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Journal of Environmental Management 355 (2024) 120545
0301-4797/© 2024 Published by Elsevier Ltd.
Research article
Go local: Enhancing sustainable production of Tenebrio molitor through
valorization of locally available agricultural byproducts
Christina Adamaki-Sotiraki
*
, Despoina Choupi , Mariastela Vrontaki , Christos I. Rumbos ,
Christos G. Athanassiou
Laboratory of Entomology and Agricultural Zoology, Department of Agriculture, Crop Production and Rural Environment, University of Thessaly, Phytokou Str., 38446,
Volos, Greece
ARTICLE INFO
Keywords:
Sustainability
Insect production
Local byproducts
Feeding substrate
Tenebrio molitor
ABSTRACT
Insects receive increasing attention as an alternative source of protein for animals and humans, and thus, the
production of low-cost insects for meeting the dietary demand on sustained basis is an ever-growing concept.
This study aims to design dietswith locally available agricultural byproducts from Greece as feed for larvae of the
yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae). This will considerably reduce the cost of
insect feed and the environmental impact of insect farming by using locally available agricultural byproducts as
economic insect feedstock. More specically, ve agricultural byproducts derived from the production of cereals
and legumes were utilized to design twelve different diets at two protein levels, i.e., 17.4 and 22.5% protein
content. All diets were evaluated both at laboratory scale, but also at pilot scale. Based on the obtained results,
both bioassays revealed that the diets contained one legume and one cereal byproduct (i.e., lupin and triticale as
well as lupin and oat) supported more efciently the growth and performance of the larvae, irrespective of the
protein level. Indicatively, individual larval weight of the best performed larvae from both groups ranged from
132 to 142 mg. Moreover, our results highlight the fact that data derived from laboratory scale bioassays are not
always easy to be extrapolated to industrial production. For instance, the total harvest of larvae, a parameter
assessed in the tray scale bioassay, exhibited a disparity between diet A2 (910 g) and diet A3 (749 g), despite
both being deemed optimal in the laboratory-scale experiment. Our study aims to promote a circular approach
for the industrial rearing of insects through integration of local agricultural byproducts into specic diets for
T. molitor larvae.
1. Introduction
The present form of agricultural production is placing pressure to the
already existing scarcity of natural resources, i.e., land, water, and en-
ergy. In particular, the increasing emissions of greenhouse gases, the
deforestation, and the soil degradation need to be tackled (Steinfeld,
2006; Thorne, 2007; Tilman et al., 2002). Livestock farming and aqua-
culture, which are two of the biggest sources of protein for Europe, are in
search of innovative and sustainable nutrient sources (Van Huis et al.,
2013). The current circumstances reveal an opportunity for insect
farming, which constitutes a promising solution for the augmented de-
mand in proteinaceous products for humans and animals.
The sector of agriculture produces a huge amount of byproducts and
organic wastes (FAO, 2013). However, the concept of wastes does not
exist in the natural world, as nature possesses mechanisms to recycle
everything. Based on the above, insect production aligns with the
principles of the circular economy, as insects efciently convert agri-
cultural byproducts and organic wastes into highly nutritious nal
products (Bodie et al., 2019; Bosch et al., 2019; Broeckx et al., 2021;
Gourgouta et al., 2022; Hars´
anyi et al., 2020). Thus, insect farming is
gaining scientic and commercial interest in Europe. Since 2017, the
yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae), has
been listed as safe nutrient source for aquaculture (EC, 2017), while
recently it has been approved as alternative protein source for livestock
animals (EC, 2021) and humans (EFSA, 2021). As one of the most
extensively mass-reared insect species for food and feed, T. molitor plays
a vital role in restoring the disrupted food chain by feeding on agricul-
tural byproducts (Kotsou et al., 2023; Ramos-Elorduy et al., 2002;
Rumbos et al., 2021). However, as insect farming sector is growing, the
design of byproduct-based diets is currently in the spotlight of research
* Corresponding author.
E-mail address: cadamaki-s@uth.gr (C. Adamaki-Sotiraki).
