固定化微生物对原油污染土壤的生物修复
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摘要
在石油勘探与开采、储运与炼制过程中,不可避免地会有大量原油和原油制品抛洒或泄漏于地面,造成大面积土壤的石油污染,对生态环境造成严重危害。由于石油烃类物质具有较高的生物学毒性、在环境中的残留时间较长且危害大,如何对受其污染的环境进行有效修复日益受到关注。微生物修复具有费用低、可现场处理污染土壤或水体、能够最大限度地降低污染物浓度和环境负面影响等特点,有关这方面的研究也越来越多。特别是在如何提高降解菌功效方面的研究已经成为国际上的研究热点。因此,提高石油烃降解菌在土壤中的繁殖效率以及便于菌剂的扩大化生产与保存、运输,采取将微生物进行固定化处理的办法具有重要意义。
本文从黄河三角洲长期受石油污染的土壤中驯化富集得到对原油具有较好降解效果的土著的石油烃降解混合菌系,在含有高浓度原油的液体介质中检验了该菌系对原油的耐受能力与降解能力,通过分离纯化培养,得到三株优势菌种,经鉴定分别为：棒状杆菌(Microbacterium foliorum)、食烷烃戈登氏菌(Gordonia alkanivorans)和中慢生根瘤菌(Mesorhizobium)
采用四种天然多糖性载体材料将石油烃降解混合菌系制备成固定化颗粒,通过对固定化颗粒的制备难易、机械强度、传质性等性能的测试,最终选取操作简便且机械强度与传质性较高的海藻酸钠作为载体,并在海藻酸钠中添加硅藻土以提高固定化颗粒的机械强度和传质性,通过测定固定化反应时间对固定化颗粒中的微生物活性的影响,最终确定固定化颗粒的制备条件,即采用6%海藻酸钠-2%硅藻土溶液(w/v)作载体,与等体积的降解菌生理盐水悬浮液混合均匀,用酸式滴定管逐滴滴入0.1M的CaCl2溶液中,固定化反应12h,并测得该方法具有较高的固定化效率,为92.25%.
用扫描电镜对固定化颗粒的微观结构进行观察,颗粒内部呈蜂窝网状结构,大部分的降解菌被包埋在孔隙内附着生长,颗粒孔隙度较为丰富,利于氧气、水分和底物的传输,且能够为固定化细菌提供充分的生长空间,为其在载体内的生长繁殖提供有利条件。
将海藻酸钠固定化颗粒和海藻酸钠+硅藻土的固定化颗粒以三种接种量添加到原油污染土壤中进行原油降解实验,调节土壤的水分和N、P含量至理想水平,分别以同等接种量的游离菌作对照。结果表明,固定化降解菌产生了较同等菌量的游离菌明显高的降解优势,特别是在实验初期,接种固定化菌的各组土壤的TPH降解率在实验20天时就已分别达到27.29%,29.83%和27.56%,分别比同等接种量的游离菌高出12.43%,10.10%和6.43%,添加固定化菌的土壤中降解菌的数量比添加游离菌的土壤高出1.20～1.53个数量级。并且低接种量的固定化菌(IL组)体现了比高接种量的游离菌(FH组)更强的降解优势,20天时,前者的降解率已比后者高出6.1%；30天后,各实验组降解菌的数量和微生物活性趋于稳定,相应的原油降解率也趋于缓慢。另外,所用载体不会破坏土壤结构,适用于长期大面积石油污染土壤的现场修复。通过对固定化颗粒的保存条件进行初步试验,将固定化降解菌于4℃下分别保存在无菌水及三种保护剂20%脱脂乳、20%甘油与液体石蜡中,结果表明,无菌水是最好的保存介质,降解菌在至少3周内都可保持较好的生物活性和颗粒性状。本实验的结果为固定化微生物在大规模土壤的实际修复中的应用提供了新的理论依据。
In oil exploration and exploitation,during the storage and transportation and refining processes,inevitably there will be a lot of crude oil and oil products spilled or leaked onto the ground, causing a large area of oil contaminated soil,soil and ecological environment on a range of hazards.As petroleum hydrocarbons have high biological toxicity,residue in the environment for long periods and cause great harm,how to rehabilitate the contaminated environment becomes increasingly concerned by the community. Bioremediation, be able to be used for in-situ treatment with low-cost, and minimize the negative effects of pollutant concentrations and environmental and other advantages, is very suitable for our present practical needs.To enhance the breeding efficiency of hydrocarbon degrading bacteria in the soil and to facilitate the enlargement of production, preservation, and transportation of the cultures, it will make great sense to make them into immobilized form.
