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Management of chicken manure using black soldier fly (Diptera: Stratiomyidae) larvae assisted by companion bacteria
Waste Management 102 (2020) 312–318
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Management of chicken manure using black soldier fly (Diptera: Stratiomyidae) larvae assisted by companion bacteria Lorenzo Mazza a,b,1, Xiaopeng Xiao a,1, Kashif ur Rehman a,c, Minmin Cai a, Dingnan Zhang a, Salvatore Fasulo b, Jeffery K. Tomberlin d, Longyu Zheng a,⇑, Abdul Aziz Soomro 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, PR China b University of Messina, Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, Messina, Italy c Livestock and Dairy Development Department, Poultry Research Institute, Rawalpindi, Pakistan d Department of Entomology, Texas A&M University, College Station, TX, USA
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Article history: Received 8 April 2019 Revised 25 October 2019 Accepted 30 October 2019
Keywords: Hermetia illucens Companion bacteria Chicken manure Environmental pollution Conversion efficiency
a b s t r a c t Black soldier fly (BSF) is used for the management of organic waste, but research has hardly explored the effect of companion bacteria when chicken manure (CHM) is converted to insect biomass. In this study, we isolated nine bacterial species (FE01, FE02, FE03, FE04, FE05, FE06, FE07, FE08, FE09) from BSF eggs and one (BSF-CL) from the larval gut. These companion bacteria were inoculated into CHM along with BSF larvae (BSFL). Larval growth and manure conversion rates were determined. Results indicated that almost all bacteria individual bacteria in this study significantly promote BSFL growth. BSFL reared in manure with the species Kocuria marina (FE01), Lysinibacillus boronitolerans (FE04), Proteus mirabilis (FE08) and Bacillus subtilis (BSF-CL) had higher weight gain and manure reduction rates compared to the control. These four strains used were then examined as a poly-bacteria community experiment to determine BSFL growth and manure conversion. Manure inoculated with the poly-bacteria Group3 (FE01:FE04:FE08:BSF-CL = 4:1:1:1) and then fed to BSFL resulted in 28.6% more weight gain than the control. The greatest manure reduction rate (52.91%) was reached when companion bacteria were mixed at a ratio of 1:1:1:4. Additionally, the companion bacteria influenced the nutritional value of BSFL. Crude protein content in Group1 (FE01:FE04:FE08:BSF-CL = 1:1:1:1) was significantly larger than that of the control. Crude fat content in Group3 was significantly larger than that of the control. BSFL companion bacteria and their poly-bacteria compound improved manure conversion efficiency and nutrient accumulation in BSFL, reduced CHM quantity, increased larvae biomass, with potential economic gains in CHM management. Ó 2019 Elsevier Ltd. All rights reserved.
1. Introduction Organic wastes, such as poultry and livestock manure, sewage sludge, kitchen and domestic wastes, have become a major global challenge, and this is expected to increase as the global population expands from 7.3 billion in 2015 to 9.7 billion in 2050 (Surendra et al., 2016). Most organic wastes generated is left untreated (Reckmann et al., 2013). Moreover, the unprocessed organic wastes, which contain valuable energy and nutrients, can contribute to an increase in the annual greenhouse gas emissions (GHG) (Dai et al., 2016) and a leaching of nutrients into the under⇑ Corresponding authors. E-mail addresses: [email protected] (L. Zheng), [email protected]. edu.cn (J. Zhang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.wasman.2019.10.055 0956-053X/Ó 2019 Elsevier Ltd. All rights reserved.
ground water table, causing eutrophication (Zhang et al., 2012). Animal manure is a very harmful organic waste, and if untreated will pose a risk for the spread of pathogens (Albihn and Vinnerås, 2007; Sun et al., 2016; ur Rehman et al., 2017). Therefore, it is important to find an effective way to recycle these organic waste streams. Insects are the most abundant and diverse group of animals on earth (Allegretti et al., 2018), and are likewise the largest underutilized valuable resources (Roffeis et al., 2018). Organic wastes may be successfully converted into biomass with insects and other invertebrates (ur Rehman et al., 2017; Wang et al., 2017), such as Hermetia illucens L. (Diptera: Stratiomyidae) (Ma et al., 2018; Xiao et al., 2018), Musca domestica L. (Diptera: Muscidae) (Zhang et al., 2012), Eisenia fetida L. (Opisthopora: Lumbricidae) (Negi and Suthar, 2018), and Tenebrio molitor L. (Coleoptera: Tenebrionidae) (Thévenot et al., 2018). These insects can digest nutrients
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Nomenclature List of abbreviations and notation BSF Black soldier fly BSFL Black soldier fly larvae BSF-CL Bacillus subtilis CHM Chicken manure CP Crude protein CF Crude fat FE01 Kocuria marina FE02 Micrococcus luteus
from food wastes (Nguyen et al., 2015) such as vegetables and fruits, agro-industrial by-products (Meneguz et al., 2018), chicken and dairy manure (Rehman et al., 2017a) and convert them into valuable proteins and fats to be used in animal feed formulations (Liu et al., 2017; Xiao et al., 2018), while the residue can be used as a fertilizer (Ma et al., 2018; Xiao et al., 2018). Insects, such as H. illucens (Jeon et al., 2011), Drosophila melanogaster L. (Diptera: Drosophilidae) (Ren et al., 2007) and M. domestica L. (Gupta et al., 2012), have a wide variety of related symbiotic microorganisms (Engel and Moran, 2013). Host associated microbes can contribute to the development of the organism as they are involved in a variety of insect physiological processes, including immune response (Chen et al., 2016). Recently, some studies indicated that symbiotic microbes play a key role in insect growth, gut microbiota development and digestive enzyme production at different life stages and depending on the environmental habitat (Jiang et al., 2019; Scott et al., 2008; ur Rehman et al., 2017; Yun et al., 2014). Moreover, some bacteria can protect insects from predators, parasites and pathogens (Laughton et al., 2011). H. illucens, commonly known as the black soldier fly (BSF), is classified as a saprophagous insect, feeding on rotten meat and decaying plant matter in nature (Makkar et al., 2014). Black soldier fly larvae (BSFL) are capable of consuming large amounts of organic side streams such as food waste, rice straw, dead animals, and animal manure (Makkar et al., 2014; Rehman et al., 2017; Xiao et al., 2018). They can reduce nitrogen-rich wastes by 75% and poultry, dairy and pig manure by 50% (Rehman et al., 2017; ur Rehman et al., 2017; Newton et al., 2005). In addition, harvested larvae contain about 40% protein and 35% fat (Liu et al., 2017). In various feeding studies, larval or prepupal meal was found to be a suitable full, or partial, replacement of fish meal, meat meal, and plant protein meal in livestock (Makkar et al., 2014), poultry (Schiavone et al., 2017), pig (Spranghers et al., 2018; Biasato et al., 2019) and aquaculture feed (Cummins et al., 2017; Renna et al., 2017; Belghit et al., 2019). Also, it was previously investigated that symbiotic microbes have synergistic effects on the development of insect biomass (Zheng et al., 2012; Ma et al., 2018). However, few studies have explored the impact of symbiotic bacteria on the accumulation of nutrients, and how they can improve the efficiency of BSF conversion of wastes to insect biomass. Therefore, this study was designed to investigate the effects of egg-associated and gut microbes of BSF on larval development, waste conversion efficiency and nutrient accumulation of BSFL, when reared in fresh chicken manure (CHM).
