Journal of Environmental Management 217 (2018) 668e676

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Journal of Environmental Management

journal homepage: www.elsevier.com/locate/jenvman

Research article

Efficient co-conversion process of chicken manure into protein feed

and organic fertilizer by Hermetia illucens L. (Diptera: Stratiomyidae)
larvae and functional bacteria
Xiaopeng Xiao a, Lorenzo Mazza a, b, Yongqiang Yu a, Minmin Cai a, Longyu Zheng a,
Jeffery K. Tomberlin c, Jeffrey Yu d, Arnold van Huis e, Ziniu Yu a, Salvatore Fasulo b,
Jibin Zhang a, *
a
State Key Laboratory of Agricultural Microbiology, National Engineering Research Center of Microbial Pesticides, College of Life Science and Technology,
Huazhong Agricultural University, Wuhan, 430070, PR China
b
University of Messina, Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, Via F. Stagno D'Alcontres n.31, 98166 Messina,
Italy
c
Department of Entomology, Texas A&M University, USA
d
Texas Academy of Mathematics and Science, University of North Texas 1155 Union Circle #311070, Denton, TX 76203-5017, USA
e
Laboratory of Entomology, Wageningen University & Research, Wageningen, The Netherlands

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

Article history: A chicken manure management process was carried out through co-conversion of Hermetia illucens L.
Received 28 December 2017 larvae (BSFL) with functional bacteria for producing larvae as feed stuff and organic fertilizer. Thirteen
Received in revised form days co-conversion of 1000 kg of chicken manure inoculated with one million 6-day-old BSFL and
17 March 2018
109 CFU Bacillus subtilis BSF-CL produced aging larvae, followed by eleven days of aerobic fermentation
Accepted 29 March 2018
inoculated with the decomposing agent to maturity. 93.2 kg of fresh larvae were harvested from the
B. subtilis BSF-CL-inoculated group, while the control group only harvested 80.4 kg of fresh larvae.
Chicken manure reduction rate of the B. subtilis BSF-CL-inoculated group was 40.5%, while chicken
Keywords:
Hermetia illucens L. larvae
manure reduction rate of the control group was 35.8%. The weight of BSFL increased by 15.9%, BSFL
Functional bacteria conversion rate increased by 12.7%, and chicken manure reduction rate increased by 13.4% compared to
Chicken manure reduction rate the control (no B. subtilis BSF-CL). The residue inoculated with decomposing agent had higher maturity
Feed stuff (germination index >92%), compared with the no decomposing agent group (germination index ~86%).
Organic fertilizer The activity patterns of different enzymes further indicated that its production was more mature and
Waste management stable than that of the no decomposing agent group. Physical and chemical production parameters
showed that the residue inoculated with the decomposing agent was more suitable for organic fertilizer
than the no decomposing agent group. Both, the co-conversion of chicken manure by BSFL with its
synergistic bacteria and the aerobic fermentation with the decomposing agent required only 24 days. The
results demonstrate that co-conversion process could shorten the processing time of chicken manure
compared to traditional compost process. Gut bacteria could enhance manure conversion and manure
reduction. We established efficient manure co-conversion process by black soldier fly and bacteria and
harvest high value-added larvae mass and biofertilizer.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction of chicken manure have been produced. Chicken manure is rich in

Chicken production has increased due to the increasing demand
for meat and eggs (Nie et al., 2015). Subsequently, massive amounts
* Corresponding author.
E-mail address: zhangjb@mail.hzau.edu.cn (J. Zhang).
readily biodegradable organic matter, nutrients, and pathogens.
However, if poorly managed, chicken manure can become an
important source of pollution (Wang et al., 2014; Nie et al., 2015).
Manure is traditionally composted to kill pathogens and fertilize
crops (Guerrini et al., 2017; Abreu-Junior et al., 2017). However, the
low C/N ratio, high moisture content, nasty odor, long decompo-
sition period, nutrient loss, and the formation of phytotoxic

