Journal of Environmental Management 196 (2017) 458e465

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Journal of Environmental Management

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

Research article

Cellulose decomposition and larval biomass production from the

co-digestion of dairy manure and chicken manure by mini-livestock
(Hermetia illucens L.)
Kashif ur Rehman a, b, Minmin Cai a, Xiaopeng Xiao a, Longyu Zheng a, Hui Wang a,
Abdul Aziz Soomro a, Yusha Zhou a, Wu Li a, Ziniu Yu a, 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, China
b
Livestock and Dairy Development Department, Government of Punjab, Pakistan

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

Article history: World trends toward the modern dairies intensification on large production units cause massive animal
Received 5 September 2016 manure production and accumulation. Improper handling of manure produced by industrial farm
Received in revised form operation greatly deteriorates the major environmental media including air, water and soil. The black
11 March 2017
soldier fly utilizes organic waste and converts it into larvae biomass to be used as livestock feed and into
Accepted 16 March 2017
Available online 22 March 2017
residues to be used as bio-fertilizer. However, due to the high ratio of cellulose, hemicellulose and lignin
in dairy manure, this conversion is difficult. Therefore, dairy manure treated with chicken manure was
digested by Hermetia illucens. In this paper, we found that the co-digestion process significantly
Keywords:
Biomass production
enhanced the larval production, waste mass reduction, rate of larvae conversion, feed conversion ratio,
Waste mass reduction nutrient reduction and fibers utilization. Whereas 40% dairy manure and 60% chicken manure group
Vermicomposting show better results than other manure mixtures and had a significantly increased the cellulose con-
Co-digestion sumption by 61.19%, hemicellulose consumption by 53.22% and lignin consumption by 42.23% compared
Cellulose with 49.89%, 49.77% and 31.95%, respectively, in the dairy-only manure group. Finally, scanning electron
Dairy manure microscopy was used to analyze the structural changes of dairy manure, chicken manure and their co-
digestion mixtures. The scan electron microscopy showed the deterioration in the structure of dairy
and chicken manure fibers by Hermetia illucens. Moreover, the carbon-nitrogen ratio was decreased in all
end products of post vermicomposting. The results suggest that the co-digestion of 40% dairy manure
with 60% chicken manure is an appropriate proportion for dairy manure management with the black
soldier fly.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Animal intensive farming, also called factory farming, has
developed rapidly due to the rise in the population and generates
large amounts of manure (Tao et al., 2016). The demand for milk
and meat is expected to be 70% and 58% higher in 2050 than their
demand in 2010 (Makkar et al., 2014). Therefore, there is a pressing
need for holistic research into strategies and technology for the
management and treatment of manure, which can ensure a sus-
tainable use of nutrients and the mitigation of environmental
* Corresponding author.
E-mail address: zhangjb@mail.hzau.edu.cn (J. Zhang).
impact while meeting the future needs of livestock industry, for
example, by reducing Greenhouse Gas Emissions, odors and NH3
emissions and by preventing the spread of diseases (Pelaez-
Samaniego et al., 2017). Mismanagement of manure often leads to
direct discharge of liquid manure into water ways, causing serious
eutrophication of rivers and lakes and increasing the risk to human
and animal health (Liu et al., 2017).
Therefore, the Insect-Manure-Digestion system is a new model
built to integrate valuable larval biomass for future protein needs
and to reduce animal manure pollution (Ogbuewu et al., 2012). The
multi beneficial Hermetia illucens, commonly called the black
soldier fly (BSF), is probably the best member of the family Strat-
iomyida and order Diptera. Black soldier fly larvae (BSFL), also
known as “phoenix worms”, are used for organic waste