FE03 FE04 FE05 FE06 FE07 FE08 FE09 LB PBS
Enterococcus faecalis Lysinibacillus boronitolerans Sporosarcina koreensis Gordonia sihwensis Enterobacter spp. Proteus mirabilis Bacillus subtilis Luria-Bertani Phosphate Buffered Saline
five days, larvae were fed a diet based on wheat bran with 75% water content. Feeding was stopped on the sixth day, and larvae were separated from the feed. 2.2. Manure biomass Fresh chicken manure (CHM) was collected from a poultry farm located in Wuhan. Water content in CHM was determined as 74.8%, and dry mass(ash), total nitrogen (TN), total organic carbon (TOC), total phosphorus (TP), cellulose, C/N ratio: carbon/nitrogen, were determined as 22.82%, 3.54%, 40.19%, 2.28%, 14.49%, 15.69 on a dry matter basis, respectively. All the compositions were analysed according to our previous research (Rehman et al., 2017; Xiao et al., 2018). 2.3. Bacterial isolation from eggs and larval gut of the black soldier fly
2. Materials and methods
Microorganisms used in this study were isolated from the egg surface of BSF within 6 h after spawning, and from the gut of 5th instar larvae. For the isolation of bacteria species from the egg surface, 0.5 g of BSF eggs were homogenized with 2 mL distilled water. The mixture was constantly stirred and incubated for 5 min at 28 °C. Following this, the supernatant was diluted and used for isolation and growth of the bacteria on Luria-Bertani (LB) agar medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 18 g/L, distilled water 1000 mL). BSF larvae were reared on a diet consisting of 50% bran and 50% corn flour. To isolate bacteria from the gut, fifth instar larvae were surface-sterilized in 75% ethanol for 3 min and then rinsed three times in sterile Phosphate Buffered Saline (PBS) at pH 7.4. The total gut (pools of 10 larvae) was dissected aseptically, transferred to 100 lL of sterile PBS, and homogenized. For isolations based on colony selection, diluted cell suspensions were plated on solid LB medium and incubated for 48 h at 28 °C. All morphologically different bacterial species were sub-cultured three times to ensure purity and stability. The bacterial species isolated from egg surfaces were identified as Kocuria marina (FE01), Micrococcus luteus (FE02), Enterococcus faecalis (FE03), Lysinibacillus boronitolerans (FE04), Gordonia sihwensis (FE06), and Proteus mirabilis (FE08). In a previous study (unpublished data) we also isolated from the gut Bacillus subtilis (BSF-CL) and this species was also used in our study. Bacterial isolates were inoculated into LB medium with aeration for 36 h at 28 °C. Following this, bacteria were washed three times in PBS for 15 min at 8000 rpm, and re-suspended in 30 mL of sterile water to obtain a final concentration of 1 108 CFU/mL determined by using a hemocytometer.
2.1. H. illucens
2.4. Co-conversion of individual bacterial isolates and BSFL
The larvae of H. illucens, Wuhan strain, used in this study were bred at the State Key Laboratory of Agricultural Microbiology of Huazhong Agricultural University, Wuhan, China. During the first
The ten BSF companion bacterial strains, namely FE01, FE02, FE03, FE04, FE05, FE06, FE07, FE08, FE09 and BSF-CL, were individually inoculated as supplements into 500 g fresh CHM with a
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proportion of 1% (v/w). Five hundred 6-day-old BSFL were inoculated into each waste-bacterial mixture, as well as into 500 g fresh CHM without bacterial supplementation, serving as the control. Each treatment was set up in three replicates and kept in the climatic chamber for approximately 14 days, at 28 °C with 60% to 70% RH. After 14 days, weight and conversion rate of BSFL, and reduction rate of CHM, were recorded using the previously described methodology (Xiao et al., 2018a). The formulae used were as follows: BSFL Conversion rate = total larval biomass(g)/ feed added(g) 100%; Weight gain of BSFL (compared to control) = (G2-G1)/G1 100%, G1: Control group BSFL dry weight (g) after conversion, G2: treatment group BSFL dry weight(g) after conversion; Reduction rate of CHM = (W1-W2)/W1 100%, W1: the initial CHM dry weight, W2: the final dry residue weight after the BSFL digestion. 2.5. Co-conversion of poly-bacterial cultures and BSFL Based on the analysis mentioned above, K. marina (FE01), L. boronitolerans (FE04), P. mirabilis (FE08), and B. subtilis (BSF-CL) were chosen to be used in the poly-bacterial study. These bacteria were combined at different ratios and inoculated into CHM. Coconversion efficiency of BSF and bacterial composites were determined. Nine poly-bacterial communities were inoculated separately into 500 g fresh CHM with a proportion of 1% (v/w) (Table 1). After this, five hundred 6-day-old BSFL were inoculated into the mixture, or into a ‘‘no-bacterial supplement” mixture as a control. Each treatment included three replicates, which were kept in the climatic chamber for 14 days, at 28 °C with 60% to 70% RH. The larvae were directly separated from the treatment residue, and larval dry weight was obtained after washing with distilled water and drying at 60 °C for 2–3 days. Dry matter of initial CHM and treatment residue were calculated after drying in an oven at 105 °C. Conversion rate and weight gain rate of BSFL, and reduction rate of CHM were observed and recorded as described by Xiao et al., 2018a. 2.6. Effects of companion microorganisms on the chemical composition of BSFL Based on the results above, group1 showed the lowest and Group3 the highest conversion rate. Therefore, only three treatments were used to evaluate the effects of companion microorganisms on the nutritional composition of BSFL: (1) BSFL without companion bacteria (control); (2) group1 (ratio at FE01:FE04: FE08:BSF-CL = 1:1:1:1); (3) group3 (ratio at FE01:FE04:FE08:BSFCL = 4:1:1:1). After co-conversion of CHM using BSFL and the bacterial community treatments, mature larvae (prepupae or 6th instar larvae Table 1 Poly-bacterial mixtures for co-conversion with BSFL. Synthetic community
Group1 Group2 Group3 Group4 Group5 Group6 Group7 Group8 Group9 Control
The ratio of bacterial strains (v/v) FE01
FE04
FE08
BSF-CL
1 2 4 1 1 1 1 1 1 0
1 1 1 2 4 1 1 1 1 0
1 1 1 1 1 2 4 1 1 0
1 1 1 1 1 1 1 4 2 0
FE01 (K. marina), FE04 (L. boronitolerans), FE08 (P. mirabilis), BSF-CL (B. subtilis).
accounting for 50% of the population) were separated from the treatment residue (Sheppard et al., 2002). Then, larvae were dried at 60 °C and pulverized with a multifunction grinder (BJ-100, BAIJIE ELECTRIC APPLIANCE CO., LTD, China). The analysis of nutritional components was performed at Hubei Academy of Agriculture Sciences, China. Crude protein (CP) was determined using the Kjeldahl nitrogen method (Bosch et al., 2014) following China National Standard for crude protein in feedstuffs: GB/T 6432-94. Crude fat (CF) content was determined with Petroleum ether extraction method following China National Standard for crude fat in feeds: GB/T 6433-2006/ISO 6492:1999. 2.7. Statistical analysis The statistical analysis was performed with SPSS 16.0 (SPSS Inc., Chicago, IL, USA). The results were analysed by one-way analysis of variance (ANOVA), followed by Tukey’s HSD post-hoc comparison. Significance was set at P < 0.05. All data were given as mean ± standard error (S.E.). The figures for CP and CF content were prepared using OriginPro 8(Origin Lab Corporation, Northampton, Massachusetts, USA). 3. Results 3.1. Influence of different BSF companion bacteria on growth property of black soldier fly larvae We inoculated endogenous strains of bacteria (FE01, FE02, FE03, FE04, FE05, FE06, FE07, FE08, FE09, BSF-CL) isolated from BSF to a sterile medium. The endogenous bacteria had an effect on the growth and development of BSFL. Average individual final weight of the larvae in treatments FE01, FE02, FE03, FE04, FE06, FE08, BSF-CL was significantly higher than that of the control (no inoculation of bacteria) (Table 2). The individual larva weight in FE05, FE07 and FE09 groups did not significantly differ from that of the sterile larvae group (Table 2). 3.2. The influence on growth of H. illucens larvae assisted by individual bacteria species The conversion rate of CHM treated with individual bacteria into BSFL biomass was significantly greater in the treatments inoculated with FE01 (8.53%), FE08 (8.80%), and BSF-CL (8.74%). Comparing with the control treatment, the treatments inoculated with FE01, FE08, and BSF-CL increased the conversion rate of BSFL
Table 2 Influence of companion bacteria on the growth of black soldier fly larvae (BSFL). Treatments
5th instar larval weight (g/each larva)
BSF only Sterile larvae (SL) SL + BSF-CL SL + FE01 SL + FE02 SL + FE03 SL + FE04 SL + FE05 SL + FE06 SL + FE07 SL + FE08 SL + FE09
0.1167 ± 0.004a 0.0893 ± 0.004b 0.1184 ± 0.004a 0.1127 ± 0.005a 0.1141 ± 0.006a 0.1100 ± 0.010a 0.1182 ± 0.012a 0.1032 ± 0.008ab 0.1112 ± 0.003a 0.1042 ± 0.003ab 0.1133 ± 0.013a 0.1046 ± 0.002ab
Each treatment has three replicates with 500 each starting with 6-day-old BSFL. Results are means ± SE. Means in the same column with different letters are significantly different. (Tukey HSD test, n = 3, P < 0.05). FE01 (K. marina), FE02 (Micrococcus luteus), FE03 (Enterococcus faecalis), FE04 (L. boronitolerans), FE05 (Sporosarcina koreensis), FE06 (Gordonia sihwensis), FE07 (Enterobacter spp.), FE08 (P. mirabilis), FE09 (Bacillus subtilis), BSF-CL (B. subtilis).
