Abstract

Introduction
The main reason for oil pollution is leakage from tanks and transmission pipelines used in oil and oil refining industries. When a leak occurs, the oil product pollutes the soil, a part of the leaked oil material is absorbed in the soil and some of it moves down through the pores of the soil, and rainfall accelerates their movement by washing it. The substances penetrate into the underground water based on the water level. Part of the pollution dissolves in the water. The solubility of some volatile petroleum, such as benzene, toluene, xylene, which are typically toxic and carcinogenic substances, is between 150 and 1800 mg/l in water.In some cases, even after environmental protection operations such as pumping, groundwater pollution is still transferred with the underground water.
(Whiteside, 1993; Aminian ,Ameri, 2000; Hokstad, 2000; Galvez, 2001).
Contamination of unsaturated areas of soil and underground water with petroleum hydrocarbons has the risk of threatening human health and negative effects on the environment, underground water, surface water, soil, agriculture, floor sediments, etc. Since drinking water and Agriculture is supplied from underground water sources, finding a solution to remove oil hydrocarbon pollution from it is necessary.(Theodorakis, Walker,2014).Due to the high cost of physical, mechanical and chemical methods and the possibility of producing secondary toxic compounds, there is a need for a safer and cost-effective method. Many researches related to bioremediation or biodegradation using microorganisms have been used to effectively remove hydrocarbon pollution from contaminated sites. (Thapa,2012). One of the types of bioremediation is biological stimulation. In this method, the pollutant-decomposing microbial population is present in the environment, but the environmental conditions are not suitable for their activity. With the improvement of the conditions, the rate of pollution decomposition increases as a result of the stimulation of native microorganisms. Due to the low level of dissolved oxygen in underground water, oxygen-releasing compounds(OCR) have been used for the biological stimulation of pollutant-consuming microorganisms (Chevalier,McCann,2008). In the research conducted, the effect of increasing calcium peroxide on the bacterial diversity of groundwater was investigated. Calcium peroxide increased the population of some native microorganisms. Also, the population of a number of microbial species decreased (Gholami, 2019). In natural conditions, the increase of OCR compounds in groundwater increases the conditions for the activities of aerobic microorganisms, but anaerobic microorganisms that decompose pollutants are also present, which have activities that prevent it. Therefore, it is necessary to investigate the effect of these compounds simultaneously on the microbial removal of pollutants such as benzene in aerobic and anaerobic conditions. Therefore, in this research, the experimental study of the microbial removal of benzene by indigenous groundwater microorganisms in the presence of calcium peroxide and without it has been investigated.

Methodology
Sampling of underground water in the area of Shiraz Refinery for microbial tests was done in accordance with the ISIRI-4208 standard. Sampling was done from one of the available wells. The samples were collected in a 1 liter autoclavable and sterile glass bottle. and was transported to the laboratory in a container containing ice and stored in a refrigerator.Benzene was prepared with a concentration of 105 mg/l as the initial concentration of the microbial removal tests and was stored in a refrigerator at 4°C. The method used in the production of calcium peroxide in this research was based on the method of producing calcium peroxide by Khodaei. (2019), with some changes. 9 grams of calcium chloride was dissolved in 90 ml of distilled water in a 250 cc Erlenmeyer flask. Then 45 ml of 25% ammonia solution was added to it. 45 ml of 35% hydrogen peroxide was added dropwise with stirring to the environment. Then 1 normal sodium hydroxide solution was added until a white precipitate was formed. After filtering the solution, the remaining sediment on the filter paper was dried at room temperature for one night and poured into a dark glass container and kept in the refrigerator until the experiments. To investigate the microbial removal of benzene by indigenous microorganisms in the underground water of the Shiraz refinery area without the presence of calcium peroxide, 10 ml of water sampled with a sterile pipette was added to 16 glass bottles containing 100 ml of benzene aqueous solution with a temperature of 40C which was prepared before. For biological stimulation, 5 cc of nutrient broth medium was added to the collection. Experiments were performed under the microbiology hood. The samples were then incubated at 22 degrees Celsius. 8 of the samples were kept in a incubator for one week and 8 of them were kept in a incubator for two weeks to measure the remaining values.Samples was sent to the certified environmental laboratory. To investigate the microbial removal of benzene by indigenous microorganisms in the underground water of the Shiraz refinery area in the presence of calcium peroxide, 10 ml of water sampled with a sterile pipette was added to 16 glass bottles containing 100 ml of benzene aqueous solution with a temperature of 40C which was prepared before. For biological stimulation, 5 cc of nutrient broth medium was added to the collection. Then 1 gram of pre-prepared calcium peroxide was poured into a sterilized test tube and 5 cc of distilled water was added to it and placed inside each of the 16 bottles from the previous step. Experiments were performed under the microbiology hood.The samples were then kept in a incubator at a temperature of 22 degrees Celsius. 8 of the samples were kept in a incubator for one week and 8 of them were kept in a incubator for two weeks to measure the remaining values.Samples was sent to the certified environmental laboratory.
Conclusion
In order to analyze the results of the experiments, the analysis of variance test with repeated measurements was used with a confidence level of 1%. In this analysis, two experimental groups were considered as between-subject factors and the variable measurement time of benzene concentration was considered as a within-subject factor. The effect of measurement time on benzene concentration was significant(F(2,28)=21303.369,P<0.01). But the interaction effect between time and group was not significant (F(2,28)=1.124, P>0.01). The effect of the group on the changes in benzene concentration was also not significant (F(1,14)=0.636, P>0/01)..According to these findings, it indicates the indigenous microorganisms of underground water can remove benzene from it.But No significant difference was observed between benzene removal in the presence of calcium peroxide and without it. This conclusion is consistent with the research conducted by Gholami. (2019). In the research conducted by them, calcium peroxide changed the indigenous microbial diversity of underground water and caused an increase in some microbial species and a decrease in some other microorganisms Therefore, more research in this field and investigating the Simultaneous role of aerobic and anaerobic microorganisms in removing groundwater pollutants will be necessary.