Contents lists available at ScienceDirect
Journal of Environmental Management
journal homepage: www.elsevier.com/locate/jenvman
https://doi.org/10.1016/j.jenvman.2024.120545
Received 30 October 2023; Received in revised form 10 February 2024; Accepted 1 March 2024
Journal of Environmental Management 355 (2024) 120545
2
(Oonincx et al., 2015; Rumbos et al., 2022; Van Broekhoven et al.,
2015).
The industrialization of T. molitor production, is becoming a reality.
However, industrial-scale production faces many challenges such as
sustainability and economic viability (Berezina, 2017). That being the
case, to make T. molitor more competitive, its mass production should be
investigated and optimized. There are only relatively few studies though
that evaluated the larval performance of T. molitor from a large-scale
production perspective (Adamaki-Sotiraki et al., 2023; Deruytter
et al., 2019, 2021). However, there is a gap in literature concerning the
simultaneously evaluation of local agricultural byproducts as diet
components at laboratory and large scale bioassays. Based on the above,
the present study aims to promote the sustainable and cost-effective
insect production via the collection of local agricultural byproducts
produced in the region of Thessaly, Greece, and their utilization as
components of various isoproteinic diets for the growth and develop-
ment of T. molitor larvae. It is expected that the design of diets composed
of a variety of different agricultural byproducts derived from the pro-
duction of cereals and legumes are suitable as feeding substrates for the
larvae of T. molitor, enhancing their economically important traits.
2. Materials and methods
2.1. Insect rearing
The stock colony of T. molitor was kept in a semi-pilot scale rearing
chambers at the Laboratory of Entomology and Agricultural Zoology at
the University of Thessaly, Greece. The present bioassay for the stock
rearing of T. molitor was performed under continuous dark on constant
environmental conditions (27 ±1 ◦C temperature and 60 ±5% relative
humidity). The adults were kept separate from larva and pupal stages,
while all stages were reared in plastic trays (60x40 ×14.5 cm) (Bee-
kenkamp Verpakkingen BV, Maasdijk, The Netherlands). As feeding
substrate, wheat bran was provided, while agar (20 g/L) was offered ad
libitum as moisture source.
2.2. Byproducts
Agricultural byproducts originated from the cleaning process of
lupin, triticale, oats, barley, and pea seeds were utilized. Byproducts
were purchased from a local company (Fyto-Animal Services (F.A.S)).
All byproducts were grinded with a grinder machine (Thermomix TM31-
96 1C, Vorwerk Elektrowerke GmbH & Co. K, Wuppertal, Germany). For
the laboratory bioassay, following to grinding, all byproducts, with the
exception of wheat bran, were sieved with a 600
μ
m sieve to achieve a
particle size <600
μ
m. For the pilot scale bioassay, byproducts were
sieved with a 2 mm sieve.
2.3. Design of diets
The byproducts tested in our study were thoroughly described and
analyzed in terms of nutrient content by Rumbos et al. (2021). Based on
the protein content of the byproducts originated from the production of
lupin, triticale, oat, barley, and pea, twelve different diets were
designed. More specically, six diets with a protein level of 17.4%, and
six diets with a protein level of 22.5% were designed (Table 1). Con-
cerning the diets with 17.4% protein content, wheat bran served as
control, while concerning the diets with 22.5% protein content, wheat
bran and yeast (9:1 ratio) served as control. All diets were evaluated for
their suitability as feeding substrates for T. molitor larvae under labo-
ratory conditions (bioassay I). As a follow up to bioassay I, two of the
diets on which larvae grew better and one of the diets on which larval
growth was poor, were selected in order to be evaluated in pilot scale
(bioassay II).
