CN101890426A

CN101890426A - Method for restoring petroleum-polluted saline alkali soil by ryegrass and high efficiency microbes

Info

Publication number: CN101890426A
Application number: CN 201010229416
Authority: CN
Inventors: 唐景春; 董健; 牛晓伟; 王敏; 王如刚; 周启星
Original assignee: Nankai University
Current assignee: Bo Chuan environmental rehabilitation (Beijing) Co., Ltd.
Priority date: 2010-07-19
Filing date: 2010-07-19
Publication date: 2010-11-24
Anticipated expiration: 2030-07-19
Also published as: CN101890426B

Abstract

The invention relates to a method for restoring petroleum-polluted saline alkali soil by ryegrass and high efficiency microbes. The invention belongs to the technical field of environment restoration, and relates to a method for restoring soil by microbe microbial inoculums and plants. The method comprises the following steps: pretreating soil, preparing different microbial inoculums, planting ryegrass, and managing according to the normal requirements for plant growth after sprouting. Microbes and microbial inoculums can cooperate to perform the synergistic action; the addition of the microbe microbial inoculums can promote the plant growth, improve the structure of soil and reduce the pH value of soil, so that environmental conditions which are beneficial to pollutant degradation are formed in the soil. Ryegrass and other garden plants have the functions of promoting petroleum hydrocarbons to degrade and greening the landscape, and can be used as a feed on the premise that the pollutants of the reaped ryegrass do not exceed the standard, thereby having certain economic benefit. The invention enhances the degrading effect of petroleum hydrocarbons, and the degrading rate of petroleum hydrocarbons after five-month growth, can reach 57%. The technique can reduce plowing required by the restoration which only uses microbes and supplement the measures for microbial inoculums, and has favorable effect of removing petroleum hydrocarbons in soil for a long time.

Description

The method of rye grass-high-effective microorganism associating restoring petroleum-polluted saline alkali soil

Technical field

The invention belongs to the environment remediation technical field, relate to the method for microbial bacterial agent and the composite rehabilitating soil of plant.

Background technology

The oil pollution problem of field area soil becomes the obstacle of oil field cleaner production and sustainable development at China's ubiquity.All being in badly area of coastal saline-alkali area and ecological environment in the many oil fields of China, is the subject matter that these areas face to the reparation of soil environment and the improvement of ecological environment.Therefore adopt the biotechnology both can remedying oil-polluted soils, can also effectively improve the field area ecological environment.The interpolation of high-effective microorganism microbial inoculum and phytoremediation are bioremediation technologies commonly used at present in the oil-polluted soils repair process.But because the special nature of salt affected soil, the use of microbial inoculum and phytoremediation effect can be affected in the biological restoration process, and particularly the growth of plant is suppressed in the saline-alkali environment.Utilize the combined remediation technology of microorganism-plant, can effectively improve negative effect saline and alkaline in the repair process.Many microorganisms can form symbiosis with plant, promote the growth of root system of plant.Therefore developing salt affected soil plant-microorganism thing combined remediation technology repairs significant to China's saline and alkaline district oil-polluted soils.

Summary of the invention

The method that the purpose of this invention is to provide a kind of rye grass-high-effective microorganism associating restoring petroleum-polluted saline alkali soil.

The method of rye grass of the present invention-high-effective microorganism associating restoring petroleum-polluted saline alkali soil comprises the steps:

1) in the experimentation soil breaking is sieved, in flowerpot, puts into oil-polluted soils, press salt with running water flushing, treat the soil natural drying after;

2) bacterial classification that will be deposited in the slant medium is transferred in the 100mL seed culture medium, and seed culture medium is formed: peptone 1%, and beef extract 0.5%, NaCl0.5%, pH are that 7.0,121 ℃ of steam sterilizing 20min are standby; Inoculum concentration by 1% inserts in the fermentation medium, and fermentation medium is formed: glucose 1%, and dusty yeast 0.5%, bean cake powder 1%, MgSO47H2O 0.03%, and NaCl 0.5%, and pH is 7.0; At airtight sterilization tank mesohigh steam sterilizing, the cooling back mixes by peat composed of rotten mosses wheat bran weight ratio at 9: 1 the peat composed of rotten mosses and wheat bran; With the gained zymotic fluid and peat composed of rotten mosses bran mixture be 1: 2 mixing by V: W, natural air drying, pack is sealed, and makes microbial inoculum;

Employed bacterial classification is: (1) bacillus subtilis (Bacillus subtillus SWH-1), or (2) Sphingobacterium multivorum (Sphingobacterium multivorum SWH-2), or (3) radioresistance acinetobacter calcoaceticus (Acinetobacter radioresistens R-1), or (4) rhodococcus erythropolis (Rhodococcus erythropolis R-5), or the Mixed Microbes of (5) above four kinds of bacterium, part by weight is 1: 1: 1: 1 or the Mixed Microbes of (6) bacillus subtilis (Bacillus subtilis AS1.504) and fluorescent pseudomonas (Pseudomonas fluorescens AS1.219), and part by weight is 1: 1; Six strain bacterial classifications are all purchased the DSMZ in Institute of Microorganism, Academia Sinica.

