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Protaetia brevitarsis larvae can efficiently convert herbaceous and ligneous plant residues to humic acids
Waste Management 83 (2019) 79–82
Contents lists available at ScienceDirect
Waste Management journal homepage: www.elsevier.com/locate/wasman
Protaetia brevitarsis larvae can efficiently convert herbaceous and ligneous plant residues to humic acids Yimei Li a,1, Tong Fu a,1, Lili Geng a, Yu Shi b, Haiyan Chu b, Fushun Liu c, Chunqin Liu c, Fuping Song a, Jie Zhang a, Changlong Shu a,⇑ a b c
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, PR China Cangzhou Academy of Agricultural and Forestry Sciences, Cangzhou 061001, PR China
a r t i c l e
i n f o
Article history: Received 31 March 2018 Revised 11 September 2018 Accepted 7 November 2018 Available online 13 November 2018 Keywords: Agricultural residues Organic fertilizer Insect manure Humic acids
a b s t r a c t Utilization of the organic residues produced after crop harvesting is currently an important issue across the world. The edible insect Protaetia brevitarsis larvae can feed various organic matters. In this paper, we investigated the potential to utilize the insect to convert herbaceous and ligneous plant residues. We feed the insect larvae with maize straw and sawdust and analyzed the produced insect manure. P. brevitarsis larval was found to be able to digest both herbaceous and ligneous straw and insect manure extract shown no phytotoxicity. The mass fractions of humic acids (HAs) in the insect manure derived from maize straw and sawdust digestion were 24.37% and 14.46%, respectively. The 13C cross-polarization magic-angle spinning nuclear magnetic resonance (CP–MAS NMR) spectra data indicated that the HAs in the insect manure were similar to those found in the soil. These data suggested that P. brevitarsis larvae can be used to convert agricultural residues and produce organic fertilizers. Ó 2018 Elsevier Ltd. All rights reserved.
1. Introduction Enormous amounts of agricultural organic residues (e.g., straws) are produced globally after crop harvesting (Chen, 2016) and the improper treatment of these organic residues can lead to pollution, warming, and ultimately a disruption of the ecological balance. In recent years, the rational and efficient utilization of straw has been extensively investigated (van Wyk, 2001). The conversion of straw to organic fertilizer is the most widely used strategy. Recently, studies have found that soil fauna can affect the stabilization of soil organic matter (Wolters, 2000), and that soildwelling invertebrates, including some earthworm and chafer species, have been used for straw degradation. Vermicomposting (Bhat et al., 2018), the biological process involving interactions between earthworms and microorganisms, can efficiently convert a variety of organic matters into nutrient-rich organic fertilizers. However, earthworms cannot feed on and convert hard straw. The larvae of Protaetia brevitarsis (Ghosh et al., 2017; Kim et al., 2017), commonly referred to as the white-spotted flower chafer (Suo et al., 2015), has strong mouthparts and can feed on humus,
⇑ Corresponding author. 1
E-mail address: [email protected] (C. Shu). Y. L. and T. F. contributed equally to this work.
https://doi.org/10.1016/j.wasman.2018.11.010 0956-053X/Ó 2018 Elsevier Ltd. All rights reserved.
crushed straw, or sawdust in an appropriate humidity, and the resulting fecal pellets (manure) have been confirmed to effectively promote plant growth (Kang et al., 2005; Tian et al., 2017). In addition, larval P. brevitarsis are reared as food in Korea (Ghosh et al., 2017; Kim et al., 2017) and used to decompose straw in China (Tian et al., 2017). Humic acids (HAs) contain one or more aromatic nuclei connecting more than one reactive functional group (Diallo et al., 2003), and have been confirmed to have effects on the water holding capacity, pH, nutrient dynamics, and heavy mental pollution remediation in soil (Chang et al., 2016; Dong et al., 2009; Iwai, 2017; Ondrasek et al., 2018; Xie et al., 2018). HAs are important components of soil and normally originate from lignin-rich organic materials (OM) via the actions of microorganisms and enzymes (Xu et al., 2017). Larval P. brevitarsis can also feed on and convert both herbaceous and ligneous plant residues, and our recent work found that the midgut and hindgut of insects contain abundant celluloseand lignin-degrading microorganisms and enzymes (Tian et al., 2017). However, the presences of HAs in insect manures have not been well studied. In the present study, we aimed to analyze (1) the HAs in larval P. brevitarsis insect manure derived from herbaceous (maize straw) and ligneous (sawdust) plants residues, and (2) the phytotoxicity of the manure, with the hypothesis that P.
