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[分享] 黑水虻与功能菌转化鸡粪(BSF-CL ,等)

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发表于 2018-5-26 09:46:49 | 显示全部楼层 |阅读模式
Chicken production has increased due to the increasing demand for meat and eggs (Nie et al., 2015). Subsequently, massive amounts of chicken manure have been produced. Chicken manure is rich in readily biodegradable organic matter, nutrients, and pathogens. However, if poorly managed, chicken manure can become an important source of pollution (Wang et al., 2014; Nie et al., 2015).
Manure is traditionally composted to kill pathogens and fertilize crops (Guerrini et al., 2017;Abreu-Junior et al., 2017). However, the low C/N ratio, high moisture content, nasty odour, long decomposition period, nutrient loss, and the formation of phytotoxic compounds have become challenges for current composting methods (Chang and Chen, 2010; Wang et al., 2013). Bulking agents, such as sawdust and rice chaff, are usually added to manure composts to reduce moisture and to achieve a suitable C/N ratio. However, these bulking agents have become increasingly expensive for composting as they are used as alternative energy resources (Chang and Chen, 2010; Zhu et al., 2012). For that reason, alternative means to convert chicken manure into fertilizer production should be explored.Many insects efficiently degrade organic waste into nutrients (Zheng et al., 2012; Čičková et al., 2015). HermetiaillucensL. (Diptera: Stratiomyidae), also known as black soldier fly (BSF), live outdoors and is often associated with livestock (Li et al., 2011). BSF can be found in decaying organic wastes, such as animal manure and plant material, and exhibits antimicrobial peptide activity against pathogens in those wastes (Liu et al., 2008; Lalander et al., 2015; Elhag et al., 2017). BSF not only reduces the accumulation of manure and the nasty smell, it also inhibits the proliferation of house flies (Zheng et al., 2013). These processes greatly reduce environmental impact caused by livestock manure. Zhou et al. (2013) found that black soldier fly larvae (BSFL) of the Wuhan strain weighed 14.4 - 37.0% more than the BSFL Guanghzhou strain and BSFL Texas strain, respectively. The BSFL Wuhan strain reduced dry matter 46.0% (swine), 40.1% (dairy), and 48.4% (chicken) more than the Guangzhou strain and 6.9%, 7.2%, and 7.9% more than the Texas strain. Gut microbes play important roles in insect nutrition and colonization resistance against invasion of exotic microbes (Dong et al., 2009). Bacteria isolated from the BSFL gut can increase the weight of prepupae and pupae, and shorten the number of days from hatching to the prepupal stage (Yu et al., 2011). Therefore, BSFL can be used as animal feed and for biodiesel production. The residue of the organic waste is a suitable substrate for aerobic fermentation during composting (Li et al., 2015; Rehman et al., 2017).Composting or aerobic fermentation accelerates the natural decomposition of organic debris by microorganisms (bacteria, actinomycetes and fungi) under controlled environmental conditions. Inoculation at the appropriate time can promote organic waste maturity and considerably shorten the period of decomposition (Zeng et al., 2010; Jurado et al., 2015). However, not much is known about the Insect-Bacteria-Manure digestionsystemin reducing manure pollution.In this study, we tried to develop an efficient and high-value co-conversion technology of chicken manure by BSFL and bacteriain a poultry farm. Our aim was to maximize BSFL harvest and waste mass reduction by using synergistic bacteria in the first stage and shorten the maturity time through inoculation with a decomposing agent to promote aerobic fermentation in the second stage. Important process parameters were monitored such as changes in: the conversion rate by the larvae; the reduction rate of chicken manure; physical and chemical properties; the occurrence of phytotoxicity, and the presence of microbial enzymes.1. Material and Methods1.1 Raw materialsHermetiaillucensL. Wuhan strain larvae used in this study were bred at Wuhan ChaoTuo Ecology Agricultural Ltd. (30.090309N, 114.349939E, Wuhan, Hubei, China). The strain was collected and domesticated by our team in the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China (Zhou et al., 2013). Eggs were incubated in a greenhouse at 28 °C with 60%-70% relative humidity.For the first 6 days, the larvae were fed with