Li Chun-rong¹，Wang Wen-ke¹ ，Cao Yu-qing²  ，Wang Li-juan¹

1 College of Environmental Science and Engineering, Chang’an University, Xi’an 710054, China;

2 College of Environmental and Resource Sciences, Jilin University, Changchun 130023, China)

 

Abstract：Bioremediation of petroleum-contaminated soil is the hot topic in current world. This paper studied corn and sunflower as the samples, did field experiment to study the effect of petroleum-contaminated soil on the physiological process and the reconditioning role of these two plants to the petroleum-contaminated soil. The reinforcing and coordinating role of DX-9 to the bioremediation of corn and sunflower were also investigated. The findings show as follows: under the contamination concentration of 10000mg/kg, the biomass of corn and sunflower reduced 14.5% and 21% respectively, but top/root ratio did not change obviously. Biodegradation rate of petroleum in the soil after 150 days were 42.5%, and 46.4%, increasing 100.5% and 118.9% compared with the controlled field. DX-9 increased the biodegradation rate of these two plants by 71.3% and 64% respectively. The degradation rate of each test area were XJ01＞YJ01 ＞X01 ＞Y01. The half-life of the petroleum hydrocarbons were 68.6d,76.2d,165d, and 177.7d respectively. Conclusion: the effect of the combined bioremediation of corn or sunflower and DX-9 is obvious.

Key words: Petroleum-contaminated Soil, Sunflower, Corn, Microorganism, Bioremediation

 

Introduction

As the rapid development of world economy, the energy human needed gradually increases. Every country accelerates the exploitation and use of oil and gas resources, so more and more oil and gas wells have emerged all over the world. During the processes of petroleum exploration and exploitation, many steps, such as well drilling, silo operating, oil extraction, oil-well blowing, leakages, and so on, will bring contamination to the soil. It is said that each time an oil field operates, the amount of ground petroleum left in the oil field is a few decades to a few hundred kilograms. The contamination area of ground petroleum per well can be up to 0.5-2.1m² and that of the down-to-floor mud is approximate 20m². The petroleum coverage area caused by a blowout is 3000-4000m². The ground petroleum entered into environment in the A’ershan Oilfield in Neimenggu in China is 0.6-2t on average per well each year. The effect area to grassland vegetation is 100×150m²；The oil content in the soil surface layer(0-20cm) in heavy contamination regions such as Daqing Oilfield or Liaohe Oilfield is 30%-50%. The petroleum-contaminated soil would worsen the soil texture，do harm to the soil entironment, reduce the rate of emergence,anti-falling over，anti-insect pest, delay the growing season, cause late ripeness and late ripeness. It may even result in the sterility in contaminated region in tens or hundreds of years ^[3]。Besides, the crude oil and petroleum products dripped or leaked to the earth’s surface and the oily water discharged during the petroleum production and processing will filter directly through aerozone or leach through precipitation to ground water, causing ground water pollution. This problem has already attracted high attention ^[4]。

Chinese tillable field resource is not enough. Recent years, the increasing soil contamination and the decrease of tillable field have been great barriers to the sustainable development of agriculture. As a result，the management and remediation to the contaminated soil becomes an urgent problem. In order to solve this problem, scientists in many countries have developed different measures: physical-chemical progress such as photocatalysis method, mechanical means and chemical washing method; original-position bioremediation method; heterotopic bioremediation method and strengthening bioremediation method.

In this paper, corn and sunflower were chosen as the remediation plants. They have resistance,, survive easily, grow quickly, have a large biomass, own a strong root system and adapt to the environment and climate in the northern region ^[5-7]. Based on the original microorganism in the loess in the north of Shaanxi province and the self-separated highly efficient petroleum-degradative bacteria used as the strengthening bacterium，we were looking for a plant-microorganism recondition technology which should be highly efficient, practical and safe. Our aim is to provide technical support to manage petroleum-contaminated soil in this locality

1.Meterials and Methods

1.1 Materials

1.1.1 Test fields

The Luochuanyuan of the Loess Plateau in the north of the Shaanxi Province, which is located at 500 meters to the west of 210 National Highway 220 kilometers, was chosen. The altitude is 1200 meters. It has a silty clay soil texture and the fertility is moderate. Former crops are vegetables. The soil pH value is 7.2. The total content of nitrogen in the soil is 0.15% and that of phosphorus is 0.13%,Na₂O 1.66%,K₂O 2.29%.

