WO2021050028A1

WO2021050028A1 - Biological improvement method of sand soils using viridibacillus arenosi k64 bacteria

Info

Publication number: WO2021050028A1
Application number: PCT/TR2020/050826
Authority: WO
Inventors: Semet ÇELİK; Özlem BARIŞ; Harun AKOĞUZ
Original assignee: Atatürk Üni̇versi̇tesi̇ Bi̇li̇msel Araştirma Projeleri̇ Bi̇ri̇mi̇
Priority date: 2019-09-12
Filing date: 2020-09-10
Publication date: 2021-03-18

Abstract

The invention relates to biologically improvement method of a sand soil applied by using the Viridibacillus arenosi bacteria, comprising the following process steps: (i) preparing the liquid growth culture, (ii) cultivating the Viridibacillus arenosi bacteria culture to the liquid growth medium, (iii) shaking the liquid growth medium cultivated at the determined time and an agitation speed, (iv) determining the hydraulic conductivity of the soil in which the application will be performed, (v) applying the culture medium to the soil at the flow rate determined according to the hydraulic conductivity of the soil, (vi) applying the urea and calcium chloride solution to the soil at the determined time and flow rate.

Description

Biological Improvement Method of Sand Soils Using Viridibacillus arenosi K64

Bacteria

Technical Field

The invention relates to soil improvement applications used in the geotechnical field in civil engineering.

In particular, the invention relates to a biologically improvement sand soil with culture medium comprising the bacteria Viridibacillus arenosi and improvement method.

The State of the Art

The biological improvement technique of soils that has emerged in recent years may be performed by microorganisms different from other techniques. With this new approach performed by microorganisms, several features of the soils may be improved. Being more environmentalist than other improvement techniques and being a new subject have increased the studies in this field.

In many studies present in the literature, silica sand and Bacillus pasteurii bacteria have been used. Other than the literature, Viridibacillus arenosi bacteria were used in this study.

In the literature, the following applications with respect to the subject were found:

In US8182604 B2, biological improvement was carried out on silica sand soil passing through 0.300 mm. During this improvement application, a flow rate of 15 mL/min was applied and the unconfined compressive strength value of this sample was found as 1.7 MPa. The relative density of the soil was not specified and the strength value obtained in the result of the application was given. In the application we performed, commercial silica sand with a relative density of 35% was applied on the sand soil and it was determined that the particle size of the soil is important in application by applying different flow rates. A unconfined compressive strength of 2.4 MPa was obtained on the sample soil. In the studies carried out within the scope of the invention, a bacterium that was never studied in the literature was used.

In US20180119185 A1 , in the applications performed on the soils, the culture medium, urea and calcium chloride were given by filtration (with the effect of gravity). In the studies carried out within the scope of the invention, this deficiency in the present patent was eliminating by specifying the flow rate. In the current patent, values between 133 kPa and 1817 kPa were obtained in the unconfined compressive strengths. In the result of our application carried out with two different sand soils, the unconfined compressive strength values were determined as 2406 kPa on the first sand soil and 1504 kPa on the second (river sand) sand soil. This increase in strength is thought to be due to the difference in bacteria and application.

Another invention is patent application No US20110027850 A1. In this application, the relationship between depth and formation of calcium carbonate was investigated. Calcite formation according to depth is an indicator of biological improvement, but the calcite grains formed must attach the soil grains together. Calcite grains that cannot attach to the soil grains contribute to the reduction of permeability, but do not have much effect on strength. In the studies carried out within the scope of the invention, the effect of the calcite percentages on the improvement was evaluated with the strength results.

The main purpose in soil improvement is to increase the strength of the soil and to decrease its permeability. In the current patent studies, parameters affecting the process such as biological activities (urea activity, ammonium concentration etc.), calcite percentages were followed. In studies carried within the scope of the invention, priority was on strength and permeability. In this context, the experiments were varied and successful results could be obtained. In the present patent studies, it is not evaluated in the application that the soils need different application method due to the grain diameters. These deficiencies were eliminated by the method of the invention. In addition, different bacteria we used affected our results.