a diet based on bran, and thenseparated from the bran residue by sieve (2.00 mm).Chicken manure was collected from a poultry farm located in Wuhan ChaoTuo Ecology Agricultural Ltd. (Wuhan, Hubei, China). The water content of chicken manure was 76%.Bacillus subtilis BSF-CL strain was isolated from the guts of surface-disinfected larvae. The bacteria of the decomposing agent (Bacillus methylotrophicus DF07H, B. subtilis DF04H, B. subtilisRX07, B. amyloliquefaciens RX01, Pseudomonas pachastrellae D-61; 1:2:1:1:2) were isolated from the thermophilic phase of chicken manure composting.Previous studies had shown that B. subtilisBSF-CL strain was the optimal one of seven strains gut bacteria on BSFL weight gain rate and manure residue rate, and the decomposing agent was the optimal one of four group agents on promoting chicken manure maturity All bacterial colonies grown on petrie dish with Luria-Bertani (LB) agar medium (tryptone 10 gL-1, yeast extract 5 gL-1, NaCl 10 gL-1, agar 18 gL-1, distilled water 1000 mL) were suspended in 100 mL of LB liquid medium (tryptone 10 g L-1, yeast extract 5 g L-1, NaCl 10 g L-1, distilled water 1000 mL) and left under stirring for 36 h at 28 °C, respectively. After 36 h, each bacterium was washed three times with phosphate-buffered saline for 15 s at 8000 r min-1, and eventually suspended in 30 mL of sterile water, until a final concentration of 1×109 CFUmL-1 was obtained.1.1 Co-conversion experiment designThe co-conversion experiment began by introducing one million larva inocula and 1 L of B. subtilis BSF-CL (1×109 CFU mL-1) to1,000 kg of fresh chicken manure in a cement pool (3.0 m×4.0 m×0.2 m), namely larvae-inoculum-treated conversion (LIC), in which the fresh chicken manure mixed with B. subtilis BSF-CL inoculum was flatly piled and the larvae was evenly inoculated into the surface of the fresh chicken manure for them to grow freely. Apart from LIC, one million larva inocula and 1 L of sterile water were inoculated into1,000 kg of fresh chicken manure (LC) as control. Each treatment was performed in triplicate.Samples weighting approximately 200 g were collected from ten points of each pool daily during the co-conversion experiment. All the samples were preserved at –20 °C for subsequent analysis.1.1 Aerobic fermentation designAfter 13 days of conversion, the aging larvae were isolated from the conversion system according to their aggregation characteristics.Six thousand kilograms chicken manure residue was collected following the way of LIC treatment and divided into six peak-shaped composts (with a bottom diameter of 2.4 m and a height of 1.0 m) on the ground in a rainproof workshop without walls. Three piles, inoculated 1 Ldecomposing agent (1×109 CFUmL-1) per pile, were named decomposing agent-inoculum-treated pile (DAITP). Another three piles with 1 L sterile water inoculated into manure residue were named water-treated pile (WP). The aerobic fermentation process continued for 11 days, during which six composts were turned upside down every day.
Samples weighing approximately 200 g were collected from 40 cm below the surface of each pile daily during the aerobic fermentation stage. All the samples were preserved at -20 °C for the subsequent analysis.1.1 Analytical method of physical and chemical propertiesDuring the co-conversion, temperature was measured daily using a 25 or 100 cm-long tubular alcohol thermometer (ZDR21, HangzhouZeda Equipment Co, Ltd., China), which was inserted into three sites of each pool 4 cm below the surface or each pile 40 cm below the surface. The moisture content and dry matter of the subsamples were obtained by drying at 105 °C in an oven for 12 h. The pH values in 1:9 (w/v) sample suspensions were assessed in an electronic pH meter (Mettler-Toledo Instruments (Shanghai) Co., Ltd., Shanghai, China). Total phosphorus, total potassium, total Kjeldahl nitrogen and total organic carbon were measured in accordance with the method of China National Standard for organic fertilizer: NY525-2012 (Zhang et al., 2012). Sample-free blank controls were also analyzed to exclude background effects.1.1 Analytical method of microbial enzyme activitiesThe assayed enzymes in all the samples collecting during the thirteen days co-conversion and eleven days aerobic fermentation included: catalases to reduce the accumulation of reactive oxygen radicals; ureases involved in the peptide bond hydrolysis of organic matter and decomposition of urea