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brevitarsis larvae can be used as a viable solution to convert agriculture residues to organic fertilizer with high HAs content. 2. Methods 2.1. Insect feeding and sampling The 3rd instar larvae of P. brevitarsis (Fig. 1A) were reared in rearing chamber that was maintained at 60–80% relative humidity and at a temperature of 25 °C. Larvae were either fed with crushed, air-dried maize straw or sawdust. After one week, fresh granulated manure (Fig. 1B) was collected and air-dried for subsequent analysis. The treatments tripled performed in plastic boxes with 41 cm wide, 65 cm long and 12 cm high. For each treatment, 1000 larvae and 1 kg (dry weight) maize straw or sawdust were applied. 2.2. Manure phytotoxicity assessment Manure phytotoxicity was assessed using the germination index (GI). Seed germination tests were conducted with manure water extracts and cucumber seeds. Manure water extracts were prepared using the methodology of Meng et al. (2017), Meng et al. (2017) with slight modifications. The air-dried manure samples were mixed with distilled water at the mass ratio of 1:10 (airdried manure:distilled water) and shaken for 2 h. The mixtures were centrifuged and the supernatants were filtered through a 0.22 lm filter membrane to obtain the extracts. The dissolved organic carbon (DOC) and pH of the water extracts were analyzed following the methodology previously described by Zhou et al. (2014). Samples (10 mL) of each extract or distilled water (the control) were then used to soak four-layered filter paper beds in sterile culture dishes (9 cm diameter). Twenty cucumber seeds were distributed on each filter paper bed and incubated in the dark at 26 °C for 3 d. Three replicate tests were conducted for each treatment. The GI of each manure sample was calculated using the following formula:
GI ð%Þ ¼ ðANT ALTÞ=ðANC ALCÞ where ANT is the average number of germinated seeds in the treatment, ALT is the average root length of the seeds in the treatment, ANC is the average number of germinated seeds in the control, and ALC is the average root length in the control. 2.3. Isolation of humic acids Briefly, the HAs in the insect manure were dissolved in 0.1 M NaOH solution at a weight:volume ratio of 1:10. After mechanical agitation for 24 h at 30 °C, the mixture was centrifuged and the supernatant was collected and acidified with 3 M HCl to a pH 1.0. The acidified supernatant was left to stand for 24 h to allow coagulation of the HA fractions. The supernatant was then centrifuged and the HA precipitates were recovered and isolated by suspension in distilled water, dialysis, and subsequent freezedrying. The HAs were weighed to calculate the extraction yield. Also, the total organic carbon of the HAs were measured according to the Chinese National Standard (NY525-2012) to represent the HAs yield. 2.4. Solid-state 13C cross-polarization magic-angle spinning nuclear magnetic resonance (CP–MAS NMR) analysis of isolated humic acids The isolated HAs were analyzed with a 600 MHz (JNM-ECZ600R, JEOL RESONANCE Inc.) NMR spectrometer equipped with a 3.2 mm HXMAS probe at 15 kHz MAS. For each sample, analysis was accu-
Fig. 1. Protaetia brevitarsis 3rd instar larvae (A) and granulated manure (B). Bars represent 1 cm.
mulated over 1 h with a relaxation time of 2 s and a contact time of 2 ms. The resulting 13C CP–MAS NMR spectra were then baseline corrected and integrated into the following chemical-shift regions: 0–45 ppm, aliphatic C; 45–110 ppm, substituted-aliphatic C, including alcohols, amines, carbohydrates, ethers, methoxyl, and acetal C; 110–160 ppm, aromatic C; and 160–220 ppm, carboxyl and carbonyl C.