https://doi.org/10.1016/j.jenvman.2018.03.122
0301-4797/© 2018 Elsevier Ltd. All rights reserved.
 X. Xiao et al. / Journal of Environmental Management 217 (2018) 668e676
compounds have become challenges for current composting
methods (Chang and Chen, 2010; Wang et al., 2013). Bulking agents,
such as sawdust and rice chaff, are usually added to manure com-
posts to reduce moisture and to achieve a suitable C/N ratio.
However, these bulking agents have become increasingly expensive
for composting as they are used as alternative energy resources
(Chang and Chen, 2010; Zhu et al., 2012). For that reason, alterna-
tive means to convert chicken manure into fertilizer production
should be explored.
Many insects efficiently degrade organic waste into nutrients
(Zheng et al., 2012; Ci ckova et al., 2015). Hermetia illucens L.
(Diptera: Stratiomyidae), also known as black soldier fly (BSF), live
outdoors and is often associated with livestock (Li et al., 2011). BSF
can be found in decaying organic wastes, such as animal manure
and plant material, and exhibits antimicrobial peptide activity
against pathogens in those wastes (Liu et al., 2008; Lalander et al.,
2015; Elhag et al., 2017). BSF not only reduces the accumulation of
manure and the nasty smell, it also inhibits the proliferation of
house flies (Zheng et al., 2013). These processes greatly reduce
environmental impact caused by livestock manure. Zhou et al.
(2013) found that black soldier fly larvae (BSFL) of the Wuhan
strain weighed 14.4e37.0% more than the BSFL Guanghzhou strain
and BSFL Texas strain, respectively. The BSFL Wuhan strain reduced
dry matter 46.0% (swine), 40.1% (dairy), and 48.4% (chicken) more
than the Guangzhou strain and 6.9%, 7.2%, and 7.9% more than the
Texas strain. Gut microbes play important roles in insect nutrition
and colonization resistance against invasion of exotic microbes
(Dong et al., 2009). Bacteria isolated from the BSFL gut can increase
the weight of prepupae and pupae, and shorten the number of days
from hatching to the prepupal stage (Yu et al., 2011). Therefore,
BSFL can be used as animal feed and for biodiesel production. The
residue of the organic waste is a suitable substrate for aerobic
fermentation during composting (Li et al., 2015; Rehman et al.,
2017).
Composting or aerobic fermentation accelerates the natural
decomposition of organic debris by microorganisms (bacteria, ac-
tinomycetes and fungi) under controlled environmental conditions.
Inoculation at the appropriate time can promote organic waste
maturity and considerably shorten the period of decomposition
(Zeng et al., 2010; Jurado et al., 2015). However, not much is known
about the Insect-Bacteria-Manure digestion system in reducing
manure pollution.
In this study, we tried to develop an efficient and high-value co-
conversion technology of chicken manure by BSFL and bacteria in a
poultry farm. Our aim was to maximize BSFL harvest and waste
mass reduction by using synergistic bacteria in the first stage and
shorten the maturity time through inoculation with a decomposing
agent to promote aerobic fermentation in the second stage.
Important process parameters were monitored such as changes in:
the conversion rate by the larvae; the reduction rate of chicken
manure; physical and chemical properties; the occurrence of
phytotoxicity, and the presence of microbial enzymes.
2. Material and methods
2.1. Raw materials
Hermetia illucens L. Wuhan strain larvae used in this study were
bred at Wuhan ChaoTuo Ecology Agricultural Ltd. (30.090309 N,
114.349939 E, Wuhan, Hubei, China). The strain was collected and
domesticated by our team in the State Key Laboratory of Agricul-
tural Microbiology, Huazhong Agricultural University, Wuhan,
China (Zhou et al., 2013). Eggs were incubated in a greenhouse at
28
C with 60%e70% relative humidity. For the first 6 days, the
larvae were fed with a diet based on bran, and then separated from
669
the bran residue by sieve (2.00 mm).
Chicken manure was collected from a poultry farm located in
Wuhan ChaoTuo Ecology Agricultural Ltd. (Wuhan, Hubei, China).
The water content of chicken manure was 76%.
Bacillus subtilis BSF-CL strain was isolated from the guts of
surface-disinfected larvae. The bacteria of the decomposing agent
(Bacillus methylotrophicus DF07H, B. subtilis DF04H, B. subtilis RX07,
B. amyloliquefaciens RX01, Pseudomonas pachastrellae D-61;
1:2:1:1:2) were isolated from the thermophilic phase of chicken
manure composting. Previous studies had shown that B. subtilis
BSF-CL strain was the optimal one of seven strains gut bacteria on
BSFL weight gain rate and manure residue rate (Zhang, 2014), and
the decomposing agent was the optimal one of four group agents
on promoting chicken manure maturity (Wang, 2013).
All bacterial colonies grown on petrie dish with Luria-Bertani
(LB) agar medium (tryptone 10 g L1, yeast extract 5 g L1, NaCl