http://dx.doi.org/10.1016/j.jenvman.2017.03.047
0301-4797/© 2017 Elsevier Ltd. All rights reserved.
 K. Rehman et al. / Journal of Environmental Management 196 (2017) 458e465
management, the bioconversion of organic waste and pest control
and may also be used in animal, poultry and fish feed supplements
(Makkar et al., 2014). BSFL are reared on substrates such as dairy
manure, poultry manure, pig manure (Myers et al., 2008; Zhou
et al., 2013), vegetable wastes and food wastes (Paz et al., 2015;
Salomone et al., 2016).
The problem regarding dairy manure (DM) utilization as feed or
growth medium for the BSF is mostly caused by its high crude fiber
content (Li et al., 2016; Pelaez-Samaniego et al., 2017). Finding
effective digestion methods of DM, such as larval biomass pro-
duction, not only effectively reduces the environmental liabilities
related to the manure management and disposal but also provides
an economic stimulus to dairy farmers. Therefore, a method of the
co-digestion of DM with less-fiber-content chicken manure (CHM)
for the development of BSFL was explored for the potential use of
rich cellulosic DM. The synergistic effects obtained with the co-
digestion of organic waste have been reported previously (Abudi
et al., 2016; Chen et al., 2016; Razaviarani et al., 2013).
Presently, no investigation has been conducted on the hydro-
lysis of cellulose, hemicellulose and lignin in DM, CHM and their co-
digestion after treatment with BSFL. This paper describes the
development of a co-digestion process of DM and CHM at different
mixing ratios after treatment with BSFL. First, the objective was to
determine the cellulose, hemicellulose and lignin degradation by
BSFL, and the cellulosic substrate of dairy manure, chicken manure
and their mixture were analyzed. Our second objective was to
evaluate the digestion performance of BSFL developed on the co-
digestion mixture of dairy and chicken manure.
2. Material and methods
2.1. Experiment site
The experiment was carried out at the greenhouse of the State
Key Laboratory of Agricultural Microbiology, Huazhong Agricultural
University (HZAU), Wuhan, Hubei, P. R. China.
2.2. Black soldier fly larvae
A colony of black soldier flies (H. illucens L.) was established in
2008 at the State Key Laboratory of Agricultural Microbiology of
HZAU, Wuhan, P. R. China. The Wuhan strain colony was main-
tained in a greenhouse for 8 years before its use in this study (Zhou
et al., 2013). The larvae were fed for 6 days on a standard colony diet
before being used in this investigation. About 1000 six-day-old
larvae were nourished with 1 kg of fresh DM and CHM, and their
mixtures at different mixing ratio levels are presented in Table 1.
2.3. Organic waste and growth conditions
The organic waste being tested in this study was DM and CHM
obtained from the breeding farm of HZAU, Wuhan, Hubei, P. R.
China. The animals at the breeding farm of the university were fed
Table 1
Percentage of dairy manure and chicken manure in different feeding mixtures.
Feeding mixtures Dairy manure (%) Chicken manure (%)
DM100 100 0 Lignin (% dry mass)
DM80 80 20
DM60 60 40
DM40 40 60
DM20 20 80
DM0 0 100
Each feeding mixture has three replicates.
459
the total standards mixed ration, and there was no known use of
pesticides in the manure. Next, 10 gm samples of DM and CHM used
in this experiment were taken and placed in three glass containers
for the estimation of the dry matter and water content of the
manure samples. The physico-chemical characteristics of the DM
and CHM used in this study are presented in Table 2.
2.4. Experimental design and operation
Four DM and CHM mixtures at different ratios, two with DM
only and CHM only, were established (Table 1). One kilogram of
fresh DM, CHM and their four mixture ratios was put in each 3 L
plastic container. The experiment was repeated three times for
each feed mixture and control. One thousand, six-day-old BSFL
were introduced into each container with the recording of the date
and time. The experimental operation condition was maintained at
a temperature of 27
C and a humidity level of 60e70% in a
greenhouse. Transformation was terminated when first prepupae
appeared in the container of each replicate; the larvae were sepa-
rated from the residues and were picked out manually using sterile
forceps. The larvae were inactivated at 105
C for 5 min after being
washed with distilled water and then were dried at 60
C for 2 days.
The development time, fresh larval weight, survival rate, and fresh
residue weight were estimated after the transformation with BSFL.
The total amount of manure consumption in each treatment
based on the dry mass, and water content of the experimental
groups pre-transformed and post-transformed were estimated as
described in the organic waste and growth conditions section by
taking 10 g of sample at the appearance of the first prepupa from all
replicates of each assorted manure ratios and their control.
2.5. Chemical analysis
The concentration of cellulose, hemicellulose and lignin from
the DM, CHM and their mixed fractions were analyzed according to
the procedures reported previously (Wang et al., 2010). The level of
total Kjeldahl nitrogen (TKN), total phosphorus (TP) and total
organic carbon (TOC) of the residual sample was determined ac-
cording to the standard method coded as NY 525-2011 Standardi-
zation Administration of People's Republic of China (Zhang et al.,
2012). The pH values of the initial feed mixture and final product
were measured in a distilled water suspension of each concentra-
tion at the ratio of 1:10 (W/V) using the Mettler Toledo FE 20 pH
system.
2.6. Scanning electron microscopy (SEM)
The structural changes in the DM and CHM fibers during
digestion with BSFL were qualitatively studied using SEM (HITACHI
Table 2
Physico-chemical characteristics of dairy manure and chicken manure.
Parameters Dairy manure Chicken manure
Water content (%) 75.46 ± 0.10 77.18 ± 0.01
Dry mass (%) 24.54 ± 0.10 22.82 ± 0.01
pH 8.22 ± 0.15 7.07 ± 0.05
Cellulose (% dry mass) 31.71 ± 0.11 14.49 ± 0.60
Hemicellulose (% dry mass) 16.79 ± 0.09 21.66 ± 0.22
13.58 ± 0.12 7.83 ± 0.12
TOC (% dry mass) 46.36 ± 1.08 40.19 ± 0.90
TN (% dry mass) 2.14 ± 0.085 3.54 ± 0.14
TP (% dry mass) 0.67 ± 0.07 2.28 ± 0.08
C/N ratio 21.77 ± 1.32 15.69 ± 2.07
TOC: total organic carbon; TN: total nitrogen; TP: total phosphorus; C/N: carbon/
nitrogen; values are in mean ± S.E; n ¼ 3.
460 K. Rehman et al. / Journal of Environmental Management 196 (2017) 458e465

