草业学报 ›› 2022, Vol. 31 ›› Issue (2): 171-181.DOI: 10.11686/cyxb2020538
• 研究论文 • 上一篇
周承福1,3,4(), 汪水平1,4(), 张佰忠2(), 张秀敏3, 王荣3, 马志远3, 王敏3
收稿日期:
2020-12-08
修回日期:
2021-03-15
出版日期:
2022-02-20
发布日期:
2021-12-22
通讯作者:
张佰忠
作者简介:
Corresponding author. E-mail: 705048333@qq.com,wangshuiping1979@sina.com基金资助:
Cheng-fu ZHOU1,3,4(), Shui-ping WANG1,4(), Bai-zhong ZHANG2(), Xiu-min ZHANG3, Rong WANG3, Zhi-yuan MA3, Min WANG3
Received:
2020-12-08
Revised:
2021-03-15
Online:
2022-02-20
Published:
2021-12-22
Contact:
Bai-zhong ZHANG
摘要:
本试验旨在研究水热处理对黄豆秸秆体外发酵、甲烷生成及微生物的影响。黄豆秸秆经水热处理后,利用全自动体外模拟瘤胃发酵系统发酵72 h,评估化学成分变化、降解特性、发酵参数、产气参数及微生物区系。结果表明:水热处理降低黄豆秸秆中性洗涤纤维(P<0.001)、酸性洗涤纤维(P<0.001)及半纤维素(P<0.001)含量,提高中性洗涤可溶物(P<0.001)、水溶性碳水化合物(P<0.001)、阿拉伯糖(P<0.001)及半乳糖(P<0.001)含量;水热处理提高黄豆秸秆干物质消失率(P<0.001)和发酵液挥发性脂肪酸浓度(P<0.05),降低乙酸/丙酸(P<0.001)、甲烷(P<0.001)与氢气产量(P<0.001)及真菌(P<0.01)、产琥珀酸丝状杆菌(P<0.05)和产甲烷菌(P<0.05)数量。结果显示:水热处理可破坏黄豆秸秆纤维素-半纤维素-木质素结构,增加中性洗涤可溶物含量,促进瘤胃降解,并减少真菌和产甲烷菌的数量,降低甲烷产量。
周承福, 汪水平, 张佰忠, 张秀敏, 王荣, 马志远, 王敏. 水热处理对黄豆秸秆体外发酵、甲烷生成及微生物的影响[J]. 草业学报, 2022, 31(2): 171-181.
Cheng-fu ZHOU, Shui-ping WANG, Bai-zhong ZHANG, Xiu-min ZHANG, Rong WANG, Zhi-yuan MA, Min WANG. Effects of hydrothermal treatment on in vitro fermentation, methanogenesis and microbiota of soybean straw[J]. Acta Prataculturae Sinica, 2022, 31(2): 171-181.
微生物种类 Microbial species | 引物序列 Primer sequence (5?-3 ?) | 大小 Size (bp) | 参考文献 References |
---|---|---|---|
原虫Protozoa | F: GCTTTCGWTGGTAGTGTATT | 223 | Sylvester等[ |
R: CTTGCCCTCYAATCGTWCT | |||
真菌Fungi | F: GAGGAAGTAAAAGTCGTAACAAGGTTTC | 121 | Denman等[ |
R: CAAATTCACAAAGGGTAGGATGATT | |||
细菌Bacteria | F: CGGCAACGAGCGCAACCC | 146 | Denman等[ |
R: CCATTGTAGCACGTGTGTAGCC | |||
产甲烷菌Methanogens | F: GGATTAGATACCCSGGTAGT | 192 | Hook等[ |
R: GTTGARTCCAATTAAACCGCA | |||
产琥珀酸丝状杆菌Fibrobacter succinogenes | F: GTTCGGAATTACTGGGCGTAAA | 121 | Denman等[ |
R: CGCCTGCCCCTGAACTATC | |||
白色瘤胃球菌Ruminococcus albus | F: CCCTAAAAGCAGTCTTAGTTCG | 176 | Koike等[ |
R: CCTCCTTGCGGTTAGAACA | |||
黄色瘤胃球菌Ruminococcus flavefaciens | F: GAACGGAGATAATTTGAGTTTACTTAGG | 132 | Denman等[ |
R: CGGTCTCTGTATGTTATGAGGTATTACC | |||
甲烷杆菌目Methanobacteriales | F: CGWAGGGAAGCTGTTAAGT | 343 | Yu等[ |
R: TACCGTCGTCCACTCCTT | |||
甲烷短杆菌Methanobrevibacter | F: CCTCCGCAATGTGAGAAATCGC | 230 | Huang等[ |
R: TCWCCAGCAATTCCCACAGTT | |||
甲烷微菌目Methanomicrobiales | F: ATCGRTACGGGTTGTGGG | 506 | Yu等[ |
R: CACCTAACGCRCATHGTTTAC |
表1 微生物qPCR引物序列
