CN1565762A - Biological method for restoring polluted soil by oil and PAH5 - Google Patents

Abstract

The invention relates to a biological application technique in environmental bioscience, specially a biological reserve method for petroleum and PAH5 contaminated soil, that is, a biological reserve system for contaminated soil constructed by mycorhiza combined symbiont. The mycorhiza combined symbiont comprises corn and Ect., VAM, Ect.+VAM or Ect.+B; marigold or soybean and VAM, VAM+B or VAM+N; white wax and Ect., Ect.+VAM or Ect.+N; weeping willow and Ect. or Ect.+B+N. The economical and practical technique provided by the invention has the following advantages: integrating the microbial reserve method with the plant reserve method, which can make the regulation and control action take effect sufficiently.

Description

A kind of biological renovation method to oil and PAH5 contaminated soil

Technical field

The present invention relates to the biologic applications technology in the environmental organism science, say the biological renovation method of a kind of mycorhiza cosymbiont specifically oil and PAH5 contaminated soil.

Background technology

In recent years because industrial or agricultural and oil industry rapid development are polluted serious day by day to farmland, river, lake etc.; Particularly along with the developing rapidly and the sharp increase of population of industrial or agricultural, then various poisonous and harmful substances also enter among atmosphere, water body and the soil thereupon; Especially be on the rise with the soil environment pollution, the repairing and treating of contaminated soil is to become the problem that people extremely pay close attention to.The mid-80, a kind of new biotechnology---biological restoration has at first obtained application in some developed country's contaminated soil reparations of Europe, and result of study for many years proves that this is not only safety but also economic method; So-called biological restoration is utilized microorganism and other biology that the dangerous pollutant scene in soil, underground water or the ocean is degraded into carbon dioxide and water exactly or is converted into the engineering system of innocuous substance.From being used for the carrier of biological restoration, it mainly is microorganism, next is a plant, and mycorhiza is as the application study (document 1: the effect of your mycorhiza in the contaminated soil biological restoration earlier of Wang Shuguang, woods, rural ecological environment 2001.17 (1): 56-59) just at the early-stage of biological restoration carrier.Mycorhiza (mycorrhiza) is a kind of general plant symbiosis phenomenon of occurring in nature, be hypha,hyphae and the formed homobium of plant nutrient root system symbiosis in the nature soil, the symbiosis fungi obtains necessary carbohydrate and other nutriment in plant, and plant also obtains required nutrition and moisture etc. there from fungi, thereby form a kind of mutually beneficial, the association of the high unity of exchanging each other's needs; The feature that it had both had general root system of plant and is had has the characteristic that the obligate fungi is had again.Mycorhiza is summarized following points to the beneficial effect of plant: the absorption area that 1) enlarges the host plant root system; 2) increase host plant absorbing to phosphorus and other element; 3) mycorrhizal fungi can produce growth stimulant; 4) improve stress resistance of plant and enhancing plant disease-resistant ability.So as the microorganism that directly connects root system of plant and soil, mycorrhizal fungi can improve and promote growth and development of plant to have more report, discovery mycorrhizal fungis such as Toner EJ can also directly influence the validity and the toxicity of heavy metal on plants, when he with 1,10, when 100ug/kgCd adds in the soil, Mycorrhizal plant absorbing Cd is higher by 10% respectively than non-Mycorrhizal plant, 127% and 131 (document 2:Joner EJ, Leyval C.Uptake of Cd by roots and hyphae of aGlomusmosseae/Trifolium subtemaneum mycorrhiza fromsoil amended with highand low conentration of cdmium (J) .New phytol.1977.135 (2): 353-360) prove thus, the Mycorrhizal plant has very strong absorbability to heavy metal, and the normal growth of plant growth is not affected.Mycorhiza be used for the domestic blank that still belongs to of degraded of oil and PAHs contaminated soil, but the effect of its PAHs in degraded soil has caused more external scholars' attention as the homobium of plant and fungi; Binet philippe (document 3:Binet philippe, Jean-morie p, Corinne L.Biodegradation of a polyaromatic hydrocarbon in therhizospere of mycorrhinal plants (A) .Abonen-Jonnarth U.DanellE.Fransson P.et.al.2nd lntl conf on Mycor (C) .Sweden:Uppo-ala1998.30) etc. the degraded of finding Mycorrhizal rye rhizosphere anthracene is apparently higher than contrast, Dittmann Jens (document 4:Dittmann Jens, Woifgang Heyser.Accumnulation of3-chlorobenzoic acid and degradation of benzoic acid and4-bydroxybenzic acid bymycorrhizal fungi (A) .Abonen-JonnarthUdanell E, Franason P.et al.2nd IntI conf on Mycor (C) .Sweden:Uppe-ala, 1998.52) etc. with two kinds of mycorrhizal fungis degraded benzoic acid tetrahydroxy benzoic acids, the concentration of discovery two kinds of materials in very short time has descended 75% respectively, illustrates that mycorrhizal fungi has the ability that the complicated organic matter of degraded pollutes.