Table 3
Survival rate, development time, and larval production of BSF reared on dairy manure and chicken manure and their co-digestion mixtures.

Feeding mixture Survival rate (%) Development time (days) Fresh larval weight (g) Dry larval weight (g)

DM100 89.45 ± 0.49c 22.03 ± 0.26c 52.77 ± 1.50d 10.29 ± 0.19e

DM80 91.86 ± 1.35bc 21.36 ± 0.39c 72.89 ± 2.29c 14.25 ± 0.44d
DM60 93.98 ± 0.28ab 20.87 ± 0.46c 81.20 ± 1.36c 16.49 ± 0.27c
DM40 95.51 ± 0.60a 20.24 ± 0.36b 92.07 ± 2.23ab 18.48 ± 0.44b
DM20 96.41 ± 0.30a 18.57 ± 0.03ab 86.46 ± 1.88b 18.32 ± 0.39b
DM0 98.35 ± 0.24a 18.34 ± 0.16a 97.54 ± 1.70a 22.56 ± 0.26a

Values are in mean ± S.E; n ¼ 3; values bearing different superscripted alphabets differ from each other at P < 0.05.

S-4800 Scanning Electron Microscopy, Japan) operated an acceler-
ating voltage of 3.0 kV. The dry samples were mounted on double
sided tape placed on aluminum stubs and a thin layer of gold
(15 nm for 10 min) was sputtered on mounted sample to reduce the
electron altering effects and to improve the conductivity of the
samples and thus the quality of images.
2.7. Calculation and statistical analysis
SPSS 16.0 (SPSS Inc., Chicago, IL, USA) was used for statistical
analyses. The results of all experiments were analyzed by one-way
analysis of variance (ANOVA) followed by Tukey's HSD (honestly
significant differences) test as a post hoc analysis. A P value < 0.05
was considered to indicate a significant difference between the
values compared. The percent mass reduction was (W1-W2)/
W1  100%, with W1 being the dry mass provided over the course
of the experiment and W2 being the dry mass amount remaining at
the conclusion of the experiment. The percent conversion of BSFL
(bioconversion) and feed conversion ratio (FCR) were estimated at
the appearance of the first prepupae.
3. Results
3.1. Survival rate, larval development and larval production
Although all the proportions of DM and CHM were accepted by
the BSFL for their development, the survival rate, development
time, larval yield and larval dry mass were strongly affected by
adding different fractions of CHM in the DM (Table 3). The survival
rate was significantly lower in the dairy manure (DM100) than in
the DM40, DM20 and chicken manure (DM0), but there was no
significant difference between these two feeding mixtures and
chicken manure. There was a fluctuation in the survival rate from
89.45 ± 0.49% in the dairy manure substrate to 96.41 ± 0.30% in
DM20. The development time in this study is the appearance of first
prepupae. The development time was 22 days for the dairy manure,
but when the feed was mixed with the DM and CHM substrate, the
time was significantly shorter than that for the dairy manure up to
21 to 18 days in Table 3.
Regarding larval production, there were significant increases in
the fresh and dry larval yield in the co-digestion mixtures. The fresh
larval weight in DM40 was significantly similar to that in DM0.
However, all other feeding mixtures were significantly lower in the
fresh yield of larvae from chicken manure but were higher in the
dairy manure (Table 3). There was a significant increase in the dry
mass content of the larvae by the addition of chicken manure to
dairy manure (Table 3). The highest increase in the dry mass of
larvae was observed in DM40 and DM20.
3.2. Percent manure mass reduction, percent conversion of the
black soldier fly larvae and feed conversion ratio
The percent manure mass reduction, also called dry mass
reduction, was significantly increased in the assorted manure feed
mixtures. Similar effects were recorded for the bioconversion and
FCR (Table 4). The highest percent dry mass reduction among the
co-digestion mixtures was observed in DM20 (53.38 ± 0.34%) with
no significant difference compared with DM0 (55.04 ± 0.31%),
while the DM40 group also showed a higher waste reduction ca-
pacity of 52.06 ± 0.70%. The lowest dry mass reduction (Table 4)
among the mixed dairy and chicken manure was found in DM80 to
be 44.32 ± 0.71%, with no significant difference in the DM0
reduction rate at 43.17 ± 0.45%. The highest bioconversion and FCR
was in the DM40 and DM20 (bioconversion: 7.86 ± 0.18% and
7.90 ± 0.17%, respectively; FCR 6.63 ± 0.20 and 6.76 ± 0.18,
respectively), which was significantly similar in both groups and
higher than that with the dairy manure only (DM100: 4.19 ± 0.09%
bioconversion and 10.29 ± 0.26 FCR, respectively) but less than the
chicken manure (DM0) bioconversion (9.88 ± 0.11%) and FCR
(5.57 ± 0.07).
3.3. Nitrogen, phosphorus and carbon reduction percentage
Nutrient analysis and reduction data are presented in Fig. 1; the
percentage nitrogen reduction was highest in DM20 (68.96 ± 1.13%)
with no significant difference among the assorted manure DM40
(56.92 ± 2.41%), DM60 (62.05 ± 0.41%), DM80 (58.63 ± 0.89%) and
chicken manure only (DM0) but significantly higher than the
DM100 (53.53 ± 1.58%). The 60.06 ± 0.68% phosphorus reduction
present in the dry mass of DM40 was removed by the BSF (Fig. 1),
which is the highest observed value that is significantly equal to the
phosphorus reduction in DM0 (57.4 ± 5.38%), DM20

