Imaging of micro-organisms on topsoil particles collected from different landscape in the Gobi Desert

. This study shows the results of field experiments of soil particles saltation and laboratory experiments of imaging of the surface structure of dust particles. In the Gobi area, dust occurs when the wind speed at ground level exceeds 7 m/s. It has been reported that bacteria are attached to dust, but the details of its attachment are unknown. It is also expected that these bacteria will fly at the time of occurrence of dust, and fundamental research is important to clarify the relationship between dust components and bacteria.


BACKGROUND OF THE STUDY
The Gobi desert of Mongolia is the main source region of Asian dust storms. Due to climatic factors and artificial development such as overgrazing and open pitting, the area has unprecedented serious environmental problems [1][2][3][4].
The destroyed topsoil is exposed to wind, making dust more likely to occur than in the past. Also, there is the possibility that various microorganisms are attached to the soil particles [5][6][7][8][9]. Dust storms in arid and semiarid areas are not only effected by weather conditions such as strong winds at the time of occurrence, but also effected by the surface condition (for example, conditions of soil dry condition, vegetation, snow cover etc.). Fig. 1 (a, b) shows the monthly average wind speed, the number of dust occurrence and the vegetation status around our study area of Sainshand (the capital of Dornogovi Province in Mongolia located in the eastern Gobi desert steppe). As shown in Fig. 1, the main dust season is in spring when the wind speed is highest and the ground surface is almost bare (indicated by a NDVI value close to zero).
Experiments on dust particles mobilization are very important to determine the threshold wind speed velocity for dust entrainment in the truly grand roughness and moisture conditions of field. It has been reported that bacteria are attached to dust, but the details of its attachment are unknown. In this paper, E. coli known as bacteria present in the arid environment (or grazing area) and similar in size to other bacteria was used as model bacteria. In this study, we used multiple precision equipment's, such as a field emission-type scanning electron microscope (FE-SEM, © JSM-7800F, JEOL Ltd.), a fluorescence microscope (BX 51, © Olympus Corporation) and a Raman image microscope (In Via, © Renishaw Ltd.), to identify microorganisms attached to the soil samples from the Mongolian Gobi desert. We examine the collected soil samples and aimed at imaging of microorganisms attached to the top soil surface. In order to investigate the relationship between wind speed and saltation of soil particles at the field site, we used a handmade equipment.

Field Experiments
The study area located in Sainshand City of Mongolian Dornogovi Province is shown in Fig. 2. As topographical features, the upper wind region has a slightly higher and the lower wind region has a slightly lower altitude, and the entire topography is a moderate basin with an altitude of 800 m to 1200 m. Dust particle saltation experiments (field experiment of blowing off the ground surface with a blower) were conducted at a total of 10 sites in the desert area, dry lake, and dry lake area. The condition of field experiment shows in Table 1. The measured dust concentration was measured for each of two particle sizes of 1 m < 3 m and 3 m < 5 m. For measurement of the number of dust saltation, a handheld particle counter Model 3886 manufactured by ©KANOMAX Co., Ltd. was used. Dimensions of measuring equipment and photographs of saltation experiments are shown in Fig. 3. In order to minimize the influence from the outside air, the air inlet of the blower was installed on the windward side, and the blower and the measurement point were sealed except for the air inlet and the air outlet. In the measurement method, an artificially generated wind is blown onto the ground surface stepwise by a blower, and the concentration of the saltation dust particles is measured. Here, stepwise means five stages of rotation speeds of the blower: 1200, 1400, 1600, 1800, 1900 rpm (revolution per minute). Air was blown for one minute at each stage, and dust particles saltation at each stage were measured. In addition, the wind speed for each rotation speed of the blower was measured. By doing this, we examined the difference in the number of dust saltation for each wind speed in the desert area and dry lake.