3. Results and discussion 3.1. Phytotoxicity assessment With respect to OM compost, phytotoxicity is the adverse effect of OM on seed germination, plant growth, and soil environment due to the decreased supply of oxygen and available nitrogen, or both, or the presence of phytotoxic compounds, possibly resulting from the further decomposition of microbially degradable OM (Luo et al., 2018). In this study, the humivorous scarabaeid beetle larvae P. brevitarsis, a sister species of Pachnoda ephippiata (Andert et al., 2008; Li and Brune, 2005) was shown to effectively convert OM. The OM digestion by insects involves a complex series of biochemical and microbiological processes. Firstly, the chewing mouthparts mechanically break down the OM to a digestible state. The mashed OM is then solubilized by the alkaline digestive juice and subjected to enzymatic hydrolysis in the midgut. The hydrolyzed monomers are absorbed directly or subjected to further microbial degradation. Finally the OM is further fermented in the hindgut—a neutral environment with a high density of microorganisms (Egert et al., 2003; Lemke et al., 2003). The useful fermentation products are absorbed by the insect (Li and Brune, 2005), and the mineralized residues and microbially degraded but persistent organic matter are reprecipitated and defecated. Therefore, most of the microbially degradable OM being digested and absorbed by the combined actions of the insect and gut microorganisms. The DOC or water-soluble carbon has been proposed by several researchers as a parameter of compost maturity, and the GI was used to assess the phytotoxicity of DOC. In this study, the DOC of the insect manure originating from maize straw and sawdust digestion was 6.37 (N = 3, SD = 0.13) and 39.70 (N = 3, SD = 0.52) g Kg1, respectively. The difference seen in the DOC values may be due to variation in the composition of maize straw and sawdust. The 13C NMR data shown that the sawdust has more Carboxyl C and less Alkyl C than maize straw (Fig. 2, Table 1) and it suggested that sawdust compositions have better water solubility. The cucumber seed GI on fresh insect manure produced from digested maize straw and sawdust was 112.19 (N = 3, SD = 4.13) and 124.53 (N = 3, SD = 8.35), respectively. The insect manure (produced from both digested maize straw and sawdust) extract was found to have no phytotoxicity. Most of the microbially degradable OM was found to have been digested by the insect and did not remain in the insect manure.
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Fig. 2. CPMAS 13C NMR spectra of maize straw (MS), sawdust (SD), humic acids (HAs) from brown coal (BC-HA) and insect manure obtained from sawdust (SD-HA) and maize straw (MS-HA) feed.
Table 1 The relative abundances of different carbons groups in sawdust (SD), maize straw (MS), the humic acids (HAs) of brown coal (BC-HA) and insect manure obtained from sawdust (SD-HA) and maize straw (MS-HA) feed.
BC-HA SD-HA MS-HA SD MS
% Alkyl C (0–45 ppm)
% O-alkyl C (45–110 ppm)
% Aromatic C (110–160 ppm)
% Carboxyl C (160–190 ppm)
% Ketone C (190–220 ppm)
5.69 26.25 22.42 9.43 19.49
28.20 49.87 47.98 78.30 70.76
49.69 15.49 21.30 2.74 2.53
3.18 8.14 8.07 8.30 7.02
13.25 0.26 0.22 1.23 0.19
3.2. Isolation and solid-state humic acids
13
C CP–MAS NMR analysis of isolated
HAs play significant roles in agriculture and in the environment, by increasing soil fertility, transferring micronutrients to plants, stimulating the microflora population in soils, accelerating the photodegradation of pesticides, and decreasing the toxicity of some toxic and heavy metals (Chang et al., 2016; Dong et al., 2009; Ondrasek et al., 2018). Generally, HAs are produced from degraded OM through the actions of microorganisms and enzymes. However, few reports have suggested that HAs also form during OM digestion in insect guts. Our data showed that the (1) organic carbon content and (2) mass fractions of the HAs in insect manure produced from digested maize straw and sawdust were 141.33 (N = 3, SD = 2.94) and 83.87 (N = 3, SD = 2.59) g Kg1, and 24.37 (N = 3, SD = 0.51)% and 14.46 (N = 3, SD = 0.45)%, respectively. The HA content of insect manure was found to be much higher than that 2.67% in composted products (Zhou et al., 2014). Solid-state CPMAS 13C NMR is a powerful technique for characterizing the structure of HAs. It can provide carbon fingerprints of solid samples and present useful information regarding the carbon skeleton. The large chemical shifts associated with this technique are helpful in distinguishing between carbon atoms with slightly different structures. Furthermore, this technique allows for quantitative evaluation. In this study, CPMAS 13C NMR data of the maize straw and sawdust, insect manure, and HAs were collected. Using the brown coal HAs as a control, the generated CPMAS 13C NMR spectra showed that HAs from the insect manure have more carbon types (Fig. 2). Generally, the CPMAS 13C NMR spectra of the HAs from the insect manure exhibited major peaks at the same positions, indicating the presence of two HAs with similar carbon skeleton structures. These HAs contained alkyl C (31 and 34 ppm), O-alkyl C (56, 72, and 105 ppm), aromatic C (117, 130,