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by 1.11%, 1.38% and 1.32%, respectively. Treatments of CHM with FE03 and FE04 increased the conversion rate by BSFL, but not significantly (Table 3). In contrast, the conversion rate of CHM treated with FE02 or FE06 was lower than that recorded for the control. CHM treated with FE01, FE03, FE04, FE08 and BSF-CL increased the individual larva weight by 14.96%, 1.21%, 0.27%, 18.60%, and 17.79%, respectively (Table 3). But, as with waste conversion, CHM treated with FE02 and FE06 significantly lowered BSFL weight gain as compared to control. 3.3. The co-conversion efficiency of H. illucens larvae assisted by individual bacteria The manure reduction rate of the treatment inoculated with BSF-CL (53.58%) was significantly higher than the control treatment (49.23%) (Table 4). Contrarily, the manure reduction rate of the treatments inoculated with FE02 (47.00%), FE03 (48.64%), and FE06 (46.66%) were lower as compared with the control treatment (49.23 ± 0.41%). We choose species for the poly-bacterial study based on the results of weight gain in Table 3 and manure reduction rate in Table 4. Weight gain and manure reduction rate in FE01, FE04, FE08 and BSF-CL were all higher than that of control. Although the conversion rate and manure reduction rate of FE04 were not significantly improved, they were both improved to a certain extent. Four strains FE01, FE04, FE08 and BSF-CL was selected for the poly-bacterial study.
Table 3 Co-conversion efficiency of black soldier fly larvae (BSFL) assisted by individual bacteria strains. Treatments
Conversion rate (%)
Total dry weight of BSFL (5th instar) after conversion (g)
Weight gain of BSFL (compared to control) (%)
BSFL + FE01 BSFL + FE02 BSFL + FE03 BSFL + FE04 BSFL + FE06 BSFL + FE08 BSFL + BSF-CL BSFL(control treatment)
8.53 ± 0.12b 7.39 ± 0.03c 7.51 ± 0.05c 7.44 ± 0.04c 7.33 ± 0.02c 8.80 ± 0.05a 8.74 ± 0.02a 7.42 ± 0.02c
10.75 ± 0.01b 9.31 ± 0.02 cd 9.46 ± 0.04c 9.37 ± 0.03 cd 9.24 ± 0.02d 11.09 ± 0.04a 11.01 ± 0.02a 9.35 ± 0.02 cd
14.96 0.40 1.21 0.27 1.21 18.60 17.79
Each treatment has three replicates with 500 each starting with 6-day-old BSFL. Results are means ± SE. Means in the same column with different letters are significantly different. (Tukey HSD test, n = 3, P < 0.05). FE01 (K. marina), FE02 (Micrococcus luteus), FE03 (Enterococcus faecalis), FE04 (L. boronitolerans), FE06 (Gordonia sihwensis), FE08 (P. mirabilis), BSF-CL (B. subtilis)
Table 4 Manure reduction efficiency of black soldier fly larvae (BSFL) assisted by individual bacteria strains. Treatments
Manure reduction rate (%)
BSFL + FE01 BSFL + FE02 BSFL + FE03 BSFL + FE04 BSFL + FE06 BSFL + FE08 BSFL + BSF-CL BSFL (control treatment)
50.73 ± 0.14ab 47.00 ± 0.19c 48.64 ± 2.62bc 50.81 ± 0.63ab 46.66 ± 0.55c 50.43 ± 0.69b 53.58 ± 0.29a 49.23 ± 0.41bc
Each treatment has three replicates with 500 larvae each starting when 6-day-old. FE01 (K. marina), FE02 (Micrococcus luteus), FE03 (Enterococcus faecalis), FE04 (L. boronitolerans), FE06 (Gordonia sihwensis), FE08 (P. mirabilis), BSF-CL (B. subtilis) *Weight gain of BSFL is in relation to the control. Results are means ± SE. Means in the same column with different letters are significantly different. (Tukey HSD test, n = 3, P < 0.05).
3.4. The influence on growth of H. illucens larvae assisted by polybacterial cultures The conversion rate of BSFL with the poly-bacterial cultures was significantly larger in the treatments inoculated with Group2 (9.51%), Group3 (10.44%), Group4 (9.85%), Group5 (10.01%) and Group8 (9.58%) than the control (8.12%) (Table 5). Compared with the control treatment, the conversion rates of BSFL inoculated with Group2, Group3, Group4, Group5 Group6 and Group8 were significantly increased by 1.39%, 2.32%, 1.73%, 1.89%, 0.47%, and 1.46%, respectively (Table 5). It was important to note the treatment inoculated with Group3 provided the greatest conversion efficiency. However, the conversion rate of BSFL in the treatment inoculated with Group1 significantly decreased by 1.06% compared to the control treatment. There was no significant difference in conversion rate for Group6, Group7, Group9 and the control. It was noted that the various complex ratios of bacteria had effects on BSFL weight increment gain over time. The different bacterial ratio used in the treatments inoculated with Group2, Groups3, Group4, Group5, Group6, and Group8 showed greater rate of weight gain as compared to the control treatment. The weight gain of BSFL in the treatment inoculated with Group2, Group3, Group4, Group5, Group6 and Group8 were higher by 17.12%, 28.57%, 21.31%, 23.28%, 5.79% and 17.98% than that recorded for the control, respectively (Table 5). Although the weight gain of BSFL in Group 3, 4 and 5 was above 20% only the one of Group3 (28.57%) was significantly larger than the control.
3.5. The co-conversion efficiency evaluation of H. illucens larvae assisted by poly-bacterial cultures BSFL inoculated with the poly-bacterial culture in treatments Group2 and Group8 showed a significantly greater manure reduction rate (51.85% and 52.91%, respectively) than the control (49.36%); the other treatments were not significantly different from that of the control (Table 6).
3.6. Influence of poly-bacterial cultures on CP and CF content of dry H. illucens after CHM co-conversion CP content of BSFL resulting from treatments Group1 was larger than that of the control (Fig. 1). CP content increased by 2.66% in the Group1 treatment when compared to the control.
Table 5 Co-conversion efficiency of black soldier fly larvae (BSFL) assisted by poly-bacterial cultures. Treatments
Conversion rate (%)
Total dry weight of BSFL after conversion (g)
Weight gain of BSFL (compared to control) (%)
BSFL + Group1 BSFL + Group2 BSFL + Group3 BSFL + Group4 BSFL + Group5 BSFL + Group6 BSFL + Group7 BSFL + Group8 BSFL + Group9 BSFL (control treatment)
7.06 ± 0.17c 9.51 ± 0.07a 10.44 ± 0.35a 9.85 ± 0.05a 10.01 ± 0.09a 8.59 ± 0.05b 7.97 ± 0.16b 9.58 ± 0.20a 8.07 ± 0.21b 8.12 ± 0.11b
8.90 ± 0.12e 11.98 ± 0.05b 13.15 ± 0.25a 12.41 ± 0.04b 12.61 ± 0.07ab 10.82 ± 0.04c 10.04 ± 0.12d 12.07 ± 0.15b 10.17 ± 0.15bc 10.23 ± 0.08bc
13.05 17.12 28.57 21.31 23.28 5.79 1.85 17.98 0.62
Each treatment has three replicates with 500 larvae starting when 6 days-old. *Weight gain of BSFL is in relation to the control. Results are means ± SE. Means in the same column with different letters are significantly different. (Tukey HSD test, n = 3, P < 0.05)
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Table 6 The manure reduction efficiency of black soldier fly larvae (BSFL) assisted by polybacterial cultures. Treatments
Manure reduction rate (%)
BSFL + Group1 BSFL + Group2 BSFL + Group3 BSFL + Group4 BSFL + Group5 BSFL + Group6 BSFL + Group7 BSFL + Group8 BSFL + Group9 BSFL (control treatment)
50.01 ± 0.24bc 51.85 ± 0.23a 49.24 ± 0.08 cd 49.22 ± 0.24 cd 48.59 ± 0.11d 49.82 ± 0.15bcd 50.62 ± 0.41b 52.91 ± 1.02a 48.68 ± 0.56d 49.36 ± 0.56bcd
Each treatment has three replicates with 500 larvae each starting when 6 days-old. Results are means ± SE. Means in the same column with different letters are significantly different. (Tukey HSD test, n = 3, P < 0.05).
Fig. 1. Crude protein (white columns) and crude fat (grey columns) contents of BSFL grown on chicken manure with companion bacteria. Bars within each column indicate the standard error (n = 3). BSFL: just black soldier fly larvae no BSF companion bacteria; BSFL + Group1: black soldier fly larvae with assisted by polybacterial cultures Group1; BSFL + Group3: black soldier fly larvae with assisted by poly-bacterial cultures Group3 (see Table 1).