Table 1 Microbial qPCR primer sequence
微生物种类 Microbial species | 引物序列 Primer sequence (5?-3 ?) | 大小 Size (bp) | 参考文献 References |
---|---|---|---|
原虫Protozoa | F: GCTTTCGWTGGTAGTGTATT | 223 | Sylvester等[ |
R: CTTGCCCTCYAATCGTWCT | |||
真菌Fungi | F: GAGGAAGTAAAAGTCGTAACAAGGTTTC | 121 | Denman等[ |
R: CAAATTCACAAAGGGTAGGATGATT | |||
细菌Bacteria | F: CGGCAACGAGCGCAACCC | 146 | Denman等[ |
R: CCATTGTAGCACGTGTGTAGCC | |||
产甲烷菌Methanogens | F: GGATTAGATACCCSGGTAGT | 192 | Hook等[ |
R: GTTGARTCCAATTAAACCGCA | |||
产琥珀酸丝状杆菌Fibrobacter succinogenes | F: GTTCGGAATTACTGGGCGTAAA | 121 | Denman等[ |
R: CGCCTGCCCCTGAACTATC | |||
白色瘤胃球菌Ruminococcus albus | F: CCCTAAAAGCAGTCTTAGTTCG | 176 | Koike等[ |
R: CCTCCTTGCGGTTAGAACA | |||
黄色瘤胃球菌Ruminococcus flavefaciens | F: GAACGGAGATAATTTGAGTTTACTTAGG | 132 | Denman等[ |
R: CGGTCTCTGTATGTTATGAGGTATTACC | |||
甲烷杆菌目Methanobacteriales | F: CGWAGGGAAGCTGTTAAGT | 343 | Yu等[ |
R: TACCGTCGTCCACTCCTT | |||
甲烷短杆菌Methanobrevibacter | F: CCTCCGCAATGTGAGAAATCGC | 230 | Huang等[ |
R: TCWCCAGCAATTCCCACAGTT | |||
甲烷微菌目Methanomicrobiales | F: ATCGRTACGGGTTGTGGG | 506 | Yu等[ |
R: CACCTAACGCRCATHGTTTAC |
项目Items | 对照Control | 水热处理Hydrothermal treatment | 标准误SEM | P值P-value |
---|---|---|---|---|
化学组成Chemical compositions | ||||
中性洗涤可溶物Neutral detergent soluble (NDS) | 251 | 418 | 42.0 | <0.001 |
中性洗涤纤维Neutral detergent fiber (NDF) | 749 | 582 | 42.0 | <0.001 |
酸性洗涤纤维Acid detergent fiber (ADF) | 627 | 563 | 14.3 | <0.001 |
水溶性碳水化合物Water soluble carbohydrates (WSC) | 13.4 | 44.7 | 5.6 | <0.001 |
半纤维素Hemicellulose (HC) | 122.0 | 15.0 | 28.7 | <0.001 |
木质素Acid detergent lignin (ADL) | 143 | 179 | 13.1 | 0.346 |
粗蛋白质Crude protein (CP) | 56.8 | 57.7 | 2.4 | 0.453 |
单糖Monosaccharides | ||||
阿拉伯糖Arabinose | 85.5 | 128.0 | 9.6 | <0.001 |
半乳糖Galactose | 353 | 513 | 35.8 | <0.001 |
葡萄糖Glucose | 1994 | 426 | 350.3 | <0.001 |
鼠李糖Rhamnose | 71.4 | 49.4 | 4.9 | <0.001 |
木糖Xylose | 231 | 633 | 89.9 | <0.001 |
果糖Fructose | 289.0 | 55.5 | 52.1 | <0.001 |
表2 发酵底物化学组成和单糖含量
Table 2 The concentrations of chemical compositions and monosaccharides for the fermentation substrates (g·kg-1, dry matter basis)
项目Items | 对照Control | 水热处理Hydrothermal treatment | 标准误SEM | P值P-value |
---|---|---|---|---|
化学组成Chemical compositions | ||||
中性洗涤可溶物Neutral detergent soluble (NDS) | 251 | 418 | 42.0 | <0.001 |
中性洗涤纤维Neutral detergent fiber (NDF) | 749 | 582 | 42.0 | <0.001 |
酸性洗涤纤维Acid detergent fiber (ADF) | 627 | 563 | 14.3 | <0.001 |