Laboratory Experiments
It is expected that these bacteria will fly at the time of dust generation, and Escherichia coli (DH 5) was used as a model of bacteria in this area to be domesticated grazed. E. coli suspended in phosphate buffer (PBS) were co-cultured with sea sand as a positive control. Soil particles from Mongolia were sterilized in an autoclave at 120°C. Samples were prepared by immobilizing the sand specimen on a sticky carbon tape on a mounting stage (1 cm in diameter). First, the specimen was observed in a fluorescence microscope and individual sand grains were identified. Next, the same particles were imaged with the FE-SEM, and energy dispersive spectroscopy (EDS) imaging was performed for mapping of the chemical elements in the samples. We also aimed to identify organic matter using Raman imaging. Used equipment's: a. Field emission scanning electron microscope (JSM-7800F); b. Fluorescent microscope (BX 51); c. Raman imaging (RENISHAW: inVia).

Result of experiments of topsoil particle saltation
The experiment result of this study has converted the rotational speed of the fan at 1 cm wind speed from the ground. As shown in the Fig. 4 (a, b), the number of soil particles blown up when the wind speed at the ground surface is 5 m/s is about 600-800 (N/m 3 ) at a particle size of 3 μm and about 100 (N/m 3 ) at a particle size of 5 μm. The OPC value (the blown up soil particles number, N/m 3 ) has increased slowly from the point of wind speed is 5 m/s (the rotation number is 1400) in the case of Wadi (DF and DF2) and from the point of wind speed is 7 m/s (the rotation number is 1600) in Dry Lake beds. In terms of wind speed, 1400 rpm is about 5 m/s and 1600 is 7 m/s at 1 cm from ground surface. In both 3 μm and 5 μm, the amount of dust sharply increases from 7 m/s (1600 rpm) in dry lake beds and Wadi. According to the results from the field experiments of topsoil particles saltation, the soil particles on the ground surface in the Gobi desert area were emitted when the wind speed reaches 5 m/s (1400 rpm), and the number of soil particles that are saltation increase abruptly at 7 m/s (1600 rpm). When the wind speed rises to 9 m/s (1800 rpm), clearly a dust storm occurred. The critical wind speed at which dust emission on the ground surface is 7 m / s.

Laboratory result of microorganisms adhering to the topsoil particles
It is expected that these bacteria will fly at the time of dust generation, and Escherichia coli (DH 5α) was used as a model of bacteria in this area to be domesticated grazed. Imaging of the sea sand attached with E. coli showed no remarkable fluorescence emission by fluorescence microscopy, but the observation of FE-SEM clearly showed adherent of E. coli on the surface (see Fig. 5).  In Raman imaging, we could observe areas on the grains where the Raman spectra were different from the Raman spectrum of pure sea sand, but it is not clear whether this change is due to microorganisms. Fig. 6A shows the Raman spectrum of sea sand only; B shows the spectrum of a sample which adsorbed E. coli on the surface of sea sand. Two broad Raman peaks at around 400 cm -1 and 1600 cm -1 can be seen, which indicate organic material. Image of Mongolian soil particles were observed by fluorescence microscope and FE-SEM in Fig. 7. The fluorescence microscope images of Gobi desert soil of Mongolia country showed some fluorescing areas.
Observation of microorganisms adhered to the particles of the Gobi desert sample in Mongolia was only slightly binding, but from the observation of E. coli attaching the sample together with sea sand, we presume that the observation of attached microorganisms is, in principle, possible. However, because the control sample contained bacteria in a high number, and desert samples should contain just a few bacteria per grain of sand, we consider that it is difficult to observe them with FE-SEM at the present state. Using fluorescent-labelled bacteria would make it possible to first identify the bacteria in the fluorescence microscope and then image the same area in an FE-SEM.

Conclusions
The result of field experiment of soil particles saltation shows, in the critical wind speed when the dust emission on the ground surface of Gobi is 7 m/s. The result of laboratory experiment of imaging of microorganisms on topsoil particles collected from different landscape in the Gobi Desert shows, it is reported that bacteria are adhering to dust derived from Mongolia, but the adhesion efficiency may not be high. In Gramnegative bacteria like E. coli, there is a difference in adhesion to sand, but it may be necessary to study with different strains such as Gram-positive bacteria. The bacterial charge is also important when adhering to dust. It is inferred that the charge state of the dust component also affects the adhesion.
Since it was impossible to observe cells using samples this time, we will aim at future work to observe microorganisms attached to desert soil particles by locating conditions under which microorganisms are easy to observe, such as fluorescence-labelled samples.