木质素降解微生物特性及其对农业废物堆肥腐殖化的影响研究
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摘要
农业废物,尤其是农作物秸秆中含有大量的木质纤维素,其中的木质素成分复杂,分解困难,导致腐殖质转化不完全,堆肥质量不高,因此加速木质素转化为腐殖质成为农业废物堆肥充分腐熟的关键。论文系统研究了木质素的生物降解特性及其对农业废物堆肥腐殖化的影响,首先研究了两种木质素降解微生物简青霉(Penicillium simplicissimum)和栗褐链霉菌(Streptomyces badius)及一组木质素降解复合菌的木质素降解特性。在此基础上,通过固态发酵实验研究了不同木质素降解微生物对腐殖质形成的影响,并在农业废物堆肥中以黄孢原毛平革菌(Phanerochaete chrysosporium)为接种剂研究了在不同发酵时期接种木质素降解微生物对堆肥腐殖化的影响,为加速堆肥腐熟、提高堆肥品质提供理论指导。
对P. simplicissimum产漆酶的调控研究发现,附加碳源抑制漆酶Lac活性,而适量氮源可显著提高酶活,常用的Lac诱导剂ABTS和二甲苯胺并不能促进简青霉产生Lac。在发酵过程中,木质素的降解率实际上可能是由Lac的聚合和解聚作用整合的结果。各种附加营养物诱使P. simplicissimum在第3d出现Lac活性峰,该峰值下漆酶聚合能力特别强,直接影响最后木质素的降解率。
S. badius对稻草木质纤维素的降解研究发现,附加碳氮源对过氧化物酶的产生及木质素的降解均有促进作用,但对半纤维素酶和纤维素酶的产生及半纤维素和纤维素的降解均有抑制作用;附加氮源-酵母膏明显促进可酸沉淀的多聚木质素(APPL)的产生,而附加氮源-氯化铵和碳源-葡萄糖均抑制APPL的产生。
分别从农业废物堆肥的升温、降温以及腐熟阶段筛选出三组木质素降解复合菌MC1、MC2和MC3。初筛和复筛的结果均表明复合菌MC1具有高效木质素降解能力。复合菌MC1的营养调控正交试验研究表明,该复合菌的最佳培养条件是:摇床转速为120r/min,第一碳源为蔗糖,氮源为蛋白胨,添加ABTS、藜芦醇、吐温80、Cu2+和Mn2+,且第二碳源碱木质素的加入时间为培养的第2d。在此最佳综合培养条件下,复合菌MC1培养6d后的LiP酶活为579.91U/L,MnP酶活为882.49U/L,Lac酶活为80.94U/L,培养10d后的木质素降解率为46.75%。
比较研究了两种不同木质素降解菌(P. chrysosporium和S. badius)及土著微生物对腐殖质形成的影响。结果表明,接种木质素降解微生物有利于腐殖质总量的形成,且S. badius更有利于腐殖质的形成。由于两种微生物具有不同的木质素降解机理,它们降解木质素形成腐殖质的过程也有所不同。P. chrysosporium转化木质素生成简单分子富里酸(FA),然后聚合FA生成复杂的分子胡敏酸(HA),而S. badius改性木质素,先生成HA,然后将HA转化成FA。
考察了在农业废物堆肥的不同发酵时期接种P. chrysosporium对堆肥腐殖组分、腐熟度及胡敏酸光学特性的影响。结果表明,在一次发酵时期接种P. chrysosporium对木质素的降解和腐殖质的形成都没有明显的促进作用,而在二次发酵时期接种P. chrysosporium既能提高木质素的降解率,也能促进腐殖质的形成。另外,在二次发酵时期接种P. chrysosporium可以使堆肥的腐熟期缩短7d,但一次发酵时期接种并没有明显缩短堆肥周期。胡敏酸光谱扫描结果进一步表明,在一次发酵时期接种黄孢原毛平革菌不能明显促进胡敏酸的腐殖化程度,但在二次发酵时期接种黄孢原毛平革菌能明显促进堆肥中胡敏酸结构的复杂化,增加其芳构化程度及缩合程度,从而明显提高胡敏酸腐殖化程度。
Agricultural wastes, especially crop straws, contain many quantities of lignocellulose. The main component, lignin, is difficult to be biodegraded due to the complex component and leads the insufficent transfomation of humus and low quality of compost. So accelerelating the humification of lignin is critical to the sufficient maturity of the agricultural wastes. Characteristics of ligninolytic microorganisms and their effects on the humification of the agricultural wastes composting were studied. Firstly, the lignin degrading characteristics of Penicillium simplicissimum, Streptomyces badius and one composite microorganism were studied. Based on these, the effect of biodelignification of rice straw by different ligninolytic organisms on humus formation was discussed in solid state fermentation. Lastly, the lignocellulolytic microorganism, Phanerochaete chrysosporium, was inoculated during different phases of agricultural wastes composting and its effects on the humification were studied. These results would give theory instruction for accelerating composting and improve the compost quality.