into ammonia and carbon dioxide; invertases relating to the levels of humus and water-soluble organic matter; and polyphenoloxidases relating to the level of humification (Zhu et al., 2012).All the samples were analyzed in triplicate assays for the following microbial enzyme activities through the methods described by Zhu et al. (2012). Sample-free blank controls were also analyzed to exclude background effects.1.1 Analytical method of phytotoxicity in samplesChinese cabbage and rape seeds were used in the germination experiments to assay the toxicity of the samples. All the samples were analyzed in triplicate assays for the following phytotoxicity analysis. Aqueous extracts were prepared by shaking 10% (w/v) sample suspensions for 30 min at 180r min-1 and then subjected to 15 min standing and 10 min centrifugation at 6,000 rmin-1. The aliquot (5 mL) of each supernatant was poured into a Petri dish (9 cm diameter) with filter paper. Twenty seeds were evenly distributed into the Petri dish. The seeds were incubated for 3 days at 25 °Cin the dark. Percent germination and mean root length in each dish were then assessed. The same volume of distilled water was used as control for seed germination. The ratios of percent germination or mean root length in each extract-inclusive dish over those in the control were defined as relative germination rate (%) and relative root growth (%), respectively. The product of the two ratios and 100% was defined as germination index (GI; Romero et al., 2013; Villar et al., 2016) for the phytotoxicityof a given sample. The indexes can reflect the overall maturity of composting materialsStatistical analysesAll the presented results are average values form triplicate experiments. The results of all experiments were analyzed bypaired Student’st-test to determine the effect of inoculation during different stages. The maximum difference among the triplicate results was 5%. The statistical analyses were performed in SPSS 19.0 software.1. Results and discussion3.1 The co-conversion by BSFL and synergistic bacteriaAfter 13 days co-conversion process, 93.2 kg of fresh larvae were harvested from the LIC group, while the LC group only harvested 80.4 kg of fresh larvae. Chicken manure reduction rate of the LIC group was 40.5%, while chicken manure reduction rate of the LC group was 35.8%(Table 1). The material reduction rate of LIC group was greater than LC group by 13.3% (P= 0.089). The weight of BSFL increased by 15.9% compared to the LC group.The BSFL conversion rate of the LIC group increased by 12.7% compared to the LC group (P= 0.031).The results of the co-conversion by BSFL and synergistic bacteriaindicated that fresh chicken manure co-converted by BSFL and B. subtilis BSF-CL inoculums, can rapidly decrease accumulated chicken manure. Also more mature larvae could be harvested as a protein source. Sheppard et al. (1994) showed that black soldier fly larvae reduced manure by ≥50% in a facility housing 460 hens. These differences may be due to different manure sources and climatic conditions. However, as in the present study, B. subtilis BSF-CL inoculums accelerated BSFL growth and reduced chicken manure accumulation, while more larvae were harvestedat the same conditions.The harvested larvae are an available protein source for production animals (Bosch et al., 2014) or biodiesel (Li et al., 2011; Zheng et al., 2012). These are emerging and environment-friendly resources. The inoculated symbiotic bacteriamaybe help insects to digest non-digestible nutrients, and protect insects from predators, parasitoids, and pathogens (Laughton et al., 2011; Douglas, 2015).Microorganisms Enzymes, small molecules, and nutrients produced by microorganisms are necessary for insects, thus insects can grow well and eat more waste. Yu et al. (2011) demonstrate that inoculating poultry manure with bacteriafrom black soldier fly larvae influences the growth and development of conspecific larvae feeding onthe manure. Lou et al. (2011) also showed that both sludge reduction and nutrient removal were enhanced simultaneously significantly within the system utilizing the symbiotic interactions of Tubificidae and microorganisms. However, the mechanism of bacteria that promotes BSFL conversion requires further research.In the co-conversion phase, nearly half of the accumulated chicken manure was consumed by BSFL and bacteria. This method may be a feasible technique to solve food, feed, energy, and