3) will add in the soil of microbial inoculum and mixing and evenly be sprinkled into ryegrass seed, application rate is a 1-1.5kg/ mu, top earthing 0.5-1.0cm, and evenly watering makes the soil conservation moisture state, treats the normal requirement management of plant germination and growth.

Technical characterstic: as rehabilitation plant, its characteristics are the fibrous root prosperity, grow thickly to choose rye grass (Lolium perenne L.), and it is a lot of to tiller, and is suitable for the reparation of topsoil oil pollution.Rye grass is moisture-proof, salt tolerant alkali relatively, containing well-grown on the soil of villaumite below 0.25%, but optimum soil pH is 6～7, is that growth in 5～8 o'clock is also better at soil pH.Rye grass is extensive to management expectancy, does not need specialty to safeguard, adapt to different environmental conditions, growth period is long, can and survive the winter in the growth in winter in the North China, thereby have repairing effect preferably.

Beneficial effect

1. the associating of microorganism and microbial inoculum can be played synergy, and the adding of microbial bacterial agent can promote the growth of plant, improves the structure of soil, reduces pH in soil (dropping to 7.8 from 8.2), makes soil form the environmental condition that is beneficial to contaminant degradation.

2. ornamental plant such as rye grass also has the effect of landscape planting except that having the effect that promotes petroleum hydrocarbon degradation, and the plantation by rye grass can beautify the oil field environment, even in the winter time also can normal growth.Simultaneously, the rye grass of harvesting also can be used as feed under the prerequisite that pollutant does not exceed standard, have certain economic benefits.

3. utilize the plant-microorganism combined remediation technology, can improve the degradation effect of petroleum hydrocarbon greatly, the growth time petroleum hydrocarbon degradation rate through five months can reach 57% (methylene chloride reflux is extracted, gravimetric method).Utilize this technology can reduce simple microorganism and repair needed turning over, replenish the measure of microbial inoculum, can in the long time, make the petroleum hydrocarbon in the soil have removal effect preferably.

Description of drawings

Fig. 1. the variation of rye grass biomass in the repair process of embodiment 1.0, rye grass; 1, rye grass+Bacillus subtilis SWH-1; 2, rye grass+Sphingobacterium multivorum SWH-2; 3, rye grass+Acinetobacter radioresistens R-1; 4, rye grass+Rhodococcus erythropolis R-5; 5, rye grass+Mixed Microbes: Bacillus subtilis SWH-1, Sphingobacteriummultivorum SWH2, Acinetobacter radioresistens R-1, Rhodococcus erythropolis R-5; 6, rye grass+Mixed Microbes: BacillussubtilisAS1.504, Pseudomonasfluorescens AS1.219. error line is represented standard deviation.

Fig. 2. the rye grass of embodiment 1 is united the comparison .1 of repairing effects, rye grass+Bacillus subtilis SWH-1 with different microbial inoculums; 2, rye grass+Sphingobacterium multivorum SWH-2; 3, rye grass+Acinetobacter radioresistens R-1; 4, rye grass+Rhodococcus erythropolis R-5; 5, rye grass+Mixed Microbes: Bacillus subtilis SWH-1, Sphingobacteriummultivorum SWH2, Acinetobacter radioresistens R-1, Rhodococcus erythropolis R-5; 6, rye grass+Mixed Microbes: BacillussubtilisAS1.504, the PseudomonasfluorescensAS1.219. error line is represented standard deviation.

Fig. 3. the repair process DGGE analysis result of embodiment 1.1 repairs preceding soil; 2-7 adds different microbial inoculum soil, and the 8-13 kind is planted rye grass and added different microbial inoculums, 14, rye grass; 15, blank.

The specific embodiment

Embodiment 1: the pot experiment of carrying out at Nankai University biological test center

The contaminated soil that picks up from Shengli Oil Field is ground after air-dry, cross the 2mm sieve.The total salt of sampling and measuring soil, physical and chemical index such as total petroleum hydrocarbons content.The total petroleum hydrocarbons content 6.19% of soil after measured, pH7.9, organic 0.58%; Total N 2.75g kg ^-1Total P, 0.11gkg ^-1The measurement result of different petroleum hydrocarbon components is as follows: saturated hydrocarbons 50.5%, aromatic hydrocarbon 20.8%, polarity thing 27.7%.

Under the different soil ratio desalinization of soil by flooding or leaching conditions to the influence of soil salt content and germination, result according to table 1, desalinization of soil by flooding or leaching soil ratio should be greater than 0.75, and this moment, soil salt content can be reduced to 0.607 by initial 4.276%, and rye grass can be at plantation normal germinating growth after 6 days.