The laccase activities of P. simplicissimum during solid state fermentation with rice straw were studied. Results showed that all supplemental carbon nutrients inhibited the laccase activity, while proper concentration of supplemental nitrogen sources remarkably enhanced the laccase activity. The enhancement of laccase activity by ordinary laccase inducers 2, 2’-azino-bis (3-ethylbenzthiazoline-6- sulfonic acid) and xylidine was not observed in this study. Lignocellulose degradation was improved when laccase activity was relatively low during the fermentation, which proved the polymerizing and depolymerizing function of laccase in lignin degradation by P. simplicissimum. All supplemental sources might induce the peak of Lac activity on day 3 when the polymerizing function was especially strong.
The lignocellulose degradation of rice straw by Streptomyces badius during solid state fermentation was researched. The results showed that nitrogen resouce-yeast extract stimulated the production of peroxidase and increased the degradation rate of lignin. However they inhibited the production of both hemicellulase and cellulase. They also decreased the degradation rate of the hemicellulose and cellulose. Nitrogen source-yeast extract obviously stimulated the production of APPL, but nitrogen source-ammonium chloride and carbon source-glucose inhibited its production.
Three composite microorganisms (MC1, MC2 and MC3) with the ability of lignin degradation were screened from three stages of agricultural waste composting and the three stages were warming phase, cooling phase and falling phase, respectively. The results of preliminary screening and further screening both indicated that the composite microorganism MC1 screened from the warming phase had the best ability to degrade lignin. And then, orthogonal experiment was adopted to study its nutritional regulation. The results showed that the optimum integrative culture conditions of MC1 were as follows: rotate speed was 120 r/min; the first carbon source was sucrose, nitrogen source was peptone. ATBS, Veratryl alcohol, Tween-80, Cu2+ and Mn2+ should be added and the second carbon source was added after 2 days. Undering the optimum integrative culture conditions, the enzyme activity of LiP, MnP and Lac after 6 days were 579.91 U/L, 882.49 U/L and 80.94 U/L, respectively. The lignin degradation rate was 46.75% after 10 days.
The effects of biodelignification of rice straw by two different ligninolytic organisms (P. chrysosporium and S. badius) and aboriginal microbe on humus formation were compared. The results indicate that lignin degrading microorganisms could promote humus formation. Furthermore, S. badius could accelerate the humus formation more than P. chrysosporium. Since the degrading mechanisms of two strains were different, the approaches of humus formation from lignin with P. chrysosporium and S. badius were also different. P. chrysosporium metabolized lignin to simple molecules (FA), which then were polymerized to complex molecules (HA). While S. badius modified lignin to HA, which then were transformed to FA.