2.4. Bioassays
2.4.1. Bioassay I – Lab scale trial
For this bioassay, plastic cylindrical vials (7.5 cm in diameter, 8.8 cm
in height) were utilized as experimental units. At the beginning of the
trial, groups pf 50 newly-hatched larvae were weighted with a precision
balance (EQUINOX, Adam Equipment Inc Fox Hollow Road, Oxford,
USA) and their initial weight was recorded. In order to collect the newly-
hatched larvae, 250 g of adults were placed in a plastic tray
(60x40x14.5 cm) on top of a rectangular sieve (50x30, 2x2 mm open-
ing). The adults were left to oviposit for a time period of 7 d. As
oviposition substrate, 2 kg of white wheat were placed in the plastic tray
under the rectangular sieve. During the oviposition period, agar (20 g/L)
was provided ad libitum to adults as moisture source. After this interval,
adults were removed and the newly-emerged larvae were left to feed
undisturbed on white wheat our for an additional time period of 14 d.
After this interval, the feeding substrate was sieved with the aid of two
different sieves (i.e., one sieve with 850
μ
m and one sieve with 600
μ
m
opening), to separate the larvae from the feeding substrate, as well as to
collect larvae of similar heal capsule size. Larvae that passed through the
850
μ
m sieve and remained at the 600
μ
m sieve were used for experi-
mentation. Each group of 50 larvae was placed in a plastic cylindrical
vial together with 4 g of each diet. Additional feed was provided if feed
was depleted and the amount of added feed was recorded. Two agar
cubes (1x1x1cm) served as moisture source and were provided to each
vial three times per week. Every two weeks larvae were separated from
the feeding substrate in order for the larval survival and weight to be
recorded. Each vial was terminated at the emergence of the rst pupa.
Each dietary treatment was replicated six times. At the end of the bio-
assays, the larval development time was calculated as the number of the
days between the initiation of the experiment and the day each vial was
terminated. The amount of feed needed (in kg) to obtain one kg of insect
biomass [feed conversion ratio (FCR)] (Equation (1)), the efciency of
conversion of ingested food [ECI (%)] (Equation (2)), and the specic
growth rate [SGR (%/day)] (Equation (3)) were calculated according to
the following formulas:
FCR =Feed consumed /Weight gained (1)
ECI =weight gained /feed consumed ×100% (2)
SGR =100 × (lnFBW – lnIBW)/days (3)
where FBW and IBW stand for nal and initial body weight, respectively.
The calculations of FCR, ECI, and SGR were performed on as is basis.
Table 1
Byproduct-based diets at two protein levels (A =17.4%; B =22.5%) tested.
A1 control A2 A3 A4 A5 A6 A7 B1 control B2 B3 B4 B5 B6 B7
Wheat bran (control) 100.0 81.88
Lupin byproduct 35.25 23.50 33.60 56.10 48.12 54.95
Triticale byproduct 64.75 55.30 43.90 28.90
Oat byproduct 76.50 68.70 51.88 35.88
Barley byproduct 66.40 57.15 45.05 29.85
Pea byproduct 44.70 31.30 42.85 71.10 64.12 70.15
Yeast 18.12
C. Adamaki-Sotiraki et al.
Journal of Environmental Management 355 (2024) 120545
3
2.4.2. Bioassay II – pilot scale trial
Bioassay II is a follow up to bioassay I. More specically, two diets on
which larvae performed good (diets A2 and A3, 17.4% protein level), as
well as one diets on which larvae did not perform so well (diet A7, 17.4%
protein level) were selected in order to be evaluated in pilot scale. Wheat
bran served as control feeding substrate (Table 1). For this bioassay,
plastic insect breeding trays (60x40x14.5 cm) served as experimental
units. Three replications were performed for each diet, whereas there
were 12 tray in total. Each plastic tray was lled with 2.1 kg of each diet.
