International Proceedings of Chemical, Biological and Environmental Engineering, Vol. 99 (2016) DOI: /IPCBEE V99. 7

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1 International Proceedings of Chemical, Biological and Environmental Engineering, Vol. 99 (2016) DOI: /IPCBEE V99. 7 Enhancement of Productive Ability of Soil and Establishment of Concept of Soil Immunity Using the Change of Distribution of Aerobic and Anaerobic Soil Bacteria Alberta Yoo, Eun Soo Lee, and Daniel Kim Yongsan International School of Seoul Abstract. As a result of culturing aerobic soil bacteria and anaerobic soil bacteria in 4 types of soil, while more types of aerobic bacteria exist than that of anaerobic bacteria in healthy soil, the types of aerobic bacteria decreased and anaerobic soil bacteria increased in huge rate in the soil near apartment or the soil contaminated with heavy metal. The growth of aerobic soil bacteria decreased greatly in contaminated soil. The growth of E.coli stimulated according to distribution of aerobic soil bacteria and anaerobic soil bacteria in contaminated soil. Throughout the confirmation of the existence of soil bacteria that lives in human skins and suppress the proliferation of other harmful bacteria and the effect of growth of this soil bacteria on growth of plants, resistance to harmful bacteria, the concept of soil immunization was defined. As a result of cultivating plants in the soil samples, the accuracy of soil immunization was checked, and the aerobic soil bacteria adapted to toxins of anaerobic soil bacteria was able to improve the soil not suitable for growth of the plants. Keywords: aerobic soil bacteria, anaerobic soil bacteria, contaminated soil, soil immunization 1. Introduction In case of soil containing growing plants, certain amount of time and various experimental methods such as analyzing the soil component and content of heavy metal should be used in order to check the condition of the soil [1]. This research was conducted to abbreviate the method to check the condition of the soil and enhance the efficiency of the process [2]. All living organisms have resistance to things that can have negative effect on them, and this process is called immunity [3]. For this research, the composition of aerobic and anaerobic soil bacteria in soil was defined as the immune system of the soil. If this composition can be differed according to the composition of the soil, it can be confirmed and defined as the immune system the soil has [4]. When the composition can be modulated, setting the standard according to contamination of soil caused by harmful bacteria such as E.coli and the contamination caused by different elements like heavy metals is possible [5]. Therefore, the hypothesis of this experiment is that enhancing the productive ability of soil is possible by applying the method to enhance the immunity of soil. The experiment was divided into two parts: confirming the hypothesis on soil immunity and enhancing the plants production using the first part [6]. First, in order to prove the purpose and necessity of this experiment, the hypothesis on soil immunity was verified. 2. Methods 2.1. Production of Nutrient Agar Media for Soil Bacteria Culture 23g of Difco Nutrient Agar (Component: Beef Extract 3.0 g, Peptone 5.0 g, Agar 15.0 g) powder purchased from Becton Dickinson Korea Inc. was measured with electronic scale and was added into 2L erlenmeyer flask. Steel spoon should be used for the powder, and pyrex glass flask must be used because Corresponding author. Tel.: address: 01357asd@gma.com 50

