English abstract
Oman is an oil producing and exporting country, hence it is vulnerable to frequent oil spills during oil production and exploration. Oil spills pose a serious threat to both terrestrial and marine environments and can have a serious impact on the economy of Oman. Oil spills can result in the contamination of underground water and can drastically affect fishing industry, public health, water desalination plants, agriculture, tourism and human health. So far, there is no national strategy in Oman of how to deal with oil spills and how to treat them. Around the world, different remediation methods were employed to clean up oil polluted sites including mechanical, physical / chemical and thermal methods. Bioremediation is one of the safest, environmentally friendly and successful method used in cleaning up oil contamination. Biostimulation is one of the bioremediation approaches that rely on stimulating indigenous microorganisms in the contaminated sites through removing the limiting factors such as nutrient, availability of oil compounds to microorganisms, and aeration. In this thesis, biostimulation was assessed using different stimulating agents. The contaminated soils were studied using physicochemical analysis, CO2 method, hydrocarbon extraction and molecular technique before and after the application of the biostimulation treatment. The work performed in this thesis will help in understanding the diversity of soil microorganisms in different oil contamination sites, and in establishing a base for future biostimulation treatments in Oman. Furthermore, the use of organic wastes as biostimulating agents will open the door for waste management. Below is a summary of the different studies performed in this thesis.
Enormous studies were carried out in oil-polluted freshwater and marine environments compared to desert soils although they are frequently exposed to oil contamination. The bacterial diversity of oil-polluted desert soils with different pollution levels was investigated in this study. The five soils from one place display a gradient of oil contamination level (S> OBM > CS > US > B3), with the highest level in S soil and lowest in B3 soil. The bacterial communities of the five soils were profoundly different (ANOSIM R = 0.45, p < 0.0001) as revealed by using automated rRNAintergenic spacer (ARISA) analysis. The shared operational taxonomic units between five soils were less than 20%. We found that soils with higher oil contamination level had highest operational taxonomic units (OTU) richness. Multivariate analyses of ARISA profiles revealed that the bacterial communities in the S soil, which contains the highest level of contamination, was different from the other soils and formed a completely separate cluster. From 454 pyrosequencing analysis, a total of 16,657 ribosomal sequences were obtained, with 42-89% of these sequences belonging to the phylum Proteobacteria. In all soils, sequences belonging to Betaproteobacteria, Gammaproteobacteria, Bacilli and Actinobacteria were found. While the sequences belonging to anaerobic bacteria from the classes Deltaproteobacteria, Clostridia and Anaerolineae were only detected in the S soil, sequences belonging to the genus Terriglobus of the class Acidobacteria were only detected in the lowest contaminated soil (B3). The variation in bacterial communities between soils was strongly determined by oil contamination level asshowen by redundancy analysis (RDA). Our conclusion is that desert soils are rich in aerobic and anaerobic bacteria that could potentially contribute to the degradation of hydrocarbons as well as oil contamination level exert a strong selective pressure on bacterial communities.
We compared the response of oil-polluted desert and marine soils to biostimulation using three nonioinc surfactants, Triton X-100, Tween-80 and Brij-35 and inorganic nutrients, nitrogen and phosphorous (N and P) at different ratios. The addition of Tween-80 and Brij-35 resulted in an increase in the produced CO2 from 2.1+0.01 to 2.5+0.1 and from 0.7+0.01 to >1.3+0.02 mg CO2 g soil in the desert and marine soil, respectively. This corresponded to 51% increase in the oil mineralization rate in the marine soil, but only to 20% increase in the desert soil. The effect of Triton X-100 was less conspicuous on the respiration activities of both soils. The addition of inorganic nutrients had a more pronounced biostimulating effect in the marine than in the desert soil. The produced CO2 in the marine soil was comparable at the different N:P ratios. Physical and chemical characterization of the two soils revealed higher oil content and lesser nitrate concentrations in the desert than in the marine soil. While the desert soil texture was classified as silt loam, the marine soil texture was sandy loam. The bacterial communities were very different, with no common genera between the two soils. While the bacterial classessAlphaproteobacteria, Gammaproteobacteria and Actinobacteria were detected in both soils, Betaproteobacteria was detected only in the desert soil and Deltaproteobacteria and Firmicutes were only found in the marine soil. We conclude that biostimulation depends very much on the physical and chemical conditions and the biological characteristics of polluted sites. Therefore, bioremediation has to be tailored specifically for each site.
Waste materials have a strong potential in the bioremediation of oilcontaminated sites, because of their richness in nutrients and their economical feasibility. We used sewage sludge, soybean meal and wheat straw to biostimulate oil degradation in a heavily contaminated desert soil. While oil degradation was assessed by following the produced CO2 and by using gas chromatography-mass spectrometry (GCMS), shifts in bacterial community composition were monitored using illuminaMi$eq. The addition of sewage sludge and wheat straw to the desert soil stimulated the respiration activities more than the addition of soybean meal. GC-MS analysis revealed that the addition of sewage sludge and wheat straw resulted in 1.7 to 1.8 fold increase in the degraded C14 to C30 alkanes, compared to only 1.3 fold increase in the case of soybean meal addition. The degradation of > 90% of the C14 to C30 alkanes were measured in the soils treated with sewage sludge and wheat straw. MiSeq sequencing revealed that the majority (76.5-86.4% of total sequences) of acquired sequences from the original soil belonged to Alphaproteobacteria, Gammaproteobacteria and Firmicutes. Multivariate analysis of operational taxonomic units (OTUS) placed the bacterial communities of the soils after the treatments in separate clusters (ANOSIM R=0.66, P=0.0001). The most remarkable shift in bacterial communities was in the wheat straw treatment, where 95-98% of the total sequences belonging to Bacilli. We conclude that sewage sludge and wheat straw are useful biostimulating agents for the cleanup of oil-contaminated desert soils.
Organic waste materials are potential stimulating agents for bioremediation of oil-polluted soils as they are cheap and environmentally safe. Spent mushroom compost (SMC) and poultry manure (PM) are by-product organic waste materials produced from
big factories in Oman. We investigated the effect of spent mushroom compost (SMC), poultry manure (PM) and urea in the stimulation of respiration activities and oil degradation in a polluted desert soil. Besides that, we studied the shifts in bacterial community structure after treating soils with organic waste materials by using MiSeq sequencing. The addition of SMC and PM resulted in a significant increase of the evolved CO2 from 8.7+1.9 to 25.7+1.6 and to 23.4–1.2 mg CO2g' soil after 96 days of incubation, respectively. However, urea addition did not significantly change respiration activities. The addition of organic wastes increased the rate of alkanes degradation (C14C30) by twofold compared to untreated soil as revealed by GC-MS analysis. Clear shifts in the bacterial community of the original soil was detected, particularly in the relative abundance of Firmicutes, Actinobacteria and Proteobacteria, which constituted 87-94% of total sequences. The relative abundance of Firmicutes remained unchanged after the addition of PM (37-48% of total sequences), but it dramatically decreased in the SMC treatment (0.5-4.5%) and increased in the urea treatment (44-87%). The addition of SMC increased the relative abundance of Alpha-, Gamma-proteobacteria and Actinobacteria. We conclude that, SMC and PM waste material are effective alternative sources of nutrient in treating oil pollution in desert areas.