An amount of approximately 10,000 larvae was also inserted in each
experimental unit. In total, approximately 120,000 larvae were utilized
for this bioassay. As mentioned at section 2.2, due to the big quantities of
byproducts that were utilized to create the diets, as well as due to the
fact that we opted to mimic an industrial scale insect farming, byprod-
ucts were grinded and sieved with a 2 mm-opening sieve. For the
collection of the young larvae, 250 g of adults were placed in a plastic
tray (60x40x14.5 cm) on top of a rectangular sieve (50x30cm, 2x2 mm
opening). The adults were left to oviposit for a time period of 7 d. As
oviposition substrate, 2 kg of white wheat our were placed under the
rectangular sieve. There were in total six oviposition trays in order to
obtain the amount of 120,000 larvae. Agar served as moisture source for
the beetles. After 7 d, adults were removed and larvae were left to grow
undisturbed for a time period of 2 weeks. After this interval, larvae were
separated from the substrate with a 600
μ
m opening sieve, were pooled
together and their number was estimated via subsampling. Three sub-
samples of larvae were taken, taking care that each subsample contained
at least 100 young larvae. After the estimation of the total number of
larvae, approximately 10,000 young larvae were transferred to each
experimental unit. Every two weeks, each experimental unit was
weighted and homogenized well. Afterwards, three subsamples with at
least 100 young larvae were taken in order to record the larval weight.
This bioassay was terminated when 10% of larvae turned into pupae. At
the termination of the bioassay, the nal individual larval weight (mg),
the total harvest of larvae (g), the total development time (weeks) were
estimated. In addition the index FCR (Equation (1)) was calculated.
3. Calculations and statistical analysis
All statistics were done using SPSS 26.0 (IBM Corporation, Armonk,
NY, USA). For bioassay I, the normality and homogeneity of variances
were tested for the nal larval survival (%), for the nal individual larval
weight (mg), for the development time (days), as well as for the indexes
FCR, ECI, and SGR, for both protein levels (17.4% and 22.5%). For the
evaluation of normality, the Shapiro-Wilk was utilized, while for the
evaluation of the homogeneity, the Levene’s test was used. The as-
sumptions were met for the FCR for the larvae reared on diets with
protein level 17.4% and for the nal larval survival (%) for the larvae
reared on diets with protein level 22.5%. Consequently, data were
submitted to ANOVA, whereas Tukey HSD test was used for the deter-
mination of differences between diets of the same proteinic level
(p<0.05), as well as of both proteinic levels (p<0.05). For the rest of the
parameters for which the assumptions were not met, the non-parametric
analysis was chosen. Specically, the Kruskal-Wallis H test was per-
formed, followed by Dunn multiple comparisons for post-hoc testing.
For bioassay II, the normality and homogeneity of variances were also
tested for the nal individual larval weight (mg) the total harvest (g),
and the FCR for the dietary treatments tested. The assumptions were met
for all the aforementioned parameters, thus data were submitted to
ANOVA and Tukey HSD test.
4. Results and discussion
In this study, it is conrmed that a variety of diets composed of
agricultural byproducts derived from the production of cereals and le-
gumes cultivated at the region of Thessaly, Greece, are suitable as
feeding substrates for the larvae of T. molitor. Indicatively, our results
showed that diets composed of lupin and triticale, as well as lupin and
oat byproducts, irrespective of their protein level, were able to ef-
ciently support the growth of T. molitor larvae. Our study is in accor-
dance with recent studies that report the suitability of byproducts
derived from the production of cereals and legumes as feeding substrates
for T. molitor larvae (Kr¨
oncke and Benning, 2023; Rumbos et al., 2021).
