Long-Term Phytoremediation Process of Diesel Oil-Contaminated Soil

Song Xueying1,a, Li Xinxin1,b, Wang Yan1,c and Hu Xiaojun1,d 1Key Laboratory of Regional Environment and Eco-Remediation (Ministry of Education), Shenyang

University, Shenyang 110044, P. R. China

asongxueying2046@163.com, blixinxin1981@126.com, cwangyan_0616@126.com, dhuxiaojun6226 @163.com(corresponding author)

Keywords: Diesel Oil, phytoremediation, soil, organic manure

Abstract. Soils contaminated with diesel oil were remediated with alfalfa (Medicago sativa) by

outdoor pot experiment over a 5-growth season treatment, with pollutant levels, special microbial

inoculators, fungi inoculators, and organic manure as control factors. The dynamics of residual

concentrations of mineral oil and PAHs in soil of different phytoremediation treatments during the 5

seasons were determined. Results showed that significant reduction of contaminant concentration

was achieved. At the end of the fifth growth season, initial concentrations of mineral oil were reduced

by 96.5% to 98.8% in the phytoremediat treatments. Among the four factors, bacterial and fungi

inoculators showed no significant effect on the contaminant removal in the process of long-term

bioremediation. Effect of organic fertilizer amendments differed depending on the diesel

concentration. In the highly contaminated treatments (15000 and 30000 mg/kg dry weight)

remarkable stimulation was detected with the increase of fertilizer amendment; however, the trend

was just reversed in the lightly contaminated treatments (5000 mg/kg dry weight) with the increase of

manure amendment.

Intorduction

Soil frequently serves as the site of petroleum spills. Petroleum contaminants impair hydrologic

conditions and physical properties of the soil, drastically reduce the contents of movable nitrogen and

phosphorus compounds, and have a toxic effect on plants, causing the destruction of chlorophylls and

carotenoids[1]. Diesel oil is a complex mixture of various petroleum hydrocarbons consisting of

everything from volatile, low molecular weight alkanes with potential phytotoxicity, to naphthalene

which may interfere with normal plant development. Of the middle-distillate fuel oils used in

terrestrial situations, diesel oil has the highest content of PAHs and aromatics[2], which makes it

increasingly more difficult to remediate.

Bioremediation is emerging as a most cost-effective procedure for hydrocarbon-contaminated

soil, especially when the contaminants are medium distillate fuel[3,4]

. The nutrient addition has been

proved to be a prime candidate as a bioremediation tool to increase the hydrocarbon degradation after

an oil spill [3], as input of large amount of carbon sources (i.e., contaminants) tends to result in rapid

depletion of the available pools of major inorganic nutrients, such as N and P. In addition, the

inoculation of special degradative microorganisms is commonly regarded as a factor accelerating the

degrading process. However, it is still a conflicting problem. Objectors conceive that indigenous

microorganisms with the ability to degrade crude oil are ubiquitously distributed in soil and rapid

growth of their biomass can occur immediately after oil contamination, and the introduced

microorganisms only have marginal effects on oil biodegradation rates.

Bioremediation of petroleum-contaminated soil were reported, however, most researches have

been carried out in short time, seldom were conducted on the long-term bioremediation. This

investigation is a follow-up of a previous laboratorial study that successfully demonstrated the

short-term stimulating action of soil microorganism inoculation in the bioremediation of diesel oil

contamination by flask experiment[5]. The main goal of this study was to discuss the effect of

introduced microbial inoculators (bacteria and fungi), and organic amendment on the long-term

bioremediation of diesel-contaminated soil.

Advanced Materials Research Vol. 414 (2012) pp 280-283Online available since 2011/Dec/06 at www.scientific.net© (2012) Trans Tech Publications, Switzerlanddoi:10.4028/www.scientific.net/AMR.414.280

All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of TTP,www.ttp.net. (ID: 128.59.222.12, Columbia University Library, New York, USA-11/11/14,17:13:53)

Materials and Methods

Soil, manure, diesel oil and microbial strains. The soil was collected from the top 0–20cm of the

wilderness soil at the Ecological Station of Chinese Academy of Sciences, Shenyang, China.

Properties of soil and organic nutrients were shown in Table 1. Concentrations of total Cu, Zn, Pb, Cd

(mg/kg dry weight) were 32.9, 28.1, 11.1, and 0.17, and the TPH was 127.5mg/kg. Test diesel oil was

with density of 821g/L. Five bacteria strains (Acetobacter sp., Alcaligenes sp., Micrococcus sp.,

Arthrobacter sp., and Bacillus sp.) and five fungi strains (Cephalosporium. sp. I, Cephalosporium sp.