10 g L1, agar 18 g L1, distilled water 1000 mL) were suspended in
100 mL of LB liquid medium (tryptone 10 g L1, yeast extract 5 g L1,
NaCl 10 g L1, distilled water 1000 mL) and left under stirring for
36 h at 28
C, respectively. After 36 h, each bacterium was washed
three times with phosphate-buffered saline for 15 s at 8000 r
min1, and eventually suspended in 30 mL of sterile water, until a
final concentration of 1  109 CFU mL1 was obtained.
2.2. Co-conversion experiment design
The co-conversion experiment began by introducing one million
larva inocula and 1 L of B. subtilis BSF-CL (1  109 CFU mL1) to
1000 kg of fresh chicken manure in a cement pool
(3.0 m  4.0 m  0.2 m), namely larvae-inoculum-treated conver-
sion (LIC), in which the fresh chicken manure mixed with B. subtilis
BSF-CL inoculum was flatly piled and the larvae was evenly inoc-
ulated into the surface of the fresh chicken manure for them to
grow freely. Apart from LIC, one million larva inocula and 1 L of
sterile water were inoculated into 1000 kg of fresh chicken manure
(LC) as control. Each treatment was performed in triplicate.
Samples weighting approximately 200 g were collected from
ten points of each pool daily during the co-conversion experiment.
All the samples were preserved at 20
C for subsequent analysis.
2.3. Aerobic fermentation design
After 13 days of conversion, the aging larvae were isolated from
the conversion system according to their aggregation characteris-
tics. Six thousand kilograms chicken manure residue was collected
following the way of LIC treatment and divided into six peak-
shaped composts (with a bottom diameter of 2.4 m and a height
of 1.0 m) on the ground in a rainproof workshop without walls.
Three piles, inoculated 1 L decomposing agent (1  109 CFU mL1)
per pile, were named decomposing agent-inoculum-treated pile
(DAITP). Another three piles with 1 L sterile water inoculated into
manure residue were named water-treated pile (WP). The aerobic
fermentation process continued for 11 days, during which six
composts were turned upside down every day.
Samples weighing approximately 200 g were collected from
40 cm below the surface of each pile daily during the aerobic
fermentation stage. All the samples were preserved at 20
C for
the subsequent analysis.
2.4. Analytical method of physical and chemical properties
During the co-conversion, temperature was measured daily
using a 25 or 100 cm-long tubular alcohol thermometer (ZDR21,
Hangzhou Zeda Equipment Co, Ltd. China), which was inserted into
three sites of each pool 4 cm below the surface or each pile 40 cm
670 X. Xiao et al. / Journal of Environmental Management 217 (2018) 668e676
below the surface. The moisture content and dry matter of the
subsamples were obtained by drying at 105
C in an oven for 12 h.
The pH values in 1:9 (w/v) sample suspensions were assessed in an
electronic pH meter (Mettler-Toledo Instruments (Shanghai) Co.
Ltd. Shanghai, China). Total phosphorus, total potassium, total
Kjeldahl nitrogen and total organic carbon were measured in
accordance with the method of China National Standard for organic
fertilizer: NY525-2012 (Zhang et al., 2012). Sample-free blank
controls were also analyzed to exclude background effects.
2.5. Analytical method of microbial enzyme activities
The assayed enzymes in all the samples collecting during the
thirteen days co-conversion and eleven days aerobic fermentation
included: catalases to reduce the accumulation of reactive oxygen
radicals; ureases involved in the peptide bond hydrolysis of organic
matter and decomposition of urea into ammonia and carbon di-
oxide; invertases relating to the levels of humus and water-soluble
organic matter; and polyphenoloxidases relating to the level of
humification (Zhu et al., 2012). All the samples were analyzed in
triplicate assays for the following microbial enzyme activities
through the methods described by Zhu et al. (2012). Sample-free
blank controls were also analyzed to exclude background effects.
2.6. Analytical method of phytotoxicity in samples
Chinese cabbage and rape seeds were used in the germination
experiments to assay the toxicity of the samples. All the samples
were analyzed in triplicate assays for the following phytotoxicity
analysis. Aqueous extracts were prepared by shaking 10% (w/v)
sample suspensions for 30 min at 180 r min1 and then subjected to
15 min standing and 10 min centrifugation at 6000 r min1. The
aliquot (5 mL) of each supernatant was poured into a Petri dish
(9 cm diameter) with filter paper. Twenty seeds were evenly
distributed into the Petri dish. The seeds were incubated for 3 days
at 25
C in the dark. Percent germination and mean root length in
each dish were then assessed. The same volume of distilled water
was used as control for seed germination. The ratios of percent
germination or mean root length in each extract-inclusive dish over
those in the control were defined as relative germination rate (%)
