PETROLEUM-CONTAMINATED SOILS

Technical Field of the Invention The present invention relates to a method which allows performing bioremediation of petroleum-contaminated soils by incorporating bio stimulation and bioaugmentation methods.

The invention particularly relates to a method which allows subjecting petroleum- contaminated soils to bioremediation using a composition in which Bjerkandera adusta and Trametes versicolor fungi are used together for bioremediation.

Background of the Invention

Although the technological background of the renewable energy generation is highly advanced, petroleum products still remain to be one of the major sources of the energy used for industrial and domestic consumption. Depending on the use of this source, more than three billion tons of petroleum is produced in the world annually.

Petroleum wastes cause health issues in human life and ecosystem degradation as contaminants comprising toxic, mutagenic, and carcinogenic compounds.

Petroleum sludge comprises water, oil emulsion, metal, tar, and many petroleum hydrocarbon compounds and has a very complex structure. A wrong petroleum sludge disposal results in serious soil, water, and groundwater pollution.

Physicochemical methods such as soil vapor extraction, soil washing and dispersion are commonly used for remediation of petroleum-contaminated sites; however, these methods fail to completely eliminate petroleum contamination and also they increase the toxicity of the contaminated sites. Bioremediation overcomes these problems as an economic and environmentally friendly treatment method. During this process, contaminant compounds are converted into either non-toxic materials or C0 ₂ and H ₂ 0 as a whole. At sites where bioremediation is applied, local microorganisms or nutrients used as inoculum may be stimulated for digesting organic contaminants by using oxygen or another additive. Bioaugmentation, as an improved bioremediation process, is to improve the degradation potential of the site at which bioremediation technique is used by adding microbial water. This process has an impact on the degradation of various contaminants. However, the concerns of genetic engineering about the use of microorganisms and the possible effects of these microorganisms on the environment are common. As an alternative to bioaugmentation and as a digestible bioremediation method, biostimulation is a more effective method of soil bioremediation than bioaugmentation. One of the important steps while applying biostimulation is to monitor the efficiency of the process and evaluate the degradation level in a contaminated environment. Effective monitoring may reduce bioremediation costs as well as the remediation time.

The prior art search revealed the International Patent Application No. WO2014031016A1, filed on 16.10.2012, wherein a method of bioremediation of soil and/or soil-based masses polluted with petroleum-derived compounds under biodegradation by subjecting the same to various processes is disclosed. Another prior art document is the U.S. Patent Application No. US5725885A, filed on 06.06.1995, wherein a composition, as well as a method of manufacturing said composition, for promoting a faster degradation of the bacteria in petroleum and petroleum-derived materials to aid in bioremediation of oil spills on water is disclosed. On the other hand, New Zealand Patent Application No. NZ519022A, filed on 17.05.2002, discloses a composition for accelerating bioremediation process, comprising particularly Trametes versicolor and other Trametes species. The composition used in this patent contains cultures A, B and C, said cultures being disclosed in three different patent documents. Nevertheless, the applications described in these documents fail to satisfy bioremediation with the desired efficiency. Therefore, developing a method for subjecting petroleum-contaminated soils to bioremediation according to the invention has been deemed necessary. Objects and Summary of the Invention

An object of the present invention is to provide an economic method for bioremediation of petroleum-contaminated soils.

Another object of the present invention is to provide a method for subjecting petroleum-contaminated soils to bioremediation, wherein biostimulation and bioaugmentation techniques are used together.

Another object of the present invention is to determine the optimum amount of biostimulation, and then to perform a method of successful biostimulation using two different types of fungi specific for biostimulation.

And another object of the present invention is to provide a method by which petroleum wastes are disposed with maximum efficiency.

Petroleum-derived wastes consist of organic molecules groups differing in terms of molecular weight, structural configuration, solubility, number of aromatic chains, volatility, adsorption coefficient, etc. These components, due to the inhibitory properties and low viscosity solubility thereof, are highly resistant against biodegradation and remain in nature without degradation. With the method according to the invention, an economic method for bioremediation of petroleum-contaminated soils has been developed. In this method, biostimulation and bioaugmentation methods are incorporated. A successful biostimulation has been achieved by first determining the optimum amount of biostimulation (C:N:P ratio, 100: 15: 1), and then using two different types of fungi specific for the determined biostimulation. Detailed Description of the Invention

The subject matter of the invention is a method which allows performing bioremediation of petroleum-contaminated soils, wherein bio stimulation and bioaugmentation steps are comprised together. The invention comprises the following specific steps: collecting petroleum-contaminated soil samples from petroleum waste lagoons and characterizing the same, determining dominant bacterial species in the collected samples by Illumina sequencing, one of the next-generation sequencing analysis methods, subjecting soil mixtures to a bio stimulation process at a C:N:P ratio of 100: 15: 1, isolating genomic DNA, total RNA, and plasmids from soil samples, measuring the degrading bacteria, functional genes alkB, phnAc and nah using alkane and polyaromatic hydrocarbons (PAH) by quantitative real time PCR method, applying bioaugmentation to the petroleum-contaminated soil, having undergone biostimulation at a C:N:P ratio of 100: 15: 1, using Trametes versicolor and Bjerkandera adusta fungi species, and

evaluating the effect of the fungi used in bioaugmentation method on local microbial community, functional genes alkB, phnAc and nah.

Petroleum-contaminated soil samples were collected from petroleum waste lagoon and they were characterized, and then dominant bacterial species were determined in the collected samples by means of Illumina sequencing method. Petroleum- contaminated samples were mixed with clean forest soil samples at different ratios and placed in glass containers, and then N:P at different ratios was added to the resulting soil mixtures and incubated at 24°C for 90 days. Genomic DNA, total RNA, and plasmids were isolated from the incubated samples. Alkane and polyaromatic hydrocarbons (PAH) were used for measuring the degrading bacteria and functional genes alkB, phnAc and nah by means of quantitative real time PCR performed with TaqMan probe. As a final treatment method, the treated contaminated soil was subject to biostimulation, followed by bioaugmentation method, using Trametes versicolor and Bjerkandera adusta fungi species.

The effect of said fungi on local microbial community and functional genes alkB, phnAc and nah was evaluated by performing bioaugmentation at 30°C for 60 days.

Throughout the degradation test, maximum amount of degradation was obtained from the microcosmoses whose C:N:P ratio was adjusted to 100: 15: 1. The amount of total organic carbon (TOC) elimination was found to increase 25% by biostimulation. The results of final treatment showed that Trametes versicolor and Bjerkandera adusta fungi were able to degrade petroleum residues remaining after biostimulation phase.

These two types of fungi were compared and it was shown that Trametes versicolor was much more effective than Bjerkandera adusta during degradation process of petroleum residues and that it eliminated petroleum residues by 36% at 30°C for 60 days.