II, Aspergillus sp. I, Aspergillus sp. II and Fusarium sp.) isolated from a long-term

petroleum-contaminated soil were utilized as the diesel degrading microbes.

Table 1. Some physical and chemical properties of the test soil and the manure

Sample pH TOC (%) TN (%) TP (%) TK (%)

Soil 6.4(6.22) 1.99 0.093 0.04 0.18

Chicken manure 6.53 14.03 0.54 0.55 0.58

Experimental design. The effects of contaminant intensity, bacteria inoculators, fungi inoculators,

and chicken manure amendment on diesel-bioremediation were carried out by L9 (34) orthogonal

design with repetition as exhibited in Table 2. Columned pots were applied, by 20cm

(diameter)×30cm (high). Each pot was filled with 3kg soil (dry weight), passing a 2mm sieve.

Table 2. L9 (34) orthogonal experiment design

Code Diesel Con. (mg/kg) Organic fertilizer (%) Bacteria (%) Fungi (%)

1 5 000 0 2 5

2 5 000 2 0 2

3 5 000 5 5 0

4 15 000 0 0 0

5 15 000 2 5 5

6 15 000 5 2 2

7 30 000 0 5 2

8 30 000 2 2 0

9 30 000 5 0 5

Results and discussion

Total degradation of mineral oil. The dynamic of the residual mineral oil concentration and total

degradation ratio was presented in table 3. A significant decrease in the mineral oil levels was

achieved in all samples after 5 growth seasons’ bioremediation. At the end of the fifth growth season,

the initial contamination levels were reduced by (96.5±0.1)% to (98.8±0.1)%. Most removal of oil in

each treatment occurred during the first two growth seasons, and at the end of the second season the

removal ratios of oil in all treatments were above 90%. Multiple comparisons of residual oil

concentration by LSD test in different treatments in each growth season were also listed in Table 3.

The final results showed that the residual oil concentration in the 15000 mg/kg and 30000 mg/kg

treatments, in general, were significantly higher than that in 5000 mg/kg treatments. Significant

correlation was detected between the final diesel oil concentration and the initially added

concentration with Pearson Correlation coefficient r=0.72 (p<0.05). This was mainly attributed to the

complex constitutes of diesel oil. N-alkanes, the major constitutes of diesel oil, was ready to be

extensively biodegraded by the multiple populations of soil microorganisms, while the high

molecular part was utilized difficultly by microbes and easily adsorbed to the soil particles (organic or

inorganic colloids), therefore, the oil residuals in highly contaminated treatments were superior to the

lower ones.

Advanced Materials Research Vol. 414 281

Effect of microbial inoculation on oil degradation. No significant correlation was detected among

the oil degradation ratio, and the microbial amendment (0%, 2%, and 5%), either bacteria or fungi

(p>0.05). A similar investigation by Cho et al. on bioremediation of oil-contaminated soil of Kuwait

with soil amendment materials confirmed the above findings[6]. Our previous studies of diesel oil

mineralization by microbial inoculators indicated that inoculation with the introduced microbial

inoculators enhanced biodegradation only in short term[5]. To be sure, microbes are primary agents

for the degradation of organic contaminants in soil, and increasing microbial density can accelerate

degradation of the contaminants. However, as was known, individual microorganisms are capable of

degrading only a limited number of crude oil components; thus, more extensive degradation of oil

depends on the presence of metabolically diverse microbial communities[7]. On the other hand, in fact,

large quantities of indigenous microbial strains capable of metabolizing petroleum components exist

in the soil ecosystem ubiquitously. Their rapid adaptability to the contaminated environment and their

strong competition capability make them able to eliminate gradually the rate predominance of

petroleum decontamination in the early days by the microbial inoculation. Therefore, the function of

introduced microbial inoculators was limited and short-termed.