2 regular erlenmeyer flask could be damaged when it was put into autoclave. Glass graduate was used to add 1L of deionized water in the erlenmeyer flask containing NA media powder. Magnetic bar was put into the flask. After the flask was sealed with aluminum foil, the tape that black line appeared after sterilization was attached on the flask. The flask was put onto a magnetic stirrer and let the magnetic bar spin so that deionized water and the powder could be mixed. After minutes of stirring, the flask was put into an autoclave, and the mixture was sterilized in 121 and 2 atm for 15 minutes. The flask was taken out from the autoclave, and whether black line appeared was checked. The flask was put onto a magnetic stirrer again and let the magnetic bar spin to that NA media can be cooled down evenly. If this is cooled down in room temperature, NA media can be solidified unevenly and cause the difference in creation of media. The UV lamp was turned on to protect the media poured in dishes from contamination caused by microorganisms during the solidification of NA media. When NA media becomes complete semisolid, the caps of the petri dishes were closed. Each 10 dishes were wrapped with plastic wrap, and was stored in 4 before use in order to prevent contamination. The dishes should be stored upside down so that the water drop caused by congelation would not touch the media. NB media (component: Beef Extract 3.0 g, Peptone 5.0 g), purchased from Becton Dickinson Korea Inc., is a media for liquid culture, as it does not contain agar component. 8g of NB media powder was mixed with 1L of deionized water, and the mixture was sterilized in autoclave. The sterilized media was poured into 50mL conical tubes and was stored in 4 before use Separation Culture of Anaerobic and Aerobic Soil Bacteria from Soil 3g of soil and 10mL of deionized water were mixed, and the mixture was put into 15mL conical tubes. Centrifugation was done with the speed of 6000 spins per minute to collect the supernatant. 5 μl of collected supernatant was injected into NA media using micropipette. Aerobic soil bacteria was cultured in regular incubator, and anaerobic soil bacteria was cultured in the incubator where CO 2 can be provided inside Growth Change of Aerobic Soil Bacteria Separated from Healthy Soil and Cultured in Contaminated Soil After contaminated soil (Apartment, heavy metal) was put into an autoclave and was sterilized so that all microorganism inside were deceased, 100 μl of NB media containing aerobic soil bacteria of healthy soil was injected into the sterilized contaminated soil. The sample was stored in a room temperature for a week to culture aerobic soil bacteria. The same experiment was repeated with the sterilized healthy soil as a control. The experiment was repeated with anaerobic bacteria Resistance Change of Aerobic Soil Bacteria Grown in Contaminated and Healthy Soil to Hydrogen Peroxide Each type of aerobic soil bacteria was separated from the healthy soil and was cultured. Platinum loop was used to collect the colonies of the soil bacteria, and the bacteria was injected NA media for separation culture of each type. Cultured bacteria was injected into contaminated soil and let the bacteria grow. After the bacteria was separated and cultured again, it was cultured in 3mL of NB media containing 100 μl of 3% hydrogen peroxide solution. The absorbance was measured in 630nm of UV length using UV- Spectrophotometer, and the change of growth was observed. (The soil was sterilized before the experiment) 2.5. Anaerobic Soil Bacteria Creating Toxin Anaerobic soil bacteria was cultured in NB media with the same method of Experiment 3. NB media where anaerobic soil bacteria was removed using 0.2 μm was prepared. Aerobic soil bacteria of Experiment 4 was injected into this NB media and was cultured. The absorbance was measured in 630nm, and the growth change was observed Pollution of Plants and Growth of E.coli in Contaminated Soil and Healthy Soil NB media containing cultured E.coli was injected into the soil used for the experiment, and the cabbage seeds were planted to observe the contamination of soil and cabbage by E.coli. 51

3 2.7. Change of Anaerobic Soil Bacteria when Aerobic Soil Bacteria Whose Resistance to Active Oxygen had decreased and Normal Aerobic Soil Bacteria were Injected into Contaminated Soil and Normal Soil 1g of normal soil and 10mL of deionized water were put into a 15mL conical tube. Centrifugation was done with the speed of 6000 spins per minute to separate the supernatant containing soil.bacteria, and with micropipette, 5 μl of NA media was injected and was smeared with a spreader. It was cultured in 28 incubator for 12 hours, and when the colonies of aerobic soil bacteria were checked, the colonies were collected with platinum loop. In order to separate the bacteria by each type, Each type of colonies were cultured in NA media. After the normal soil and the soil containing heavy metal were sterilized, aerobic soil bacteria that was separated and cultured was injected into each soil sample. After 7 days, the supernatant of the soil was separated again and was cultured in 3mL of NB media. 100 μl of 3% hydrogen peroxide solution was injected into the media, and the absorbance was measured in 630nm to observe the growth change. Anaerobic soil bacteria was cultured with the same method, and the changes of aerobic and anaerobic soil bacteria were compared Toxicity of Anaerobic Soil Bacteria in the Soil Containing Aerobic Soil Bacteria who s Resistance to Oxygen Decreased 1g of normal soil was collected and put into 15mL conical tube with 5mL of sterilized distilled water. Centrifugation was done with the speed of 6000 spins per minute to collect the supernatant containing soil bacteria. 5 μl of the supernatant was injected into NA media and was smeared with a spreader. Gas Pak-EZ Anaerobic Container System was used to culture the bacteria in 28 incubator under anaerobic condition. When the colonies of anaerobic soil bacteria were checked, the colonies were collected with platinum loop and was injected into 15mL conical tube containing 3Ml of NB media. The sample was cultured in 28 shaking incubator. With UV-spectrophotometer, the absorbance was measured in 630nm to check the culture state of anaerobic soil bacteria. The bacteria was injected into NA media again, and the separation culture of each type was confirmed. After this was injected into normal soil and the soil contaminated with heavy metal, the samples were stored in room temperature for a week. The soil supernatant was separated following the process above to culture anaerobic soil bacteria, and NB media where each type of anaerobic soil bacteria was cultured were penetrated through 0.2 μm filter to remove all anaerobic soil bacteria. After normal soil bacteria was injected into these NB media, it was cultured in 28 shaking incubator for 12 hours. The absorbance was measured in 630nm to check the toxicity of anaerobic soil bacteria throughout the growth change of aerobic soil bacteria Change of Aerobic Soil Bacteria with Reduced Resistance to Oxygen According to Temperature After NB media, where normal aerobic soil bacteria and aerobic soil bacteria whose ability to endure oxygen decreased by toxin anaerobic soil bacteria produces in the soil contaminated by heavy metal, was injected into sterilized soil, the samples were stored in 20, 28, 40. 1g of soil was collected and put into 15mL conical tube, and 5mL of sterilized distilled water was mixed. Centrifugation was done with the speed of 6000 spins per minute for 10 minutes to collect the supernatant containing soil bacteria. 5 μl was injected into NA media and was smeared with a spreader. After 5 μl was injected into 3mL of NB media, it was cultured in 28 shaking incubator for 12 hours, and the absorbance was measured in 630nm to observe the growth change of aerobic soil bacteria Method to Strengthen Aerobic Soil Bacteria Using the Toxin of Anaerobic Soil Bacteria Among culture media containing toxin of anaerobic soil bacteria from Experiment 8>, all media that show severe toxicity were all mixed, and NB media was also added to dilute the solution into the concentration of 1/ μl of NB media containing normal aerobic bacteria and NB media containing aerobic bacteria with reduced oxygen-enduring ability was injected, and the samples were cultured in room temperature for 12 hours. After the culture, the absorbance was measured to check the growth of aerobic soil bacteria, and this was injected into culture media of anaerobic bacteria diluted into concentration of 1/90-1/10 to select the aerobic soil bacteria group that can endure the toxins produced by anaerobic soil bacteria. 52