水溶性碳水化合物Water soluble carbohydrates (WSC) | 13.4 | 44.7 | 5.6 | <0.001 |
半纤维素Hemicellulose (HC) | 122.0 | 15.0 | 28.7 | <0.001 |
木质素Acid detergent lignin (ADL) | 143 | 179 | 13.1 | 0.346 |
粗蛋白质Crude protein (CP) | 56.8 | 57.7 | 2.4 | 0.453 |
单糖Monosaccharides | ||||
阿拉伯糖Arabinose | 85.5 | 128.0 | 9.6 | <0.001 |
半乳糖Galactose | 353 | 513 | 35.8 | <0.001 |
葡萄糖Glucose | 1994 | 426 | 350.3 | <0.001 |
鼠李糖Rhamnose | 71.4 | 49.4 | 4.9 | <0.001 |
木糖Xylose | 231 | 633 | 89.9 | <0.001 |
果糖Fructose | 289.0 | 55.5 | 52.1 | <0.001 |
项目 Items | 对照 Control | 水热处理 Hydrothermal treatment | 标准误 SEM | P值 P-value |
---|---|---|---|---|
干物质降解率Dry matter degradation (DMD,g·kg-1) | 553 | 616 | 6.700 | <0.001 |
产气量Gas volume (VDM,mL·g-1) | 199 | 184 | 1.300 | <0.001 |
可消化干物质产气量Gas volume per g of digestible dry matter (VDDM,mL·g-1) | 361 | 299 | 3.700 | <0.001 |
潜在最大产气量Final asymptotic gas volume (VF,mL·g-1) | 194 | 184 | 2.500 | 0.054 |
总产气速率Fractional rate of gas production (kGP,h-1) | 0.07 | 0.07 | 0.002 | 0.329 |
起始底物降解速率Initial fractional rate of degradation (FRD0,mmol·g-1·h-1) | 0.03 | 0.02 | 0.001 | 0.042 |
表3 HT对黄豆秸秆72 h体外模拟瘤胃发酵底物降解与总产气参数的影响
Table 3 Effects of HT on thesubstrate degradation and the parameters of gas production of soybean straw after 72 h in vitro ruminal incubation
项目 Items | 对照 Control | 水热处理 Hydrothermal treatment | 标准误 SEM | P值 P-value |
---|---|---|---|---|
干物质降解率Dry matter degradation (DMD,g·kg-1) | 553 | 616 | 6.700 | <0.001 |
产气量Gas volume (VDM,mL·g-1) | 199 | 184 | 1.300 | <0.001 |
可消化干物质产气量Gas volume per g of digestible dry matter (VDDM,mL·g-1) | 361 | 299 | 3.700 | <0.001 |
潜在最大产气量Final asymptotic gas volume (VF,mL·g-1) | 194 | 184 | 2.500 | 0.054 |
总产气速率Fractional rate of gas production (kGP,h-1) | 0.07 | 0.07 | 0.002 | 0.329 |
起始底物降解速率Initial fractional rate of degradation (FRD0,mmol·g-1·h-1) | 0.03 | 0.02 | 0.001 | 0.042 |
项目 Items | 对照 Control | 水热处理 Hydrothermal treatment | 标准误SEM | P值 P-value | |
---|---|---|---|---|---|
甲烷 Methane | 甲烷产量Methane volume (VM·DM,mL·g-1) | 23.2 | 19.8 | 0.230 | <0.001 |
可消化干物质甲烷产量Methane volume per g of digestible dry matter (VM·DDM,mL·g-1) | 42.0 | 32.1 | 0.520 | <0.001 | |
甲烷浓度Methane concentration (CM,%) | 13.0 | 10.9 | 0.140 | <0.001 | |
甲烷潜在最大产量Final asymptotic methane volume (VMF,mL·g-1) | 21.8 | 20.1 | 0.230 | 0.006 | |
甲烷产气速率Fractional rate of methane production (kM,h-1) | 0.01 | 0.06 | 0.002 | <0.001 | |