Table 3. Dynamics of the residual oil concentration (mg/kg) and degradation ratio (%) in

bioremediation

NO. First season Second season Third season Fourth season Fifth season

1 584.8±10.5a

(88.3±0.2)

370.7±13.6a

(92.6±0.3)

240.4±10.8a

(95.2±0.2)

123.3±8.7a

(97.5±0.2)

102.9±10.6a

(98.1±0.2)

2 645.1±21.3a

(87.1±0.4)

475.3±32.4a

(90.5±0.6)

311.7±15.3a

(93.8±0.3)

102.6±6.3a

(97.9±0.1)

64.7±4.1a

(98.8±0.1)

3 690.4±15.8a

(86.2±0.3)

485.6±8.9a

(90.3±0.2)

297.3±12.4a

(94.1±0.2)

137.4±12.8a

(97.3±0.3)

75.6±13.5a

(98.3±0.3)

4 6181.3±84.2e

(58.8±0.6)

1109.8±35.9c

(92.6±0.2)

680.6±25.3c

(95.5±0.2)

549.6±19.6c

(96.3±0.1)

528.9±22.1d

(96.5±0.1)

5 2849.6±61.5c

(81.0±0.4)

641.1±44.2b

(95.7±0.3)

504.9±36.9b

(96.6±0.2)

458.2±47.8b

(97.0±0.3)

453.1±35.1cd

(97.0±0.2)

6 2338.9±54.3b

(84.4±0.4)

615.3±20.7b

(95.9±0.1)

519.7±47.1bc

(96.5±0.3)

515.3±24.5bc

(96.6±0.2)

484.7±8.6d

(96.8±0.1)

7 9360.7±124.1f

(68.8±0.4)

2170.5±36.5f

(92.8±0.1)

1119.3±62.3d

(96.3±0.2)

763.8±33.6d

(97.5±0.1)

693.7±54.1e

(97.7±0.2)

8 9567.7±187.2f

(68.1±0.6)

2065.6±62.7e

(93.1±0.2)

995.6±29.5c

(96.7±0.1)

584.5±28.4c

(98.1±0.1)

391.7±13.0bc

(98.7±0.0)

9 4290.5±88.3d

(85.7±0.3)

1268.8±41.5d

(95.8±0.1)

844.9±33.8c

(97.2±0.1)

551.6±36.5c

(98.2±0.1)

362.6±26.2b

(98.8±0.1)

*Different letters (a, b… f) in a row indicated statistically significant differences (LSD- test) between

treatments; the same letters indicated that values were not significantly different. The significant

difference was at the 0.05 level.

Effect of manure amendment on oil degradation. After one-growth season, among the three diesel

concentration levels, the degradation was markedly stimulated by the increase of organic fertilizer

addition (p<0.05) in the initial treatment of 30000mg/kg. For instance, the oil degradation rate with

5% fertilizer amendment was 85.7%, which was superior to the natural fertilization treatment 68.8%,

and 2% fertilizer amendment 68.1% (see Table 3). Similarly, in the mediate contaminated soil, the

15000 mg/kg treatment, the oil degradation ratio with the increase of fertilizer amendment of natural

fertilization, 2% and 5% amendment, ranged from 58.8% to 81.0% and 84.4%, respectively. On the

contrary, in the lightly contaminated soil with 5000 mg/kg, there was obvious decreasing tendency

instead of stimulation to oil degradation with the increased fertilization amendments, the degradation

ratios were 88.3%, 87.1%, and 86.2% respectively. Significant positive correlations (r=0.87) were

found between the mineral oil degradation ratio and the organic fertilizer amendment (p<0.05) at the

282 Contaminated Sites Remediation

heavily contaminated treatments (15000 and 30000 mg/kg). In fact, addition of organic amendments

can facilitate degradation of organic contaminants, especially for the heavily contaminated soils,

because they play a role in supplementing abundant nutrients and carbon source in contaminated

soil[8]. After an oil spill, large amount of carbon sources were input, which resulted in rapid depletion

of the available pools of major inorganic nutrients, such as N and P; and the more heavier the

contamination was, the more the content of N and P was ready to become a limiting factor. The

tendency of the effect of organic fertilizer amendment on the oil degradation was the same in the

second growth season as that in the first growth season; however, the tendency became more and

more ambiguous in the last three growth seasons, as shown in Table 3. This can be mainly attributed

to the depletion of nutrients available to oil degradation. As shown in Table 5, the concentrations of

quick-N were not significantly different in different treatments, which became the most essential

limited factor for the bioremediation of petroleum-contaminated soil[8].

Conclusions

Microbial inoculators showed no significant effect on the contaminant removal in the process of

long-term bioremediation. Organic fertilizer amendments remarkably stimulated the degradation of

diesel oil in the relatively heavily contaminated treatments (15000 and 30000 mg/kg), however,

showed no action or negative action in the lightly contaminated treatments (5000 mg/kg) with the

manure amendment.

Acknowledgements

We gratefully acknowledge for the grant of National Natural Science Foundation of China

(No.41101289; 20807029) and the National Science and Technology Supporting Project (No.

2011BAJ06B02).

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