4 2.11. Reaction to Hydrogen Peroxide and Condition Change of Aerobic Soil Bacteria Enduring the Toxins of Anaerobic Soil Bacteria After NB media containing cultured aerobic bacteria produced to endure the toxins of anaerobic soil bacteria was injected in 3mL of NB media, 100 μl of 3% hydrogen peroxide solution was injected, and the bacteria was cultured in 28 shaking incubator for 12 hours. The absorbance was measured in 630nm with UV-Spectrophotometer to observe the change of growth. Then the experiment was conducted based on the experiment conditions above Change of Plants Grown in the Soil Contaminated with Heavy Metal and Growth Change of Plants Grown in Contaminated Soil after the Injection of Aerobic Soil Bacteria and Diluted Anaerobic Soil Bacteria Solution (Starch Combination and DNA Change) Diluted solution(1/50) of toxins of aerobic soil bacteria was injected to 3g of soil, and after 100 μl of NB media containing aerobic soil bacteria whose vitality increased by toxins of anaerobic soil bacteria was injected into the soil, arabidopsis seeds were planted into the soil. The arabidopsis was grown for a week. After a week, the leaves were collected and were put into liquid nitrogen to grind the leaves. Grinded leaves were put into 5 micro tubes. 500uL of solution of 99% ethanol and formic acid in ratio of 2 to 8 was added in and was treated in 80. The caps were opened in order to prevent tubes from bursting. After ethanol was evaporated for 1 hour, 500uL of deionized water was added in, and they were mixed for 20 minutes. Centrifugation was done with the speed of 14,000 rpm for 5 minutes. The supernatant was separated and was stored in uL of deionized water was added again, then the mixture was put into an autoclave and was sterilized for 3 hours. 450uL of α-amylase (1U) 4ul + α-glucosidase (1U) 7ul + 0.1M NaoAc (ph 4.8) was added into 50uL of supernatant, and after each enzyme was added, the absorbance was measured in 520nm Change of Contamination of Plants and Soil by E.coli in Sterilized Soil after the Injection of Aerobic Soil Bacteria Adapted to Toxins of Anaerobic Soil Bacteria The soil containing aerobic soil bacteria adapted to toxins of anaerobic soil bacteria was put into an autoclave and was sterilized in 121 and 2atm. After sterilization, it was moved to clean bench, and 100 μl of NB media containing cultured E.coli was injected. The soil sample containing NB media was stored in a room temperature for a week. Based on the prior experiments, the soil supernatant from the soil sample was collected and cultured to observe the growth change of E.coli. The experiment was repeated with contaminated soil and the normal soil Confirmation of Soil Immunity in Different Contaminated Soils and Analysis on the Effect of Injection of Aerobic Bacteria Adapted to Toxins of Anaerobic Soil Bacteria on Growth of the Plants 6 samples of soil were collected from 6 certain locations and was stored in 4 before the experiment. 0.1g of each sample was put into each 1.5mL micro tubes. 1mL of deionized water was added into each tube. Centrifugation was done with the speed of 6000 rpm to separate the supernatant containing soil bacteria. 5 μl of separated supernatant was injected into NA media and was cultured for 12 hours in 28 incubator. Following the method of prior experiments, anaerobic culture was conducted after the 5 μl of separated supernatant was injected into NA media to culture anaerobic soil bacteria. 5 μl of separated supernatant was injected into 3mL of NB media and was cultured in 28 shaking incubator to culture aerobic soil bacteria. Following the method of prior experiments, anaerobic soil bacteria was cultured, and with the use of UV- Spectrophotometer, the absorbance was measured in 630nm to compare the growth of aerobic and anaerobic soil bacteria. 5g of each soil sample was put into each small petri dish, and cabbage seeds were planted in. The dishes were put into airtight plastic container, and the growth of cabbages were observed. In the soil considered to be not suitable to grow plants by the decrease of soil immunity throughout the experiment from 1)-5), the aerobic soil bacteria from Experiment 13, the bacteria adapted to toxins of anaerobic bacteria, was injected, and the experiment from 1)-5) was repeated to observe the change of soil and growth of the plants. 3. Results 53