Oil is the complex mixture of chain hydrocarbon, cycloalkane, aromatic hydrocarbon and a small amount of non-hydrocarbon compound; Oil exploitation is smelted and the pollution and the spill incident of transportation, and various petroleum products volatilize, and the discharging of oily waste water, sewage irrigation etc. cause the problem of a series of soil oil pollution.PAHs (polycyclic aromatic hydrocarbon) pollutes most relevant with oil pollution, although the content of PAHs in crude oil (oil) is less, but because various surrounding mediums, the effect of factors such as physical chemistry and biology, make each component in the oil be transformed and biodegradation and photodissociation etc. by physics, chemical factor, the PAHs that is difficult to degrade but continues and extensively is distributed among the environment, and in some heavily contaminated districts, PAHs content can reach hundreds of, thousands of even tens thousand of milligram/kilograms in the soil.Because PAHs has carcinogenicity, teratogenesis, causes prominent property, EPA lists 16 kinds of PAHs in the priority pollutants blacklist in (document 5: Song Yufang, the biological restoration research 2001 of being permitted soil mineral oil in fluid and polycyclic aromatic hydrocarbon (PAHs) under two kinds of plant conditions such as China, 12 (1): 108-112) the PAHs major part belongs to the organic matter of difficult degradation, its degraded difficulty generally increases with the increase of molecular weight and the increase of number of rings, and the environmental problem of therefore repairing and administering this class pollution is imperative.

Summary of the invention

The object of the present invention is to provide a kind ofly, combine, with the mycorhiza cosymbiont of plant mutualism biological renovation method oil and PAHs contaminated soil with other microbe species based on mycorrhizal fungi.

The technical solution used in the present invention is to achieve these goals:

Be built into biological restoration system to contaminated soil with the mycorhiza cosymbiont, described mycorhiza cosymbiont is combined as, corn and Ect., VAM, Ect.+VAM or Ect.+B;

Marigold or soybean and VAM, VAM+B or VAM+N;

White cured and Ect., Ect.+VAM or Ect.+N;

Weeping willow and Ect. or Ect.+B+N.

Described VAM is: Glomus mosseae Glomus mosseae (to call G.M in the following text), G.geospsra ground sacculus mould (to call G.g in the following text), G.cowstrictum and follow sacculus mould (to call G.c in the following text); Ect. be: the sliding rust of Hebeloma mesophaeum burr umbrella (to call E1 in the following text) Lactaria insulsus kakgeusia breast mushroom (to call E2 in the following text) and Cortinarius russus cortina bacterium (to call E3 in the following text); The B bacterium is: Bacillus sp bacillus and Zoogloea sp move the glue Bacillus; The N nitrogen-fixing bacteria are: Azotobacter vinelandii azotobacter vinelandii.

Described bacterial strain to choose process as follows:

(1) from the typical vegetation soil of pollution sources environment of living in, separates bacterial classification;

(2) pure culture screening in chamber has the bacterial strain of degradability by experiment, and is prepared into microbial inoculum;

(3) the domestication experiment of simulation variable concentrations oil, mineral oil polluted soil degrading speed confirms to screen bacterial classification dyes soil to greasy dirt degradation rate with this.

The present invention has following advantage:

1. concentrate the microorganism repairing method of modern bioremediation technology, the phytoremediation method is an one.

