Influence of necrophytoremediation as a bioremediation tool on the activity, composition and diversity of the microbial community in petrogenic hydrocarbon contaminated soils
Creators: name: KOSHLAF, Eman Mohammed
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310703
subject: Microbial ecology
classificationCode: 410303
subject: Bioremediation
classificationCode: 4104
subject: Environmental management
classificationCode: 410404
subject: Environmental management

description: <br>The vast extraction of crude oil and the widespread use of petroleum and its by-products in daily life has led to the release of petroleum hydrocarbons into various ecosystems including water and soil. In recent decades, attention has focused on the application of microbial bioremediation as an efficient, cost-effective and environmentally friendly technology for the degradation of petroleum hydrocarbons in polluted environments. The overall aim of the research presented in this thesis was to develop a rapid, efficient, cost-effective, environmentally-friendly, easily managed and applicable bioremediation approach for the remediation of different petroleum hydrocarbon-polluted soils based on the application of plant residues (pea straw). This research was conducted to: (i) investigate the potential of an economical and readily available bioremediation strategy (necro-phytoremediation) using pea straw on the degradation of Total Petroleum Hydrocarbons (TPHs) in a Libyan agricultural
soil under laboratory conditions and evaluate changes in soil microbial community abundance, growth and activity during the bioremediation process; (ii) evaluate the impact of pea straw on the degradation of Poly Aromatic Hydrocarbons (PAH) and soil microbial diversity and composition and determine changes in soil microbial population including bacteria, fungi and functional gene communities [PAHring hydroxylating dioxygenase genes (PAH-RHDa)] present in an Australian landfill soil; and (iii) examine changes in The microbial community composition and diversity of TPH contaminated Australian soil exposed to necrophytoremediation under greenhouse conditions and assess the potential of pea straw on petroleum hydrocarbon degradation and ecotoxicity.<br>

<br>The presence of petroleum hydrocarbons in the soil was assessed using comprehensive two-dimensional gas chromatography and RemScan technologies. Most probable number (MPN), heterotrophic plate counts and quantitative PCR (qPCR) techniques were used to assess the abundance of total bacteria, fungi and bacterial genera possessing functional genes such as alkane monooxygenase (alkB) encoding for enzymes capable of breaking down different hydrocarbon complexes. The number
of gene copies encoding the α-subunit of the ring hydroxylating dioxygenase (PAH RHDα) gene from Gram-negative (GN) and Gram-positive (GP) bacteria were also quantified. Soil microbial activity was assessed using fluorescein diacetate (FDA) hydrolysis. The effect of necrophytoremediation on the diversity and composition of bacterial communities in the soil was examined through DGGE and high throughput 16S rRNA (Illumina MiSeq) sequencing.<br>

<br>The efficacy of pea straw on the degradation of diesel contaminated Libyan soil was evaluated in comparison to and together with two other bioremediation processes, bioaugmentation (the addition of <i>Bacillus</i> spp. isolates) and natural attenuation. The highest degradation of total petroleum hydrocarbons (TPH) was obtained in soil amended with pea straw alone (96.1%) or in combination with the Bacillus consortium (95%). Higher numbers of hydrocarbon-degrading bacteria (5.7 log10 CFU g-1
dry soil) were present following pea straw treatment when compared to untreated soil (4.4 log10 CFU g-1 dry soil). The addition of pea straw also stimulated microbial growth and activity, factors which may have been responsible for the improved degradation.<br>

<br>Following this initial demonstration of the positive impact of pea straw amendments, the potential of pea straw to enhance the degradation of more complicated and persistent weathered petroleum contaminants from a landfill site in New South Wales was investigated. Pea straw led to rapid PAH degradation (66.6%) at the beginning of the treatment, although the degradation had slowed or stopped by the end of incubation. Necrophytoremediation resulted in an increase in the numbers of 16S ribosomal RNA (rRNA) genes (12.1 log10 g soil-1) and of internal transcribed spacer (ITS) sequences (8.0 log10 copies g soil−1) as well as genes associated with Gram-positive PAH-degrading bacteria (11.3 log10 g soil−1) indicating an increase in the number of hydrocarbon-degrading organisms. The most significant shift in the bacterial community was observed in the pea straw-amended soils with increased abundance of hydrocarbon-degrading bacteria including <i>Pseudomonas, Pseudoxanthomonas, Alcanivorax and Parvibaculum</i>. By the end of the experiment (Day 102), <i>Pseudomonas</i> and <i>Pseudoxanthomonas</i> were notably decreased/ not detected, possibly explaining the slower degradation that occurred at the end of incubation.<br>

<br>Maintaining the highest level of growth of microorganisms with high potential for hydrocarbon degradation and metabolic diversity is imperative for a sustainable and more efficient bioremediation process. This could be accomplished through the provision of enhanced nutrients and increased soil porosity, as provided by the addition of pea straw. However, it is also possible that petroleum hydrocarbons are converted to recalcitrant dead-end metabolites that are more toxic than the parent contaminants.<br>

<br>Therefore, evaluating the activity and diversity of the soil microbial community together with the ecotoxicity of petroleum hydrocarbons during necro-phytoremediation is essential for a better understanding of the fate of petroleum hydrocarbons in soil. Both natural attenuation and pea strawtreated soils displayed effective biodegradation (&gt;80 %), assessed in terms of total petroleumhydrocarbons reduction by the end of the incubation. After 5 months remediation, pea straw amendedoil exhibited a significant reduction in soil toxicity EC50 = 91 mg diesel/kg when compared to the natural attenuation treatment (EC50 = 52.9 mg diesel kg-1 soil). An increased number of 16S rRNA (8.8 log10 g kg-1) and alkB genes (6.6 log10 g kg-1) was observed in the PS-amended soil compared to 7.7 log10 g kg-1 and 5.6 log10 g kg-1 in natural attenuation samples at the end of the incubation. Interestingly, pea straw itself was found to harbour a diverse hydrocarbonoclastic community including <i>Luteimonas, Achromobacter, Sphingomonas, Rhodococcus and Microbacterium</i>. During the bioremediation process, substantial shifts in the soil bacterial community including an increase in the abundance of several hydrocarbon-degrading bacteria such as <i>Dokdonella, Nocardioides, Sphingomonas, Pseudoxanthomonas and Achromobacter</i> were observed. Necrophytoremediation treatment using pea straw exhibited reduced ecotoxicity and increased bacterial diversity. These findings suggest the rapid growth of species stimulated by the bioremediation treatment and strong selection for hydrocarbonoclastic bacteria capable of degrading petroleum hydrocarbons during necrophytoremediation.<br>

<br>Overall, this research has demonstrated that pea straw addition can accelerate and increase bioremediation in hydrocarbon polluted soils. Pea straw simultaneously provides microflora (bioaugmentation) and nutrients (biostimulation) when applied to petroleum contaminated soils. The substantial capacity for pea straw to stimulate the soil microbial activity, improving degradation and reducing the toxicity of petrogenic hydrocarbons suggest that necrophytoremediation using pea straw is a sustainable and an effective management tool to enhance the degradation of petroleum hydrocarbons in contaminated soils. The application of pea straw in petroleum hydrocarbon
contaminated soil can significantly reduce the time and therefore the cost associated with the bioremediation process. Thus, pea straw may be a suitable candidate for the remediation of other types of contaminants in the environment.<br>