In conclusion, it has been necessary to make a development in the technical field due to the above-mentioned negativities and the inadequacy of the present solutions about the subject.

Brief Description of the Invention

The present invention relates to a biologically improved sand soil and improvement method meeting the above-mentioned requirements, eliminating all the disadvantages and bringing some additional advantages.

The primary object of the invention is to increase the strength of the sand soil to be improved and to decrease the hydraulic conductivity. An object of the invention is to provide improvement in the application method which will be prepared in accordance with the hydraulic conductivity of the soil by using chemicals both in a shorter time and more effectively.

Another object of the invention is to obtain higher strength values than the present applications by using a different bacteria and method.

Another object of the invention is to form a biologically improved sand soil by using the culture medium containing Viridibacillus arenosi bacteria.

Sand soil improvement method developed to perform the objects described above comprises the following process steps: i. Preparing the liquid growth culture, ii. Cultivating the Viridibacillus arenosi bacteria to the liquid growth medium, iii. Shaking the liquid growth medium cultivated at the determined time and agitation speed, iv. Determining the hydraulic conductivity of the soil in which the application will be performed, v. Applying the culture medium to the soil at the flow rate determined according to the hydraulic conductivity of the soil, vi. Applying the urea and calcium chloride solution to the soil at the determined time and flow rate.

The structural and characteristic features and all of the advantages of the invention will be more clearly understood by means of the figures given below and the detailed description written with reference to these figures and therefore, evaluation should be performed by taking these figures and detailed description into account. Drawings Assisting to the Understanding of the Invention

Figure 1, is the view of the particle size distribution curve of the sand soils applied by the method of the invention.

The drawings do not necessarily have to be scaled, and details that are not required to understand the present invention may have been neglected. Furthermore, elements that are at least substantially identical or at least have substantially identical functions are indicated by the same number.

Detailed Description of the Invention

In this detailed description, the sand soil improvement method according to the invention is described only for a better understanding of the subject and without any limiting effect.

The sand soil improvement method of the invention comprises the following process steps: i. Preparing the liquid growth culture, ii. inoculating the Viridibacillus arenosi bacteria to the liquid growth medium, iii. Shaking the liquid growth medium cultivated at the determined time and agitation speed, iv. Determining the hydraulic conductivity of the soil in which the application will be performed, v. Applying the culture medium to the soil at the flow rate determined according to the hydraulic conductivity of the soil, vi. Applying the urea and calcium chloride solution to the soil at the determined time and flow rate.

According to a preferred embodiment of the invention, the growth medium includes Tryptic Soy Broth, Urea, Manganese Sulfate Monohydrate (MnSCVHbO) and Nickel Chloride Hexahydrate (NiCI_2*6H₂0). This medium includes 30 g/L Tryptic Soy Broth, 20 g/L urea, 12 mg/L Manganese Sulfate Monohydrate (MnSO HhO) and 24 mg/L Nickel Chloride Hexahydrate (NiCI_2*6H₂0) by weight. According to a preferred embodiment of the invention, the pH of the medium is between 7 and 9 during the cultivation of Viridibacillus arenosi bacteria into the liquid growth medium. Preferably the average pH is 8.3.

According to a preferred embodiment of the invention, the growth medium average temperature is 25±5^tC.

According to a preferred embodiment of the invention, the medium temperature of the culture medium during shaking is 25± 5TT

According to a preferred embodiment of the invention, the medium temperature during applications to the soil (v. and vi. process steps) is between 15 and SSTT

According to a preferred embodiment of the invention, the pH of the culture medium taken from the shaker after 40 to 50 hours is 8 to 9 in average. Preferably, the pH of the culture medium taken from the shaker after 48 hours is 8.3 in average.