and relative root growth (%), respectively. The product of the two
ratios and 100% was defined as germination index (GI; Romero
et al., 2013; Villar et al., 2016) for the phytotoxicity of a given
sample. The indexes can reflect the overall maturity of composting
materials (Ravindran et al., 2016).
2.7. Statistical analyses
All the presented results are average values form triplicate ex-
periments. The results of all experiments were analyzed by paired
Student's t-test to determine the effect of inoculation during
different stages. The maximum difference among the triplicate
results was 5%. The statistical analyses were performed in SPSS 22.0
software.
3. Results and discussion
3.1. The co-conversion by BSFL and synergistic bacteria
After 13 days co-conversion process, 93.2 kg of fresh larvae were
harvested from the LIC group, while the LC group only harvested
80.4 kg of fresh larvae. Chicken manure reduction rate of the LIC
group was 40.5%, while chicken manure reduction rate of the LC
group was 35.8% (Table 1). The material reduction rate of LIC group
was greater than LC group by 13.3% (P ¼ 0.089). The weight of BSFL
Table 1
Conversion effects during the co-conversion stage.
Groups Material reduction BSFL conversion BSFL weight gain
rate (%) rate (%) rate (%)
LC 35.8 ± 1.1a 10.2 ± 0.1a 0
LIC 40.5 ± 0.8a 11.5 ± 0.2b 15.9
Results are all indicated as means ± SE. Means in the same row with different letters
are significantly different. (paired Student's t-test, n ¼ 3, P < 0.05).
increased by 15.9% compared to the LC group. The BSFL conversion
rate of the LIC group increased by 12.7% compared to the LC group
(P ¼ 0.031).
The results of the co-conversion by BSFL and synergistic bacteria
indicated that fresh chicken manure co-converted by BSFL and
B. subtilis BSF-CL inoculums, can rapidly decrease accumulated
chicken manure. Also more mature larvae could be harvested as a
protein source. Sheppard et al. (1994) showed that black soldier fly
larvae reduced manure by  50% in a facility housing 460 hens.
These differences may be due to different manure sources and
climatic conditions. However, as in the present study, B. subtilis BSF-
CL inoculums accelerated BSFL growth and reduced chicken
manure accumulation, while more larvae were harvested at the
same conditions. The harvested larvae are an available protein
source for production animals (Bosch et al., 2014) or biodiesel
(Zheng et al., 2012). These are emerging and environment-friendly
resources. The inoculated symbiotic bacteria maybe help insects to
digest non-digestible nutrients, provide nutrition that the black
soldier fly could not synthesize, and protect insects from predators,
parasitoids, and pathogens (Laughton et al., 2011; Douglas, 2015).
Yu et al. (2011) demonstrated that inoculating poultry manure with
bacteria from black soldier fly larvae influences the growth and
development of conspecific larvae feeding on the manure. Micro-
organisms enzymes, small molecules, and nutrients produced by
microorganisms are necessary for insects, thus insects can grow
well and eat more waste. There is growing evidence that intestinal
bacteria are important beneficial partners of their metazoan hosts.
Shin et al. (2011) showed that the pyrroloquinoline
quinoneedependent alcohol dehydrogenase activity of a
commensal bacterium, Acetobacter pomorum, modulates insulin/
insulin-like growth factor signaling in Drosophila to regulate host
homeostatic programs controlling developmental rate, body size,
energy metabolism, and intestinal stem cell activity. Storelli et al.
(2011) revealed that Lactobacillus plantarum, a commensal bacte-
rium of the Drosophila intestine, promotes its host systemic growth
by modulating hormonal signals through TOR-dependent nutrient
sensing. Yamada et al. (2015) showed that Issatchenkia orientalis
promotes Drosophila melanogaster amino acid harvest to rescue the
lifespan of undernourished flies. Hermetia illucens L. maybe have
the same or similar mechanism, however, the mechanism of in-
testinal bacteria (for example B. subtilis BSF-CL) that promotes BSFL
conversion requires further research. In the co-conversion phase,
nearly half of the accumulated chicken manure was consumed by
BSFL and bacteria. This method may be a feasible technique to solve
food, feed, energy, and environmental crises in the future.
3.2. Physical and chemical parameter
During the co-conversion phase, daily temperature increased
rapidly in the group inoculated with larvae and B. subtilis BSF-CL
strain on the third day (Fig. 1A). It was greater than the group
inoculated with larvae only. At the fourth to eighth day, the tem-
perature of the LIC group was approximately 45
C. Its daily tem-
perature then decreased steadily to 30
C. However, the daily
temperature of the LC group increased slowly, and the highest
 X. Xiao et al. / Journal of Environmental Management 217 (2018) 668e676 671