environmental crises in the future.Physical and chemical parameterDuring the co-conversion phase, daily temperature increased rapidly in the group inoculated with larvae and B. subtilis BSF-CL strain on the third day (Fig. 1A). It was greater than the group inoculated with larvae only. At the fourth to eighth day, the temperature of the LIC group was approximately 45 °C. Its daily temperature then decreased steadily to 30 °C. However, the daily temperature of the LC group increased slowly, and the highest temperature was observed on the ninth day (43 °C), and then decreased steadily to approximately 30 °C (P = 0.005). The ambient temperature was approximately 25 °C during the conversion stage. After 13 days of co-conversion, the manure residue became granular, and dark brown and its water content decreased. During the first eighth days, the moisture of the manure slowly decreased in both treatments. After the eighth day of conversion, the moisture of the LIC group decreased significantly compared with that in the LC group (P< 0.001). The moisture of the LIC group decreased to 52.3% at the 12th day and remained stable. The LC group decreased to 58.6% on the 13th day (Fig. 1B). The pH value trends of the treatments were similar (P= 0.132). The pH value of the LC group was high before the fifth day of conversion, and then it decreased, with a value approximately 8.4 on the 13th day (Fig. 1C). The organic matter content of the LIC group decreased by 13.2%, while that of the LC group decreased by 8.8% (P= 0.034, Fig. 1D). After 13 days co-conversion, the total nutrient content of the LIC group was lower than the LC group (P< 0.001), but organic matter contents in both groups were more than 8.8% (Fig. 1E).During the aerobic fermentation stage, the daily temperature of the DAITP group increased rapidly from the initial temperature. Moreover, it increased to 56°Con the 3rd day. The temperature was maintained at 55-66°C during days three to eight. Then, the temperature decreased to the bottom and fluctuated at low levels after the 11th day. However, the daily temperature of the WP group increased relatively slow (P= 0.186). It increased to 56 °C on the fifth day. Moreover, the temperature was maintained at 55-65 °C during days 5 to 12. After 15 days of fermentation, the temperature decreased at its lowest level (Fig. 2A). The moisture content in the DAITP group decreased faster from the initial level by 24–25% compared with that in the WPgroup (P< 0.001).The moisture content of the WP group was maintained at 29–30% in the end (Fig. 2B). The pH value of the DAITPgroup also increased faster at days one to three. However, it decreased at the fifth day to remain at 8.0–8.1. The pH value of the WP group was higher than that of the DAITP group (P= 0.001), and remained at 8.6–8.9 from days 3 to 11 (Fig. 2C). The organic matter content of the DAITP group was lower than that of the WP group (P< 0.001). However, the values of the two treatments were larger than 45% (Fig. 2D). The total nutrient content of the DAITP group was also lower than that of the WP group (P< 0.001). Both groups remained at a steady state after seven days of fermentation, and the nutrient values were more than 9.2% after the aerobic fermentation stage (Fig. 2E). The C/N ratio of DAITP group was also lower than that of the WP group (P= 0.009). The C/N ratio of DAITP group decreased from 62.65% to 15.58%, While WP group decreased from 60.33% to 16.36% (Fig. 2F).Physical and chemical parameters are important for organic fertilizers.Insect larvae, like BSFL and Musca domesticaL. (Diptera: Muscidae) maggots, can efficiently convert organic waste to nutrients. However, the prepupae of BSF have the advantage that they separate themselves from the organic waste residue. During co-conversion the physical and chemical parameters of organic waste changed and BSFL and bacteria metabolism led to an increase in manure temperature and release of ammonia. The temperature was higher in the treatment without inoculation and also in the treatment using M.domestica maggots (Zhu et al., 2012, 2015).Inoculant decomposition considerably shortened the aerobic fermentation period to maturity by altering the physical and chemical parameters of chicken manure. The aerobic fermentation stage temperature maintained at 55-66°C for six consecutive days was sufficient for pathogen elimination from the DAITP group. This because a composting temperature exceeding 55 °C for