Seeing it mainly is microorganism from the carrier that is used for the contaminated soil biological restoration, secondly is plant; And mycorhiza has as the carrier distinctive feature of biological restoration:

1) enlarges the host plant root absorbing area, thereby promote pollutant to absorb and degraded, as fungi and plant symbiosis, formed cosymbiont mycorhiza, the crisscross extension mycelia net that forms in soil has increased the contact area of root system and contaminated soil; Studies show that, mycorhiza is bigger respectively 10 times and 1000 times than root system at the middle absorption area and the absorption length of soil, the formation of mycorhiza simultaneously, can promote the absorption of plant to nutrient, improved micro-ecological environment, thereby improved plant increment and resistance, and then promoted absorption and the degraded of plant pollutant;

2) biological restoration of mycorhiza cosymbiont can overcome the deficiency in microorganism recovery technique, the phytoremediation technology, shows as:

A. can better solve the engineered strain of microorganism in repairing in the field the short problem of its time to live when on probation;

B. the concentration height handled than microorganism repairing method engineered strain of the pollutant levels of this technical finesse, this be because the plant of Mycorrhizal to the tolerance degree of pollutant than edaphon height such as bacterium, actinomyces, saccharomycete;

Though c. phytoremediation is more economical, be more suitable for the removal environmental contaminants and the technology of execute-in-place, but most plants of occurring in nature are difficult to survive barren, in the noxious material contaminated soil and under the condition of arid climate, and the plant of Mycorrhizal can overcome the restriction of this adverse environmental factor.

2. give full play to the regulating and controlling effect of mycorhiza rhizosphere.

Light and the product of the plant of mycorhiza rhizosphere (promptly being subjected to the soil region of root system of plant and mycorrhizal fungi double influence to be called the mycorhiza rhizosphere) Mycorrhizal are transferred to root from blade, pass on the light of root and the variation that product causes the root rhizosphere secreta value volume and range of product simultaneously, flora that can repairing polluted soil around the cast prop root of these secretion and plant.There are a lot of associating bacteriums on the extension mycelia surface of the Pinus syloestris seedling of Mycorrhizal according to observations, and the bacterium border composition of this extension has been supported the bacterium that quantity is huge; In addition, the every gram exotrophic mycorrhizas of trees (fresh weight) can support 10 ⁶With 10 ²Aerobic bacteria and saccharomycete.And the micro organism quantity of mycorhiza rhizosphere is higher 1000 times than surrounding soil, obviously the huge micropopulation of these quantity can play sizable effect to the reparation of contaminated soil, the mycorhiza root exudates also can be used as the common metabolism substrate of degraded simultaneously, thereby promotes the degraded of pollutant.

3. use easy, economical and practical, suitable large-area original position reparation.

Because mycorrhizal fungi and plant symbiosis, its history of life and plant also are more than synchronously, therefore, make the biological renovation method of mycorhiza cosymbiont to be suitable for the original position reparation equally; And add after microbial inoculum then that mycorrhizal fungi breeds with the growth of plant, so that continuous infection host plant and increase its quantity at soil, thus with the microorganism species synergy of its rhizosphere, unite the reparation contaminated soil.

4. the present invention (plant-microorganism is united reparation) in view of mycorhiza to the effect of the health center of plant and the life metabolic activity can reduce the concentration of poisonous and harmful substance in the soil environment, ability with cleaning and recovery contaminated soil, as multidimensional one, synergistic biological restoration method, be in the frontier of biological restoration research, it will be the first-selected factor of contaminated soil recovery technique, be very promising research direction; It possesses biodegradable whole advantage, and secondary pollution is little.

