含镉堆肥施用对红壤微生物量、酶活性及微生物多样性的影响
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摘要
堆肥是我国农业生产的重要肥源,施用堆肥对于提供作物养分,维持土壤肥力发挥了重要作用。长期以来堆肥一直被认为是一种安全肥料,在无公害食品、绿色食品、有机食品等安全农产品生产中,均强调堆肥的施用。然而,堆肥的重要作用是以其本身无污染为前提。近年来,随着社会经济的发展和环境压力的日益增加,城市生活垃圾、畜禽粪便等堆肥原料的重金属污染问题已经非常普遍,堆肥重金属污染问题日益突出。而重金属镉在土壤中又具有较强的生物活性,容易被植物根系吸收而进入人类食物链,危害人体健康。迄今,关于含重金属的堆肥对土壤环境的污染研究较少。因此,研究重金属污染堆肥施用后对土壤微生物量、酶活性及微生物多样性的影响具有重要的意义。
本文选取了我国南方广泛分布的旱地红壤为供试材料,通过室内培养实验,采用连续提取法、PCR-DGGE技术等,探讨含Cd堆肥施用后,对红壤中微生物量(C、P)、酶活性(脲酶、过氧化氢酶、脱氢酶和酸性磷酸酶活性)、微生物多样性的影响,以及Cd在红壤中的赋存形态(可交换态、碳酸盐结合态、有机结合态、Fe-Mn氧化态、残渣态)的迁移转化及生物有效性。在理论上,进一步完善并丰富堆肥中重金属的基础研究；在实践上,为生产中堆肥的合理安全使用提供科学参考。
使用化学分析方法分析施用含Cd堆肥(浓度分别为7.8 mg/kg、8.8 mg/kg、12.8 mg/kg、17.8 mg/kg、27.8 mg/kg、67.8 mg/kg、107.8 mg/kg)对红壤微生物量、及酶活性的影响。研究结果表明：施用含Cd堆肥使红壤微生物量C、微生物量P、以及脱氢酶、过氧化氢酶、酸性磷酸酶的活性随着培养时间的延长而不断降低。相同培养时间,与对照相比,含Cd堆肥对微生物生物量P、酸性磷酸酶活性有抑制作用。然而,Cd浓度小于27.8 mg/kg堆肥,对微生物生物量C有激活作用,其激活率为0.26～98.46%；Cd浓度小于12.8 mg/kg堆肥,对过氧化氢酶活性有激活作用,其激活率为0.17～4.49%；含Cd浓度为8.8 mg/kg堆肥使红壤脱氢酶活性增加1.6～16.1%；脲酶活性在Cd浓度小于17.8 mg/kg堆肥时,其活性随着Cd浓度的增加而增加；然而,第20天时,Cd浓度大于27.8 mg/kg的堆肥对脲酶活性有抑制作用,其抑制率为0.81～2.65%。
应用连续提取法和PCR-DGGE技术,探讨了含Cd堆肥(浓度分别为7.8mg/kg、12.8 mg/kg、107.8 mg/kg)施用后,Cd在红壤中各赋存形态的迁移转化以及对微生物多样性的影响。研究发现：相同培养时间内,12.8 mg/kg Cd和107.8mg/kg Cd堆肥处理中可交换态Cd、碳酸盐结合态Cd、铁锰氧化态Cd含量均大于对照处理,其含量分别增加0.88～10.12 mg/kg、0.19～2.58 mg/kg、0.47～1.9 mg/kg;而有机结合态Cd随着堆肥中Cd浓度的增加而逐渐减少,其降低了0.05～0.34mg/kg;残渣态Cd变化不明显。DGGE指纹图谱表明：含Cd堆肥对红壤微生物有一定程度的影响。相同培养时间时,含Cd堆肥对微生物多样性影响小。随着培养时间的延长,微生物多样性发生变化,培养前14天,DGGE指纹图谱中的条带如e、g、l、n亮度增强,且在培养第7天,出现新增条带(h、r、A、B、C、D)；培养28天后,A、B、C等条带逐渐消失,说明含Cd堆肥对微生物种群有毒害作用。各泳道条带进行相似性(Cs)分析发现,培养前期不同处理中微生物DNA基因相似性逐渐增加,最大值为94%；而培养后期不同处理之间红壤微生物DNA基因型相似性不断减低,减低到76.4%.Shannon指数和Cd赋存各形态之间的相关系数表明,对照处理对微生物多样性的影响不明显；12.8 mg/kg Cd堆肥处理中,碳酸盐结合态Cd对微生物有激活作用；107.8 mg/kg Cd堆肥处理中,可交换态、碳酸盐结合态对微生物有抑制作用。
综上所述,含Cd堆肥对微生物生物量P、酸性磷酸酶活性有抑制作用；小于27.8 mg/kg Cd堆肥对微生物生物量C有激活作用；小于12.8 mg/kg Cd堆肥对过氧化氢酶活性有激活作用；8.8 mg/kg Cd堆肥对脱氢酶活性有激活作用；脲酶活性在Cd浓度小于17.8 mg/kg堆肥时,其活性随着Cd浓度的增加而增加。Cd在红壤中各赋存形态发生了迁移转化,DGGE指纹图谱说明碳酸盐结合态Cd对微生物有激活作用在12.8 mg/kg Cd堆肥处理中；可交换态Cd、碳酸盐结合态Cd在107.8mg/kg Cd堆肥处理中对微生物有抑制作用。
Cadmium is a kind of dispensable chemical element for plant growth. It has a strong toxicity to livings. So people pay more attention to environment pollution caused by cadmium. Compost is an important fertilizer in agricultural production. The use of compost plays an important role in the aspects of offering plant nutrients and keeping soil fertilizer efficiency. Compost is regarded as a very safe fertilizer for a long time in the production of safe agricultural products as no environmental