Table 4
Manure mass reduction, bioconversion and feed conversion ratio of Hermetia illucens converting dairy manure, chicken manure and co-digestion mixtures.

Feeding mixture Dry larval weight (g) Feed added dry mass (g) Residues dry weight (g) Dry mass consumed (g) Dry mass reduction (%) Bioconversion FCR
(%)

DM100 10.29 ± 0.19e 245.40 ± 1.00 139.43 ± 1.08 105.96 ± 1.31 43.17 ± 0.45c 4.19 ± 0.09e 10.29 ± 0.26c
DM80 14.25 ± 0.44d 241.96 ± 0.41 134.74 ± 1.92 107.24 ± 1.60 44.32 ± 0.71c 5.88 ± 0.18d 7.54 ± 0.33b
DM60 16.49 ± 0.27c 238.52 ± 1.50 121.37 ± 2.82 117.15 ± 3.42 49.11 ± 1.28b 6.92 ± 0.11c 7.11 ± 0.28b
DM40 18.47 ± 0.44b 235.08 ± 0.35 112.68 ± 1.64 122.28 ± 1.67 52.06 ± 0.70ab 7.86 ± 0.18b 6.63 ± 0.20ab
DM20 18.32 ± 0.39b 231.64 ± 0.30 107.98 ± 0.86 123.65 ± 0.77 53.38 ± 0.34a 7.90 ± 0.17b 6.76 ± 0.18b
DM0 22.56 ± 0.26a 228.20 ± 0.18 102.58 ± 0.73 125.61 ± 0.73 55.04 ± 0.31a 9.88 ± 0.11a 5.56 ± 0.07a