1.1.2 Test plants:corn sunflower

1.1.3 Test crude oil:obtained from Yan’an Oil Refinery

1.1.4 Test bacteria and their sources: test bacteria: Arthrobacter sp.,the highly efficient petroleum-degradative bacteria(DX-9) which was separated from the substrate sludge in the sewage lagoon in Yan’an Oil Refinery.

1.2 Research methods:

1.2.1 The design of test field and the process methods of the soil:

The test field was divided into eight pieces, 4×4m² each., One of them was blank control （K00）without vegetation and one was a contamination control（K01）without vegetation; Three pieces were test areas of corn（Y00，Y01，YJ01）and another three were test areas of sunflower（X00，X01，XJ01）. According to the dry density of soil, 1%(w/w) crude oil was added in“01”test area in the 15cm depth of the cultivable soil layer. Except for that, the highly efficient petroleum-degradative bacteria was added in “J01” test area at the ear stage(or blossom stage) and flowered grain stage (or period of bloom)of corn (or sunflower).The dosage of bacteria is 1L/m²  while the concentration of bacterial suspension is larger than10¹⁰/mL. The test field was irrigated properly before seeding, during growing season. and after the addition of bacteria. Conventional field management was adopted.

1.2.2 Microorganism count: flat plant counting process ^[8]

1.2.3 Microorganism separation: flat plate separation method by dilution ^[8]

1.2.4 The determination of the content of petroleum hydrocarbon:  gravimetric method ^[9]

2 Results and Discussion:

2.1The observed results of the physiological process of test plants

2.1.1The physiological process of corn:

The observation results of the growth of corn was shown in table 1  

Table 1    Effect of petroleum contaminated soil on growth of corn

Test region	Rate of Emergence （%）	Stalk Diameter （cm）	One Hundred-grain Weight（g）	Grain Weight of per bar （g）	Plant Height （㎝）	Root Weight （g）	Biomass per Plant（g）
Y00	100	3.5	38.8	214.5	260.2	31.7	680.4
Y01	100	3.0	38.3	201.2	228.5	26.8	581.9
YJ01	100	3.2	38.7	208.1	247.2	29.8	639.6

※The composition factor of biomass: stalk weight + leaves weight + root weight。

It is obvious that when the concentration of petroleum contamination was up to 10000mg/kg，the growth of corn was restrained to some extent. At this time, the restrain rate of the root system was 15.5% and the organism biomass was reduced by 14.5%.

2.1.2 The physiological process of sunflower:

The plant height, sunflower-plate diameter, number of full seeds per plate, number of empty seed, one hundred-grain weight, root weight and biomass per plant in each test area were determined before and after harvest. The results is shown in Table 2.

Table 2   Effect of petroleum contaminated soil on growth of sunflower

Test Region	Rate of Emergence （％）	Plant Height（㎝）	Sunflower-plate diameter （㎝）	Number of full seeds	Number of empty seeds	One Hundred-grain Weight（g）	Biomass per Plant (g）	Top/Root Ratio
X00	100	321.1	20.6	712.3	143.2	11.08	265.8	2.86
X01	100	298.0	18.4	541.3	256.4	9.24	210.9	2.85
XJ01	100	305.2	19.2	602.7	202.2	9.38	237.9	2.86

※The composition factor of biomass: stalk weight + sunflower plate weight +seed weight（The leaves weight was ignored cause it takes very little ratio when the sunflower is mature.）

It is shown in table 2 that the sunflower plate diameter in contaminated soil(X01) dwindles 10.8% compared with that in uncontaminated soil. The rate of empty seeds increased 2.4 times，while the one hundred-grain weight reduced 16.7%. The reason may be as follows: After the soil contamination, the sunflowers have screened the inorganic and some organic nourishing substance due to the hydrophobicity of the petroleum hydrocarbon which affects the nutriment transportation, inversion and distribution functions of sunflowers. The results of that appear in the restraint of the protraction of the sunflower-plate cells, the cut of the transportation of light and products to sunflower seeds, the reduction of dry matter accumulation, the weakening in fertilization ability of tubular flower and the increasing of empty-grain rate. The sunflower’s plant height and biomass per plant reduced 7.0% and 21% respectively. There was no obvious difference in the top/root ratio between the contaminated area and uncontaminated control area(X00，Y00). All the results mentioned above means that under that contamination concentration, the growth of the root system of corn and sunflower were not restricted specially compared with the growth of parts over earth.