pollution food, green food and organic food. However, no pollution of compost itself is the premise of the great effect of compost. With the development of social economy and the increasing of environment pressure in recent years, the pollution of compost itself is becoming outstanding. The appearance of environmental pollution caused by unsuitable use of compost is frequent. However, because cadmium has a strong bioavailability in the soil, it is easy for plant to absorb by roots and then get into human food chain which is bad for human beings healthy. Up to now, there was few studies about heavy mental contaminated compost on red soil environmental pollution. Therefore, researches on heavy mental contaminated compost influencing on red soil properties and microbial community diversity had significant importance.
In this paper, dryland red soil which was wide distributed in South China was selected in this study. Chemical analysis, sequential extraction method and PCR-DGGE were used with indoor culture condition. The effects of Cd-contaminated compost on red soil microbial biomass C、P, urease, catalase, dehydrogenase and acid phosphatase activity and the transformation of different fractions of Cd, microbial community diversity were studied in red soil. The research could provide futher information on effects of heavy metals contaminated compost on soil, and could provide a scientific reference for the production of reasonable safety compost.
Chemical analysis methods was used to assess changes of microbial biomass and enzyme activities between compost and cadmium-polluted compost (8.8 mg/kg、12.8 mg/kg、17.8mg/kg、27.8 mg/kg、67.8 mg/kg、107.8 mg/kg Cd) applied to red soil. Results showed that soil microbial biomass C and P, activities of catalase, dehydrogenase and acid phosphatase decreased in all treatments during the incubation time. Compared with all treatments at the same time, Cd-polluted compost had an inhibitory effect on microbial biomass P and acid phosphatase activity than control. Microbial biomass C was activated by 0.26～98.46% in<27.8 mg/kg Cd treatment than that of control. Catalase activity was stimulated by 0.17～4.49% in treatments with< 12.8 mg/kg Cd. When treated with 8.8 mg/kg Cd, dehydrogenase activity increased by 1.6～16.1% than that of control. Urease activity increased by 0.56～65.90% with an increase of Cd in compost with<17.8 mg/kg Cd, but in treatments with> 27.8 mg/kg Cd, the urease activity was inhibited by 0.81～2.65% at the 20th day.
