Survey of oil and gas geomicrobial anomalies in mud volcano Seyvan, around Marand city - East Azerbaijan

Introduction
Mud Volcano are one of strangest and most fascinating geomorphologic phenomena. They have important for a wide Spectrum of disciplines, including the oil industry (Stewart and Davies, 2009). Since the mud volcanoes originate from deeper depths of the earth, they act as regional indications for hydrocarbon exploration (Shnyukow and Yanko-Hombach, 2020). One of the recent methods of hydrocarbon exploration is geomicrobial exploration that is based on surface excavation technique to identify leaking gases relevant to hydrocarbon microseepage. According, they shows detection of seepage trend and their migration from subsurface oil reservoirs to surface environments. A direct and positive relationship has been observed between microbial populations and hydrocarbon concentrations in the soil of production various reservoirs in around the world. (Wanger et al, 2002).
 
Materials and Methods
The sampling site is located in the village of Seyvan, which is part of the central district of Marand County in the northwestern region of East Azerbaijan Province. The collected samples include water and soil samples. The samples were gathered from the site of a currently inactive mud volcano, as well as from the hill surrounding the mud volcano, based on its color and geomorphology, and from locations where water and gas were periodically and intermittently released. Each soil sample, weighing approximately 1 kilogram, was collected from a depth of 0.5 meters in plastic bags that had been sterilized in an autoclave beforehand, while the water samples were collected in glass containers. One of the acceptable and common methods for survey of microbial population of oil-eating bacteria to study of oil and gas geomicrobial anomaly in the Seyvan mud volcano is counting method on culture plates that is performed in the form of Plate Count. The Geomicrbi survey method includes collecting soil samples from the study area, packing, maintenance and storing samples in pre-sterilized sample bags in conditions without microbe and cold to sample preparation for culture, Analysis and separation and counting of hydrocarbon user bacteria such as methane, ethane, propane and butane oxidizers. The results of bacteria count results for each sample were calculated based on the number of bacteria colonies per gram soil or 1 ml liquid.
 
Results and Discussion
After the incubation period for assessing the total number of colonies formed by the target bacteria, each of the colonies formed at different dilutions of a sample was examined visually and under a stereomicroscope on culture medium plates. A light stereomicroscope is used to confirm the target microorganism and verify the characteristics of the colonies.
Consequently, the target bacteria were distinguished from other potential bacteria, and the number of desired colonies on the culture medium was counted, with calculations made per gram of the soil sample examined. The assessments included distinguishing the target bacteria from other potential bacteria, counting the desired colonies, and calculating this per gram of the soil sample examined. To calculate the number of methanotrophic, ethanotrophic, and propanotrophic bacteria in each soil sample, the number of target bacteria counted on each culture medium plate is multiplied by the inverse of the dilution factor applied to each plate. Additionally, since 0.1 mL of each suspension was used for culturing, the resulting number is multiplied by 10. The microbial population is expressed as "Colony Forming Units (CFU) per milliliter of microbial suspension" (Liu et al, 2016).
Inverse of dilution factor * 10 * number of colonies = cfu/mg (number of microbes in 1 milliliter of suspension)
Finally, to calculate the number of microorganisms per gram of soil, the CFU/mL calculated for each microbial suspension is multiplied by 5, as 20 grams of the initial soil were suspended in 50 mL of serum. The final number represents the target microorganisms per gram of soil.
Number of bacteria in 1 milligram * 10/50 (Table 1) 10/50 * number of bacteria in 1 milligram = number per gram of soil.
 
Conclusion
In order to determine population of methanotorphic, ethanotrophic and propanotrophic bacteria in the Seyvan mud volcanoes (Figure 1), a total of seven soil and mud samples together with two water samples were collected from 8 points of the study area. Due to the sampling points of Seyvan mud volcanoes in three points of the study area, methane and ethane oxidizing bacteria are present at the same time, those points include the main crater of mud volcanoes and in points from the hill which contain of mud volcanoes that from they were coming out water and gas periodic and intermittently. The only place where only ethanotrophic bacteria were present, was point sv2. while to confirm the presence of propanotroph bacteria, supplementary experiments are needed (Figure 2). Considering presence of methane and ethane user bacteria, two results can be taken, in the first place, methane can be of the type of biogenetic methane and the presence of ethane in the area can be due to the fusion of two methane affected by pressure. On the other hand, can be argued due to the limited number of methanotrophic and ethanotrophic bacterias that the presence of hydrocarbons is possible but it does not have economic value.