Results for the CF content of BSFL varied depending on treatment (Fig. 1). The larvae resulting from the Gourp1 treatment had a lower CF content ( 3.06%) than those resulting from the control treatment. In contrast, the CF content of BSFL resulting from the Group3 treatment was larger than that of the control (1.53%). 4. Discussion The present study describes the effects of BSF egg-associated and gut companion bacteria on manure conversion efficiency, larval weight gain, manure mass reduction, and nutritional accumulation of BSFL reared on CHM. Still, little research has explored whether bacteria from BSF could influence BSFL growth and efficacy of converting substrates to insect biomass. Yu et al. (2011) isolated companion bacteria from BSFL and demonstrated that inoculating poultry manure with these agents could enhance larval growth and development. Manure inoculated with bacteria resulted in accelerated development. Herein, results from this study clearly indicate that the companion bacteria can enhance the BSFL conversion efficiency, BSFL weight, manure reduction and nutrient accumulation of BSFL. Results from our study showed that the bacteria from BSF eggs (FE01, FE02, FE03, FE04, FE06, FE08) and from BSFL gut (BSF-CL)
could be used to enhance BSFL weight. Especially, strains FE01, FE08 and BSF-CL have obviously role in obtaining more BSFL biomass. We speculated the reason is that strains FE01, FE08 and BSF-CL could produce some substances to stimulate BSFL growth. Maybe others produce some substances to inhibit BSFL growth. In our study, after 14 days of co-conversion of CHM by BSFL and individual bacterial isolates, conversion efficiency was not different from the control. It would have been beneficial for the industrial production of BSFL, if shortening the production cycle would allow for an increased harvest of BSFL biomass. However, various ratios of bacteria had different influences on BSFL conversion efficiency. In particular, the conversion rate of group3 inoculated with the poly-bacterial community mixture at a ratio of 4:1:1:1 was 10.44% compared to 8.12% for the control, an increase of 28.6%. How the bacteria of eggs and larval gut enhance BSFL growth and convert CHM is unclear. There is growing evidence that intestinal bacteria are important beneficial partners of their metazoan hosts (Colman et al., 2012; ur Rehman et al., 2017). Abduh et al. (2017) used Aspergillus niger and a Biotaff solution containing a consortium of microbes to assist BSFL in converting rubber seeds into protein and oil-rich biomass. The primary mechanism is that microbes are capable of degrading lignocellulosic materials in rubber seeds which facilitates further bioconversion by BSFL. Zheng et al. (2012) also used microbes (Rid-X) to first degrade lignocellulose in rice straw and then allowing BSFL to gain access to previously unavailable bound nutrients. Shin et al. (2011) reported that the pyrroloquinoline quinone–dependent alcohol dehydrogenase activity of a commensal bacterium, Acetobacter pomorum, modulates insulin/insulin-like growth factor signalling 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 bacterium of the Drosophila intestine, promotes its host systemic growth by modulating hormonal signals through TOR-dependent nutrient sensing. Yamada et al. (2015), demonstrated that Issatchenkia orientalis promotes Drosophila melanogaster amino acid harvest to replenish undernourished flies. Therefore, we hypothesize similar physiological processes are occurring in BSFL. For the nutrient accumulation in BSFL after co-conversion with poly-bacterial cultures, we selected two treatments, Group1 (lowest conversion rate) and Group3 (highest conversion rate) to analyze the CP and CF in order to compare them with the control group. Interestingly, the content of CP is highest and CF lowest in the Group1 treatment. On the contrary, content of CF is the highest in Group3. The ratio of poly-bacterial cultures is FE01:FE04:FE08: BSF-CL = 4:1:1:1, FE01 being the most important species, while in Group1 all bacterial species were equally represented (1:1:1:1). This means that that FE01 must be responsible for the CF accumulation in the BSF larval body. However, FE01 must then also be responsible for the lower crude protein fraction in Group3 compared to Group1. Some recent studies have been undertaken to analyse the nutrient accumulation of BSFL reared on different organic waste substrates (Bosch et al., 2014; Spranghers et al., 2017; Julita et al., 2018; Meneguz et al., 2018). In our study, the CP content of dry larvae was close to the values reported by Xiao et al., 2018a. The fat content of dry larvae was lower than that of Xiao et al., 2018a, but this may be related to the different organic waste stream converted and, therefore, to the different nutritional content (Yu et al., 2011). Wang et al. (2017b) showed that in mammals the intestinal microbiota regulates body composition through the circadian transcription factor NFIL3. However, similar mechanisms in H. illucens need to yet be investigated. In terms of industrial economics, in our previous research, we used B. subtilis (BSF-CL) in BSFL large-scale conversion systems to enhance manure conversion. The weight gain of the BSFL increased
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by 15.9% compared to the control group (no B. subtilis BSF-CL) (Xiao et al., 2018a). In the present study, results were even better with a weight gain increase of 28.57% higher than the control. In other words, by converting the same amount of CHM, treatments are able to produce more larval mass. Harvesting more larval mass potentially means more income in the CHM conversion process. Given that adult BSF does not need to feed, the BSFL accumulate a large fat reserve for larval development and adult survival (Nguyen et al., 2015). Companion bacteria can help insects to digest non-digestible nutrients, and protect them from predators, parasites and pathogens (Engel and Moran, 2013). After pretreatment with companion bacteria, BSFL can digest small nutrient molecules, such as peptides, amino acids, and fatty acids directly. The digestive process is enhanced by the presence of bacteria since they have enzymes such as lipases, pectinases and others, which promote digestion of macromolecules that would otherwise be indigestible to insects (Ruokolainen et al., 2016). 5. Conclusion Findings from this study revealed that BSF egg-associated and larval gut bacteria have significant effects, in most cases positive, on the conversion rate, larvae weight gain, manure reduction, and nutrient accumulation in BSF, which are important benefits for the insect farming industry in terms of protein and oil production. Overall, this research could open new pathways since the elucidation of the co-conversion mechanisms in BSF might enhance the efficiency of organic waste disposal centres and industrialscale BSFL production facilities. Therefore, it is highly recommended to BSFL producers to implement specific BSF eggassociated and larval gut bacterial strains to help in the nutrient accumulation (CP and CF) in the larvae and to improve chicken manure reduction management. Declaration of Competing Interest None. Acknowledgements This work was supported by the National Key Technology R & D Program of China (2018YFD0500203 and 2017YFD0800200). Thanks to our cooperation friend Dr. Heather R. Jordan from Mississippi State University, USA, for revising the language. References Abduh, M.Y., Jamilah, M., Istiandari, P., Manurung, S., Manurung, R., 2017. Bioconversion of rubber seeds to produce protein and oil-rich biomass using black soldier fly larva assisted by microbes. J. Entomol. Zool. Stud. 5, 591–597. Albihn, A., Vinnerås, B., 2007. Biosecurity and arable use of manure and biowaste treatment alternatives. Livest. Sci. 112, 232–239. https://doi.org/10.1016/j. livsci.2007.09.015. Allegretti, G., Talamini, E., Schmidt, V., Bogorni, P.C., Ortega, E., 2018. Insect as feed: an emergy assessment of insect meal as a sustainable protein source for the Brazilian poultry industry. J. Clean. Prod. 171, 403–412. https://doi.org/10.1016/ j.jclepro.2017.09.244. Belghit, I., Liland, N.S., Gjesdal, P., Biancarosa, I., Menchetti, E., Li, Y., Waagbo, R., Krogdahl, A., Lock, E.J., 2019. Black soldier fly larvae meal can replace fish meal in diets of sea-water phase Atlantic Salmon (Salmo salar). Aquaculture 503, 609–619. https://doi.org/10.1016/j.aquaculture.2018.12.032. Biasato, I., Renna, M., Gai, F., Dabbou, S., Meneguz, M., Perona, G., Martinez, S., Lajusticia, A.C.B., Bergana, S., Sardi, L., Teresa, M., Capucchio, M.T., Bressan, E., Dama, A., Schiavone, A., Gasco, L., 2019. Partially defatted black soldier fly larva meal inclusion in piglet diets: effects on the growth performance, nutrient digestibility, blood profile, gut morphology and histological features. J. Anim. Sci. Biotechno. 10 (1), 12. https://doi.org/10.1186/s40104-019-0325-x. Bosch, G., Zhang, S., Oonincx, D.G.A.B., Hendriks, W.H., 2014. Protein quality of insects as potential ingredients for dog and cat foods. J. Nutr. Sci. 3,. https://doi. org/10.1017/jns.2014.23 e29.