Sequential extraction procedure and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis were used to study dynamic speciation of cadmium and changes in microbial community diversity, respectively, in red soil stressed by different concentration of cadmium-polluted compost (7.8 mg/kg、12.8 mg/kg、107.8 mg/kgCd). The resulted showed that exchangeable, carbonate, and Fe-Mn oxides fractions of Cd were higher in 12.8 mg/kg Cd and 107.8 mg/kg Cd treated (polluted) compost treatments than that in the controls. The percentage of exchangeable fraction, carbonate fraction, Fe-Mn oxides fraction were higher in Cd-contaminated compost treatments than that in the controls, they were18.49%～56.95%、18.02%～35.35%、15.04%～25.07%,respectively. The organic fraction of Cd decreased with increased Cd in compost applied to red soil. Residual formation did not change obviously in any treatment. DGGE profiles indicated that bacteria communities were affected by Cd-polluted compost to some extent. New bands (h, r, A, B, C, and D) emerged at early incubation stage, and some bands (A, B, and C) disappeared at late incubation stage. Similarity coefficient (Cs) of DGGE profiles showed that genetic similarity increased before 14 days (highest of 93.8%), and decreased after 28 days (lowest of 74.6%). High Shannon's diversity index (H) revealed activation effect of Cd to bacteria communities, which mainly attributed to the increase of carbonate fraction in 12.8 mg/kg treatment. Low H implied an inhibition of bacteria communities in 107.8 mg/kg Cd compost, which possibly due to the increase of exchangeable and carbonate fractions.
In summary, soil microbial biomass C、P, urease, catalase, dehydrogenase, acid phosphtatase activity had a certain change with the incubation time after application of high Cd concentration compost to red soil. Lower than 27.8 mg/kg Cd-contaminated compost have an activation impacts on soil microbial biomass C, urease, catalase, and dehydrogenase activity. Genetic similarity increased in the early incubation stage, and decreased in the late incubation period. Bacteria communities were activated by carbonate fraction in 12.8 mg/kg Cd compost, and inhibited by exchangeable and carbonate fractions in 107.8 mg/kg Cd-contaminated compost.
本文选取了我国南方广泛分布的旱地红壤为供试材料,通过室内培养实验,采用连续提取法、PCR-DGGE技术等,探讨含Cd堆肥施用后,对红壤中微生物量(C、P)、酶活性(脲酶、过氧化氢酶、脱氢酶和酸性磷酸酶活性)、微生物多样性的影响,以及Cd在红壤中的赋存形态(可交换态、碳酸盐结合态、有机结合态、Fe-Mn氧化态、残渣态)的迁移转化及生物有效性。在理论上,进一步完善并丰富堆肥中重金属的基础研究；在实践上,为生产中堆肥的合理安全使用提供科学参考。