and 152 ppm), and carboxyl C (174 ppm) (Fig. 2, SD-HA and MSHA). The data indicated that the insect manure HAs have a structures similar to those of the HAs from soil OM (Xu et al., 2017). However, the HAs from the brown coal exhibited fewer peaks, and only contained carbons with chemical shifts of 31 ppm (alkyl C), 130 ppm (aromatic C) and 177 ppm (carboxyl C) (Fig. 2, BCHA). The missing 56, 72, and 105 ppm peaks indicated that the brown coal HAs lacked O-alkyl C or hydroxy; the indistinctness of the peaks at the 117 and 152 ppm suggested that the substitutional groups of the aromatic carbon in lignin were lost during the brown coal forming process. The HAs from insect manure, therefore, have more active groups than those from brown coal and possibly exhibit a similar activity as those found in the soil. To compare the abundance of each carbon group among the HAs from brown coal and insect manures, the CPMAS 13C NMR spectra of these HAs were divided into five regions according to their organic functional groups, and the relative abundances of different carbons groups were calculated by integrating the areas of the corresponding peaks in the solid NMR spectrum (Table 1). The data indicated that the BC-HA contained more aromatic C and ketone C, while the HAs from insect mature contained more alkyl C, O-alkyl C, and carboxyl C. The alkyl C, O-alkyl C, and carboxyl C, and the relative reactive functional groups always have strong interactions with other molecules and ions, and, therefore, insect manure HAs may have better activities, for example, chelate fertilizer molecules.
4. Conclusion Assessment of the HAs in P. brevitarsis insect manure showed that the insect larvae can convert herbaceous (maize straw) and ligneous (sawdust) plant residues to HAs in efficiency much higher
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than the current composting technology. In addition to being edible and having high medicinal value, P. brevitarsis larvae promises to provide a viable solution to convert agriculture residues into organic fertilizer and play important role in agricultural waste recycling and agricultural economy development. Conflict of interest The authors declare no commercial or financial conflict of interest. Funding This study was supported by the National Key Research and Development Program of China (No. 2018YFD0800906), the Hebei Key Research and Development Program (No. 18273809D) and National Natural Science Foundation of China (No. 41701298). References Andert, J., Geissinger, O., Brune, A., 2008. Peptidic soil components are a major dietary resource for the humivorous larvae of Pachnoda spp. (Coleoptera: Scarabaeidae). J. Insect Physiol. 54, 105–113. Bhat, S.A., Singh, S., Singh, J., Kumar, S., Bhawana, Vig AP., 2018. Bioremediation and detoxification of industrial wastes by earthworms: vermicompost as powerful crop nutrient in sustainable agriculture. Bioresour. Technol. 252, 172–179. Chang, C.H., Wei, C.C., Lin, L.H., Tu, T.H., Liao, V.H., 2016. Humic acids enhance the microbially mediated release of sedimentary ferrous iron. Environ. Sci. Pollut. Res. Int. 23, 4176–4184. Chen, X., 2016. Economic potential of biomass supply from crop residues in China. Appl. Energy 166, 141–149. Diallo, M.S., Simpson, A., Gassman, P., Faulon, J.L., Johnson Jr., J.H., Goddard 3rd, W. A., Hatcher, P.G., 2003. 3-D structural modeling of humic acids through experimental characterization, computer assisted structure elucidation and atomistic simulations. 1. Chelsea soil humic acid. Environ. Sci. Technol. 37, 1783–1793. Dong, L., Cordova-Kreylos, A.L., Yang, J., Yuan, H., Scow, K.M., 2009. Humic acids buffer the effects of urea on soil ammonia oxidizers and potential nitrification. Soil Biol. Biochem. 41, 1612–1621. Egert, M., Wagner, B., Lemke, T., Brune, A., Friedrich, M.W., 2003. Microbial community structure in midgut and hindgut of the humus-feeding larva of