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Management of chicken manure using black soldier fly (Diptera: Stratiomyidae) larvae assisted by companion bacteria Lorenzo Mazza a,b,1, Xiaopeng Xiao a,1, Kashif ur Rehman a,c, Minmin Cai a, Dingnan Zhang a, Salvatore Fasulo b, Jeffery K. Tomberlin d, Longyu Zheng a,⇑, Abdul Aziz Soomro 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, PR China b University of Messina, Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, Messina, Italy c Livestock and Dairy Development Department, Poultry Research Institute, Rawalpindi, Pakistan d Department of Entomology, Texas A&M University, College Station, TX, USA
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Article history: Received 8 April 2019 Revised 25 October 2019 Accepted 30 October 2019
Keywords: Hermetia illucens Companion bacteria Chicken manure Environmental pollution Conversion efficiency
a b s t r a c t Black soldier fly (BSF) is used for the management of organic waste, but research has hardly explored the effect of companion bacteria when chicken manure (CHM) is converted to insect biomass. In this study, we isolated nine bacterial species (FE01, FE02, FE03, FE04, FE05, FE06, FE07, FE08, FE09) from BSF eggs and one (BSF-CL) from the larval gut. These companion bacteria were inoculated into CHM along with BSF larvae (BSFL). Larval growth and manure conversion rates were determined. Results indicated that almost all bacteria individual bacteria in this study significantly promote BSFL growth. BSFL reared in manure with the species Kocuria marina (FE01), Lysinibacillus boronitolerans (FE04), Proteus mirabilis (FE08) and Bacillus subtilis (BSF-CL) had higher weight gain and manure reduction rates compared to the control. These four strains used were then examined as a poly-bacteria community experiment to determine BSFL growth and manure conversion. Manure inoculated with the poly-bacteria Group3 (FE01:FE04:FE08:BSF-CL = 4:1:1:1) and then fed to BSFL resulted in 28.6% more weight gain than the control. The greatest manure reduction rate (52.91%) was reached when companion bacteria were mixed at a ratio of 1:1:1:4. Additionally, the companion bacteria influenced the nutritional value of BSFL. Crude protein content in Group1 (FE01:FE04:FE08:BSF-CL = 1:1:1:1) was significantly larger than that of the control. Crude fat content in Group3 was significantly larger than that of the control. BSFL companion bacteria and their poly-bacteria compound improved manure conversion efficiency and nutrient accumulation in BSFL, reduced CHM quantity, increased larvae biomass, with potential economic gains in CHM management. Ó 2019 Elsevier Ltd. All rights reserved.
1. Introduction Organic wastes, such as poultry and livestock manure, sewage sludge, kitchen and domestic wastes, have become a major global challenge, and this is expected to increase as the global population expands from 7.3 billion in 2015 to 9.7 billion in 2050 (Surendra et al., 2016). Most organic wastes generated is left untreated (Reckmann et al., 2013). Moreover, the unprocessed organic wastes, which contain valuable energy and nutrients, can contribute to an increase in the annual greenhouse gas emissions (GHG) (Dai et al., 2016) and a leaching of nutrients into the under⇑ Corresponding authors. E-mail addresses: [email protected] (L. Zheng), [email protected]. edu.cn (J. Zhang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.wasman.2019.10.055 0956-053X/Ó 2019 Elsevier Ltd. All rights reserved.
ground water table, causing eutrophication (Zhang et al., 2012). Animal manure is a very harmful organic waste, and if untreated will pose a risk for the spread of pathogens (Albihn and Vinnerås, 2007; Sun et al., 2016; ur Rehman et al., 2017). Therefore, it is important to find an effective way to recycle these organic waste streams. Insects are the most abundant and diverse group of animals on earth (Allegretti et al., 2018), and are likewise the largest underutilized valuable resources (Roffeis et al., 2018). Organic wastes may be successfully converted into biomass with insects and other invertebrates (ur Rehman et al., 2017; Wang et al., 2017), such as Hermetia illucens L. (Diptera: Stratiomyidae) (Ma et al., 2018; Xiao et al., 2018), Musca domestica L. (Diptera: Muscidae) (Zhang et al., 2012), Eisenia fetida L. (Opisthopora: Lumbricidae) (Negi and Suthar, 2018), and Tenebrio molitor L. (Coleoptera: Tenebrionidae) (Thévenot et al., 2018). These insects can digest nutrients
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Nomenclature List of abbreviations and notation BSF Black soldier fly BSFL Black soldier fly larvae BSF-CL Bacillus subtilis CHM Chicken manure CP Crude protein CF Crude fat FE01 Kocuria marina FE02 Micrococcus luteus
from food wastes (Nguyen et al., 2015) such as vegetables and fruits, agro-industrial by-products (Meneguz et al., 2018), chicken and dairy manure (Rehman et al., 2017a) and convert them into valuable proteins and fats to be used in animal feed formulations (Liu et al., 2017; Xiao et al., 2018), while the residue can be used as a fertilizer (Ma et al., 2018; Xiao et al., 2018). Insects, such as H. illucens (Jeon et al., 2011), Drosophila melanogaster L. (Diptera: Drosophilidae) (Ren et al., 2007) and M. domestica L. (Gupta et al., 2012), have a wide variety of related symbiotic microorganisms (Engel and Moran, 2013). Host associated microbes can contribute to the development of the organism as they are involved in a variety of insect physiological processes, including immune response (Chen et al., 2016). Recently, some studies indicated that symbiotic microbes play a key role in insect growth, gut microbiota development and digestive enzyme production at different life stages and depending on the environmental habitat (Jiang et al., 2019; Scott et al., 2008; ur Rehman et al., 2017; Yun et al., 2014). Moreover, some bacteria can protect insects from predators, parasites and pathogens (Laughton et al., 2011). H. illucens, commonly known as the black soldier fly (BSF), is classified as a saprophagous insect, feeding on rotten meat and decaying plant matter in nature (Makkar et al., 2014). Black soldier fly larvae (BSFL) are capable of consuming large amounts of organic side streams such as food waste, rice straw, dead animals, and animal manure (Makkar et al., 2014; Rehman et al., 2017; Xiao et al., 2018). They can reduce nitrogen-rich wastes by 75% and poultry, dairy and pig manure by 50% (Rehman et al., 2017; ur Rehman et al., 2017; Newton et al., 2005). In addition, harvested larvae contain about 40% protein and 35% fat (Liu et al., 2017). In various feeding studies, larval or prepupal meal was found to be a suitable full, or partial, replacement of fish meal, meat meal, and plant protein meal in livestock (Makkar et al., 2014), poultry (Schiavone et al., 2017), pig (Spranghers et al., 2018; Biasato et al., 2019) and aquaculture feed (Cummins et al., 2017; Renna et al., 2017; Belghit et al., 2019). Also, it was previously investigated that symbiotic microbes have synergistic effects on the development of insect biomass (Zheng et al., 2012; Ma et al., 2018). However, few studies have explored the impact of symbiotic bacteria on the accumulation of nutrients, and how they can improve the efficiency of BSF conversion of wastes to insect biomass. Therefore, this study was designed to investigate the effects of egg-associated and gut microbes of BSF on larval development, waste conversion efficiency and nutrient accumulation of BSFL, when reared in fresh chicken manure (CHM).