使用化学分析方法分析施用含Cd堆肥(浓度分别为7.8 mg/kg、8.8 mg/kg、12.8 mg/kg、17.8 mg/kg、27.8 mg/kg、67.8 mg/kg、107.8 mg/kg)对红壤微生物量、及酶活性的影响。研究结果表明：施用含Cd堆肥使红壤微生物量C、微生物量P、以及脱氢酶、过氧化氢酶、酸性磷酸酶的活性随着培养时间的延长而不断降低。相同培养时间,与对照相比,含Cd堆肥对微生物生物量P、酸性磷酸酶活性有抑制作用。然而,Cd浓度小于27.8 mg/kg堆肥,对微生物生物量C有激活作用,其激活率为0.26～98.46%；Cd浓度小于12.8 mg/kg堆肥,对过氧化氢酶活性有激活作用,其激活率为0.17～4.49%；含Cd浓度为8.8 mg/kg堆肥使红壤脱氢酶活性增加1.6～16.1%；脲酶活性在Cd浓度小于17.8 mg/kg堆肥时,其活性随着Cd浓度的增加而增加；然而,第20天时,Cd浓度大于27.8 mg/kg的堆肥对脲酶活性有抑制作用,其抑制率为0.81～2.65%。
应用连续提取法和PCR-DGGE技术,探讨了含Cd堆肥(浓度分别为7.8mg/kg、12.8 mg/kg、107.8 mg/kg)施用后,Cd在红壤中各赋存形态的迁移转化以及对微生物多样性的影响。研究发现：相同培养时间内,12.8 mg/kg Cd和107.8mg/kg Cd堆肥处理中可交换态Cd、碳酸盐结合态Cd、铁锰氧化态Cd含量均大于对照处理,其含量分别增加0.88～10.12 mg/kg、0.19～2.58 mg/kg、0.47～1.9 mg/kg;而有机结合态Cd随着堆肥中Cd浓度的增加而逐渐减少,其降低了0.05～0.34mg/kg;残渣态Cd变化不明显。DGGE指纹图谱表明：含Cd堆肥对红壤微生物有一定程度的影响。相同培养时间时,含Cd堆肥对微生物多样性影响小。随着培养时间的延长,微生物多样性发生变化,培养前14天,DGGE指纹图谱中的条带如e、g、l、n亮度增强,且在培养第7天,出现新增条带(h、r、A、B、C、D)；培养28天后,A、B、C等条带逐渐消失,说明含Cd堆肥对微生物种群有毒害作用。各泳道条带进行相似性(Cs)分析发现,培养前期不同处理中微生物DNA基因相似性逐渐增加,最大值为94%；而培养后期不同处理之间红壤微生物DNA基因型相似性不断减低,减低到76.4%.Shannon指数和Cd赋存各形态之间的相关系数表明,对照处理对微生物多样性的影响不明显；12.8 mg/kg Cd堆肥处理中,碳酸盐结合态Cd对微生物有激活作用；107.8 mg/kg Cd堆肥处理中,可交换态、碳酸盐结合态对微生物有抑制作用。
综上所述,含Cd堆肥对微生物生物量P、酸性磷酸酶活性有抑制作用；小于27.8 mg/kg Cd堆肥对微生物生物量C有激活作用；小于12.8 mg/kg Cd堆肥对过氧化氢酶活性有激活作用；8.8 mg/kg Cd堆肥对脱氢酶活性有激活作用；脲酶活性在Cd浓度小于17.8 mg/kg堆肥时,其活性随着Cd浓度的增加而增加。Cd在红壤中各赋存形态发生了迁移转化,DGGE指纹图谱说明碳酸盐结合态Cd对微生物有激活作用在12.8 mg/kg Cd堆肥处理中；可交换态Cd、碳酸盐结合态Cd在107.8mg/kg Cd堆肥处理中对微生物有抑制作用。
Cadmium is a kind of dispensable chemical element for plant growth. It has a strong toxicity to livings. So people pay more attention to environment pollution caused by cadmium. Compost is an important fertilizer in agricultural production. The use of compost plays an important role in the aspects of offering plant nutrients and keeping soil fertilizer efficiency. Compost is regarded as a very safe fertilizer for a long time in the production of safe agricultural products as no environmental pollution food, green food and organic food. However, no pollution of compost itself is the premise of the great effect of compost. With the development of social economy and the increasing of environment pressure in recent years, the pollution of compost itself is becoming outstanding. The appearance of environmental pollution caused by unsuitable use of compost is frequent. However, because cadmium has a strong bioavailability in the soil, it is easy for plant to absorb by roots and then get into human food chain which is bad for human beings healthy. Up to now, there was few studies about heavy mental contaminated compost on red soil environmental pollution. Therefore, researches on heavy mental contaminated compost influencing on red soil properties and microbial community diversity had significant importance.