5 3.1. Production of Nutrient Agar Media for Soil Bacteria Culture Fig. 1: NA media and NB media produced with autoclave NA and NB media, media that can culture most types of bacteria including soil bacteria and E.coli, were produced and stored in 4 before use. They can be used in 1-2 months, and old media should be discarded due to the possibility of contamination Separation Culture of Anaerobic and Aerobic Soil Bacteria from Soil Fig. 2: Culturing soil bacteria in anaerobic and aerobic condition As a result of culturing aerobic and anaerobic soil bacteria in 4 types of soil, in healthy soil where plants can grow, more types of aerobic soil bacteria existed than those of anaerobic soil bacteria. However, in case of soil contaminated with heavy metal and the soil near apartment which has possibility of contamination, the types of aerobic soil bacteria decreased drastically, and the types of anaerobic bacteria increased greatly. It is normal for soil bacteria that fixes nitrogen and provides inorganic nutrients to plants grows under anaerobic condition, soil itself is exposed to aerobic environment, and in order to overcome this, most of the soil bacteria related to nitrogen fixation and carbon cycle has ability to endure aerobic condition. Throughout the experiment, it was found that the healthy soil and contaminated soil showed difference in types and distribution of aerobic bacteria and anaerobic bacteria Growth Change of Aerobic Soil Bacteria Separated from Healthy Soil and Cultured in Contaminated Soil. Fig. 3: Change of aerobic and anaerobic soil bacteria in contaminated soil and healthy soil 54

6 As a result, in case of aerobic soil bacteria, the growth greatly decreased in contaminated soil, but the growth of anaerobic bacteria did not show significant difference between that in contaminated soil and healthy soil. This result showed that the aerobic soil bacteria could be the standard of immunity improvement Resistance Change of Aerobic Soil Bacteria Grown in Contaminated and Healthy Soil to Hydrogen Peroxide Fig. 4: Resistance change of aerobic soil bacteria in soil contaminated with heavy metal to hydrogen peroxide Despite the same type of aerobic soil bacteria, when the bacteria was grown in contaminated soil, the resistance on hydrogen peroxide decreased in great amount. In other word, when aerobic soil bacteria was exposed to contaminated soil, its ability to resist on active oxygen greatly decreased. Fig. 5: Growth relationship between aerobic soil bacteria and generated substance of anaerobic soil bacteria 3.5. Anaerobic Soil Bacteria Creating Toxin 55