FE03 FE04 FE05 FE06 FE07 FE08 FE09 LB PBS
Enterococcus faecalis Lysinibacillus boronitolerans Sporosarcina koreensis Gordonia sihwensis Enterobacter spp. Proteus mirabilis Bacillus subtilis Luria-Bertani Phosphate Buffered Saline
five days, larvae were fed a diet based on wheat bran with 75% water content. Feeding was stopped on the sixth day, and larvae were separated from the feed. 2.2. Manure biomass Fresh chicken manure (CHM) was collected from a poultry farm located in Wuhan. Water content in CHM was determined as 74.8%, and dry mass(ash), total nitrogen (TN), total organic carbon (TOC), total phosphorus (TP), cellulose, C/N ratio: carbon/nitrogen, were determined as 22.82%, 3.54%, 40.19%, 2.28%, 14.49%, 15.69 on a dry matter basis, respectively. All the compositions were analysed according to our previous research (Rehman et al., 2017; Xiao et al., 2018). 2.3. Bacterial isolation from eggs and larval gut of the black soldier fly
2. Materials and methods
Microorganisms used in this study were isolated from the egg surface of BSF within 6 h after spawning, and from the gut of 5th instar larvae. For the isolation of bacteria species from the egg surface, 0.5 g of BSF eggs were homogenized with 2 mL distilled water. The mixture was constantly stirred and incubated for 5 min at 28 °C. Following this, the supernatant was diluted and used for isolation and growth of the bacteria on Luria-Bertani (LB) agar medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 18 g/L, distilled water 1000 mL). BSF larvae were reared on a diet consisting of 50% bran and 50% corn flour. To isolate bacteria from the gut, fifth instar larvae were surface-sterilized in 75% ethanol for 3 min and then rinsed three times in sterile Phosphate Buffered Saline (PBS) at pH 7.4. The total gut (pools of 10 larvae) was dissected aseptically, transferred to 100 lL of sterile PBS, and homogenized. For isolations based on colony selection, diluted cell suspensions were plated on solid LB medium and incubated for 48 h at 28 °C. All morphologically different bacterial species were sub-cultured three times to ensure purity and stability. The bacterial species isolated from egg surfaces were identified as Kocuria marina (FE01), Micrococcus luteus (FE02), Enterococcus faecalis (FE03), Lysinibacillus boronitolerans (FE04), Gordonia sihwensis (FE06), and Proteus mirabilis (FE08). In a previous study (unpublished data) we also isolated from the gut Bacillus subtilis (BSF-CL) and this species was also used in our study. Bacterial isolates were inoculated into LB medium with aeration for 36 h at 28 °C. Following this, bacteria were washed three times in PBS for 15 min at 8000 rpm, and re-suspended in 30 mL of sterile water to obtain a final concentration of 1 108 CFU/mL determined by using a hemocytometer.
2.1. H. illucens
2.4. Co-conversion of individual bacterial isolates and BSFL
The larvae of H. illucens, Wuhan strain, used in this study were bred at the State Key Laboratory of Agricultural Microbiology of Huazhong Agricultural University, Wuhan, China. During the first
The ten BSF companion bacterial strains, namely FE01, FE02, FE03, FE04, FE05, FE06, FE07, FE08, FE09 and BSF-CL, were individually inoculated as supplements into 500 g fresh CHM with a
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proportion of 1% (v/w). Five hundred 6-day-old BSFL were inoculated into each waste-bacterial mixture, as well as into 500 g fresh CHM without bacterial supplementation, serving as the control. Each treatment was set up in three replicates and kept in the climatic chamber for approximately 14 days, at 28 °C with 60% to 70% RH. After 14 days, weight and conversion rate of BSFL, and reduction rate of CHM, were recorded using the previously described methodology (Xiao et al., 2018a). The formulae used were as follows: BSFL Conversion rate = total larval biomass(g)/ feed added(g) 100%; Weight gain of BSFL (compared to control) = (G2-G1)/G1 100%, G1: Control group BSFL dry weight (g) after conversion, G2: treatment group BSFL dry weight(g) after conversion; Reduction rate of CHM = (W1-W2)/W1 100%, W1: the initial CHM dry weight, W2: the final dry residue weight after the BSFL digestion. 2.5. Co-conversion of poly-bacterial cultures and BSFL Based on the analysis mentioned above, K. marina (FE01), L. boronitolerans (FE04), P. mirabilis (FE08), and B. subtilis (BSF-CL) were chosen to be used in the poly-bacterial study. These bacteria were combined at different ratios and inoculated into CHM. Coconversion efficiency of BSF and bacterial composites were determined. Nine poly-bacterial communities were inoculated separately into 500 g fresh CHM with a proportion of 1% (v/w) (Table 1). After this, five hundred 6-day-old BSFL were inoculated into the mixture, or into a ‘‘no-bacterial supplement” mixture as a control. Each treatment included three replicates, which were kept in the climatic chamber for 14 days, at 28 °C with 60% to 70% RH. The larvae were directly separated from the treatment residue, and larval dry weight was obtained after washing with distilled water and drying at 60 °C for 2–3 days. Dry matter of initial CHM and treatment residue were calculated after drying in an oven at 105 °C. Conversion rate and weight gain rate of BSFL, and reduction rate of CHM were observed and recorded as described by Xiao et al., 2018a. 2.6. Effects of companion microorganisms on the chemical composition of BSFL Based on the results above, group1 showed the lowest and Group3 the highest conversion rate. Therefore, only three treatments were used to evaluate the effects of companion microorganisms on the nutritional composition of BSFL: (1) BSFL without companion bacteria (control); (2) group1 (ratio at FE01:FE04: FE08:BSF-CL = 1:1:1:1); (3) group3 (ratio at FE01:FE04:FE08:BSFCL = 4:1:1:1). After co-conversion of CHM using BSFL and the bacterial community treatments, mature larvae (prepupae or 6th instar larvae Table 1 Poly-bacterial mixtures for co-conversion with BSFL. Synthetic community
Group1 Group2 Group3 Group4 Group5 Group6 Group7 Group8 Group9 Control
The ratio of bacterial strains (v/v) FE01
FE04
FE08
BSF-CL
1 2 4 1 1 1 1 1 1 0
1 1 1 2 4 1 1 1 1 0
1 1 1 1 1 2 4 1 1 0
1 1 1 1 1 1 1 4 2 0
FE01 (K. marina), FE04 (L. boronitolerans), FE08 (P. mirabilis), BSF-CL (B. subtilis).
accounting for 50% of the population) were separated from the treatment residue (Sheppard et al., 2002). Then, larvae were dried at 60 °C and pulverized with a multifunction grinder (BJ-100, BAIJIE ELECTRIC APPLIANCE CO., LTD, China). The analysis of nutritional components was performed at Hubei Academy of Agriculture Sciences, China. Crude protein (CP) was determined using the Kjeldahl nitrogen method (Bosch et al., 2014) following China National Standard for crude protein in feedstuffs: GB/T 6432-94. Crude fat (CF) content was determined with Petroleum ether extraction method following China National Standard for crude fat in feeds: GB/T 6433-2006/ISO 6492:1999. 2.7. Statistical analysis The statistical analysis was performed with SPSS 16.0 (SPSS Inc., Chicago, IL, USA). The results were analysed by one-way analysis of variance (ANOVA), followed by Tukey’s HSD post-hoc comparison. Significance was set at P < 0.05. All data were given as mean ± standard error (S.E.). The figures for CP and CF content were prepared using OriginPro 8(Origin Lab Corporation, Northampton, Massachusetts, USA). 3. Results 3.1. Influence of different BSF companion bacteria on growth property of black soldier fly larvae We inoculated endogenous strains of bacteria (FE01, FE02, FE03, FE04, FE05, FE06, FE07, FE08, FE09, BSF-CL) isolated from BSF to a sterile medium. The endogenous bacteria had an effect on the growth and development of BSFL. Average individual final weight of the larvae in treatments FE01, FE02, FE03, FE04, FE06, FE08, BSF-CL was significantly higher than that of the control (no inoculation of bacteria) (Table 2). The individual larva weight in FE05, FE07 and FE09 groups did not significantly differ from that of the sterile larvae group (Table 2). 3.2. The influence on growth of H. illucens larvae assisted by individual bacteria species The conversion rate of CHM treated with individual bacteria into BSFL biomass was significantly greater in the treatments inoculated with FE01 (8.53%), FE08 (8.80%), and BSF-CL (8.74%). Comparing with the control treatment, the treatments inoculated with FE01, FE08, and BSF-CL increased the conversion rate of BSFL
Table 2 Influence of companion bacteria on the growth of black soldier fly larvae (BSFL). Treatments
5th instar larval weight (g/each larva)
BSF only Sterile larvae (SL) SL + BSF-CL SL + FE01 SL + FE02 SL + FE03 SL + FE04 SL + FE05 SL + FE06 SL + FE07 SL + FE08 SL + FE09
0.1167 ± 0.004a 0.0893 ± 0.004b 0.1184 ± 0.004a 0.1127 ± 0.005a 0.1141 ± 0.006a 0.1100 ± 0.010a 0.1182 ± 0.012a 0.1032 ± 0.008ab 0.1112 ± 0.003a 0.1042 ± 0.003ab 0.1133 ± 0.013a 0.1046 ± 0.002ab
Each treatment has three replicates with 500 each starting with 6-day-old BSFL. Results are means ± SE. Means in the same column with different letters are significantly different. (Tukey HSD test, n = 3, P < 0.05). FE01 (K. marina), FE02 (Micrococcus luteus), FE03 (Enterococcus faecalis), FE04 (L. boronitolerans), FE05 (Sporosarcina koreensis), FE06 (Gordonia sihwensis), FE07 (Enterobacter spp.), FE08 (P. mirabilis), FE09 (Bacillus subtilis), BSF-CL (B. subtilis).