Data are expressed as mean ± S.E; n ¼ 3; values with different superscripts differ from each other at P < 0.05.
 K. Rehman et al. / Journal of Environmental Management 196 (2017) 458e465
Fig. 1. Reduction of nitrogen, phosphorus and carbon during the digestion of pure
dairy manure (DM100), chicken manure (DM0) and in-between mixing ratios by black
soldier fly larvae; values with the same superscripts do not differ from each other at
P<0.05; bars indicate the standard error of the means (n¼ 3).
(59.07 ± 0.65%).
The carbon reduction was approximately 68% in DM0, DM20
and DM40, which was the greatest recorded value among the
experimental groups, but the smallest reduction rate of approxi-
mately 56.71 ± 0.87% was observed in dairy manure DM100 (Fig. 1).
However, the carbon reduction rates of the co-digestion mixtures
were significantly higher than those in the dairy manure (DM100).
3.4. Cellulose, hemicellulose and lignin hydrolysis by black soldier
fly larvae
Fig. 2 shows the cellulose hydrolysis of dairy manure, chicken
manure and their mixtures by BSFL. The highest cellulose reduction
was in chicken manure at 67.77 ± 1.65%, while the lowest value
observed in dairy manure (DM100) was 49.89 ± 1.73%. The cellulose
reduction was significantly similar among DM0, DM20 and DM40;
whereas the other mixed manure groups had significantly lower
reduction rates than those of the DM20 and DM40 groups.
The hemicellulose content of the dairy manure was reduced by
49.77 ± 2.36% (DM100). However, this reduction was slightly more
intense in chicken manure (DM0), being highest among all of the
experimental groups at 58.69 ± 1.51%. However, the hemicellulose
degradation was significantly parallel in DM20 (53.74 ± 0.51%),
DM40 (53.22 ± 0.98%) and pure chicken manure with the highest
value of hemicellulose reduction. It was particularly evident that,
Fig. 2. Comparison of fiber utilization during the digestion of pure dairy manure
(DM100), chicken manure (DM0) and in-between mixing ratios by black soldier fly
larvae; bars indicate the standard error of the means (n¼ 3).
461
among the assorted manure feeding groups DM40 and DM20, the
reduction rate was not significantly different as presented in Fig. 2.
The lignin reduction rate is lower than cellulose and hemicel-
lulose. The highest reduction was found in DM40 (42.23 ± 1.02%)
with no significant difference between DM20 (38.55 ± 0.74%) and
DM0 (37.81 ± 0.86%). However, the lignin reduction rate in the dairy
manure and mixture with a high dairy manure percentage was
significantly lower, approximately 31% in DM100, 32% in DM80 and
30% in DM60. Whereas, the comparison of the fiber content
reduction (cellulose, hemicellulose and lignin) by BSFL during the
co-digestion and single digestion of DM and CHM is displayed in
Fig. 2.
3.5. Fibers surface profile changes of dairy manure and chicken
manure during BSFL digestion
Scan electron microscopy to compare changes in the lignocel-
lulose structure of diary manure, chicken manure and their co-
digestion mixture before and after being BSFL treatment. The
sample of original DM (Fig. 3A), post-treated DM (Fig 3B) and co-
digestion mixtures (Fig. 3 C, D, E, F) were amplified 3500 times to
analyze surface profile of fibers. It can be seen that the texture of
predigested DM was compact and well-organize, however the
surface structure of the digested DM and co-digestion mixtures
with CHM were hollow and cracked. Moreover the cellulose pre-
sent in CHM used in this study as co-digest waste with DM by BSFL
was examined by SEM displayed in Fig. 4 predigested CHM (A) and
post BSFL digested CHM (B) both at magnification 3500  . The
surface morphology of CHM fibers showed relatively compact and
rigid surface before BSFL hydrolysis but after digestion they
exhibited a corrugated and more porous surface.
3.6. Physico-chemical characteristics of the manure mixtures
The decrease in the water content was observed, and the initial
moisture content was highest in the chicken manure 77.18 ± 0.21%
and dairy manure 75.46 ± 0.10%. Additionally, in the assorted
manure groups, DM80 (75.81 ± 0.04%), DM60 (76.15 ± 0.15%),
DM40 (76.49 ± 0.03%) and DM20 (77.18 ± 0.21%), the water con-
tents were all high, above 75% (Table 5). However, in the residues,
the water content was reduced to between 55.27 ± 0.80% and
66.08 ± 0.77% in all of the manure mixtures (Table 5), confirming
the optimum water content percentage in the compost. A decrease
in pH was witnessed in the manure mixture and dairy manure;
however, the carbon to nitrogen ratio (C:N) ratio was found to
decrease significantly in all feeding ratios (Table 5) of the dairy
manure and chicken manure. The C:N ratio shows the waste sta-
bilization and mineralization during the process of BSF digestion.
4. Discussion
The DM and the mixtures with CHM enhanced all parameters of
life history traits of the BSF in comparison to pure dairy manure as
feed (Table 3). The survival rate and development time in our study
were compared with those in previous reported studies (Table 6).
The survival rate and development time were determined using
various factors, feed composition and availability (Diener et al.,
2009; Oonincx et al., 2015b), temperature and diet (Harnden and
Tomberlin, 2016), fresh and old animal manure (Oonincx et al.,
2015a) and relative humidity (Holmes et al., 2012). All of the
assorted manure groups showed a significant increase in the larval
production (fresh and dry larval weights) from the DM (Table 3)
with a similar finding that co-digestion increases the yield (Li et al.,
2009; Serrano et al., 2014) and unstable parameters depend on the
dietary quality (Gobbi et al., 2013), the similar findings from
462 K. Rehman et al. / Journal of Environmental Management 196 (2017) 458e465

Fig. 3. Scan electron microscope micrographs (3500 magnification): (A) cellulose in dairy manure before digestion; (B) cellulose in dairy manure after digestion DM100; (C-F)
cellulose in feeding mixtures DM80, DM60, DM40 and DM20 respectively after digestion with black soldier fly larvae.