All parameters evaluated at bioassay I, i.e., nal larval survival (%),
nal individual larval weight (mg), development time (days), FCR, ECI,
and SGR for both groups of diets are summarized in Fig. 1, Fig. 2, and
Table 2. More specically, for most of the larvae reared on diets of group
A, high survival rates (>63%) were recorded, with the exception of
larvae reared on the diets A4 and A7, for which almost half of the larvae
died at the termination of the bioassay (54% and 50%, respectively)
(Table 2; Fig. 1).Regarding group B, the highest survival rate was
recorded for larvae reared on the diets B2, B3, and B6 (56–67%),
whereas on diets B4, B5 and B7 larval survival rate was signicantly
lower (<40%) (Table 2; Fig. 2). The individual larval weight ranged
from 84.74 mg (A5) to 142.41 mg (A3) for treatments of group A (Fig. 3)
and from 39.90 mg (B7) to 136.82 mg (B3) for treatments of group B
(Fig. 4). Larvae reared on A2, A3, and A4 resulted in high nal indi-
vidual larval weight (>129.32 mg). The lowest nal individual larval
weight of diets of group A was recorded for the larvae reared on the diets
A6, A7, and A5 (<101.19 mg). Larvae reared on B2, B3, B4, and B5 had a
high nal individual larval weight (>93.01 mg). However, larvae that
consumed diets B6 and B7 grew poorly, with nal individual larval
weights ranging from 55.03 to 39.97 mg, respectively. The diets of group
A on which larvae performed better in terms of survival and weight
contained lupin, triticale, and oat byproduct. The same pattern was also
observed for larvae reared on diets of group B. These similarities reveal
that economic characteristics of T. molitor larvae (i.e., larval survival and
weight) do not depend only on the protein content of the diets provided
as feeding substrates. Concerning the byproducts of the lupin and triti-
cale production, Rumbos et al. (2021) highlighted that the same
by-products gave good results with respect to nal individual larval
weight and development time for T. molitor larvae. Rumbos et al. (2022)
also reported that oat byproduct was suitable as feeding substrate for
T. molitor larvae either singly or as diet component. Insects require eight
to ten essential amino acids (i.e., methionine, threonine, tryptophan,
valine, isoleucine, leucine, phenylalanine, lysine, arginine, and histi-
dine) and lupin contains all of them (Cohen, 2003; Khalid et al., 2016).
Apart from its high content in amino acids, lupin has a favorable
nutritional prole (B¨
ahr et al., 2015; Khan et al., 2015; Rumiyati et al.,
2013; Thambiraj et al., 2015). Moreover, cereal grains such as triticale,
constitute a good source of several vitamins and minerals, including
manganese, folic acid, calcium, phosphorus, zinc, copper, magnesium,
iron, the B vitamins, and vitamin E (Mergoum et al., 2009; Zhu, 2018).
In addition, oat is rich in vitamin E and has high levels of lipids,
Fig. 1. Average survival (mg) of Tenebrio molitor larvae reared on six diets or
wheat bran (control) with 17.4% protein content (n =6) (Bioassay I). Refer to
Table 1 for the diets composition.
C. Adamaki-Sotiraki et al.
Journal of Environmental Management 355 (2024) 120545
4
providing high energy along with good fatty acid composition for live-
stock animals (Rasane et al., 2015).
Concerning the larval development time for the larvae reared on
diets of group A, the larvae reared on A2, A3, A4 and A5 developed
faster (<50 d), while for the larvae reared on A6 and A7 longer devel-
opment time was recorded (>68 d). The larvae reared on diet B3 grew
fast (46 d), while the slower development time was recorded for larvae
reared on diets B5, B6 and B7 (>65 d). Concerning the larval develop-
ment time for both diets of group A and group B, the shortest larval
development time was observed for the larvae reared on diets A2, A3,
B3, and B4 (<50 days), while the longest larval development time was
recorded for the larvae reared on diets B7, A5, A7, B6, and B5 (>65
days). These variations indicate the inuence of diet composition on the
duration of larval development. Relevant studies reported that larvae
are able to adjust the consumption of nutrients by selecting from a va-
riety of ingredients in substrates (Morales-Ramos et al., 2011, 2020).
Thus, in specic substrates more time may be needed to uptake the
mandatory nutrients.
After the termination of the experiment, feed conversion ratio (FCR),
efciency of ingested food conversion (ECI, %) and specic growth rate
Fig. 2. Average survival (mg) of Tenebrio molitor larvae reared on six diets or
wheat bran and yeast (control) with 22.5% protein content (n =6) (Bioassay I).
Refer to Table 1 for the diets composition.
Table 2
Final larval survival (%), nal individual larval weight (mg), development time (days), feed conversion ratio (FCR), efciency of ingested food conversion (ECI, %) and
specic growth rate (SGR, %) of Tenebrio molitor larvae reared on six diets with 17.4% protein content or wheat bran (control) (group A) and on six diets with 22.5%
protein content or wheat bran and yeast (control) (group B) (n =6) (Bioassay I). Refer to Table 1 for the diets composition of diets. The upper case letters represent
statistical differences among diets of the same protein group, while the lowercase letters indicate differences among all diets from both protein groups.