7 Anaerobic soil bacteria excretes different substances while growing, and when anaerobic bacteria grown in normal soil was left in soil polluted with heavy metal, the excretion of toxin increased, thus suppressing the growth of aerobic soil bacteria. The toxin is thought to be generated because anaerobic bacteria produces different toxins in soil such as hydrogen sulfide and methane gas Pollution of Plants and Growth of E.coli in Contaminated Soil and Healthy Soil The growth of plants decreased abnormally in contaminated soil, possibly because the nitrogen, whose provision is directly related to soil bacteria, could not be provided well. In case of contaminated soil, the proliferation of E.coli was so active that the plants grown in the soil were severely contaminated by E.coli as well. Fig. 6: Changes of growth of plants in different types of soil Fig. 7: Change of growth of E.coli in different types of soil 3.7. Change of Anaerobic Soil Bacteria when Aerobic Soil Bacteria whose Resistance to Active Oxygen had Decreased and Normal Aerobic Soil Bacteria were Injected into Contaminated Soil and Normal Soil As a result of the experiment, when aerobic bacteria whose resistance to active oxygen had decreased was injected into the soil, in both normal soil and the soil contaminated with heavy metal, the number of aerobic soil bacteria decreased in great amount. Compared to the number of types of anaerobic soil bacteria contained in the soil where aerobic soil bacteria whose resistance to oxygen decreased was injected, the number of types of anaerobic soil bacteria in the soil where normal aerobic soil bacteria was injected into was reduced. This shows that aerobic soil bacteria whose resistance to oxygen was reduced has lower ability to living in the soil or has weaker resistance to different toxins anaerobic soil bacteria produces. The number of anaerobic bacteria increased in a great amount when the bacteria was in the soil where aerobic soil bacteria whose resistance to oxygen decreased was injected. On the other hand, the number of 56

8 anaerobic bacteria decreased when the bacteria was contained in the soil where normal aerobic soil bacteria was injected. In conclusion, when aerobic soil bacteria whose resistance to oxygen decreased was injected into the soil, proliferation in the soil was suppressed, while the number and types of anaerobic soil bacteria greatly increased. Fig. 8: Changes of aerobic soil bacteria after aerobic soil bacteria, whose resistance to active oxygen has decreased, was injected into the soil Fig. 9: Growth change of anaerobic bacteria after the injection of aerobic soil bacteria whose resistance to active oxygen has decreased (NA media) Fig. 10: Growth change of anaerobic bacteria after the injection of aerobic soil bacteria whose resistance to active oxygen has decreased (NB media) 3.8. Toxicity of Anaerobic Soil Bacteria in the Soil Containing Aerobic Soil Bacteria whose Resistance to Oxygen Decreased 57

9 Fig. 11: Colonies of anaerobic soil bacteria grown in normal soil Fig. 12: Separation culture of anaerobic soil bacteria by type Fig. 13: Change of toxicity of anaerobic soil bacteria in contaminated soil and normal soil As a result of comparing toxicity of 32 types of anaerobic soil bacteria separated from the soil where normal aerobic soil bacteria or aerobic soil bacteria whose resistance to oxygen decreased was injected in, the toxicity of anaerobic soil bacteria in the soil whose resistance to oxygen was reduced showed big amount of increase. Therefore, it was checked that when aerobic bacteria cannot grow and proliferate normally in the soil, the growth of toxin is encourage, which causes the negative influence on plants growing in the soil Change of Aerobic Soil Bacteria with Reduced Resistance to Oxygen According to Temperature Fig. 14: Growth change of aerobic bacteria, whose resistance to oxygen was reduced, according to the temperature change 58

10 As a result, in case of aerobic bacteria whose ability to endure oxygen was reduced, the bacteria grew normally in 28, the normal growth temperature, but in condition of relatively high or low temperature, its number and type greatly decreased. Therefore, it was confirmed that aerobic bacteria whose ability endure oxygen was reduced showed weaker surviving ability Method to Strengthen Aerobic Soil Bacteria Using the Toxin of Anaerobic Soil Bacteria As a result of culturing the toxins produced by anaerobic soil bacteria by concentrations, all aerobic soil bacteria were deceased in the concentration higher than 1/40, and the aerobic bacteria which can endure the toxin concentration of 1/50 was able to be produced. However, the toxins of this experiment was very highly concentrated; as the concentration that actually exist in real soil was much lower than the concentration from the experiment, the aerobic soil bacteria produced from this experiment is expected to resist to toxic material produced by anaerobic bacteria in soil. Fig. 15: Adaptation of aerobic soil bacteria on toxic substances of anaerobic soil bacteria Fig. 16: Changes of colonies of aerobic soil bacteria before and after adaptation to toxins of anaerobic soil bacteria Throughout the experiment, the aerobic soil bacteria that can endure the toxin of anaerobic soil bacteria diluted into the concentration of 1/40 was confirmed to have no differences in types and number from that before adaptation to toxins. In other words, aerobic soil bacteria adapted to toxins was expected to show effect enough to be added into the soil, as the number and types of the bacteria was not reduced Reaction to Hydrogen Peroxide and Condition Change of Aerobic Soil Bacteria Enduring the Toxins of Anaerobic Soil Bacteria Fig. 17: Resistance change of aerobic soil bacteria adapted to toxins of anaerobic soil bacteria to hydrogen peroxide 59