氢气 Hydrogen | 干物质氢气产量Hydrogen volume (VH·DM,mL·g-1) | 0.05 | 0.02 | 0.001 | <0.001 |
可消化干物质氢气产量Hydrogen volume per g of digestible dry matter (VH·DDM,mL·g-1) | 0.09 | 0.03 | 0.002 | <0.001 | |
氢气浓度Hydrogen concentration (CH,%) | 0.02 | 0.01 | 0.001 | <0.001 | |
氢气潜在最大产量Final asymptotic hydrogen volume (VHF,mL·g-1) | 0.06 | 0.03 | 0.003 | 0.001 | |
氢气产气速率Fractional rate of hydrogen production (kH,h-1) | 0.13 | 0.16 | 0.097 | 0.807 |
表4 HT对黄豆秸秆72 h体外模拟瘤胃发酵甲烷和氢气产气参数的影响
Table 4 Effects of HT on the parameters of methane and hydrogen gas production of soybean straw after 72 h in vitro ruminal incubation
项目 Items | 对照 Control | 水热处理 Hydrothermal treatment | 标准误SEM | P值 P-value | |
---|---|---|---|---|---|
甲烷 Methane | 甲烷产量Methane volume (VM·DM,mL·g-1) | 23.2 | 19.8 | 0.230 | <0.001 |
可消化干物质甲烷产量Methane volume per g of digestible dry matter (VM·DDM,mL·g-1) | 42.0 | 32.1 | 0.520 | <0.001 | |
甲烷浓度Methane concentration (CM,%) | 13.0 | 10.9 | 0.140 | <0.001 | |
甲烷潜在最大产量Final asymptotic methane volume (VMF,mL·g-1) | 21.8 | 20.1 | 0.230 | 0.006 | |
甲烷产气速率Fractional rate of methane production (kM,h-1) | 0.01 | 0.06 | 0.002 | <0.001 | |
氢气 Hydrogen | 干物质氢气产量Hydrogen volume (VH·DM,mL·g-1) | 0.05 | 0.02 | 0.001 | <0.001 |
可消化干物质氢气产量Hydrogen volume per g of digestible dry matter (VH·DDM,mL·g-1) | 0.09 | 0.03 | 0.002 | <0.001 | |
氢气浓度Hydrogen concentration (CH,%) | 0.02 | 0.01 | 0.001 | <0.001 | |
氢气潜在最大产量Final asymptotic hydrogen volume (VHF,mL·g-1) | 0.06 | 0.03 | 0.003 | 0.001 | |
氢气产气速率Fractional rate of hydrogen production (kH,h-1) | 0.13 | 0.16 | 0.097 | 0.807 |
图3 HT对黄豆秸秆72 h体外模拟瘤胃发酵氢气产量和生成速率曲线的影响
Fig.3 Effects of HT on the kinetic of hydrogen production of soybean straw after 72 h in vitro ruminal incubation
项目 Items | 对照 Control | 水热处理 Hydrothermal treatment | 标准误 SEM | P值 P-value |
---|---|---|---|---|
pH | 6.47 | 6.44 | 0.012 | 0.132 |
氨态氮NH3-N (mmol·L-1) | 13.6 | 11.2 | 0.580 | 0.012 |
总挥发性脂肪酸Total volatile fatty acids (TVFA,mmol·L-1) | 77.9 | 82.8 | 3.240 | 0.043 |
乙酸/丙酸Acetate/propionate | 3.83 | 2.06 | 0.021 | <0.001 |
VFA产氢量Net hydrogen produced per VFA (RNH2,mol·100 mol-1) | 136 | 105 | 0.400 | <0.001 |
挥发性脂肪酸 Volatile fatty acid (VFA,mol·100 mol-1) | ||||
乙酸Acetate | 72.9 | 61.3 | 0.110 | <0.001 |
丙酸Propionate | 19.1 | 29.8 | 0.150 | <0.001 |
丁酸Butyrate | 5.12 | 6.03 | 0.021 | <0.001 |
异丁酸Iso-butyrate | 0.86 | 0.87 | 0.009 | 0.308 |
戊酸Valerate | 0.93 | 0.99 | 0.015 | 0.011 |