Fig. 1. Changes of physical and chemical parameters of chicken manure in the conversion phase. B, Larvae-inoculum-treated. △, Only larvae treated,  , Ambient temperature.
(A) Daily temperature. (B) Moisture. (C) pH value. (D) Organic matter content. (E) Total nutrient content. Error bars: SE of the mean from three replicates (paired Student's t-test,
n ¼ 3, P < 0.05).

temperature was observed on the ninth day (43
C), and then
decreased steadily to approximately 30
C (P ¼ 0.005). The ambient
temperature was approximately 25
C during the conversion stage.
After 13 days of co-conversion, the manure residue became gran-
ular, and dark brown and its water content decreased. During the
first eighth days, the moisture of the manure slowly decreased in
both treatments. After the eighth day of conversion, the moisture of
the LIC group decreased significantly compared with that in the LC
group (P < 0.001). The moisture of the LIC group decreased to 52.3%
at the 12th day and remained stable. The LC group decreased to
58.6% on the 13th day (Fig. 1B). The pH value trends of the treat-
ments were similar (P ¼ 0.132). The pH value of the LC group was
high before the fifth day of conversion, and then it decreased, with
a value approximately 8.4 on the 13th day (Fig. 1C). The organic
matter content of the LIC group decreased by 13.2%, while that of
the LC group decreased by 8.8% (P ¼ 0.034, Fig. 1D). After 13 days
co-conversion, the total nutrient content of the LIC group was lower
than the LC group (P < 0.001), but organic matter contents in both
groups were more than 8.8% (Fig. 1E).
During the aerobic fermentation stage, the daily temperature of
the DAITP group increased rapidly from the initial temperature.
Moreover, it increased to 56
C on the 3rd day. The temperature was
maintained at 55e66
C during days three to eight. Then, the
temperature decreased to the bottom and fluctuated at low levels
after the 11th day. However, the daily temperature of the WP group
increased relatively slow (P ¼ 0.186). It increased to 56
C on the
fifth day. Moreover, the temperature was maintained at 55e65
C
during days 5e12. After 15 days of fermentation, the temperature
decreased at its lowest level (Fig. 2A). The moisture content in the
DAITP group decreased faster from the initial level by 24e25%
compared with that in the WP group (P < 0.001). The moisture
content of the WP group was maintained at 29e30% in the end
(Fig. 2B). The pH value of the DAITP group also increased faster at
days one to three. However, it decreased at the fifth day to remain
at 8.0e8.1. The pH value of the WP group was higher than that of
the DAITP group (P ¼ 0.001), and remained at 8.6e8.9 from days
3e11 (Fig. 2C). The organic matter content of the DAITP group was
lower than that of the WP group (P < 0.001). However, the values of
the two treatments were larger than 45% (Fig. 2D). The total
nutrient content of the DAITP group was also lower than that of the
WP group (P < 0.001). Both groups remained at a steady state after
seven days of fermentation, and the nutrient values were more
than 9.2% after the aerobic fermentation stage (Fig. 2E). The C/N
ratio of DAITP group was also lower than that of the WP group
672 X. Xiao et al. / Journal of Environmental Management 217 (2018) 668e676

