Molecular aspects of rhizodegradation and bioremediation of hydrocarbon-contaminated soil

Shahsavari, E 2013, Molecular aspects of rhizodegradation and bioremediation of hydrocarbon-contaminated soil, Doctor of Philosophy (PhD), Applied Sciences, RMIT University.

Document type: Thesis
Collection: Theses

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Title Molecular aspects of rhizodegradation and bioremediation of hydrocarbon-contaminated soil
Author(s) Shahsavari, E
Year 2013
Abstract Petrogenic hydrocarbons represent ubiquitous pollutants which threaten both human and environmental health. In recent years, there has been increasing interest in the use of sustainable, cost effective technologies to remediate these petrogenic hydrocarbons in the environment. This PhD research was designed to enhance and develop these clean technologies using phytoremediation (plant-assisted bioremediation) and necrophytoremediation (plant dead biomass-assisted bioremediation) methods. Traditional microbiological methods were used in conjunction with the molecular ecological methods throughout this research to better understand the response of the natural microbial community to phytoremediation and necrophytoremediation.

In the first part of this study a variety of plants including alfalfa, arrowleaf clover, balansa clover, berseem clover, French serradella, maize, pea, Persian clover, wallaby grass, wheat and wheat grass were screened for their ability to grow in a soil contaminated with 1% w/w (10,000 mg kg−1) aliphatic hydrocarbons (60% diesel/40% engine oil) or 1% w/w crude sludge. Based on emergence, shoot and root length and root shoot weight ratio, maize and wheat were selected as both showed the greatest performance. A further 90 day greenhouse study was carried out to evaluate the microbial aspect of rhizodegradation, using a contaminated soil containing a diesel/engine oil mix (1% w/w). The results showed that the presence of maize and wheat plants increased the degradation of total petroleum hydrocarbons (TPH), with soil containing maize and wheat showing a significant TPH reduction of 72% (5,880 mg kg−1) and 66% (5,390 mg kg−1) respectively, compared with a reduction of 57% (4,633 mg kg−1) in contaminated soil only. Microbial community analyses using DNA-PCR-DGGE for both bacterial and fungal communities confirmed that the presence of the plants influenced the structure of the soil microbial community. In addition to changes in microbial community, quantification of alkB genes (encoding alkane monooxygenase; a key enzyme in the degradation of aliphatic hydrocarbon) using MPN-qPCR demonstrated that the planting of maize and wheat on contaminated soil led to a 20 and 16-fold increase in alkB gene copy numbers respectively, relative to the control soil.

In the second part of this study, necrophytoremediation (using alfalfa hay, pea straw, wheat straw and various residues including 20% hay, 37.5% pea straw, 37.5% wheat straw and 5% gypsum) was evaluated as a technique for use in the bioremediation of aliphatic hydrocarbons. The presence of plant residues was found to have a significant impact on the utilisation rate of TPH when compared to the control. The highest TPH reduction, up to 83% (6,800 mg kg-1) was observed in soil mixed with pea straw, compared to a TPH utilisation of 57% (4,633 mg kg-1) in the control soil. The abundance of petrogenic hydrocarbon-utilising microorganisms increased in the contaminated soil amended with plant residues. For example, a 12-fold increase in hydrocarbon utilising microorganisms was observed when pea straw was mixed with contaminated soil. Microbial community analyses using DNA-PCR-DGGE of both bacterial and fungal communities showed that amending the contaminated soil with plant residues led to significant changes in the soil microbial community diversity in the most of the treatments (e.g. pea straw in terms of bacterial community). Sequencing of the bands of interest again showed the presence of some hydrocarbonoclastic microorganisms only in soil amended with plant residues in the bacterial and fungal DGGE-profiles, confirming that the presence of plant residues changed the activity and diversity of hydrocarbon utilising-microorganisms. Interestingly the presence of plant residues (e.g. pea straw) led to the detection of fungus Trichurus spiralis, which exhibits both hydrocarbonoclastic and necrophytic properties.

In the third part of this study, the effect of necrophytoremediation using pea and wheat straws (found to promote degradation in the second part of the study) on the remediation of polycyclic aromatic hydrocarbons (PAHs) was investigated using soil contaminated with phenanthrene (500 mg kg−1) and pyrene (500 mg kg−1), alone or in a combination. The results showed that adding pea or wheat straw to contaminated soil significantly increased PAH reduction in the all treatments except in phenanthrene-contaminated soil amended with wheat straw. The results also showed that the beneficial effects of necrophytoremediation were most evident in pyrene contamination. For example, pyrene-contaminated soil amended with pea straw led to an increase in the degradation of pyrene from 15% (64 mg kg−1) in the corresponding control to 70% (301 mg kg−1). The results also showed that pea straw generally resulted in greater beneficial effects compared with wheat straw in terms of PAH degradation. Microbiological analysis of the soils using MPN in conjunction with PCR-DGGE-sequencing methods demonstrated that both straws promoted microbial hydrocarbonoclastic biomass rather than changes in microbial diversity.
In summary, this study has shown that both phytoremediation and necrophytoremediation can be used in the successful bioremediation of petrogenic hydrocarbons. However, the plant toxicity associated with many hydrocarbon products may limit the use of phytoremediation. In contrast, necrophytoremediation represents an effective toxic-independent remediation protocol. In addition, using plant residues in general and pea straw in particular represents a cost effective, green technology which can be applied as a biostimulator for the remediation of crude oils, diesel oils and PAHs in contaminated soils.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Applied Sciences
Keyword(s) Petrogenic hydrocarbons
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