the highly positive results of COMM and FOOD.F arise due to
the syntrophic activities of the diverse bacteria which evolve into
different communities with the ability to carry out activities that
promote the growth of plants. As a note of caution, from the com-
pounded heatmap (Fig. 6b), it is discerned that not all bacteria aris-
ing from a certain phylum are beneficial, as the poorly performing
Group Q (CONT and OKARA.T) do present a multitude of Proteobac-
teria. These may be detrimental to lettuce growth, as they are pre-
sent in minimal amounts in the best performance Group R and in
intermediate amounts in the corresponding intermediate perfor-
mance Group S. Penultimately, there is also the curious case of Ter-
rimonas being present in significant although not copious amounts
in Group R. Little is known of this genus, although it has putative
novel nitrogen-fixing abilities (Xie and Yokota, 2006) and was
found co-located with a nitrogen-fixing microbial consortium
(Khan et al., 2015). If proven to be a microbe of interest, future
optimizing strategies could focus on increasing its abundance for
concomitant enhancement of lettuce yields or by bioaugmentation.
Finally, analyses of the bacterial genus of the different media
samples were not able to detect common foodborne pathogens like
Campylobacter, Listeria, Salmonella and Yersinia. Whilst not an
exhaustive list, these results reinforce the idea that BSFL frass
derived from okara or food is sanitary.
5. Conclusions
We showed that 10% (v/v) surplus food-derived BSF larval frass
can be used effectively as a compost when incorporated into waste
wood-derived biochar and fortified with inorganic fertilizer. The
frass-biochar mixture is a promising soilless growing medium
and its performance relative to peat could be tested in future stud-
ies. When applied as a fertilizer to a soilless growing medium com-
prising of biochar and coir, direct application of the BSF larval frass
as a side-dress fertilizer was more effective in promoting crop
growth than application of the same frass as a frass-tea drench.
Also, fertilizers derived from surplus food performed better than
164
Waste Management 130 (2021) 155–166
those derived from okara. These results are possibly because of
the differences in nutrient availability and the presence of bacterial
communities. The BSF larval frass and frass teas were found to con-
tain negligible levels of heavy metals and the biochar-coir growing
media after BSF larval frass and frass-tea fertilizations (one growth
cycle) were not found to contain common foodborne pathogens.
The implications of our findings are that BSF frass growing media
and organic fertilizers derived from food waste could replace
unsustainable agricultural inputs for leafy vegetable cultivation,
including soil and inorganic fertilizers.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgements
This research was supported by the National University of Sin-
gapore (NUS)-Office of the Deputy President (Research and Tech-
nology) under the grant R-154-000-A97-133 and the National
Research Foundation Singapore under its Campus for Research
Excellence and Technological Enterprise (CREATE) programme. It
was made possible through collaboration with the black soldier
fly research team of the Evolutionary Biology Laboratory, National
University of Singapore. We thank Dr Erik Arthur Smith, Phira
Unadirekkul, Nicholas Lim Weishou, Tan Cheng Lin and Eugene
Tok Wei Xian for the advice and assistance pertaining to the pro-
duction of the black soldier fly larval frass used in our experiments.
We also appreciate the suggestions by Deon Lum Wen Hao to
improve the manuscript.
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.wasman.2021.05.025.
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