Efficient biodegrading microorganism consortium of crude oil of the Yellow River Delta were acclimated and enriched from oil contaminated soil.Their tolerance and biodegradability was tested by liquid culture medium which contains high concentrations of crude oil.Three dominant species were obtained through isolation and purifying culture, and were identified as Microbacterium foliorum, Gordonia alkanivorans and Mesorhizobium.
Four kinds of natural polysaccharide were chosen for carrier materials to make the consortium into immobilized beads.The handling difficulty, mechanical strength, and mass transfer abilities of bead were examined, and sodium alginate was selected as carrier, for its simple handling, strong mechanical strength and mass transfer ability.And then diatomite particles were added to sodium alginate to enhance the mechanical strength and mass transfer of the immobilized beads,and the impact of immobilization time on microorganism's activity was tested as well. Finally the preparation conditions of the immobilized beads were set as follows: carrier solution consists of 6% sodium alginate-2% diatomite (w/v),mixed with an equal volume of degrading bacteria saline suspension, and then be dropped into 0.1M CaCl2 solution with acid buret and incubated for 12h. The immobilization efficiency of this method is 92.25%.
Scanning electron microscopy microstructures of the immobilized beads were observed,showing a cellular network structure within the particles.Most of the bacteria were embedded in the pores or adhered on the walls of the pores.Particle porosity inside bead is abundant, which is beneficial for oxygen, water and substrate transfer, and can provide sufficient space for the embedded cells'growth. There for, favorable conditions were provided for microorganism cells'growth and multiplication inside the carrier.
The immobilized hydrocarbon degrading bacteria in three inoculums were added to the crude oil contaminated soil for biodegradation experiment, and soil moisture and N, P levels were controlled to the desired level.the same amount of free bacteria were inoculated respectively as control.The immobilized consortium achieved much higher reproductive rate TPH degradation efficiency in soil, especially in the initial period of the experiment. After inoculated for 20 days,the immobilized consortium inoculated groups achieved high TPH degradation rate of 27.29%,29.83% and 27.56%, respectively, higher than that of free bacteria at 12.43%,10.10% and 6.43% respectively. And the populations of biodegrading bacteria in soils inoculated immobilized consortium were higher than that of free consortium at 1.20～1.53 magnitude.
Finally the storage conditions for the immobilized beads were tested.When the beads are kept in sterilized water at 4℃, they can maintain better biological activity for at least there weeks. The results of this study provides new theoretical basis for the application of immobilized microorganisms in bioremediation of large areas of contaminated soil.
本文从黄河三角洲长期受石油污染的土壤中驯化富集得到对原油具有较好降解效果的土著的石油烃降解混合菌系,在含有高浓度原油的液体介质中检验了该菌系对原油的耐受能力与降解能力,通过分离纯化培养,得到三株优势菌种,经鉴定分别为：棒状杆菌(Microbacterium foliorum)、食烷烃戈登氏菌(Gordonia alkanivorans)和中慢生根瘤菌(Mesorhizobium)
采用四种天然多糖性载体材料将石油烃降解混合菌系制备成固定化颗粒,通过对固定化颗粒的制备难易、机械强度、传质性等性能的测试,最终选取操作简便且机械强度与传质性较高的海藻酸钠作为载体,并在海藻酸钠中添加硅藻土以提高固定化颗粒的机械强度和传质性,通过测定固定化反应时间对固定化颗粒中的微生物活性的影响,最终确定固定化颗粒的制备条件,即采用6%海藻酸钠-2%硅藻土溶液(w/v)作载体,与等体积的降解菌生理盐水悬浮液混合均匀,用酸式滴定管逐滴滴入0.1M的CaCl2溶液中,固定化反应12h,并测得该方法具有较高的固定化效率,为92.25%.