The specific embodiment

1. from the ecological environment of pollution sources, separate bacterial classification.Comforting dirty irrigated area with Liaohe Oil Field and Shen serves as to investigate target, chooses typical pollution sources ecotype, 1) choose the other typical plant (reed, willow, the fluffy grass of alkali and green bristlegrass) of oil production machine soil sample 1-2 kilogram down in the Liaohe Oil Field; 2) comfort dirty irrigated area in Shen and choose in the milpa that intermediate pollution district and semilate rice field, serious pollution district and paddy field change dry land 1～2 kilogram of each soil sampling, adopt the wet screening decantation of Gerdemann and Nicolson, from this contaminated soil, separate the mycorrhizal fungi of gaining the upper hand, Preliminary Identification has Glomus mosseae Glomus mosseae G.qeospsra ground sacculus mould for the VAM bacterium, and it is mould that G.Constrictum follows sacculus; The separation method of Raymand is adopted in the separation of other microbe groups, and the microbiology class of separation is mainly based on bacterium, and bacterium mainly is that secondly bacillus Baciecus sp. is glue Bacillus, pseudomonad, Xanthomonas campestris etc., and fungi and actinomyces are less; And fungi is that cephalo is mould, aspergillus, Fusarium and Penicillium, and actinomyces are then based on streptomycete.

2. the bacterial strain (screening of oil resistant, degraded bacterial classification) with degradability is screened in the chamber pure culture by experiment: select for use the oil of variable concentrations or mineral oil quantitative respectively, add in the culture medium (routine) of cultivating different strain, make for the examination bacterial classification and grow therein, after stopping cultivating, the Residual oil of cultivating in the strain substrate is measured with ultrasonic extraction ultraviolet spectrometry, confirmed to screen the degradation rate of bacterial classification oleaginous base with this; For example: the screening that the Ect. bacterial classification is cultivated through firsts and seconds filters out Hebeloma mesopsra burr Weihe rust umbrella (E1) Lactaria insulsus newborn mushroom (E that distinguishes the flavor of ₂) Cortinarius russus cortina bacterium (E3) three strains have the bacterial classification of certain endurance and degradability to oil; The bacterial strain that is screened is made microbial inoculum according to a conventional method.

3. the domestication experiment of simulation variable concentrations oil, Dormant oils polluted soil degrading speed

1) microorganism and floristic choosing:

Bacterial classification is selected VAM mycorhiza bacterium, ectotrophic mycorrhiza (Ect.), nitrogen-fixing bacteria (Azotobacteria) and bacterium (Bacilus) for use;

The VAM bacterium is: Glomus mosseae Glomus mosseae (to call GM in the following text), and G, geospsra ground sacculus mould (to call G.g in the following text), G.cowstrictum follows sacculus mould (to call G.c in the following text);

Ect. bacterium is: the sliding rust of Hebeloma mesopsra burr umbrella (to call E1 in the following text), and Lactariainsulsus kakgeusia breast mushroom (to call E2 in the following text), Cortinarius russus cortina bacterium (to call E3 in the following text), the three is mixed into Ect.;

Bacterium is: Bacillus sp bacillus, the moving glue Bacillus of Zoogloea sp, and the microbial inoculum of the two mixing is B during application,

Nitrogen-fixing bacteria are: Azotobacter vinandii azotobacter vinelandii (to call N in the following text).

Host plant is chosen the soybean of crops corn gramineous, pulse family, marigold, xylophyta weeping willow and the Bai La of flowers;

2) structure of cosymbiont

The structure of cosymbiont see Table 1 with table 2, all with microbial inoculum form inoculation, inoculum concentration is a 100-150 gram/rice for the bacterial classification of examination ²

After bacterial classification and the host plant various combination collocation symbiosis, observe the syntaxial system of each combination collocation, greasy dirt dyes the biological effect of representative host plant of soil and the degradation rate of rhizosphere contaminated soil detects to adding, confirm united symbiosis system biological restoration efficient, filter out their advantage combination at last;

The degradation rate that all kinds of advantages combination of screening all shows soil pollution oil reaches 53.8～75%, and the biomass of plant is proportionate with degradation rate to oil; Structure with different its advantage combination homobiums of mycorhiza type also is not quite similar; The advantage of the corns of crops and soybean made up be Ect., Ect.+B and VAM; The soybean advantage of marigold of flowers and pulse family is combined as VAM+B, VAM+N; The white cured advantage of xylophyta is combined as Ect., Ect.+N.