11 The aerobic bacteria, which can endure the toxins of anaerobic soil bacteria, produced from the experiment did not show the resistance reduction to oxygen, and it was able to function normally in other different portions as well. When aerobic soil bacteria adapted to toxins of anaerobic soil bacteria was injected into the soil polluted by heavy metal, the number and types of aerobic soil bacteria was able to be recovered to the amount in normal soil even in contaminated condition (A), and the number of anaerobic bacteria decreased greatly (B). When this result is compared to the result that the number and types of aerobic soil bacteria that did not adapt to toxin of anaerobic soil bacteria decreased greatly when it was injected into contaminated soil (C) and the number of anaerobic soil bacteria increased dramatically, this shows that aerobic soil bacteria adapted to toxins of anaerobic soil bacteria was strengthened enough to survive and proliferate even in contaminated soil. Fig. 18: Distribution change of aerobic and anaerobic soil bacteria after aerobic bacteria adapted to toxin of anaerobic soil bacteria was injected into contaminated soil Change of Plants Grown in the Soil Contaminated with Heavy Metal and Growth Change of Plants Grown in Contaminated Soil After the Injection of Aerobic Soil Bacteria and Diluted Anaerobic Soil Bacteria Solution (Starch Combination and DNA Change) After leaves of arabidopsis were collected, put into liquid nitrogen, and grinded, RNA of the laves was extracted, and this RNA was converted into cdna with the use of RT-PCR kit to compare and analyze the occurrence difference of TPS1, cab1, SPase, and TUB gene. TPS1 is a gene related to stress of plants, cab1 is a gene related to photosynthesis, SPase is a gene related to sucrose synthesis, and TUB is a standard of gene occurrence. The experiment fails if same amount of this gene does not appear in every condition. As a result, in case of arabidopsis grown in the soil contaminated with heavy metal, the content of glucose increased in great amount, and the arabidopsis in normal condition had relatively low content. In other word, the plants grown in contaminated soil encountered problems in metabolism, causing the increase of glucose concentration which should maintain low level. However, when aerobic soil bacteria adapted to toxin of anaerobic soil bacteria was injected into the soil, the glucose content decreased to the level of the glucose content of normal arabidopsis. Conclusively, it was confirmed that aerobic soil bacteria adapted to toxins of anaerobic soil bacteria helps both soil and plants stay healthy. Sucrose is a material that plants produce in order to decompose starch produced through photosynthesis and transform into other material. Plants should maintain minimum certain concentration of sucrose unlike glucose, in order to conduct normal metabolism. Arabidopsis grown in contaminated soil showed low sucrose concentration; on the other hand, when aerobic soil bacteria adapted to toxins of anaerobic soil bacteria was injected into the contaminated soil, the arabidopsis was able to maintain the normal sucrose concentration. Starch is a material plants produce through photosynthesis, and as it is synthesized at night and decomposed to become other material in the daytime, its amount decreased in the daytime and increased at night. This pattern could not be maintained in case of arabidopsis grown in contaminated soil, and the content of starch was relatively high; this result shows that the plants cannot metabolize, producing and decomposing starch for function and growth of plants, well enough. However, when the aerobic soil bacteria adapted to toxins of anaerobic soil bacteria was injected into the contaminated soil, the ability to produce and decompose starch was recovered. 60