2.2 The distribution of bacteria flora in test region and the result of the determination of petroleum content:

The petroleum contents in the soil samples were determined in the very day, the 35^th, 80^th, 120^th and the 150^th day after seeding; In the 140^th day, the distribution of bacteria in soil in every test field was analyzed. The results are shown in table 3 and graph 1,2

          

Table 3 Distribution of bacteria from soil in 140^th day

From table3, it is known that the total numbers of microorganism in the areaY00, X00 are 150 times and 240 times as many as those in the control area K00 respectively. The numbers in the area Y01,X01 are 22.7 times and 18 times more than those in area K01. It means that the rhizosphere effect of corn and sunflower have improved the growth and reproduction of microorganism in soil；After the contamination of soil, the Oil-philic bacteria in soil increased in evidence，which would become more obvious under the rhizosphere effect. The reason is that after the soil contamination, the microorganism in the polluted spot can metabolize pollutant after being acclimated, the result of which is to make the pollutant which can not be transformed or has a very slow transformation rate when overflow drain occurred be metabolized and degradated. The adaptability of microorganism increases the quantities of Oil-philic bacteria ^[10]；The addition of DX-9 makes the total amount of Oil-philic bacteria increased by 1/1000 to 4/10000. It means that DX-9 adapts the soil environment basically. It has competitive power in growth and reproduction when coexisting with the original microorganism. As it is indicated in table 1From graph1and2,Corn and sunflower have obvious remediation ability to the petroleum-contaminated soil. The degeneration rate in the 150^th day reached 42.5% and 46.4% respectively, which is more than two times higher than the control area (K01). The highly efficient petroleum-degradative bacteria ( DX-9) enhances the plant biomass and improves other biocharacters, It enhanced the biomass by 10.1%, plant height by 8.2% and root weight by 11.2% in YJ01 area compared with Y01. The biomass and sunflower-plate diameter increased by 12.8% and 4.5% in XJ01 area compared with X01, while the rate of empty seeds decreased by 20.6%；The speed of biodegradation is accelerated in evidence. In the 150^th day, the petroleum degradation rates in YJ01 and XJ01 test areas increased 71.3% and 64.0% respectively compared with the test areas in which the bacteria was not addedThe degradation curves in area Y01, YJ01, X01, XJ01 were analyzed through the linear regression, The results are shown in graph 3 to graph 6.It can be seen that the petroleum degradation curve accords with the equation of kinetics of first-order reaction:

-dc/dt=kc      ……             (1)

From（1）we could get:   lnC=lnC₀ –kt    (2)

C is the residual concentration of petroleum at any time（mg/L）. C₀ is the original concentration and k is the velocity constant（The constant was divided by the ground substance，d^-1）. The horizontal axis is time and the vertical axis is lnC. Test points were analyzed through the linear regression. The intercept of the straight line is lnC₀ and the slope is k. Using the values of k, the degradation rates under different test conditions could be obtained^[11] . The degradation rates in four test areas are : XJ01＞ YJ01＞ X01＞Y01.

From equation (2), the half-time formula of petroleum degradation is obtained:

t_1/2=ln2/k       ……               （3）

The half-time of the degradation of petroleum in area Y01, YJ01, X01, XJ01 is 177.7d, 76.1d, 165d, and 68.6d respectively. The half-time in the area YJ01 is about 2.3 times shorter than that in area Y01 and the half time in area XJ01 is approximate 2.4 times shorter than area X01. It means that the method using sunflower and adding highly efficient petroleum-degradative bacteria(DX-9) has a fast remediation rate and terrific effects.

          

           

 

 

3 Conclusion:

(1) The petroleum-contaminated soil restrains the growth of corn and sunflower. When the concentration of petroleum contaminated soil was up to 10000mg/kg， the organism biomass was reduced by 14.5% and 21% respectively. However, under that concentration, the petroleum didn’t have special restraints to the roots and the part over earth.

(2)The corn and sunflower have obvious remediation function to the   petroleum-contaminated soil. The degeneration rate in the 150^th day reached 42.5% and 46.4% respectively increasing 100.5% and 118.9% compared with the control areas

(3)The reinforcing and coordinating functions of DX-9 to plant reconditioning of petroleum-contaminated soil were also obvious.  The degradation rates in areaYJ01 in the 150^th day is 72.8% ,increased by 71.3% compared with control area Y01 and the rate in area XJ01 is 76.1% ,increased by 64% compared with area X01 .

(4) Under the conditions of field test, the degradation curve accords with the equation of kinetics of first-order reaction. The degradation rates are: XJ01＞ YJ01 ＞X01 ＞Y01 and the half-life of the petroleum hydrocarbons were 68.6d,76.2d,165d, and 177.7d respectively. The effect of the technology of DX-9 corn(or sunflower) reconditioning on  petroleum-contaminated soil is good.

(5) The residuum of petroleum hydrocarbon in plant needs a further study.

 

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