In this paper, dryland red soil which was wide distributed in South China was selected in this study. Chemical analysis, sequential extraction method and PCR-DGGE were used with indoor culture condition. The effects of Cd-contaminated compost on red soil microbial biomass C、P, urease, catalase, dehydrogenase and acid phosphatase activity and the transformation of different fractions of Cd, microbial community diversity were studied in red soil. The research could provide futher information on effects of heavy metals contaminated compost on soil, and could provide a scientific reference for the production of reasonable safety compost.
Chemical analysis methods was used to assess changes of microbial biomass and enzyme activities between compost and cadmium-polluted compost (8.8 mg/kg、12.8 mg/kg、17.8mg/kg、27.8 mg/kg、67.8 mg/kg、107.8 mg/kg Cd) applied to red soil. Results showed that soil microbial biomass C and P, activities of catalase, dehydrogenase and acid phosphatase decreased in all treatments during the incubation time. Compared with all treatments at the same time, Cd-polluted compost had an inhibitory effect on microbial biomass P and acid phosphatase activity than control. Microbial biomass C was activated by 0.26～98.46% in<27.8 mg/kg Cd treatment than that of control. Catalase activity was stimulated by 0.17～4.49% in treatments with< 12.8 mg/kg Cd. When treated with 8.8 mg/kg Cd, dehydrogenase activity increased by 1.6～16.1% than that of control. Urease activity increased by 0.56～65.90% with an increase of Cd in compost with<17.8 mg/kg Cd, but in treatments with> 27.8 mg/kg Cd, the urease activity was inhibited by 0.81～2.65% at the 20th day.
Sequential extraction procedure and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis were used to study dynamic speciation of cadmium and changes in microbial community diversity, respectively, in red soil stressed by different concentration of cadmium-polluted compost (7.8 mg/kg、12.8 mg/kg、107.8 mg/kgCd). The resulted showed that exchangeable, carbonate, and Fe-Mn oxides fractions of Cd were higher in 12.8 mg/kg Cd and 107.8 mg/kg Cd treated (polluted) compost treatments than that in the controls. The percentage of exchangeable fraction, carbonate fraction, Fe-Mn oxides fraction were higher in Cd-contaminated compost treatments than that in the controls, they were18.49%～56.95%、18.02%～35.35%、15.04%～25.07%,respectively. The organic fraction of Cd decreased with increased Cd in compost applied to red soil. Residual formation did not change obviously in any treatment. DGGE profiles indicated that bacteria communities were affected by Cd-polluted compost to some extent. New bands (h, r, A, B, C, and D) emerged at early incubation stage, and some bands (A, B, and C) disappeared at late incubation stage. Similarity coefficient (Cs) of DGGE profiles showed that genetic similarity increased before 14 days (highest of 93.8%), and decreased after 28 days (lowest of 74.6%). High Shannon's diversity index (H) revealed activation effect of Cd to bacteria communities, which mainly attributed to the increase of carbonate fraction in 12.8 mg/kg treatment. Low H implied an inhibition of bacteria communities in 107.8 mg/kg Cd compost, which possibly due to the increase of exchangeable and carbonate fractions.
In summary, soil microbial biomass C、P, urease, catalase, dehydrogenase, acid phosphtatase activity had a certain change with the incubation time after application of high Cd concentration compost to red soil. Lower than 27.8 mg/kg Cd-contaminated compost have an activation impacts on soil microbial biomass C, urease, catalase, and dehydrogenase activity. Genetic similarity increased in the early incubation stage, and decreased in the late incubation period. Bacteria communities were activated by carbonate fraction in 12.8 mg/kg Cd compost, and inhibited by exchangeable and carbonate fractions in 107.8 mg/kg Cd-contaminated compost.
引文
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