12 In case of gene expression as well, when aerobic soil bacteria adapted to toxins of anaerobic soil bacteria was injected into the contaminated soil, expression of TPS1, gene related to stress, decreased. This shows that arabidopsis exposed to stress by the contaminated environment was able to recover its stress level by the injection of aerobic soil bacteria adapted to toxins of anaerobic soil bacteria. Furthermore, as expression of cab1, gene related to photosynthesis, increased, it was confirmed that photosynthesis efficiency increased. The expression of SPase, gene synthesizing sucrose, increase, the result showing that the injection of aerobic soil bacteria adapted to toxins of anaerobic soil bacteria recover the metabolism of plants. In conclusion, injection of aerobic soil bacteria adapted to toxins of anaerobic soil bacteria was confirmed to help enhance the immunity of soil and also help plants grow normally even in a negative condition. A: Arabidopsis grown in the soil contaminated with heavy metal B: Arabidopsis grown in the soil, contaminated with heavy metal, containing aerobic soil bacteria adapted to toxins of anaerobic soil bacteria C: Arabidopsis grown in normal soil where all aerobic soil bacteria was removed D: Arabidopsis grown in normal soil containing aerobic soil bacteria adapted to tosins of anaerobic soil bacteria Fig. 19: Metabolism change of plants grown in the soil containing aerobic soil bacteria adapted to toxins of anaerobic soil bacteria 61

13 A: Arabidopsis grown in soil contaminated by heavy metal B: Arabidopsis grown in contaminated soil where aerobic soil bacteria adapted to toxins of anaerobic soil bacteria was injected in C: Arabidopsis grown in normal soil where all aerobic soil bacteria was removed D: Arabidopsis grown in normal soil where where aerobic soil bacteria adapted to toxins of anaerobic soil bacteria was injected in Fig. 20: Change in Gene Expression Related to Stress and Metabolism of Plants after the Injection of Aerobic Soil Bacteria Adapted to Toxins of Anaerobic Bacteria into the Soil Contaminated with Heavy Metal Change of Contamination of Plants and Soil by E.coli in Sterilized Soil After the Injection of Aerobic Soil Bacteria Adapted to Toxins of Anaerobic Soil Bacteria Fig. 21: Change of E.coli proliferation after the injection of aerobic soil bacteria adapted to toxins of anaerobic soil bacteria into contaminated soil E.coli was not able to grow in the soil where aerobic soil bacteria adapted to toxins of anaerobic soil bacteria was injected. On the other hand, E.coli proliferated in great amount in the soil without aerobic soil bacteria. This is the same principle as vaccination; the leftover of aerobic bacteria stimulated the growth of aerobic soil bacteria, thus suppressing the growth of E.coli Confirmation of Soil Immunity in Different Contaminated Soils and Analysis on the Effect of Injection of Aerobic Bacteria Adapted to Toxins of Anaerobic Soil Bacteria on Growth of the Plants Fig. 22: Culture of aerobic and anaerobic soil bacteria in 6 types of soil samples (NA) As a result of separating and culturing the aerobic and anaerobic soil bacteria in 6 types of soil, the soil from A and D showed lower number of anaerobic soil bacteria colonies compared to aerobic soil bacteria 62

14 colonies, and these types of soil were the types considered to be suitable for growth of the plants based on the soil immunization addressed from this experiment. On the other hand, in case of other types of soil (B, C, E, F), the number of colonies of anaerobic soil bacteria was lot higher than that of aerobic soil bacteria, and these types of soil were assumed to be not suitable for plants growth according to soil immunization. Fig. 23: Culture of aerobic and anaerobic soil bacteria in 6 soil samples (NB) In order to check the total number of aerobic and anaerobic soil bacteria in 6 types of soil, those bacteria were cultured in NB media. As a result, the soil from A and D had a lot less number of anaerobic soil bacteria compared to that of aerobic bacteria, while the other types of soil (B,C,E,F) showed opposite result. Therefore, same as the result from culturing in NA media, soil from B, C, E, F was considered to be the types that can have negative impact on growth of plants. Fig. 24: Growth of plants in 6 soil samples All types of soil except for A and D were hypothesized to be not suitable for plants growth, and the result of the experiment confirmed the hypothesis. This result shows the accuracy of the soil immunization addressed in this experiment. Fig. 25: Plants grown in 6 soil samples after the injection of aerobic soil bacteria adapted to toxins of anaerobic soil bacteria 63