more than 3 days (Yu et al., 2007) or 50 °C for 8 consecutive days (Bao et al., 2010) was recommended to disinfect animal and plant pathogens in waste materials, BSFL antimicrobial peptides exhibited anti-pathogenic in organic wastes (Liu et al., 2008; Lalander et al., 2015; Elhag et al., 2017). Thus, this results in an extra mortality factor for some pathogens. The other parameter results indicated that they maintained a steady state, in agreement with the China National Standard for organic fertilizer: NY 525-2012. Changes in the phytotoxicityIn the germination experiments, the Chinese cabbage and rape seeds in the LIC groupsample extracts started to germinate rapidly after five days (Fig. 3A and B). After 13 days of co-conversion, the germination index (GI) of Chinese cabbage and rape seeds were 66.6% and 70.5%, respectively. However, the Chinese cabbage and rape seeds in the LC group sample extracts both failed to germinate before day seven. And the GIs of both seeds were significantly lower than the LIC group (P< 0.001).The Chinese cabbage seeds GI of the DAITP group increased faster from the initial value of 66.2% to the final value of 92.3% than that of the LP group (P< 0.001, Fig. 3C). The Chinese cabbage seeds GI of the WP group increased from 66.2% to 86.3% during the aerobic fermentation stage. This increase was also observed in the rape seeds GI (Fig. 3D). The GI of the DAITP group increased from 69.4% at day one to 97.2% at day ten and remained stable, while the WP group increased from 69.4% to 87.0% in the same period (P< 0.001). At the end of the aerobic fermentation stage, the chicken manure residue was completely decomposed (GI > 85%) in both treatments. Therefore, the residue can be used as organic fertilizer.Germination index is one of the commonly used parameters to indicate the maturity of the material (Zhu et al., 2012; Zorpas et al., 2017). Chinese cabbage seeds were more sensitive to the sample extracts, irrespective of the LIC group or the LC group, than rape seeds. Zhu et al. (2012) reported that Chinese cabbage seeds were more sensitive to sample extracts than cucumber seeds. Given that a GI above 50% is indicative of the maturity of composting; the observations indicated that LIC was mature for the germination of Chinese cabbage seeds (66.6%) and rape seeds (70.5%) on day 13.To our knowledge it was shown the first time to obtain so higher maturity at so short time using insect and bacteria. Specifically, the chicken manure residue reached prime maturity after co-conversion by BSFL and B. subtilis BSF-CL. Including the co-conversion of BSFL and B. subtilisBSF-CL, and the aerobic fermentation with decomposing agent, fresh chicken manure into organic fertilizer only took 24 days. This process is more efficient than natural composting (Wang et al., 2014; Vázquez et al., 2015), and also more than other biological treatment methods for organic waste (Zhu et al., 2012; Lleó et al., 2013; Cestonaro et al., 2017; Li et al., 2017; Rodrigues et al., 2017). Therefore, this process saves more time, space, and labor.Activity patterns of different enzymesIn order to further verify the stability of the residue, we monitored the microbial enzyme activity in the residue. Catalase activity in the LIC group increased and then decreased during the co-conversion stage (Fig. 4A) earlier than that of the LC group, and the final value was lower than that of the LC group (P= 0.118). Polyphenoloxidase activity increased from the initial level of 82.6 mg PPg-1 3h-1 to a maximum in the LIC group faster than in the LC group during the co-conversion stage (P= 0.032). The initial and final values were extremely close (Fig. 4B). The urease activity of the LIC group increased from 5,549 mg NH4+-N g-1 3h-1 to the peak 12,757 mg NH4+-N g-1 3h-1three days from the start, but became consistently underdectable from day fiveonward. The LC group increased from 5,549.5 mg NH4+-N g-1 3h-1 to the peak 12,198.5 mg NH4+-N g-1 3h-1three days from the start and became consistently undetectable from day seven onward. The LC group delayed for two days compared with the LIC group(P=0.096,Fig. 4C). The invertase activity in the LIC group decreased faster from an initial level of 270.9 (mL 0.1 M Na2S2O3g-1 24h-1) compared with that in the LC group during the co-conversion stage (P< 0.001, Fig. 4D).After 11 days of fermentation, enzymes activity further confirmed that the chicken manure residue reached maturity. Catalase