Fig. 2. Changes of physical and chemical parameters of residue in aerobic fermentation stage. B, Decomposing agent-inoculum-treated. △, water-treated.  , Ambient
temperature. (A) Daily temperature. (B) Moisture. (C) pH value. (D) Organic matter content. (E) Total nutrient content. (F) C/N. Error bars: SE of the mean from three replicates
(paired Student's t-test, n ¼ 3, P < 0.05).

(P ¼ 0.009). The C/N ratio of DAITP group decreased from 62.65% to
15.58%, While WP group decreased from 60.33% to 16.36% (Fig. 2F).
Physical and chemical parameters are important for organic
fertilizers. Insect larvae, like BSFL and Musca domestica L. (Diptera:
Muscidae) maggots, can efficiently convert organic waste to nu-
trients. However, the prepupae of BSF have the advantage that they
separate themselves from the organic waste residue. During co-
conversion the physical and chemical parameters of organic
waste changed and BSFL and bacteria metabolism led to an increase
in manure temperature and release of ammonia. The temperature
was higher in the treatment without inoculation and also in the
treatment using M. domestica maggots (Zhu et al., 2012). Inoculant
decomposition considerably shortened the aerobic fermentation
period to maturity by altering the physical and chemical parame-
ters of chicken manure. The aerobic fermentation stage tempera-
ture maintained at 55e66
C for six consecutive days was sufficient
for pathogen elimination from the DAITP group. This because a
composting temperature exceeding 55
C for more than 3 days (Yu
et al., 2007) or 50
C for 8 consecutive days (Bao et al., 2010) was
recommended to disinfect animal and plant pathogens in waste
materials. And we believe that BSFL antimicrobial peptides
exhibited in organic wastes (Elhag et al., 2017) also is an extra
mortality factor for some pathogens. The other parameter results
indicated that they maintained a steady state, in agreement with
the China National Standard for organic fertilizer: NY 525e2012.
3.3. Changes in the phytotoxicity
In the germination experiments, the Chinese cabbage and rape
seeds in the LIC group sample extracts started to germinate rapidly
after five days (Fig. 3A and B). After 13 days of co-conversion, the
germination index (GI) of Chinese cabbage and rape seeds were
66.6% and 70.5%, respectively. However, the Chinese cabbage and
rape seeds in the LC group sample extracts both failed to germinate
before day seven. And the GIs of both seeds were significantly lower
than the LIC group (P < 0.001).
The Chinese cabbage seeds GI of the DAITP group increased
faster from the initial value of 66.2% to the final value of 92.3% than
that of the LP group (P < 0.001, Fig. 3C). The Chinese cabbage seeds
GI of the WP group increased from 66.2% to 86.3% during the aer-
obic fermentation stage. This increase was also observed in the rape
seeds GI (Fig. 3D). The GI of the DAITP group increased from 69.4%
at day one to 97.2% at day ten and remained stable, while the WP
group increased from 69.4% to 87.0% in the same period (P < 0.001).
At the end of the aerobic fermentation stage, the chicken manure
residue was completely decomposed (GI > 85%) in both treatments.
 X. Xiao et al. / Journal of Environmental Management 217 (2018) 668e676
Fig. 3. GI trends of Chinese cabbage seeds (A, C) and Rape seeds (B, D) in the aqueous extracts of 10% sample suspensions. A and B: GI trends during conversion phase, B,
Larvae-inoculum-treated. △, Only larvae treated. C and D: GI trends during aerobic fermentation stage, B, Decomposing agent-inoculum-treated. △, water-treated. Error bars: SE
of the mean from three replicates (paired Student's t-test, n ¼ 3, P < 0.05).
Therefore, the residue can be used as organic fertilizer.
Germination index is one of the commonly used parameters to
indicate the maturity of the material (Zorpas et al., 2018). Chinese
cabbage seeds were more sensitive to the sample extracts, irre-
spective of the LIC group or the LC group, than rape seeds. Zhu et al.