Fig. 4. Scan electron microscope images of chicken manure fibers: (A) chicken manure before digestion; (B) chicken manure after digestion with black soldier fly larvae at 3500
magnification.

previous studies are presented in Table 6. The feed quality of larval the reported value of 17e40% (Table 6). This indicates that the
resources affects insects and is positively correlated with the variation in the dry mass content of BSFL can occur in the diet
percent survival and weight (Oonincx et al., 2015b). The average dry provided; therefore, the yields should be reported in both the fresh
mass content of BSFL in our study was 19e23%, within the range of and dry weight.
 K. Rehman et al. / Journal of Environmental Management 196 (2017) 458e465 463

Table 5
Physico-chemical characteristic of dairy manure, chicken manure and their co-digestion mixtures before and after treatment with black soldier fly larvae.

Feeding Water content pH value Cellulose in dry Hemicellulose in dry Lignin in dry TOC in dry mass TN in dry mass TP in dry mass C/N ratio
mixture (%) mass (%) mass (%) mass (%) (%) (%) (%)

DM100 Initial 75.46 ± 0.10 8.22 ± 0.15 31.70 ± 0.90 16.79 ± 0.35 13.58 ± 0.12 46.36 ± 0.06 2.14 ± 0.04 0.67 ± 0.03 21.67 ± 0.42
Final 56.29 ± 0.24 7.78 ± 0.02 27.90 ± 0.36 14.81 ± 0.34 16.26 ± 0.18 35.32 ± 0.47 1.75 ± 0.08 0.58 ± 0.02 20.22 ± 0.84
DM80 Initial 75.81 ± 0.04 8.12 ± 0.01 28.36 ± 0.42 17.54 ± 0.41 12.55 ± 0.04 45.27 ± 0.13 2.36 ± 0.02 1.01 ± 0.02 19.11 ± 0.04
Final 55.27 ± 0.80 7.79 ± 0.06 23.33 ± 0.37 16.06 ± 0.31 15.13 ± 0.36 30.58 ± 0.73 1.76 ± 0.05 0.98 ± 0.05 17.39 ± 0.46
DM60 Initial 76.15 ± 0.15 7.95 ± 0.01 24.89 ± 0.21 18.54 ± 0.18 11.26 ± 0.04 43.54 ± 0.17 2.63 ± 0.02 1.34 ± 0.02 16.56 ± 0.43
Final 58.79 ± 1.77 7.44 ± 0.01 21.63 ± 0.84 17.46 ± 0.23 15.55 ± 0.36 30.48 ± 0.52 1.96 ± 0.01 1.24 ± 0.07 15.52 ± 0.24
DM40 Initial 76.49 ± 0.03 7.81 ± 0.02 21.47 ± 0.86 19.42 ± 0.60 10.15 ± 0.03 42.83 ± 0.59 3.02 ± 0.01 1.61 ± 0.03 14.20 ± 0.17
Final 59.83 ± 0.70 7.42 ± 0.01 17.3 ± 0.58 18.93 ± 0.24 12.22 ± 0.14 28.39 ± 0.55 2.71 ± 0.16 1.34 ± 0.01 10.52 ± 0.51
DM20 Initial 76.84 ± 0.03 7.75 ± 0.02 18.01 ± 0.33 20.96 ± 0.84 9.16 ± 0.09 41.69 ± 0.07 3.32 ± 0.05 1.92 ± 0.06 12.56 ± 0.21
Final 65.63 ± 0.70 7.18 ± 0.03 15.28 ± 0.16 20.79 ± 0.79 12.08 ± 0.24 27.16 ± 1.15 2.21 ± 0.07 1.68 ± 0.02 12.33 ± 0.83
DM0 Initial 77.18 ± 0.21 7.07 ± 0.05 14.49 ± 0.20 21.66 ± 0.15 7.83 ± 0.13 40.19 ± 0.90 3.54 ± 0.14 2.28 ± 0.08 11.41 ± 0.68
Final 66.08 ± 0.77 7.31 ± 0.01 10.41 ± 0.67 19.89 ± 0.53 12.35 ± 0.36 28.41 ± 1.50 2.81 ± 0.15 2.15 ± 0.13 10.13 ± 0.43

TOC: total organic carbon; TN: total nitrogen; TP: total phosphorus; C/N: carbon/nitrogen; data are expressed as mean ± S.E; n ¼ 3.

Table 6
Comparative data from previous reported black soldier fly digestion experiments.