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by 1.11%, 1.38% and 1.32%, respectively. Treatments of CHM with FE03 and FE04 increased the conversion rate by BSFL, but not significantly (Table 3). In contrast, the conversion rate of CHM treated with FE02 or FE06 was lower than that recorded for the control. CHM treated with FE01, FE03, FE04, FE08 and BSF-CL increased the individual larva weight by 14.96%, 1.21%, 0.27%, 18.60%, and 17.79%, respectively (Table 3). But, as with waste conversion, CHM treated with FE02 and FE06 significantly lowered BSFL weight gain as compared to control. 3.3. The co-conversion efficiency of H. illucens larvae assisted by individual bacteria The manure reduction rate of the treatment inoculated with BSF-CL (53.58%) was significantly higher than the control treatment (49.23%) (Table 4). Contrarily, the manure reduction rate of the treatments inoculated with FE02 (47.00%), FE03 (48.64%), and FE06 (46.66%) were lower as compared with the control treatment (49.23 ± 0.41%). We choose species for the poly-bacterial study based on the results of weight gain in Table 3 and manure reduction rate in Table 4. Weight gain and manure reduction rate in FE01, FE04, FE08 and BSF-CL were all higher than that of control. Although the conversion rate and manure reduction rate of FE04 were not significantly improved, they were both improved to a certain extent. Four strains FE01, FE04, FE08 and BSF-CL was selected for the poly-bacterial study.
Table 3 Co-conversion efficiency of black soldier fly larvae (BSFL) assisted by individual bacteria strains. Treatments
Conversion rate (%)
Total dry weight of BSFL (5th instar) after conversion (g)
Weight gain of BSFL (compared to control) (%)
BSFL + FE01 BSFL + FE02 BSFL + FE03 BSFL + FE04 BSFL + FE06 BSFL + FE08 BSFL + BSF-CL BSFL(control treatment)
8.53 ± 0.12b 7.39 ± 0.03c 7.51 ± 0.05c 7.44 ± 0.04c 7.33 ± 0.02c 8.80 ± 0.05a 8.74 ± 0.02a 7.42 ± 0.02c
10.75 ± 0.01b 9.31 ± 0.02 cd 9.46 ± 0.04c 9.37 ± 0.03 cd 9.24 ± 0.02d 11.09 ± 0.04a 11.01 ± 0.02a 9.35 ± 0.02 cd
14.96 0.40 1.21 0.27 1.21 18.60 17.79
Each treatment has three replicates with 500 each starting with 6-day-old BSFL. Results are means ± SE. Means in the same column with different letters are significantly different. (Tukey HSD test, n = 3, P < 0.05). FE01 (K. marina), FE02 (Micrococcus luteus), FE03 (Enterococcus faecalis), FE04 (L. boronitolerans), FE06 (Gordonia sihwensis), FE08 (P. mirabilis), BSF-CL (B. subtilis)
Table 4 Manure reduction efficiency of black soldier fly larvae (BSFL) assisted by individual bacteria strains. Treatments
Manure reduction rate (%)
BSFL + FE01 BSFL + FE02 BSFL + FE03 BSFL + FE04 BSFL + FE06 BSFL + FE08 BSFL + BSF-CL BSFL (control treatment)
50.73 ± 0.14ab 47.00 ± 0.19c 48.64 ± 2.62bc 50.81 ± 0.63ab 46.66 ± 0.55c 50.43 ± 0.69b 53.58 ± 0.29a 49.23 ± 0.41bc
Each treatment has three replicates with 500 larvae each starting when 6-day-old. FE01 (K. marina), FE02 (Micrococcus luteus), FE03 (Enterococcus faecalis), FE04 (L. boronitolerans), FE06 (Gordonia sihwensis), FE08 (P. mirabilis), BSF-CL (B. subtilis) *Weight gain of BSFL is in relation to the control. Results are means ± SE. Means in the same column with different letters are significantly different. (Tukey HSD test, n = 3, P < 0.05).
3.4. The influence on growth of H. illucens larvae assisted by polybacterial cultures The conversion rate of BSFL with the poly-bacterial cultures was significantly larger in the treatments inoculated with Group2 (9.51%), Group3 (10.44%), Group4 (9.85%), Group5 (10.01%) and Group8 (9.58%) than the control (8.12%) (Table 5). Compared with the control treatment, the conversion rates of BSFL inoculated with Group2, Group3, Group4, Group5 Group6 and Group8 were significantly increased by 1.39%, 2.32%, 1.73%, 1.89%, 0.47%, and 1.46%, respectively (Table 5). It was important to note the treatment inoculated with Group3 provided the greatest conversion efficiency. However, the conversion rate of BSFL in the treatment inoculated with Group1 significantly decreased by 1.06% compared to the control treatment. There was no significant difference in conversion rate for Group6, Group7, Group9 and the control. It was noted that the various complex ratios of bacteria had effects on BSFL weight increment gain over time. The different bacterial ratio used in the treatments inoculated with Group2, Groups3, Group4, Group5, Group6, and Group8 showed greater rate of weight gain as compared to the control treatment. The weight gain of BSFL in the treatment inoculated with Group2, Group3, Group4, Group5, Group6 and Group8 were higher by 17.12%, 28.57%, 21.31%, 23.28%, 5.79% and 17.98% than that recorded for the control, respectively (Table 5). Although the weight gain of BSFL in Group 3, 4 and 5 was above 20% only the one of Group3 (28.57%) was significantly larger than the control.
3.5. The co-conversion efficiency evaluation of H. illucens larvae assisted by poly-bacterial cultures BSFL inoculated with the poly-bacterial culture in treatments Group2 and Group8 showed a significantly greater manure reduction rate (51.85% and 52.91%, respectively) than the control (49.36%); the other treatments were not significantly different from that of the control (Table 6).
3.6. Influence of poly-bacterial cultures on CP and CF content of dry H. illucens after CHM co-conversion CP content of BSFL resulting from treatments Group1 was larger than that of the control (Fig. 1). CP content increased by 2.66% in the Group1 treatment when compared to the control.
Table 5 Co-conversion efficiency of black soldier fly larvae (BSFL) assisted by poly-bacterial cultures. Treatments
Conversion rate (%)
Total dry weight of BSFL after conversion (g)
Weight gain of BSFL (compared to control) (%)
BSFL + Group1 BSFL + Group2 BSFL + Group3 BSFL + Group4 BSFL + Group5 BSFL + Group6 BSFL + Group7 BSFL + Group8 BSFL + Group9 BSFL (control treatment)
7.06 ± 0.17c 9.51 ± 0.07a 10.44 ± 0.35a 9.85 ± 0.05a 10.01 ± 0.09a 8.59 ± 0.05b 7.97 ± 0.16b 9.58 ± 0.20a 8.07 ± 0.21b 8.12 ± 0.11b
8.90 ± 0.12e 11.98 ± 0.05b 13.15 ± 0.25a 12.41 ± 0.04b 12.61 ± 0.07ab 10.82 ± 0.04c 10.04 ± 0.12d 12.07 ± 0.15b 10.17 ± 0.15bc 10.23 ± 0.08bc
13.05 17.12 28.57 21.31 23.28 5.79 1.85 17.98 0.62
Each treatment has three replicates with 500 larvae starting when 6 days-old. *Weight gain of BSFL is in relation to the control. Results are means ± SE. Means in the same column with different letters are significantly different. (Tukey HSD test, n = 3, P < 0.05)
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Table 6 The manure reduction efficiency of black soldier fly larvae (BSFL) assisted by polybacterial cultures. Treatments
Manure reduction rate (%)
BSFL + Group1 BSFL + Group2 BSFL + Group3 BSFL + Group4 BSFL + Group5 BSFL + Group6 BSFL + Group7 BSFL + Group8 BSFL + Group9 BSFL (control treatment)
50.01 ± 0.24bc 51.85 ± 0.23a 49.24 ± 0.08 cd 49.22 ± 0.24 cd 48.59 ± 0.11d 49.82 ± 0.15bcd 50.62 ± 0.41b 52.91 ± 1.02a 48.68 ± 0.56d 49.36 ± 0.56bcd
Each treatment has three replicates with 500 larvae each starting when 6 days-old. Results are means ± SE. Means in the same column with different letters are significantly different. (Tukey HSD test, n = 3, P < 0.05).
Fig. 1. Crude protein (white columns) and crude fat (grey columns) contents of BSFL grown on chicken manure with companion bacteria. Bars within each column indicate the standard error (n = 3). BSFL: just black soldier fly larvae no BSF companion bacteria; BSFL + Group1: black soldier fly larvae with assisted by polybacterial cultures Group1; BSFL + Group3: black soldier fly larvae with assisted by poly-bacterial cultures Group3 (see Table 1).
