The effect of lead as co-contaminant with petrogenic hydrocarbons on soil bioremediation, ecotoxicity and diversity of the microbial community

Khudur, L 2019, The effect of lead as co-contaminant with petrogenic hydrocarbons on soil bioremediation, ecotoxicity and diversity of the microbial community, Doctor of Philosophy (PhD), Science, RMIT University.

Document type: Thesis
Collection: Theses

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Title The effect of lead as co-contaminant with petrogenic hydrocarbons on soil bioremediation, ecotoxicity and diversity of the microbial community
Author(s) Khudur, L
Year 2019
Abstract The global demand for petroleum products has increased, reaching 4,488 Mtoe in 2018. Total Petroleum Hydrocarbons (TPH), the main component of crude oil, comprise a broad family of chemicals consisting largely of carbon and hydrogen. During the exploration, recovery, storage and transport of petroleum products, TPH enters the environment causing serious land contamination. TPH contamination is of major concern worldwide due to its high toxicity to the soil biota which, causes damage to the ecosystem if left untreated. Bioremediation represents a simple, cost-effective and environmentally safe approach to treat TPH contaminated soils. However, the traditional approaches of measuring TPH concentration during the bioremediation process are relatively expensive and time-consuming. Additionally, assessing the reduction in TPH concentration, which is usually used to evaluate the efficacy of bioremediation, is not itself an indication of a decrease in the associated ecotoxicity. Co-contamination, often with lead (Pb) which was commonly added to gasoline until 16 years ago is one of the biggest challenges affecting the effectiveness of bioremediation. The presence of heavy metals, as co-contaminants negatively impacts the rate of TPH biodegradation, the associated ecotoxicity as well as the activity and diversity of the natural microbial communities which are the backbone of any bioremediation process. 

The overarching aim of this study was to design appropriate bioremediation strategies for heavy metals co-contaminated soils by evaluating the impact of lead co-contamination on the ecotoxicity and bacterial community in TPH-contaminated soils.

The aim of the work carried out in Chapter 3 of the thesis (the first results chapter) was to assess the potential of RemScan as a fast, accurate and cost-effective portable device to be used as a tool to monitor the bioremediation process. Quantitative analysis of TPH was performed using RemScan on a variety of TPH-contaminated soils as an alternative to slow and expensive analyses currently being used, such as Gas Chromatography applications. The TPH values obtained were validated and compared with the results obtained from an accredited external laboratory, which uses Gas Chromatography/Mass Spectrometry (GC/MS) for TPH analysis. RemScan showed a correlation coefficient (R2) of 0.998 in comparison with GC/MS but importantly results in a significant reduction in both time and cost.

The aim of the research conducted in Chapter 4 was to evaluate the effect of heavy metals as co-contaminant, together with total petroleum hydrocarbons (TPH), in terms of the remaining soil toxicity and the structure of the microbial communities in weathered contaminated soils. Contaminated soil samples from a relatively hot and dry climate in Western Australia were collected (n=27). The Microtox test confirmed elevated ecotoxicity in the co-contaminated soils. Toxicity was correlated with the presence of lead, zinc and TPH (0.893, 0.599 and 0.488), respectively. 16S rRNA amplicon-based sequencing showed a lack of dominant genera; however, many genera of hydrocarbon-degrading bacteria were identified in all soil samples. Streptomyces spp. were present in 93% of the samples with abundance between 7% and 40%. In contrast, Acinetobacter spp. were found in only one sample but were a dominant member (45%) of the microbial community. In addition, some bacterial genera were correlated to the presence of heavy metals, such as Geodermatophilus spp., Rhodovibrio spp. and Rubrobacter spp. which were correlated with copper, lead and zinc, respectively. 

The research conducted in Chapter 5 aimed to investigate the impact of co-contamination with lead on the efficacy of two bioremediation processes, natural attenuation and biostimulation of TPH. The biostimulated treatment, using RemActiv (a commercial biostimulator), resulted in 96% and 84% reduction in TPH concentration in a single and a co-contamination scenario, respectively over 28 weeks of a mesocosm study. This reduction was significantly higher in comparison to soils undergoing natural attenuation in a single and a co-contamination scenario (56% and 59% respectively). In contrast, earthworm toxicity tests resulted in a decrease of 72% of the naturally attenuated toxicity compared to only 62% in soils undertaking the biostimulated treatment of a single contamination scenario. In a co-contamination scenario, the toxicity decreased by only 30% and 8% in natural attenuation and biostimulation treatments, respectively. 16S rRNA amplicon-based sequencing revealed major bacterial dominance by Nocardioides spp., which reached 40% in week 20 of the natural attenuation treatment. In the biostimulated soil samples, more than 50% of the bacterial community was dominated by Alcanivorax spp. in week 12. The presence of Pb in soils undergoing the natural attenuation treatment resulted in an increased abundance of several Pb-resistance genera in addition to Nocardioides spp. such as Sphingopyxis spp., Thermomonas spp. In contrast, in the presence of Pb, the microbial community was completely dominated by Pseudomonas spp., comprising approximately 45% of the bacterial profile in week 12.

Overall, it can be concluded from the work that (i) RemScan can be used as an accurate, fast and cost-effective tool to measure the TPH concentration during bioremediation of TPH contaminated soils; (ii) biostimulation is more effective than natural attenuation in remediating TPH and TPH-Pb contaminated soils; (iii) the presence of co-contaminants (e.g. heavy metals) alongside TPH causes serious complications impacting the efficacy of bioremediation of TPH in co-contaminated soils. These complications, which include elevating the associated ecotoxicity and changing the structure of soil microflora, must be fully assessed and considered in designing an effective bioremediation strategy.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Science
Subjects Environmental Management
Keyword(s) bioremediation
petroleum hydrocarbons
microbial diversity
next generation sequencing
Lead (Pb)
soil microbial community
land contamination
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