Diets Final larval survival (%) Final Individual larval weight (mg) Development time (days) FCR ECI SGR
A1 72.00 ±8.20 ABab 132.59 ±11.98 ABab 45.17 ±1.95 Aab 2.69 ±0.26 Aab 37.47 ±3.43 Aa 10.70 ±0.30 Aab
A2 78.33 ±5.72 Aa 142.30 ±4.83 Aa 46.33 ±1.70 ABab 3.31 ±0.36 Aabc 30.48 ±3.38 ABab 10.57 ±0.36 Aabc
A3 70.67 ±11.08 ABab 142.41 ±13.59 Aa 45.17 ±2.03 Aab 4.09 ±0.68 Abcde 24.97 ±3.84 Babc 10.82 ±0.47 Aab
A4 54.00 ±12.77 BCbcde 129.32 ±17.43 ABab 50.83 ±5.01 ABbc 4.79 ±1.23 ABcdef 22.08 ±5.80 BCbcd 9.82 ±0.70 ABbcde
A5 63.33 ±13.31 ABCabc 84.74 ±6.12 Ccde 73.50 ±0.50 Cde 7.51 ±1.82 BCef 13.90 ±2.95 Ccde 6.11 ±0.17 Cf
A6 77.67 ±11.96 Aa 101.19 ±5.48 BCbcd 53.50 ±2.57 ABbcd 3.79 ±0.55 Aabcd 26.84 ±3.47 ABab 8.54 ±0.57 BCcdef
A7 50.00 ±17.48 Cbcde 94.56 ±13.62 Cbcde 68.33 ±7.25 Ccde 9.47 ±4.02 Cf 12.55 ±5.89 Cde 6.66 ±0.56 Cdef
B1 81.00 ±10.18 Aa 128.20 ±6.62 Aabc 41.50 ±0.50 a 2.26 ±0.23 Aa 44.67 ±4.19 Aa 11.84 ±0.20 Aa
B2 65.00 ±10.86 Aabc 133.51 ±15.87 Aa 50.50 ±4.72 b 3.59 ±0.82 ABabc 28.96 ±6.11 ABab 9.86 ±0.75 BCbcd
B3 67.33 ±13.78 Aab 136.82 ±17.47 Aa 46.50 ±3.55 ab 3.94 ±0.87 ABbcde 26.71 ±7.61 ABCabc 10.81 ±0.67 ABab
B4 40.67 ±19.00 BCcde 132.73 ±10.70 Aa 49.50 ±3.95 ab 7.59 ±3.25 BCef 15.06 ±5.69 BCDcde 9.94 ±0.80 BCbcd
B5 38.33 ±12.36 BCde 67.05 ±6.40 Bcd 65.83 ±8.09 cde 12.80 ±2.53 CDf 8.11 ±1.82 De 6.64 ±0.70 CDef
B6 56.33 ±12.23 ABbcd 55.03 ±5.27 BCde 67.67 ±7.93 de 9.23 ±2.11 BCDf 11.26 ±2.26 CDde 6.05 ±0.93 Df
B7 22.33 ±8.43 Ce 39.97 ±1.94 Ce 76.83 ±1.77 e 42.46 ±19.24 Df 2.72 ±1.04 Ee 4.89 ±0.22 Df
Statistical analysis among diets of the same protein group (Group A)
df 6 6 6 6 6 6
P 0.005 <0.001 <0.001 <0.001 <0.001 <0.001
Statistical analysis among diets of the same protein group (Group B)
df 6 6 6 6 6 6
P <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
Statistical analysis among all diets from both protein groups
df 13 13 13 13 13 13
P <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
Fig. 3. Average individual larval weight (mg) of Tenebrio molitor larvae reared
on six diets or wheat bran (control) with 17.4% protein content (n =6)
(Bioassay I). Refer to Table 1 for the diets composition.