Total salt content and rye grass germination under the different soil ratio conditions of table 1 after the desalinization of soil by flooding or leaching

Select for use six kinds of different microbial inoculums composed as follows: (1) bacillus subtilis, (2) Sphingobacterium multivorum, (3) radioresistance acinetobacter calcoaceticus, (4) rhodococcus erythropolis, the Mixed Microbes of (5) above each bacterium, the Mixed Microbes of (6) bacillus subtilis and fluorescent pseudomonas.Each bacterium is cultured to the logarithmic growth after date separately or be mixed to join and make microbial inoculum in the peat composed of rotten mosses at its optimal culture condition, bacterium liquid and peat composed of rotten mosses ratio are: 1: 2～1: 3 (w/w). each bacterium is cultured to the logarithmic growth after date separately or be mixed to join and make microbial inoculum in the peat composed of rotten mosses at its optimal culture condition, bacterium liquid and peat composed of rotten mosses ratio are: 1: 2～1: 3 (w/w). after adding the microbial inoculum of different proportion, soil and microbial inoculum are fully mixed.

1000g soil is put into 12cm * 12cm flowerpot, microbial inoculum fully mixes back sowing rye grass seeds with soil, spraying running water behind the earthing 0.5-1cm makes soil fully moistening but do not have excessive moisture to flow out from the flowerpot bottom, the maintenance ground moistening regularly waters, the biomass of rye grass is measured in harvesting after 162 days, measures the petroleum hydrocarbon content in the soil and measure soil microbial community with the DGGE technology to change.

The biomass that results obtain different disposal the results are shown in Figure 1, and the 6th kind of processed group promptly added Mixed Microbes and the rye grass of 4 kinds of bacterium and united when repairing rye grass biomass maximum, and the biomass minimum of single cropping rye grass as can be seen from Figure.Find out the biomass that adds the rye grass after the microbial inoculum among the figure all than the biomass height of single cropping rye grass, i.e. the interpolation of microbial inoculum has promoted the growth of rye grass plant.

Fig. 2 has provided six kinds of microbial inoculums and rye grass is united the degradation effect of reparation to oil-polluted soils.Six kinds of different microbial inoculums and rye grass are united reparation the soil petroleum hydrocarbon are all had repairing effect preferably, and the petroleum hydrocarbon degradation rate is 53.03%-58.73% after 162 days.Wherein the associating repairing effect of four kinds of Mixed Microbes and rye grass is best, and degradation rate reaches 58.73%, and is all higher than the associating repairing effect of single bacterium and rye grass, and exceeds 18% than blank.Uniting in the reparation of four kinds of single bacterium and rye grass, the order of degradation effect is rhodococcus erythropolis＞bacillus subtilis＞Sphingobacterium multivorum＞radioresistance acinetobacter calcoaceticus, the repairing effect difference of these four kinds of microbial inoculums and rye grass is little, and maximum difference only is 1.9%.Four kinds of single bacterium and rye grass unite that all the repairing effect than Mixed Microbes 2 (bacillus subtilis and Pseudomonas fluorescens) is good in the reparation.The single reparation height of degradation rate than rye grass repaired in uniting of microbial inoculum and rye grass, and the single reparation oil degradation of rye grass rate is 48.36%.

Among Fig. 3 in the different disposal soil DGGE band more all have than untreated samples and increase.G1-G7 is a general band that processing all exists, the band of S1-S11 for only occurring in individual samples.Result among the figure can illustrate the Combined Treatment by plant and microorganism, can increase the diversity of edaphon.

Claims

1. the method for rye grass-high-effective microorganism associating restoring petroleum-polluted saline alkali soil comprises the steps:

2) bacterial classification that will be deposited in the slant medium is transferred in the 100mL seed culture medium, and seed culture medium is formed: peptone 1%, and beef extract 0.5%, NaCl0.5%, pH are that 7.0,121 ℃ of steam sterilizing 20min are standby; Inoculum concentration by 1% inserts in the fermentation medium, and fermentation medium is formed: glucose 1%, and dusty yeast 0.5%, bean cake powder 1%, MgSO47H2O 0.03%, and NaCl0.5%, pH are 7.0; At airtight sterilization tank mesohigh steam sterilizing, the cooling back mixes by peat composed of rotten mosses wheat bran weight ratio at 9: 1 the peat composed of rotten mosses and wheat bran; With the gained zymotic fluid and peat composed of rotten mosses bran mixture be 1: 2 mixing by V: W, natural air drying, pack is sealed, and makes microbial inoculum;

Employed bacterial classification is: (1) bacillus subtilis (Bacillussubtillus SWH-1), or (2) Sphingobacterium multivorum (Sphingobacterium multivorum SWH-2), or (3) radioresistance acinetobacter calcoaceticus (Acinetobacter radioresistens R-1), or (4) rhodococcus erythropolis (RhodococcuserythropolisR-5), or the Mixed Microbes of (5) above four kinds of bacterium, part by weight is 1: 1: 1: 1 or the Mixed Microbes of (6) bacillus subtilis (Bacillus subtilis AS1.504) and fluorescent pseudomonas (Pseudomonas fluorescens AS1.219), and part by weight is 1: 1;