Pachnoda ephippiata (Coleoptera: Scarabaeidae). Appl. Environ. Microbiol. 69, 6659–6668. Ghosh, S., Lee, S., Jung, C., Meyer-Rochow, V.B., 2017. Nutritional composition of five commercial edible insects in South Korea. J. Asia-Pacific Entomol. 20, 686–694. Iwai, H., 2017. Removal of trace levels of Cu(II) from seawater by co-precipitation with humic acids. Anal. Sci. 33, 1231–1236. Kang, S., Park, C., Han, S., Yi, Y., Kim, Y., 2005. A grub (Protaetia brevitarsis seulensis) rearing technique using cellulose-digesting bacteria and natural recycling of rearing byproduct to an organic fertilizer. Kor. J. Appl. Entomol. 44, 189–197. Kim, S., Weaver, C.M., Choi, M., 2017. Proximate composition and mineral content of five edible insects consumed in Korea. CyTA – J. Food 15, 143–146. Lemke, T., Stingl, U., Egert, M., Friedrich, M.W., Brune, A., 2003. Physicochemical conditions and microbial activities in the highly alkaline gut of the humusfeeding larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae). Appl. Environ. Microbiol. 69, 6650–6658. Li, X., Brune, A., 2005. Digestion of microbial biomass, structural polysaccharides, and protein by the humivorous larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae). Soil Biol. Biochem. 37, 107–116. Luo, Y., Liang, J., Zeng, G., Chen, M., Mo, D., Li, G., Zhang, D., 2018. Seed germination test for toxicity evaluation of compost: Its roles, problems and prospects. Waste Manag. 71, 109–114. Meng, L., Li, W., Zhang, S., Wu, C., Lv, L., 2017. Feasibility of co-composting of sewage sludge, spent mushroom substrate and wheat straw. Bioresour. Technol. 226, 39–45. Ondrasek, G., Rengel, Z., Romic, D., 2018. Humic acids decrease uptake and distribution of trace metals, but not the growth of radish exposed to cadmium toxicity. Ecotoxicol. Environ. Saf. 151, 55–61. Suo, Z., Bai, M., Li, S., Yang, H., Li, T., Ma, D., 2015. A geometric morphometric analysis of the morphological variations among Chinese populations of Protaetia brevitarsis (Coleoptera: Scarabaeidae) with an inference of the invading source of its Xinjiang populations. Acta Entomol. Sin. 58, 408–418. Tian, X., Song, F., Zhang, J., Liu, R., Zhang, X., Duan, J., Shu, C., 2017. Diversity of gut bacteria in larval Protaetia brevitarsis (Coleoptera: Scarabaedia) fed on corn stalk. Acta Entomol. Sin. 60, 632–641. van Wyk, J.P., 2001. Biotechnology and the utilization of biowaste as a resource for bioproduct development. Trends Biotechnol. 19, 172–177. Wolters, V., 2000. Invertebrate control of soil organic matter stability. Biol. Fert. Soils 31, 1–19. Xie, J., Liang, W., Lin, J., Zhou, X., Li, M., 2018. Humic acids facilitated microbial reduction of polymeric Pu(IV) under anaerobic conditions. Sci. Total Environ. 610–611, 1321–1328. Xu, J., Zhao, B., Chu, W., Mao, J., Zhang, J., 2017. Chemical nature of humic substances in two typical Chinese soils (upland vs paddy soil): a comparative advanced solid state NMR study. Sci. Total Environ. 576, 444–452. Zhou, Y., Selvam, A., Wong, J.W., 2014. Evaluation of humic substances during cocomposting of food waste, sawdust and Chinese medicinal herbal residues. Bioresour. Technol. 168, 229–234.
Contents lists available at ScienceDirect
Waste Management journal homepage: www.elsevier.com/locate/wasman
Protaetia brevitarsis larvae can efficiently convert herbaceous and ligneous plant residues to humic acids Yimei Li a,1, Tong Fu a,1, Lili Geng a, Yu Shi b, Haiyan Chu b, Fushun Liu c, Chunqin Liu c, Fuping Song a, Jie Zhang a, Changlong Shu a,⇑ a b c
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, PR China Cangzhou Academy of Agricultural and Forestry Sciences, Cangzhou 061001, PR China
a r t i c l e
i n f o
Article history: Received 31 March 2018 Revised 11 September 2018 Accepted 7 November 2018 Available online 13 November 2018 Keywords: Agricultural residues Organic fertilizer Insect manure Humic acids
a b s t r a c t Utilization of the organic residues produced after crop harvesting is currently an important issue across the world. The edible insect Protaetia brevitarsis larvae can feed various organic matters. In this paper, we investigated the potential to utilize the insect to convert herbaceous and ligneous plant residues. We feed the insect larvae with maize straw and sawdust and analyzed the produced insect manure. P. brevitarsis larval was found to be able to digest both herbaceous and ligneous straw and insect manure extract shown no phytotoxicity. The mass fractions of humic acids (HAs) in the insect manure derived from maize straw and sawdust digestion were 24.37% and 14.46%, respectively. The 13C cross-polarization magic-angle spinning nuclear magnetic resonance (CP–MAS NMR) spectra data indicated that the HAs in the insect manure were similar to those found in the soil. These data suggested that P. brevitarsis larvae can be used to convert agricultural residues and produce organic fertilizers. Ó 2018 Elsevier Ltd. All rights reserved.