Fig. 4. Average individual larval weight (mg) of Tenebrio molitor larvae reared
on six diets or wheat bran and yeast (control) with 22.5% protein content (n =
6) (Bioassay I). Refer to Table 1 for the diets composition.
C. Adamaki-Sotiraki et al.
Journal of Environmental Management 355 (2024) 120545
5
(SGR, %/day) were calculated (Table 2). Regarding the larvae reared on
diets of group A, FCR ranged from 2.7 to 9.5, ECI ranged from 12.5 to
37.5% and SGR ranged from 6.1 to 10.8%/day. Regarding the larvae
reared on diets of group B, FCR ranged between 2.3 and 42.5, ECI ranged
between 2.7 and 44.7%, and SGR ranged between 4.9 and 11.8 %/day.
Comparing FCR, ECI, and SGR for larvae reared on both groups of diets
(group A and group B) the larvae reared on diets A2, B2, and A6 pre-
sented the lowest FCR (<3.8), however the larvae reared on B7, B5, A7,
B6, B4, A5 and A4 presented the highest FCR (>4.8). Concerning the
ECI, the most efcient feed conversion was achieved for larvae fed on
diets A2, B2, A6 B3 and A3 (30.5–25%), while the less efcient feed
conversion was observed for larvae reared on diets B7, B5, B6, A7, A5
and B4 (<15%). The highest SGR was recorded for larvae reared on diets
A3, B3 and A2 (10.6–10.8 %/day), while the lowest SGRs were recorded
for larvae raised on diets B6, A5, B5 A7 and A6 (<8.5 %/day). In general,
insects feed on highly proteinaceous substrates to support the growth
and survival of the larval stage (Cohen, 2003). However, Rumbos et al.
(2020) recently reported that high amount of protein in the substrates
tested may not necessarily promote the growth of T. molitor larvae. Our
results showed that comparing all diets from both proteinic levels, the
performance of T. molitor larvae reared on A2 diet signicantly
augmented for all the parameters tested, i.e., individual larval weight
(mg), larval survival (%), development time (days), FCR, ECI, and SGR.
Our results also showed that diets with high protein content for which
one of the ingredients was the pea byproduct, were not able to efciently
support larval growth and performance. Pea is known to contain sapo-
nins that are associated with insecticidal effects (Ellen et al., 2007; Singh
and Kaur, 2018). Similarly, other researchers attributed the poor per-
formance of T. molitor larvae reared on diets that contained pea
byproducts to antinutritional factors that pea may contain (Kr¨
oncke and
Benning, 2023; Rumbos et al., 2022). Interestingly, in our study, the
comparison of larval growth on diets of both proteinic levels revealed
that diets contained lower percentage of pea (<50%) performed better
in terms of nal individual larval weight and FCR compared to diets that
contained an increased amount of pea byproducts.
The increasing market demand for insect-based products propel the
insect production to upscale from small farming to industrial scale. The
second bioassay conrmed the data of the rst bioassay (Table 3; Fig. 5),
as the diets that contained lupin, triticale, and oat byproducts were
suitable for the rearing of T. molitor larvae, resulting in high nal larval
weight. Indicatively, the highest weights were recorded for larvae
reared on the diets A2 and A3 (95.6 and 97.6 mg, respectively). The diet
A7 which contained barley and pea byproducts was not able to support
larval growth. Signicant differences among the tested diets were also
recorded for the total harvest of larvae (g). The highest total harvest was
recorded for A2 diet (895.3 g and 910.1 g, respectively), followed by A3
diet (749.3 g). The lowest total harvest was recorded for diet A7 (410.2
g). In addition, for all diets tested, the FCR ranged from 2.3 to 5.2. The
lowest FCR values were recorded for the larvae reared on diets A1, A2
and A3 while the higher FCR was calculated for the larvae reared on A7
diet. Our results reveal that, although laboratory bioassays are able to
provide a general overview of larval growth on specic byproducts and
diets, the results are not always easy to be extrapolated to industrial
production. Indicatively, diets A2 and A3 seemed to support larval
growth in terms of individual larval weight, at laboratory scale bioassay,
however, the total nal harvest of larvae was signicantly higher only
for the diet that contained lupin and triticale (diet A2). Although the
need for larval performance evaluation from a large-scale production
perspective is mandatory, data related to this topic are scarce (Deruytter
et al., 2019, 2021; Adamaki-Sotiraki et al., 2023). In the few relative
studies, Deruytter et al. (2019), highlighted the importance of optimi-
zation of the number of produced mealworms in each crate in an insect
industry early and accurately. While, Adamaki-Sotiraki et al. (2023)
aimed to draw the attention on large scale bioassays concerning the
genetic material of T. molitor for the production of insects at industrial
scale.