(2012) reported that Chinese cabbage seeds were more sensitive to
sample extracts than cucumber seeds. Given that a GI above 50% is
indicative of the maturity of composting (Ravindran et al., 2016);
the observations indicated that LIC was mature for the germination
of Chinese cabbage seeds (66.6%) and rape seeds (70.5%) on day 13.
To our knowledge it was shown the first time to obtain so higher
maturity at so short time using insect and bacteria. Specifically, the
chicken manure residue reached prime maturity after co-
conversion by BSFL and B. subtilis BSF-CL. Including the co-
conversion of BSFL and B. subtilis BSF-CL, and the aerobic fermen-
tation with decomposing agent, fresh chicken manure into organic
fertilizer only took 24 days. This process is more efficient than
natural composting, and also more than other biological treatment
methods for organic waste (Table 2). Therefore, this process saves
more time, space, and labor.
3.4. Activity patterns of different enzymes
In order to further verify the stability of the residue, we moni-
tored the microbial enzyme activity in the residue. Catalase activity
in the LIC group increased and then decreased during the co-
conversion stage (Fig. 4A) earlier than that of the LC group, and
the final value was lower than that of the LC group (P ¼ 0.118).
Polyphenoloxidase activity increased from the initial level of
82.6 mg PPg1 3 h1 to a maximum in the LIC group faster than in
the LC group during the co-conversion stage (P ¼ 0.032). The initial
and final values were extremely close (Fig. 4B). The urease activity
of the LIC group increased from 5549 mg NHþ 4 eN g
1
3 h1 to the
peak 12,757 mg NHþ 4 eN g1
3 h1
three days from the start, but
became consistently undetectable from day five onward. The LC
673
group increased from 5549.5 mg NHþ 4 eN g
1
3 h1 to the peak
þ 1 1
12,198.5 mg NH4 eN g 3 h three days from the start and became
consistently undetectable from day seven onward. The LC group
delayed for two days compared with the LIC group (P ¼ 0.096,
Fig. 4C). The invertase activity in the LIC group decreased faster
from an initial level of 270.9 (mL 0.1 M Na2S2O3g1 24 h1)
compared with that in the LC group during the co-conversion stage
(P < 0.001, Fig. 4D).
After 11 days of fermentation, enzymes activity further
confirmed that the chicken manure residue reached maturity.
Catalase activity of the DAITP group increased faster than in the WP
group. Furthermore, the peak value of the DAITP group was
significantly higher than the WP group. The value was lower than
that of the WP group (P < 0.001, Fig. 5A). Polyphenoloxidase activity
of the DAITP group increased faster from the initial level to the peak
than the WP group. Moreover, it also decreased faster from its
highest value to the lowest value than the WP group (P ¼ 0.811,
Fig. 5B). Urease activity of the DAITP group increased from 267.1 mg
NHþ 4 eN g
1
3 h1 to the peak at 2719.8 mg NHþ 4 eN g
1
3 h1 on
days one to three, but became consistently undetectable from day
five onward. Urease activity of the WP group increased from
267.1 mg NHþ 4 eN g
1
3 h1 to the peak 2517.4 mg NHþ4 eN g
1
3 h1
three days from the start and became consistently undetectable
from day seven onward. It was delayed for two days in comparison
with the DAITP group (P ¼ 0.148, Fig. 5C). Invertase activity of the
DAITP group was lower than the WP group throughout the aerobic
fermentation stage. The peaks appeared on the third day, and then
decreased to stable level (P < 0.001, Fig. 5D).
The enzyme activity is a reflection of microbe metabolic in-
tensity in the fermentation process (Xue and Huang, 2013). Our
observations are in agreement with the enzyme activity trends
from the initial to the mature stage in composts of other materials
(Zhu et al., 2012). At the end of aerobic fermentation, each enzyme
detected in our study became consistently undetectable or main-
tained low activity. This result indicated that the microbes in
674 X. Xiao et al. / Journal of Environmental Management 217 (2018) 668e676