15 As a result of injecting aerobic soil bacteria adapted to toxins of anaerobic soil bacteria into the soils not suitable for the growth of the plants decided by soil immunization, the number of aerobic soil bacteria in the soil recovered, thus the plants were able to grow normally in this soil. Fig. 26: Change of anaerobic and aerobic soil bacteria in 6 soil samples where plants were grown after the injection of aerobic soil bacteria adapted to toxins of anaerobic soil bacteria 4. Conclusion As a result of culturing aerobic soil bacteria and anaerobic soil bacteria in 4 types of soil, while more types of aerobic bacteria exist than that of anaerobic bacteria in healthy soil, the types of aerobic bacteria decreased and anaerobic soil bacteria increased in huge rate in the soil near apartment or the soil contaminated with heavy metal. The growth of aerobic soil bacteria decreased greatly in contaminated soil, and the growth of anaerobic soil bacteria showed differences in contaminated soil and normal soil, but it showed growth change as significant as aerobic soil bacteria. Thus, this result showed that aerobic soil bacteria are the standard of immunity improvement of soil. Even if they are the same type of aerobic soil bacteria, when it is grown in contaminated soil, the resistance to hydrogen peroxide greatly decreased. The growth of E.coli stimulated according to distribution of aerobic soil bacteria and anaerobic soil bacteria in contaminated soil. Throughout the confirmation of the existence of soil bacteria that lives in human skins and suppresses the proliferation of other harmful bacteria and the effect of growth of these soil bacteria on growth of plants, resistance to harmful bacteria, the concept of soil immunization was defined. Based on this, aerobic soil bacteria containing resistance to toxic materials of anaerobic soil bacteria was separated and applied to contaminated soil; and as a result, the immunity of soil could be enhanced. Moreover, it was found that growth change of aerobic and anaerobic soil bacteria could be the standard of soil immunity. NB media containing anaerobic soil bacteria was diluted into low concentration to induce the growth of aerobic soil bacteria that can resist to toxins, and this was cultured in NB media treated with hydrogen peroxide to culture the aerobic soil bacteria whose function was recovered. Cultured aerobic soil bacteria were injected into the soil, and the growth of aerobic soil bacteria in contaminated soil was checked. By this process, the contamination of soil caused by the harmful bacteria was prevented and the plants production of the soil was enhanced. As a result of experiment using Arabidopsis, in case of Arabidopsis grown in contaminated soil containing aerobic soil bacteria adapted to the toxins of anaerobic soil bacteria, its starch and other different substances were recovered to the level of metabolism of normal plants. Moreover, the reduction of stress provided by heavy metal pollution and improvement of photosynthesis efficiency were also confirmed throughout DNA analysis. Therefore, the aerobic soil bacteria adapted to toxins of anaerobic bacteria was help plants maintain the growth level even in contaminated environment. If the aerobic soil bacteria adapted to toxins of anaerobic soil bacteria was injected into the soil once, it could consistently protect the soil from contamination by E.coli. In order to prove the soil immunization of this research, the experiment was conducted with the soil collected from 6 random locations; the types suitable for plants were selected based on the soil immunization. As a result of cultivating plants in the soil samples, the accuracy of soil immunization was checked, and the aerobic soil bacteria adapted to toxins of anaerobic soil bacteria was able to improve the soil not suitable for growth of the plants. Throughout confirming the hypothesis of this 64

16 research, the result of the research would help soil increase the production ability of the soil not by chemical fertilizer or immense soil addition but by simple and convenient method. 5. References [1] J. S. Suh, H. J. Noh, J. S. Kwon, H. Y. Weon, and S. Y. Hong, Distribution Map of Microbial Diversity in Agricultural Land, Korean Journal of Soil Science and Fertilizer, vol. 43, no. 6, pp , [2] K. J. Park, B. C. Lee, J. S. Lee, C. S. Park, and M. H. Cho, Dominant-species Variation of Soil Microbes by Temperate Change, Korean Journal of Environmental Biology, vol. 29, no. 1, pp 52-60, [3] J. H. Joo, K. A. Hussein, S. A. Hassan, Bacteria and Fungi as Alternatives for Remediation of Water Resources Polluting Heavy Metals, Korean Journal of Soil Science and Fertilizer, vol. 44, no. 4, pp , [4] H. J. Jang, S. C. Jee, S. H. Kang, J. S. Sung, Biocontrol of Red Clay Soil Processed Nano Material against Phytopathogenic Bacteria: A. tumefaciens, E. carotovora, X. campestris, Korean Society of Mycology News Letter, vol. 25, no. 2, pp , [5] N. H. Moon, K. Kim, S. H. Choi, Isolation of Soil Bacteria Secreting Raw-Starch-Digesting Enzyme and the Enzyme Production, Journal of Microbiology and Biotechnology, vol. 3, no. 2, pp , [6] T. M. Sa and P. S. Chauhan, Research Trends on Plant Associated Beneficial Bacteria as Biofertilizers for Sustainable Agriculture: An Overview, Korean Journal of Soil Science and Fertilizer, vol. 42, no. 2, pp ,