1. Introduction Enormous amounts of agricultural organic residues (e.g., straws) are produced globally after crop harvesting (Chen, 2016) and the improper treatment of these organic residues can lead to pollution, warming, and ultimately a disruption of the ecological balance. In recent years, the rational and efficient utilization of straw has been extensively investigated (van Wyk, 2001). The conversion of straw to organic fertilizer is the most widely used strategy. Recently, studies have found that soil fauna can affect the stabilization of soil organic matter (Wolters, 2000), and that soildwelling invertebrates, including some earthworm and chafer species, have been used for straw degradation. Vermicomposting (Bhat et al., 2018), the biological process involving interactions between earthworms and microorganisms, can efficiently convert a variety of organic matters into nutrient-rich organic fertilizers. However, earthworms cannot feed on and convert hard straw. The larvae of Protaetia brevitarsis (Ghosh et al., 2017; Kim et al., 2017), commonly referred to as the white-spotted flower chafer (Suo et al., 2015), has strong mouthparts and can feed on humus,
⇑ Corresponding author. 1
E-mail address: [email protected] (C. Shu). Y. L. and T. F. contributed equally to this work.
https://doi.org/10.1016/j.wasman.2018.11.010 0956-053X/Ó 2018 Elsevier Ltd. All rights reserved.
crushed straw, or sawdust in an appropriate humidity, and the resulting fecal pellets (manure) have been confirmed to effectively promote plant growth (Kang et al., 2005; Tian et al., 2017). In addition, larval P. brevitarsis are reared as food in Korea (Ghosh et al., 2017; Kim et al., 2017) and used to decompose straw in China (Tian et al., 2017). Humic acids (HAs) contain one or more aromatic nuclei connecting more than one reactive functional group (Diallo et al., 2003), and have been confirmed to have effects on the water holding capacity, pH, nutrient dynamics, and heavy mental pollution remediation in soil (Chang et al., 2016; Dong et al., 2009; Iwai, 2017; Ondrasek et al., 2018; Xie et al., 2018). HAs are important components of soil and normally originate from lignin-rich organic materials (OM) via the actions of microorganisms and enzymes (Xu et al., 2017). Larval P. brevitarsis can also feed on and convert both herbaceous and ligneous plant residues, and our recent work found that the midgut and hindgut of insects contain abundant celluloseand lignin-degrading microorganisms and enzymes (Tian et al., 2017). However, the presences of HAs in insect manures have not been well studied. In the present study, we aimed to analyze (1) the HAs in larval P. brevitarsis insect manure derived from herbaceous (maize straw) and ligneous (sawdust) plants residues, and (2) the phytotoxicity of the manure, with the hypothesis that P.
80
Y. Li et al. / Waste Management 83 (2019) 79–82
brevitarsis larvae can be used as a viable solution to convert agriculture residues to organic fertilizer with high HAs content. 2. Methods 2.1. Insect feeding and sampling The 3rd instar larvae of P. brevitarsis (Fig. 1A) were reared in rearing chamber that was maintained at 60–80% relative humidity and at a temperature of 25 °C. Larvae were either fed with crushed, air-dried maize straw or sawdust. After one week, fresh granulated manure (Fig. 1B) was collected and air-dried for subsequent analysis. The treatments tripled performed in plastic boxes with 41 cm wide, 65 cm long and 12 cm high. For each treatment, 1000 larvae and 1 kg (dry weight) maize straw or sawdust were applied. 2.2. Manure phytotoxicity assessment Manure phytotoxicity was assessed using the germination index (GI). Seed germination tests were conducted with manure water extracts and cucumber seeds. Manure water extracts were prepared using the methodology of Meng et al. (2017), Meng et al. (2017) with slight modifications. The air-dried manure samples were mixed with distilled water at the mass ratio of 1:10 (airdried manure:distilled water) and shaken for 2 h. The mixtures were centrifuged and the supernatants were filtered through a 0.22 lm filter membrane to obtain the extracts. The dissolved organic carbon (DOC) and pH of the water extracts were analyzed following the methodology previously described by Zhou et al. (2014). Samples (10 mL) of each extract or distilled water (the control) were then used to soak four-layered filter paper beds in sterile culture dishes (9 cm diameter). Twenty cucumber seeds were distributed on each filter paper bed and incubated in the dark at 26 °C for 3 d. Three replicate tests were conducted for each treatment. The GI of each manure sample was calculated using the following formula:
GI ð%Þ ¼ ðANT ALTÞ=ðANC ALCÞ where ANT is the average number of germinated seeds in the treatment, ALT is the average root length of the seeds in the treatment, ANC is the average number of germinated seeds in the control, and ALC is the average root length in the control. 2.3. Isolation of humic acids Briefly, the HAs in the insect manure were dissolved in 0.1 M NaOH solution at a weight:volume ratio of 1:10. After mechanical agitation for 24 h at 30 °C, the mixture was centrifuged and the supernatant was collected and acidified with 3 M HCl to a pH 1.0. The acidified supernatant was left to stand for 24 h to allow coagulation of the HA fractions. The supernatant was then centrifuged and the HA precipitates were recovered and isolated by suspension in distilled water, dialysis, and subsequent freezedrying. The HAs were weighed to calculate the extraction yield. Also, the total organic carbon of the HAs were measured according to the Chinese National Standard (NY525-2012) to represent the HAs yield. 2.4. Solid-state 13C cross-polarization magic-angle spinning nuclear magnetic resonance (CP–MAS NMR) analysis of isolated humic acids The isolated HAs were analyzed with a 600 MHz (JNM-ECZ600R, JEOL RESONANCE Inc.) NMR spectrometer equipped with a 3.2 mm HXMAS probe at 15 kHz MAS. For each sample, analysis was accu-
Fig. 1. Protaetia brevitarsis 3rd instar larvae (A) and granulated manure (B). Bars represent 1 cm.
mulated over 1 h with a relaxation time of 2 s and a contact time of 2 ms. The resulting 13C CP–MAS NMR spectra were then baseline corrected and integrated into the following chemical-shift regions: 0–45 ppm, aliphatic C; 45–110 ppm, substituted-aliphatic C, including alcohols, amines, carbohydrates, ethers, methoxyl, and acetal C; 110–160 ppm, aromatic C; and 160–220 ppm, carboxyl and carbonyl C.
3. Results and discussion 3.1. Phytotoxicity assessment With respect to OM compost, phytotoxicity is the adverse effect of OM on seed germination, plant growth, and soil environment due to the decreased supply of oxygen and available nitrogen, or both, or the presence of phytotoxic compounds, possibly resulting from the further decomposition of microbially degradable OM (Luo et al., 2018). In this study, the humivorous scarabaeid beetle larvae P. brevitarsis, a sister species of Pachnoda ephippiata (Andert et al., 2008; Li and Brune, 2005) was shown to effectively convert OM. The OM digestion by insects involves a complex series of biochemical and microbiological processes. Firstly, the chewing mouthparts mechanically break down the OM to a digestible state. The mashed OM is then solubilized by the alkaline digestive juice and subjected to enzymatic hydrolysis in the midgut. The hydrolyzed monomers are absorbed directly or subjected to further microbial degradation. Finally the OM is further fermented in the hindgut—a neutral environment with a high density of microorganisms (Egert et al., 2003; Lemke et al., 2003). The useful fermentation products are absorbed by the insect (Li and Brune, 2005), and the mineralized residues and microbially degraded but persistent organic matter are reprecipitated and defecated. Therefore, most of the microbially degradable OM being digested and absorbed by the combined actions of the insect and gut microorganisms. The DOC or water-soluble carbon has been proposed by several researchers as a parameter of compost maturity, and the GI was used to assess the phytotoxicity of DOC. In this study, the DOC of the insect manure originating from maize straw and sawdust digestion was 6.37 (N = 3, SD = 0.13) and 39.70 (N = 3, SD = 0.52) g Kg1, respectively. The difference seen in the DOC values may be due to variation in the composition of maize straw and sawdust. The 13C NMR data shown that the sawdust has more Carboxyl C and less Alkyl C than maize straw (Fig. 2, Table 1) and it suggested that sawdust compositions have better water solubility. The cucumber seed GI on fresh insect manure produced from digested maize straw and sawdust was 112.19 (N = 3, SD = 4.13) and 124.53 (N = 3, SD = 8.35), respectively. The insect manure (produced from both digested maize straw and sawdust) extract was found to have no phytotoxicity. Most of the microbially degradable OM was found to have been digested by the insect and did not remain in the insect manure.