References Feed source Survival Development Mean fresh larval Dry mass (%) of live Dry mass Temperature Humidity
rate (%) time (day) weight (g) weight BSFL reduction (%) (
C) (%)

Present study Dairy manure and 89.5e98.4 18e22 0.06e0.10 19.5e23.2 43e55 27 60e70
chicken manure
Manurung et al. Rice straw 51.2e98.3 38e54 e e ~9e31 28 70
(2016)
Oonincx et al. Artificial feed 75e85 21e37 e 32.9e35.6 e 28 70
(2015b)
Oonincx et al. Cow manure 87.8 214.5 0.07 20.3 e 28 70
(2015a) Chicken manure 82.2 144 0.05 20.6 e 28 70
Pig Manure 97 144 0.06 20.2 e 28 70
Gobbi et al. (2013) Hen feed 93 15 e e ~70 27 e
Gobbi et al. (2013) Meat meal 40 33 e e ~31 25 e
Zhou et al. (2013) Swine manure e e e e ~29e53 28 75
Chicken manure e e e e ~32e62 28 75
Dairy manure e e e e ~34e58 28 75
Li et al. (2011) Dairy manure e 31 e e 53 27 e
Sealey et al. (2011) Dairy manure e ~120 e e e 25 e
Diener et al. (2009) Chicken feed e 16e42 0.08e0.15 33e40 23e44 27 e
Myers et al. (2008) Dairy manure 71e85 26e30 0.14e0.17 e 33e58 e e
Dierenfeld and King Commercial diet e e e 27 e e e
(2008)

e ¼ not reported; ~ ¼ approximately.