Table 2
Organic waste traditional treatment methods and processing cycle.

Methods Processing cycle Reference

Swine, cow and chicken manures composting 60 days Wang et al., 2014
Pig manure composting ~200 days Vazquez et al., 2015
House fly conversion and composting 90 days Zhu et al., 2012
Home and vermicomposting ~200 days  et al., 2013
Lleo
Sheep bedding and cattle manure vermicomposting 148 days Cestonaro et al., 2017
Chicken manure composting with municipal sludge and solid waste 53 days Li et al., 2017
Organic waste vermicomposting 120 days Rodrigues et al., 2017

Fig. 4. Temporal activity patterns of different types of microbial enzymes of chicken manure in the conversion phase. B, Larvae-inoculum-treated. △, Only larvae treated. (A)
Catalase activity. (B) Polyphenoloxidase activity. (C) Urease activity. (D) Invertase activity. Error bars: SE of the mean from three replicates (paired Student's t-test, n ¼ 3, P < 0.05).

Fig. 5. Temporal activity patterns of different types of microbial enzymes of residue in aerobic fermentation stage. B, Decomposing agent-inoculum-treated. △, water-
treated. (A) Catalase activity. (B) Polyphenoloxidase activity. (C) Urease activity. (D) Invertase activity. Error bars: SE of the mean from three replicates (paired Student's t-test,
n ¼ 3, P < 0.05).
 X. Xiao et al. / Journal of Environmental Management 217 (2018) 668e676
chicken manure residue were maintained at a steady state. The
present study shown that enzyme activity, physical and chemical
parameters changed following BSFL and synergistic bacteria ac-
tivity. Compared Zhu et al. (2012) reported, the changes of enzyme
activity, physical and chemical parameters in the co-conversion
stage and aerobic fermentation stage were rapid, and that was
agree with their report on variation tendency, but need a shorter
time to reach a steady state.
4. Conclusions
Black soldier fly larvae (BSFL) conversion rate and chicken
manure reduction rate were improved by BSFL and B. subtilis BSF-CL
co-conversion. The incremental percentage of BSFL weight, BSFL
conversion rate and chicken manure reduction rate was 15.9%,
12.7% and 13.4%, respectively. The decomposing agent could effi-
ciently accelerate chicken manure residue decomposition. Activity
patterns of different enzymes further indicated that the production
process was completely mature and at a stable aerobic fermenta-
tion stage. Physical and chemical production parameters indicated
that the residue was suitable as organic fertilizer. The total time of
conversion from fresh chicken manure to organic fertilizer was
shortened to 24 days. To our knowledge it is shown for the first time
that co-conversion of chicken manure by composting through BSFL
and their synergistic bacteria, which is high-efficient and need less
time. The process will be a new strategy in chicken manure and
other organic wastes management.
Acknowledgements
This study was supported by the National Key R&D Program of
China (2017YFD0800200), the Fundamental Research Funds for the
Central Universities, China (Grant No. 2662016PY110), the Major
projects of Technological Innovation in Hubei Province, China
(2017ABA072), and the “Yellow Crane Talent Plan” project of
Wuhan City.
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