The lignocellulolytic microorganism, P. chrysosporium, was inoculated during diifferent fermentation phases of agricultural wastes composting and its effects on humus, compost maturity and spectroscopic characterization of humic acid fraction were investigated. The results showed that it has no obvious effect on the lignin degradation and humus formation when P. chrysosporium was inoculated during the first fermentation phase of composting, but it can increase the lignin degradation and stimulate the humus release when it was inoculated during the second fermentation phase of composting. When inoculated during the second fermentation phase, P. chrysosporium shorten the period of the composting, while it did not short the period of the composting when inoculated during the first fermentation phase. Spectroscopic characterization of humic acid fraction showed that the inoculation P. chrysosporium increased the degree of the aromatization and the molecular polymerization of humic acid when inoculated during second fermentation phase, while it did not produce an obvious change on the humification degree of humic acid when inoculated during the first fermentation phase.
对P. simplicissimum产漆酶的调控研究发现,附加碳源抑制漆酶Lac活性,而适量氮源可显著提高酶活,常用的Lac诱导剂ABTS和二甲苯胺并不能促进简青霉产生Lac。在发酵过程中,木质素的降解率实际上可能是由Lac的聚合和解聚作用整合的结果。各种附加营养物诱使P. simplicissimum在第3d出现Lac活性峰,该峰值下漆酶聚合能力特别强,直接影响最后木质素的降解率。
S. badius对稻草木质纤维素的降解研究发现,附加碳氮源对过氧化物酶的产生及木质素的降解均有促进作用,但对半纤维素酶和纤维素酶的产生及半纤维素和纤维素的降解均有抑制作用;附加氮源-酵母膏明显促进可酸沉淀的多聚木质素(APPL)的产生,而附加氮源-氯化铵和碳源-葡萄糖均抑制APPL的产生。
分别从农业废物堆肥的升温、降温以及腐熟阶段筛选出三组木质素降解复合菌MC1、MC2和MC3。初筛和复筛的结果均表明复合菌MC1具有高效木质素降解能力。复合菌MC1的营养调控正交试验研究表明,该复合菌的最佳培养条件是:摇床转速为120r/min,第一碳源为蔗糖,氮源为蛋白胨,添加ABTS、藜芦醇、吐温80、Cu2+和Mn2+,且第二碳源碱木质素的加入时间为培养的第2d。在此最佳综合培养条件下,复合菌MC1培养6d后的LiP酶活为579.91U/L,MnP酶活为882.49U/L,Lac酶活为80.94U/L,培养10d后的木质素降解率为46.75%。
比较研究了两种不同木质素降解菌(P. chrysosporium和S. badius)及土著微生物对腐殖质形成的影响。结果表明,接种木质素降解微生物有利于腐殖质总量的形成,且S. badius更有利于腐殖质的形成。由于两种微生物具有不同的木质素降解机理,它们降解木质素形成腐殖质的过程也有所不同。P. chrysosporium转化木质素生成简单分子富里酸(FA),然后聚合FA生成复杂的分子胡敏酸(HA),而S. badius改性木质素,先生成HA,然后将HA转化成FA。
考察了在农业废物堆肥的不同发酵时期接种P. chrysosporium对堆肥腐殖组分、腐熟度及胡敏酸光学特性的影响。结果表明,在一次发酵时期接种P. chrysosporium对木质素的降解和腐殖质的形成都没有明显的促进作用,而在二次发酵时期接种P. chrysosporium既能提高木质素的降解率,也能促进腐殖质的形成。另外,在二次发酵时期接种P. chrysosporium可以使堆肥的腐熟期缩短7d,但一次发酵时期接种并没有明显缩短堆肥周期。胡敏酸光谱扫描结果进一步表明,在一次发酵时期接种黄孢原毛平革菌不能明显促进胡敏酸的腐殖化程度,但在二次发酵时期接种黄孢原毛平革菌能明显促进堆肥中胡敏酸结构的复杂化,增加其芳构化程度及缩合程度,从而明显提高胡敏酸腐殖化程度。
Agricultural wastes, especially crop straws, contain many quantities of lignocellulose. The main component, lignin, is difficult to be biodegraded due to the complex component and leads the insufficent transfomation of humus and low quality of compost. So accelerelating the humification of lignin is critical to the sufficient maturity of the agricultural wastes. Characteristics of ligninolytic microorganisms and their effects on the humification of the agricultural wastes composting were studied. Firstly, the lignin degrading characteristics of Penicillium simplicissimum, Streptomyces badius and one composite microorganism were studied. Based on these, the effect of biodelignification of rice straw by different ligninolytic organisms on humus formation was discussed in solid state fermentation. Lastly, the lignocellulolytic microorganism, Phanerochaete chrysosporium, was inoculated during different phases of agricultural wastes composting and its effects on the humification were studied. These results would give theory instruction for accelerating composting and improve the compost quality.