activity of the DAITP group increased faster than in the WP group. Furthermore, the peak value of the DAITP group was significantly higher than the WP group. The value was lower than that of the WP group(P< 0.001, Fig. 5A). Polyphenoloxidase activity of the DAITP group increased faster from the initial level to the peak than the WP group. Moreover, it also decreased faster from its highest value to the lowest value than the WP group (P=0.811, Fig. 5B). Urease activity of the DAITP group increased from 267.1 mg NH4+-N g-1 3h-1 to the peak at 2,719.8 mg NH4+-N g-1 3h-1 on days one to three, but became consistently undetectable from day five onward. Urease activity of the WP group increased from 267.1 mg NH4+-N g-1 3h-1 to the peak 2,517.4 mg NH4+-N g-1 3h-1three days from the start and became consistently undetectable from day seven onward. It was delayed for two days in comparison with the DAITP group (P=0.148, Fig. 5C). Invertase activity of the DAITP group was lower than the WP group throughout the aerobic fermentation stage. The peaks appeared on the third day, and then decreased to stable level (P< 0.001, Fig. 5D).The enzyme activity is a reflection of microbe metabolic intensity in the fermentation process (Xue and Huang, 2013). Our observations are in agreement with the enzyme activity trends from the initial to the mature stage in composts of other materials (Zhu et al., 2012). At the end of aerobic fermentation, each enzyme detected in our study became consistently undetectable or maintained low activity. This result indicated that the microbes in chicken manure residue were maintained at a steady state.The present study shown that enzyme activity, physical and chemical parameters changed following BSFL and synergistic bacteria activity. Compared Zhu et al. (2013) reported, the changes of enzyme activity, physical and chemical parameters in the co-conversion stage and aerobic fermentation stage were rapid, and that was agree with their report on variation tendency, but need a shorter time to reach a steady state.ConclusionsBSFL conversion rate and chicken manure reduction rate were improved by BSFL and B. subtilis BSF-CL co-conversion. The incremental percentage of BSFL weight, BSFL conversion rate and chicken manure reduction rate was 15.9%, 12.7% and 13.4%, respectively. The decomposing agent could efficiently accelerate chicken manure residue decomposition. Activity patterns of different enzymes further indicated that the production process was completely mature and at a stable aerobic fermentation stage. Physical and chemical production parameters indicated that the residue was suitable as organic fertilizer. The total time of conversion from fresh chicken manure to organic fertilizer was shortened to 24 days. To our knowledge it is shown for the first time that co-conversion of chicken manure by composting through BSFL and their synergistic bacteria, which is high-efficient and need less time. The process will be a new strategy in chicken manure and other organic wastes management.ReferencesAbreu-Junior, Cassio Hamilton, et al. 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Results are all indicated as means ± SE. Means in the same row with different letters are significa ...

Results are all indicated as means ± SE. Means in the same row with different letters are significa ...

Fig.1Changes of physical and chemical parameters of chicken manure in the conversion phase.○, Larva ...

Fig.1Changes of physical and chemical parameters of chicken manure in the conversion phase.○, Larva ...

Fig. 2Changes of physical and chemical parameters of residue in aerobic fermentation stage.○, Decom ...

Fig. 2Changes of physical and chemical parameters of residue in aerobic fermentation stage.○, Decom ...

Fig. 3GI trends of Chinese cabbage seeds (A, C) and Rape seeds (B, D) in the aqueous extracts of 10% ...

Fig. 3GI trends of Chinese cabbage seeds (A, C) and Rape seeds (B, D) in the aqueous extracts of 10% ...

Fig. 4Temporal activity patterns of different types of microbial enzymes of chicken manure in the co ...

Fig. 4Temporal activity patterns of different types of microbial enzymes of chicken manure in the co ...

Fig. 5Temporal activity patterns of different types of microbial enzymes of residue in aerobic ferme ...

Fig. 5Temporal activity patterns of different types of microbial enzymes of residue in aerobic ferme ...
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