Below be based on the cosymbiont of external mycorrhizal fungi biological effect and degradation rate to contaminated soil:

Biological effect and the degradation rate of table 1 mycorhiza cosymbiont in the oil crops contaminated soil

Plant	Bacterial classification	Plant height (cm)	Footpath thick (cm)	Complete stool heavy (g)	Knot appetite (g)	Degradation rate (%)
							Corn	Ect.	124.83	1.717	285.71	135.94	60.6
VAM	138.86	2.124	392.86	166.56	61.8
						Ect.+B		126.57	2.267	612.86	213.26	53.8
Ect.+VAM	127.25	1.944	287.86	129.49	52.0
						CK		118.43	1.807	307.14	130.81	29.2
Soybean	Ect.	63.7	0.953	88.33	62.27								70.7
						VAM	61.73	0.777	67.47	53.47	62.4
	Ect.+B	65.33	1.001	87.45	72.40							75.0
						CK	50.87	0.763	60.00	56.80	44.8

Annotate: oily assay method is that (" environmental monitoring and analysis method " Bureau for Environmental Protection of Ministry of Urban and Rural Construction and Environmental Protection environmental monitoring and analysis method is write group to gravimetric method; the 1986:329_332 of Chinese environmental publishing house) and ultrasonic extraction ultraviolet spectrometry determination method (" environment PetroChina Company Limited. Analysis of contaminant technology " Xie Chongge compile, China Environmental Science Press 1986).

Table 2 mycorhiza cosymbiont dyes biological effect and degradation rate in the soil at the xylophyta greasy dirt

Plant	Bacterial classification	Plant height (cm)	Footpath thick (cm)	Survival rate (%)	Degradation rate (%)
						Cured in vain	Ect	16.58	0.335	100	54.8
Ect+VAM	10.79	O.301	75	48.9
					Ect+N		5.12	0.172	60	70.86
CK	3.00	0.044	20	36.2
					Weeping willow		Ect	104.5	8.76	100	53.3
Ect+N+B	173.0	10.78	50	55.0
						CK	70.0	4.61	50	29.3

Simultaneously, the present invention serves as the main cosymbiont that makes up with the VAM mycorrhizal fungi again, in simulation reparation oil and PAHS contaminated soil, add mineral oil 5000mg/kg, for the VAM bacterium and the bacterium collocation (VAM+B) of examination, the biological effect that makes its symbiosis of host plant marigold flowers is that the growth rate of high growth, leading thread, lateral root number, branch amount, number of flowers and total biomass is followed successively by: 5.5%, 11.9%, 17.8%, 114.3%, 75%, 20.8%; VAM and solid N bacterium collocation, the biological effect that makes the host plant soybean is that stem is heavy, root nodule is heavy, the heavy growth rate of beanpod is followed successively by 26.7%, 11.1%, 27.1%, 4.0%..

Claims (6)

1. biological renovation method to oil and PAH5 contaminated soil is characterized in that: be built into biological restoration system to contaminated soil with the mycorhiza cosymbiont; Described mycorhiza cosymbiont is combined as corn and Ect., VAM, Ect.+VAM or Ect.+B; Marigold or soybean and VAM, VAM+B or VAM+N; White cured and Ect., Ect.+VAM or Ect.+N; Weeping willow and Ect. or Ect.+B+N.

2. according to the described biological renovation method to oil and PAH5 contaminated soil of claim 1, it is characterized in that: described VAM is: Glomus mosseae, sacculus mould and to follow sacculus mould.

3. according to the described biological renovation method to oil and PAH5 contaminated soil of claim 1, it is characterized in that: described Ect. is: the sliding rust of burr umbrella, kakgeusia breast mushroom and cortina bacterium.

4. according to the described biological renovation method to oil and PAH5 contaminated soil of claim 1, it is characterized in that: described B bacterium is: bacillus and moving glue Bacillus.

5. according to the described biological renovation method to oil and PAH5 contaminated soil of claim 1, it is characterized in that: described N nitrogen-fixing bacteria are: azotobacter vinelandii.

6. according to the described biological renovation method of claim 1, it is characterized in that oil and PAH5 contaminated soil: described bacterial strain to choose process as follows:

(1) from the typical vegetation soil of pollution sources environment of living in, separates bacterial classification;

(2) bacterial strain with degradability is screened in the chamber pure culture by experiment;

(3) the domestication experiment of simulation variable concentrations oil, mineral oil polluted soil degrading speed confirms to screen bacterial classification dyes soil to greasy dirt degradation rate with this.