5. Conclusions
In conclusion, this study underscores the signicance of integrating
circular economy principles into insect farming practices. The incorpo-
ration of byproducts from cereal and legume production into compound
diets has been demonstrated to efciently support the growth and per-
formance of T. molitor larvae. The inclusion of lupin and triticale or lupin
and oat byproducts, irrespective of protein levels, has emerged as
particularly effective in promoting larval development. Furthermore,
our ndings emphasize the importance of considering broader factors,
such as total larval harvest, when evaluating the suitability of diets for
industrial insect production. The transition from small-scale farming to
large-scale industrial production demands a nuanced understanding of
key performance indicators to ensure the scalability and efciency of the
chosen diets. Moving forward, it is mandatory that future studies delve
deeper into the performance of T. molitor larvae on compound diets
composed of locally sourced agricultural byproducts. This approach will
enable researchers to address emerging challenges and rene diet for-
mulations for optimal outcomes in industrial-scale insect production.
Funding
This work was supported by the EU-PRIMA program project
ADVAGROMED (Prima, 2021- Section 2). The project is funded by the
General Secretariat for Research and Innovation of the Ministry of
Development and Investments of Greece under the PRIMA Program.
PRIMA is an Art.185 initiative supported and co-funded under Horizon
2020, the European Union’s Program for Research and Innovation.
CRediT authorship contribution statement
Christina Adamaki-Sotiraki: Conceptualization, Data curation,
Formal analysis, Funding acquisition, Investigation, Methodology,
Project administration, Resources, Software, Supervision, Validation,
Table 3
Final individual larval weight (mg), total harvest (g), and feed conversion ratio
(FCR) of Tenebrio molitor larvae reared on three diets or wheat bran (control)
with 17.4% protein content (n =3) (Bioassay II). Refer to Table 1 for the diets
composition.
Diets Final Individual larval weight (mg) Total harvest (g) FCR
A1 113.36 ±1.64 a 895.27 ±40.40 a 2.35 ±0.13 a
A2 95.59 ±2.44 b 910.07 ±5.73 a 2.31 ±0.02 a
A3 97.58 ±2.89 b 749.34 ±22.05 b 2.80 ±0.08 a
A7 27.70 ±4.48 c 410.20 ±58.83 c 5.17 ±0.74 c
df 3 3 3
P <0.001 <0.001 <0.001
Fig. 5. Average individual larval weight (mg) of Tenebrio molitor larvae reared
on three diets or wheat bran (control) with 17.4% protein content (n =3)
(Bioassay II). Refer to Table 1 for the diets composition.
C. Adamaki-Sotiraki et al.
Journal of Environmental Management 355 (2024) 120545
6
Visualization, Writing – original draft, Writing – review & editing.
Despoina Choupi: Data curation, Formal analysis, Methodology, Soft-
ware, Validation, Writing – original draft, Writing – review & editing.
Mariastela Vrontaki: Data curation, Formal analysis, Methodology,
Software, Writing – original draft, Writing – review & editing. Christos
I. Rumbos: Conceptualization, Data curation, Formal analysis, Meth-
odology, Software, Supervision, Validation, Visualization, Writing –
original draft, Writing – review & editing. Christos G. Athanassiou:
Conceptualization, Data curation, Formal analysis, Investigation,
Methodology, Resources, Software, Supervision, Validation, Visualiza-
tion, Writing – original draft, Writing – review & editing.
Declaration of competing interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Data availability
Data will be made available on request.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jenvman.2024.120545.
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