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Fig. 2. CPMAS 13C NMR spectra of maize straw (MS), sawdust (SD), humic acids (HAs) from brown coal (BC-HA) and insect manure obtained from sawdust (SD-HA) and maize straw (MS-HA) feed.
Table 1 The relative abundances of different carbons groups in sawdust (SD), maize straw (MS), the humic acids (HAs) of brown coal (BC-HA) and insect manure obtained from sawdust (SD-HA) and maize straw (MS-HA) feed.
BC-HA SD-HA MS-HA SD MS
% Alkyl C (0–45 ppm)
% O-alkyl C (45–110 ppm)
% Aromatic C (110–160 ppm)
% Carboxyl C (160–190 ppm)
% Ketone C (190–220 ppm)
5.69 26.25 22.42 9.43 19.49
28.20 49.87 47.98 78.30 70.76
49.69 15.49 21.30 2.74 2.53
3.18 8.14 8.07 8.30 7.02
13.25 0.26 0.22 1.23 0.19
3.2. Isolation and solid-state humic acids
13
C CP–MAS NMR analysis of isolated
HAs play significant roles in agriculture and in the environment, by increasing soil fertility, transferring micronutrients to plants, stimulating the microflora population in soils, accelerating the photodegradation of pesticides, and decreasing the toxicity of some toxic and heavy metals (Chang et al., 2016; Dong et al., 2009; Ondrasek et al., 2018). Generally, HAs are produced from degraded OM through the actions of microorganisms and enzymes. However, few reports have suggested that HAs also form during OM digestion in insect guts. Our data showed that the (1) organic carbon content and (2) mass fractions of the HAs in insect manure produced from digested maize straw and sawdust were 141.33 (N = 3, SD = 2.94) and 83.87 (N = 3, SD = 2.59) g Kg1, and 24.37 (N = 3, SD = 0.51)% and 14.46 (N = 3, SD = 0.45)%, respectively. The HA content of insect manure was found to be much higher than that 2.67% in composted products (Zhou et al., 2014). Solid-state CPMAS 13C NMR is a powerful technique for characterizing the structure of HAs. It can provide carbon fingerprints of solid samples and present useful information regarding the carbon skeleton. The large chemical shifts associated with this technique are helpful in distinguishing between carbon atoms with slightly different structures. Furthermore, this technique allows for quantitative evaluation. In this study, CPMAS 13C NMR data of the maize straw and sawdust, insect manure, and HAs were collected. Using the brown coal HAs as a control, the generated CPMAS 13C NMR spectra showed that HAs from the insect manure have more carbon types (Fig. 2). Generally, the CPMAS 13C NMR spectra of the HAs from the insect manure exhibited major peaks at the same positions, indicating the presence of two HAs with similar carbon skeleton structures. These HAs contained alkyl C (31 and 34 ppm), O-alkyl C (56, 72, and 105 ppm), aromatic C (117, 130,
and 152 ppm), and carboxyl C (174 ppm) (Fig. 2, SD-HA and MSHA). The data indicated that the insect manure HAs have a structures similar to those of the HAs from soil OM (Xu et al., 2017). However, the HAs from the brown coal exhibited fewer peaks, and only contained carbons with chemical shifts of 31 ppm (alkyl C), 130 ppm (aromatic C) and 177 ppm (carboxyl C) (Fig. 2, BCHA). The missing 56, 72, and 105 ppm peaks indicated that the brown coal HAs lacked O-alkyl C or hydroxy; the indistinctness of the peaks at the 117 and 152 ppm suggested that the substitutional groups of the aromatic carbon in lignin were lost during the brown coal forming process. The HAs from insect manure, therefore, have more active groups than those from brown coal and possibly exhibit a similar activity as those found in the soil. To compare the abundance of each carbon group among the HAs from brown coal and insect manures, the CPMAS 13C NMR spectra of these HAs were divided into five regions according to their organic functional groups, and the relative abundances of different carbons groups were calculated by integrating the areas of the corresponding peaks in the solid NMR spectrum (Table 1). The data indicated that the BC-HA contained more aromatic C and ketone C, while the HAs from insect mature contained more alkyl C, O-alkyl C, and carboxyl C. The alkyl C, O-alkyl C, and carboxyl C, and the relative reactive functional groups always have strong interactions with other molecules and ions, and, therefore, insect manure HAs may have better activities, for example, chelate fertilizer molecules.
4. Conclusion Assessment of the HAs in P. brevitarsis insect manure showed that the insect larvae can convert herbaceous (maize straw) and ligneous (sawdust) plant residues to HAs in efficiency much higher
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