用扫描电镜对固定化颗粒的微观结构进行观察,颗粒内部呈蜂窝网状结构,大部分的降解菌被包埋在孔隙内附着生长,颗粒孔隙度较为丰富,利于氧气、水分和底物的传输,且能够为固定化细菌提供充分的生长空间,为其在载体内的生长繁殖提供有利条件。
将海藻酸钠固定化颗粒和海藻酸钠+硅藻土的固定化颗粒以三种接种量添加到原油污染土壤中进行原油降解实验,调节土壤的水分和N、P含量至理想水平,分别以同等接种量的游离菌作对照。结果表明,固定化降解菌产生了较同等菌量的游离菌明显高的降解优势,特别是在实验初期,接种固定化菌的各组土壤的TPH降解率在实验20天时就已分别达到27.29%,29.83%和27.56%,分别比同等接种量的游离菌高出12.43%,10.10%和6.43%,添加固定化菌的土壤中降解菌的数量比添加游离菌的土壤高出1.20～1.53个数量级。并且低接种量的固定化菌(IL组)体现了比高接种量的游离菌(FH组)更强的降解优势,20天时,前者的降解率已比后者高出6.1%；30天后,各实验组降解菌的数量和微生物活性趋于稳定,相应的原油降解率也趋于缓慢。另外,所用载体不会破坏土壤结构,适用于长期大面积石油污染土壤的现场修复。通过对固定化颗粒的保存条件进行初步试验,将固定化降解菌于4℃下分别保存在无菌水及三种保护剂20%脱脂乳、20%甘油与液体石蜡中,结果表明,无菌水是最好的保存介质,降解菌在至少3周内都可保持较好的生物活性和颗粒性状。本实验的结果为固定化微生物在大规模土壤的实际修复中的应用提供了新的理论依据。
In oil exploration and exploitation,during the storage and transportation and refining processes,inevitably there will be a lot of crude oil and oil products spilled or leaked onto the ground, causing a large area of oil contaminated soil,soil and ecological environment on a range of hazards.As petroleum hydrocarbons have high biological toxicity,residue in the environment for long periods and cause great harm,how to rehabilitate the contaminated environment becomes increasingly concerned by the community. Bioremediation, be able to be used for in-situ treatment with low-cost, and minimize the negative effects of pollutant concentrations and environmental and other advantages, is very suitable for our present practical needs.To enhance the breeding efficiency of hydrocarbon degrading bacteria in the soil and to facilitate the enlargement of production, preservation, and transportation of the cultures, it will make great sense to make them into immobilized form.
Efficient biodegrading microorganism consortium of crude oil of the Yellow River Delta were acclimated and enriched from oil contaminated soil.Their tolerance and biodegradability was tested by liquid culture medium which contains high concentrations of crude oil.Three dominant species were obtained through isolation and purifying culture, and were identified as Microbacterium foliorum, Gordonia alkanivorans and Mesorhizobium.
Four kinds of natural polysaccharide were chosen for carrier materials to make the consortium into immobilized beads.The handling difficulty, mechanical strength, and mass transfer abilities of bead were examined, and sodium alginate was selected as carrier, for its simple handling, strong mechanical strength and mass transfer ability.And then diatomite particles were added to sodium alginate to enhance the mechanical strength and mass transfer of the immobilized beads,and the impact of immobilization time on microorganism's activity was tested as well. Finally the preparation conditions of the immobilized beads were set as follows: carrier solution consists of 6% sodium alginate-2% diatomite (w/v),mixed with an equal volume of degrading bacteria saline suspension, and then be dropped into 0.1M CaCl2 solution with acid buret and incubated for 12h. The immobilization efficiency of this method is 92.25%.
Scanning electron microscopy microstructures of the immobilized beads were observed,showing a cellular network structure within the particles.Most of the bacteria were embedded in the pores or adhered on the walls of the pores.Particle porosity inside bead is abundant, which is beneficial for oxygen, water and substrate transfer, and can provide sufficient space for the embedded cells'growth. There for, favorable conditions were provided for microorganism cells'growth and multiplication inside the carrier.
The immobilized hydrocarbon degrading bacteria in three inoculums were added to the crude oil contaminated soil for biodegradation experiment, and soil moisture and N, P levels were controlled to the desired level.the same amount of free bacteria were inoculated respectively as control.The immobilized consortium achieved much higher reproductive rate TPH degradation efficiency in soil, especially in the initial period of the experiment. After inoculated for 20 days,the immobilized consortium inoculated groups achieved high TPH degradation rate of 27.29%,29.83% and 27.56%, respectively, higher than that of free bacteria at 12.43%,10.10% and 6.43% respectively. And the populations of biodegrading bacteria in soils inoculated immobilized consortium were higher than that of free consortium at 1.20～1.53 magnitude.
Finally the storage conditions for the immobilized beads were tested.When the beads are kept in sterilized water at 4℃, they can maintain better biological activity for at least there weeks. The results of this study provides new theoretical basis for the application of immobilized microorganisms in bioremediation of large areas of contaminated soil.
引文
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