异戊酸Iso-valerate | 1.14 | 1.04 | 0.009 | <0.001 |
表5 HT对黄豆秸秆72 h体外模拟瘤胃发酵参数的影响
Table 5 Effects of HT on the fermentation parameters of soybean straw after 72 h in vitro ruminal incubation
项目 Items | 对照 Control | 水热处理 Hydrothermal treatment | 标准误 SEM | P值 P-value |
---|---|---|---|---|
pH | 6.47 | 6.44 | 0.012 | 0.132 |
氨态氮NH3-N (mmol·L-1) | 13.6 | 11.2 | 0.580 | 0.012 |
总挥发性脂肪酸Total volatile fatty acids (TVFA,mmol·L-1) | 77.9 | 82.8 | 3.240 | 0.043 |
乙酸/丙酸Acetate/propionate | 3.83 | 2.06 | 0.021 | <0.001 |
VFA产氢量Net hydrogen produced per VFA (RNH2,mol·100 mol-1) | 136 | 105 | 0.400 | <0.001 |
挥发性脂肪酸 Volatile fatty acid (VFA,mol·100 mol-1) | ||||
乙酸Acetate | 72.9 | 61.3 | 0.110 | <0.001 |
丙酸Propionate | 19.1 | 29.8 | 0.150 | <0.001 |
丁酸Butyrate | 5.12 | 6.03 | 0.021 | <0.001 |
异丁酸Iso-butyrate | 0.86 | 0.87 | 0.009 | 0.308 |
戊酸Valerate | 0.93 | 0.99 | 0.015 | 0.011 |
异戊酸Iso-valerate | 1.14 | 1.04 | 0.009 | <0.001 |
项目Items | 对照Control | 水热处理Hydrothermal treatment | 标准误SEM | P值P-value |
---|---|---|---|---|
原虫Protozoa | 7.23 | 6.91 | 0.102 | 0.067 |
真菌Fungi | 8.31 | 7.51 | 0.124 | 0.004 |
细菌Bacteria | 10.17 | 10.15 | 0.046 | 0.764 |
产甲烷菌Methanogen | 8.65 | 8.49 | 0.034 | 0.020 |
产琥珀酸丝状杆菌Fibrobacter succinogenes | 8.15 | 7.75 | 0.102 | 0.033 |
白色瘤胃球菌Ruminococcus albus | 5.97 | 6.37 | 0.151 | 0.109 |
黄色瘤胃球菌Ruminococcus flavefaciens | 6.48 | 6.26 | 0.116 | 0.237 |
甲烷杆菌目Methanobacteriales | 8.28 | 8.19 | 0.039 | 0.185 |
甲烷短杆菌Methanobrebacteria | 8.07 | 7.98 | 0.053 | 0.271 |
甲烷微菌目Methanomicrobiales | 5.90 | 6.28 | 0.122 | 0.075 |
表6 HT对黄豆秸秆72 h体外模拟瘤胃发酵微生物种群的影响
Table 6 Effects of HT on the populations of major microorganisms of soybean straw after 72 h in vitro ruminal incubation (log10, copies·mL-1)
项目Items | 对照Control | 水热处理Hydrothermal treatment | 标准误SEM | P值P-value |
---|---|---|---|---|
原虫Protozoa | 7.23 | 6.91 | 0.102 | 0.067 |
真菌Fungi | 8.31 | 7.51 | 0.124 | 0.004 |
细菌Bacteria | 10.17 | 10.15 | 0.046 | 0.764 |
产甲烷菌Methanogen | 8.65 | 8.49 | 0.034 | 0.020 |
产琥珀酸丝状杆菌Fibrobacter succinogenes | 8.15 | 7.75 | 0.102 | 0.033 |
白色瘤胃球菌Ruminococcus albus | 5.97 | 6.37 | 0.151 | 0.109 |
黄色瘤胃球菌Ruminococcus flavefaciens | 6.48 | 6.26 | 0.116 | 0.237 |
甲烷杆菌目Methanobacteriales | 8.28 | 8.19 | 0.039 | 0.185 |
甲烷短杆菌Methanobrebacteria | 8.07 | 7.98 | 0.053 | 0.271 |
甲烷微菌目Methanomicrobiales | 5.90 | 6.28 | 0.122 | 0.075 |
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