Results for the CF content of BSFL varied depending on treatment (Fig. 1). The larvae resulting from the Gourp1 treatment had a lower CF content ( 3.06%) than those resulting from the control treatment. In contrast, the CF content of BSFL resulting from the Group3 treatment was larger than that of the control (1.53%). 4. Discussion The present study describes the effects of BSF egg-associated and gut companion bacteria on manure conversion efficiency, larval weight gain, manure mass reduction, and nutritional accumulation of BSFL reared on CHM. Still, little research has explored whether bacteria from BSF could influence BSFL growth and efficacy of converting substrates to insect biomass. Yu et al. (2011) isolated companion bacteria from BSFL and demonstrated that inoculating poultry manure with these agents could enhance larval growth and development. Manure inoculated with bacteria resulted in accelerated development. Herein, results from this study clearly indicate that the companion bacteria can enhance the BSFL conversion efficiency, BSFL weight, manure reduction and nutrient accumulation of BSFL. Results from our study showed that the bacteria from BSF eggs (FE01, FE02, FE03, FE04, FE06, FE08) and from BSFL gut (BSF-CL)
could be used to enhance BSFL weight. Especially, strains FE01, FE08 and BSF-CL have obviously role in obtaining more BSFL biomass. We speculated the reason is that strains FE01, FE08 and BSF-CL could produce some substances to stimulate BSFL growth. Maybe others produce some substances to inhibit BSFL growth. In our study, after 14 days of co-conversion of CHM by BSFL and individual bacterial isolates, conversion efficiency was not different from the control. It would have been beneficial for the industrial production of BSFL, if shortening the production cycle would allow for an increased harvest of BSFL biomass. However, various ratios of bacteria had different influences on BSFL conversion efficiency. In particular, the conversion rate of group3 inoculated with the poly-bacterial community mixture at a ratio of 4:1:1:1 was 10.44% compared to 8.12% for the control, an increase of 28.6%. How the bacteria of eggs and larval gut enhance BSFL growth and convert CHM is unclear. There is growing evidence that intestinal bacteria are important beneficial partners of their metazoan hosts (Colman et al., 2012; ur Rehman et al., 2017). Abduh et al. (2017) used Aspergillus niger and a Biotaff solution containing a consortium of microbes to assist BSFL in converting rubber seeds into protein and oil-rich biomass. The primary mechanism is that microbes are capable of degrading lignocellulosic materials in rubber seeds which facilitates further bioconversion by BSFL. Zheng et al. (2012) also used microbes (Rid-X) to first degrade lignocellulose in rice straw and then allowing BSFL to gain access to previously unavailable bound nutrients. Shin et al. (2011) reported that the pyrroloquinoline quinone–dependent alcohol dehydrogenase activity of a commensal bacterium, Acetobacter pomorum, modulates insulin/insulin-like growth factor signalling 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 bacterium of the Drosophila intestine, promotes its host systemic growth by modulating hormonal signals through TOR-dependent nutrient sensing. Yamada et al. (2015), demonstrated that Issatchenkia orientalis promotes Drosophila melanogaster amino acid harvest to replenish undernourished flies. Therefore, we hypothesize similar physiological processes are occurring in BSFL. For the nutrient accumulation in BSFL after co-conversion with poly-bacterial cultures, we selected two treatments, Group1 (lowest conversion rate) and Group3 (highest conversion rate) to analyze the CP and CF in order to compare them with the control group. Interestingly, the content of CP is highest and CF lowest in the Group1 treatment. On the contrary, content of CF is the highest in Group3. The ratio of poly-bacterial cultures is FE01:FE04:FE08: BSF-CL = 4:1:1:1, FE01 being the most important species, while in Group1 all bacterial species were equally represented (1:1:1:1). This means that that FE01 must be responsible for the CF accumulation in the BSF larval body. However, FE01 must then also be responsible for the lower crude protein fraction in Group3 compared to Group1. Some recent studies have been undertaken to analyse the nutrient accumulation of BSFL reared on different organic waste substrates (Bosch et al., 2014; Spranghers et al., 2017; Julita et al., 2018; Meneguz et al., 2018). In our study, the CP content of dry larvae was close to the values reported by Xiao et al., 2018a. The fat content of dry larvae was lower than that of Xiao et al., 2018a, but this may be related to the different organic waste stream converted and, therefore, to the different nutritional content (Yu et al., 2011). Wang et al. (2017b) showed that in mammals the intestinal microbiota regulates body composition through the circadian transcription factor NFIL3. However, similar mechanisms in H. illucens need to yet be investigated. In terms of industrial economics, in our previous research, we used B. subtilis (BSF-CL) in BSFL large-scale conversion systems to enhance manure conversion. The weight gain of the BSFL increased
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by 15.9% compared to the control group (no B. subtilis BSF-CL) (Xiao et al., 2018a). In the present study, results were even better with a weight gain increase of 28.57% higher than the control. In other words, by converting the same amount of CHM, treatments are able to produce more larval mass. Harvesting more larval mass potentially means more income in the CHM conversion process. Given that adult BSF does not need to feed, the BSFL accumulate a large fat reserve for larval development and adult survival (Nguyen et al., 2015). Companion bacteria can help insects to digest non-digestible nutrients, and protect them from predators, parasites and pathogens (Engel and Moran, 2013). After pretreatment with companion bacteria, BSFL can digest small nutrient molecules, such as peptides, amino acids, and fatty acids directly. The digestive process is enhanced by the presence of bacteria since they have enzymes such as lipases, pectinases and others, which promote digestion of macromolecules that would otherwise be indigestible to insects (Ruokolainen et al., 2016). 5. Conclusion Findings from this study revealed that BSF egg-associated and larval gut bacteria have significant effects, in most cases positive, on the conversion rate, larvae weight gain, manure reduction, and nutrient accumulation in BSF, which are important benefits for the insect farming industry in terms of protein and oil production. Overall, this research could open new pathways since the elucidation of the co-conversion mechanisms in BSF might enhance the efficiency of organic waste disposal centres and industrialscale BSFL production facilities. Therefore, it is highly recommended to BSFL producers to implement specific BSF eggassociated and larval gut bacterial strains to help in the nutrient accumulation (CP and CF) in the larvae and to improve chicken manure reduction management. Declaration of Competing Interest None. Acknowledgements This work was supported by the National Key Technology R & D Program of China (2018YFD0500203 and 2017YFD0800200). Thanks to our cooperation friend Dr. Heather R. Jordan from Mississippi State University, USA, for revising the language. References Abduh, M.Y., Jamilah, M., Istiandari, P., Manurung, S., Manurung, R., 2017. Bioconversion of rubber seeds to produce protein and oil-rich biomass using black soldier fly larva assisted by microbes. J. Entomol. Zool. Stud. 5, 591–597. Albihn, A., Vinnerås, B., 2007. Biosecurity and arable use of manure and biowaste treatment alternatives. Livest. Sci. 112, 232–239. https://doi.org/10.1016/j. livsci.2007.09.015. Allegretti, G., Talamini, E., Schmidt, V., Bogorni, P.C., Ortega, E., 2018. Insect as feed: an emergy assessment of insect meal as a sustainable protein source for the Brazilian poultry industry. J. Clean. Prod. 171, 403–412. https://doi.org/10.1016/ j.jclepro.2017.09.244. Belghit, I., Liland, N.S., Gjesdal, P., Biancarosa, I., Menchetti, E., Li, Y., Waagbo, R., Krogdahl, A., Lock, E.J., 2019. Black soldier fly larvae meal can replace fish meal in diets of sea-water phase Atlantic Salmon (Salmo salar). Aquaculture 503, 609–619. https://doi.org/10.1016/j.aquaculture.2018.12.032. Biasato, I., Renna, M., Gai, F., Dabbou, S., Meneguz, M., Perona, G., Martinez, S., Lajusticia, A.C.B., Bergana, S., Sardi, L., Teresa, M., Capucchio, M.T., Bressan, E., Dama, A., Schiavone, A., Gasco, L., 2019. Partially defatted black soldier fly larva meal inclusion in piglet diets: effects on the growth performance, nutrient digestibility, blood profile, gut morphology and histological features. J. Anim. Sci. Biotechno. 10 (1), 12. https://doi.org/10.1186/s40104-019-0325-x. Bosch, G., Zhang, S., Oonincx, D.G.A.B., Hendriks, W.H., 2014. Protein quality of insects as potential ingredients for dog and cat foods. J. Nutr. Sci. 3,. https://doi. org/10.1017/jns.2014.23 e29.
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