The dry mass reduction in this experiment was significantly
enhanced by BSFL with the addition of CHM (Table 4), and the re-
sults are comparable with the previous findings (Table 6) However,
the dry mass reduction seems very variable according to the feed
source as shown for the previous data in Table 6. Moreover, the life
history traits and waste reduction data from former study by Zhou
et al. (2013) demonstrate phenotypic plasticity among three BSF
strains may be due to climate variation. The co-digestion of dairy
and chicken manure has very beneficial effects on the bioconver-
sion and FCR (Table 4). The previously reported bioconversion re-
sults 4% in swine manure (Newton et al., 2005) and 12% in
municipality organic waste (Diener et al., 2011) were comparable
with the current finding (Table 4). The FCR on the dry mass base
was not reported formerly in DM and CHM; however, in swine
manure, it was reported to be 9.6 and 5.8 in municipality organic
waste (Diener et al., 2011; Newton et al., 2005). The presently
discussed outcome is comparable to previous results. The diet
composition is the major variable determining the feed conversion
efficiency (FCR) for insect species (Oonincx et al., 2015b).
The total N content in CHM in our study was 3.54% (Table 2) of
dry mass within the range of the previously reported value of
3.05%e5.86% of dry mass (Li et al., 2017; Savala et al., 2016) and DM
(2.14% vs 1.42%e3.30% of dry mass) (Pelaez-Samaniego et al., 2017).
The finding confirms that BSFL reduce the nitrogen content
efficiently in the co-digestion process in the dairy manure only
group DM100 (Fig. 1). The results are in agreement with the report
that the BSF reduces the N content in CHM (24%e56% and 82%)
more competently than dairy manure (30%e50%) (Myers et al.,
2008; Oonincx et al., 2015a; Zhou et al., 2013). The total P content
in the presently discussed study was 2.28% of dry mass in chicken
manure (Table 2) compared with 1.25e2.80% in a previous report
(Li et al., 2017; Savala et al., 2016). Phosphorus reduction was 50% in
the dairy manure group (DM100) and 57% in the chicken manure
group (DM0). The phosphorus reduction was comparable to the
rates found in the previous literature, 61e70% in dairy manure
(Myers et al., 2008) and 44% in swine manure (Newton et al., 2005).
The TOC content in our experiment was 46.36% in dairy manure
and 40% in chicken manure (Table 2), results that were almost
identical to those in diary manure described by Bhat et al. (2015)
and chicken manure by Abouelenien et al. (2014). The reduction
rate of carbon in the co-digestion mixture was significantly higher
in comparison with dairy manure DM100 (Fig. 1). The utilization of
carbon by the BSF was recorded by a 62% reduction of the TOC in
swine manure (Newton et al., 2005). The utilization of TOC during
the process of vermicomposting was also reported previously by
Bhat et al. (2014). The ingested manure mixtures would be broken
down and homogenized by insects through their muscular activity,
leading to the increase in their surface area for microbial action and
464 K. Rehman et al. / Journal of Environmental Management 196 (2017) 458e465
leading to the decline of TOC.
The undigested DM was found to have a percent dry mass of
31.71% for cellulose, that of 16.79% for hemicellulose, and that of
13.58% for lignin. However, in CHM, the cellulose, hemicellulose
and lignin contents were 14.49%, 21.66% and 7.83%, respectively
(Table 2). The reduction rates were elevated significantly in the
assorted manure groups DM80, DM60, DM40 and DM20 (Fig. 2).
The oxidation of fiber is an important part in the metabolic activity
of black soldier fly as it provides material and energy for Hermetia
illuucens life activities reported previously in insects (Li et al., 2016).
Therefore balance nutrition (C/N ratio) and buffering capacity (pH)
of waste material will enhance the total conversion efficiency and
help in the digestion of material which cannot be well utilized by
black soldier fly (Table 5). Our study is also in agreement with
previous studies that co-digestion increases the yield due to a
better nutrient balance and buffer capacity, which is necessary to
establish positive synergism for biological growth (Anjum et al.,
2016; Li et al., 2009). The degradation of cellulose, hemicellulose
and lignin by the BSF was previously recorded in rice straw and
corncob (Li et al., 2015a, 2015b; Zheng et al., 2012) and DM (Li et al.,
2011) was similar to the results found in the present study. SEM was
used to study the morphological of the original DM, CHM and co-
digestion mixtures of both environmentally hazardous waste
mass (Figs. 3 and 4). The presence of BSF shows more surface ir-
regularities in cellulosic mass this may be due to BSFL and their gut
microbiome symbioses, because it was formerly confirmed that
insects and their microbial symbionts digest cellulose in gut
(Watanabe and Tokuda, 2010). The finding of changes in the surface
structure profile of cellulose in manure was confirmed by Cunault
et al. (2013).
The water content percentage at the start of experiment was
between 75% and 78%; however, after H. illucens L. digestion, the
water content of the residual material (Table 5) in the acceptable
range for compost (Lim et al., 2016). The BSFL-digested manure has
a pH (Table 5), which is within the optimal range of plant growth
(He et al., 2016). The plummeting in the pH during vermi-
composting is due to the mineralization of nitrogen and phos-
phorus into nitrites/nitrates and bioconversion of organic material
into intermediate species of organic acids that may have decreased
the pH (Alidadi et al., 2016; Bhat et al., 2015). However, rises in the
pH value noted in DM0 suggest the alkalization of CHM because the
release of ammonium ions (NHþ 4 ) and ammonia (Alidadi et al.,
2016) also reported the same phenomena during composting
with the earthworm (Majlessi et al., 2012). The decline in the per-
centage of total nitrogen, total phosphorus, and total organic car-
bon was recorded in the present investigation as found in the swine
manure digestion by the BSF (Newton et al., 2005). The carbon to
nitrogen ratio C:N was found to be decreased in all of the feed ratios
of DM and CHM. The C:N ratio shows the waste stabilization and
mineralization during the process of vermicomposting, and this
declining pattern in C:N was also recorded during vermicompost-
ing in cow dung (Bhat et al., 2015). The decline in the C:N ratio was
due to the higher loss of carbon through microbial respiration in the
form of CO2 (Alidadi et al., 2016; Hait and Tare, 2011; Vig et al.,
2011).
5. Conclusions
In summary, the present study has clearly demonstrated that
the black soldier fly has the potential to degrade cellulose, hemi-
cellulose and lignin. The co-digestion of dairy manure and chicken
manure brings better digestion performance for BSFL. There is no
significant difference between the DM20 and DM40 groups.
Although DM20 and DM40 show better results than other manure
mixtures, it was concluded that DM40 met both the demand of the
nutrient-rich larval output and high organic mass reduction
compared with dairy manure and the other mixtures. Moreover it
has been demonstrated that a balanced nutrition and buffering
capacity enhances the total waste conversion efficiency and helps
with the digestion of material which cannot be well utilized by BSF.
Therefore a carbon/nitrogen ratio of 14.2 and a pH of 7.8 for
bioconversion of DM and CHM using BSF are recommended. Ulti-
mately, H. illucens have the potential to solve the problem of high-
fiber dairy manure pollution by co-digestion with the benefits of
producing larval biomass that can be used directly or after drying in
the animal feed industry and that could provide a source of income
for dairy farmers and local entrepreneurs; additionally, the diges-
ted residues can be used as bio-fertilizer. The disposal of livestock
manure mainly relies on traditional methods, which include
organic fertilizer (direct application, compost and microbial
fermentation); therefore, the present studied technology is novel
and highly beneficial for the management of livestock manure and
for the economic generation of livestock farmers by the production
of valuable biomass.
Further study should focus on the implementation of the high-
fiber-content organic waste co-digestion process on a large scale in
situ leading to the new challenges under changing environmental
conditions. The mechanisms and magnitude of the effects of BSFL
on the cellulose in fiber-rich organic waste need to be explored.
Acknowledgments
This study was supported by the Special Fund for Agro-scientific
Research in the Public Interest, China (201303094), and National
Natural Science Funds of China (grant 31301913).
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