The laccase activities of P. simplicissimum during solid state fermentation with rice straw were studied. Results showed that all supplemental carbon nutrients inhibited the laccase activity, while proper concentration of supplemental nitrogen sources remarkably enhanced the laccase activity. The enhancement of laccase activity by ordinary laccase inducers 2, 2’-azino-bis (3-ethylbenzthiazoline-6- sulfonic acid) and xylidine was not observed in this study. Lignocellulose degradation was improved when laccase activity was relatively low during the fermentation, which proved the polymerizing and depolymerizing function of laccase in lignin degradation by P. simplicissimum. All supplemental sources might induce the peak of Lac activity on day 3 when the polymerizing function was especially strong.
The lignocellulose degradation of rice straw by Streptomyces badius during solid state fermentation was researched. The results showed that nitrogen resouce-yeast extract stimulated the production of peroxidase and increased the degradation rate of lignin. However they inhibited the production of both hemicellulase and cellulase. They also decreased the degradation rate of the hemicellulose and cellulose. Nitrogen source-yeast extract obviously stimulated the production of APPL, but nitrogen source-ammonium chloride and carbon source-glucose inhibited its production.
Three composite microorganisms (MC1, MC2 and MC3) with the ability of lignin degradation were screened from three stages of agricultural waste composting and the three stages were warming phase, cooling phase and falling phase, respectively. The results of preliminary screening and further screening both indicated that the composite microorganism MC1 screened from the warming phase had the best ability to degrade lignin. And then, orthogonal experiment was adopted to study its nutritional regulation. The results showed that the optimum integrative culture conditions of MC1 were as follows: rotate speed was 120 r/min; the first carbon source was sucrose, nitrogen source was peptone. ATBS, Veratryl alcohol, Tween-80, Cu2+ and Mn2+ should be added and the second carbon source was added after 2 days. Undering the optimum integrative culture conditions, the enzyme activity of LiP, MnP and Lac after 6 days were 579.91 U/L, 882.49 U/L and 80.94 U/L, respectively. The lignin degradation rate was 46.75% after 10 days.
The effects of biodelignification of rice straw by two different ligninolytic organisms (P. chrysosporium and S. badius) and aboriginal microbe on humus formation were compared. The results indicate that lignin degrading microorganisms could promote humus formation. Furthermore, S. badius could accelerate the humus formation more than P. chrysosporium. Since the degrading mechanisms of two strains were different, the approaches of humus formation from lignin with P. chrysosporium and S. badius were also different. P. chrysosporium metabolized lignin to simple molecules (FA), which then were polymerized to complex molecules (HA). While S. badius modified lignin to HA, which then were transformed to FA.
The lignocellulolytic microorganism, P. chrysosporium, was inoculated during diifferent fermentation phases of agricultural wastes composting and its effects on humus, compost maturity and spectroscopic characterization of humic acid fraction were investigated. The results showed that it has no obvious effect on the lignin degradation and humus formation when P. chrysosporium was inoculated during the first fermentation phase of composting, but it can increase the lignin degradation and stimulate the humus release when it was inoculated during the second fermentation phase of composting. When inoculated during the second fermentation phase, P. chrysosporium shorten the period of the composting, while it did not short the period of the composting when inoculated during the first fermentation phase. Spectroscopic characterization of humic acid fraction showed that the inoculation P. chrysosporium increased the degree of the aromatization and the molecular polymerization of humic acid when inoculated during second fermentation phase, while it did not produce an obvious change on the humification degree of humic acid when inoculated during the first fermentation phase.
引文
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