According to a preferred embodiment of the invention, in the process step (v), culture medium is applied to the soil for 15 to 30 hours at a flow rate determined by the hydraulic conductivity of the soil. Preferably, culture medium is applied to the soil for 24 hours.

Biologically improved sand soil according to the invention includes the culture medium containing the Viridibacillus arenosi bacteria, calcium chloride and urea.

According to a preferred embodiment of the invention, the improved sand soil of the invention includes an average of 0.5-2% by weight of culture medium, 3-8% calcium chloride, 3-6% urea and 84-93.5% sand. Preferably it includes an average of 1% by weight of culture medium, 5% calcium chloride, 4% urea and 90% sand.

According to a preferred embodiment of the invention, the sand soil improvement according to the invention includes 0.5-1 M calcium chloride, 1-2 M urea. Preferably, it includes 0.75 M calcium chloride, 1 .5 M urea.

According to a preferred embodiment of the invention, the relative density of the sand soil according to the invention is 25-50%. Preferably the relative density of the sand soil is 35%.

The sand soil improvement method of the invention is described in detail below:

Preparing the growth medium As the culture, 15 g Tryptic Soy Broth, 20 g Urea, 0.012 g NiCh, 0.006 g MnSC>4 are weighed with precision balance. Other chemicals except urea are dissolved in 350 mL of pure water and placed in an autoclave by mixing. Urea is dissolved in 150 mL of pure water and sterilized under aseptic environment conditions by using a 0.40 pm filter. Urea that is sterilized by a filter with a 350 mL mixture removed from the autoclave is combined under the aseptic environment conditions. The medium temperature needs to be approximately 250.

- Cultivating the bacteria to the growth medium

Bacteria taken from the petri plate are cultivated in the prepared liquid growth medium under the aseptic environment conditions. The medium temperature needs to be approximately 20- 300.

- Preparing the culture medium

Bacterial cultivation broth is cultivated is placed in the shaker set at the speed of 100-125 rpm and shaken for 40-50 hours. The medium temperature should be approximately 20- 300.

Preparing the application solution (calcium chloride and urea)

0.5 - 1 M (molar) calcium chloride is weighed with the precision scales and dissolved in 160 ml. of pure water. Then, it is sterilized by placing it in an autoclave. After weighing 1-2 M urea with the precision balance, it is dissolved in 80 ml. of pure water and sterilized under the aseptic environment conditions by using a 0.40 pm filter. Then, 160 ml. of calcium chloride removed from the autoclave and urea sterilized with the filter are combined under the aseptic environment conditions. The medium temperature needs to be approximately 20-30^tC.

- Performing application to the ground

The particle size distribution curve is given in Figure 1 ; application was performed to two different soils; the first sand soil no 1 and the second sand soil no 2. Approximately 128 g of the prepared culture medium is applied to the first sand soil (relative density 35%) or 108 g of the second sand soil (relative density 35%) for 15-30 hours at a flow rate of 0.400 mL/sec. Then, the application solution is given to the first sand soil at a flow rate of 0.400 mL/sec and to the second sand soil at a flow rate of 0.266 mL/sec for 10 days by means of the peristaltic pumps. The application solution filtered by the bottom and overflowing from the upper part is re-accumulated in the lower container and this continues during the application. When the application is over, the soils are placed in the drying oven set at QOΌ. It is recommended that the medium temperature is between duri ng the application.

While applying the invention, attention must be paid to the medium temperature. Since the calcite formed within the soil can be affected by the difference in the temperature, the medium temperature of approximately 15 - 35Ό will provide better results. Attention must be paid to cultivate the bacteria in the liquid growth medium under the aseptic environment conditions. The correct adjustment of the flow rate during the soil application is also an important situation for the improvement to be successful.

Calcium carbonate precipitations that connect the soil grains were analyzed on the soils having different grain. Since the calcium chloride and urea applied in the soil matrix move among the grains, more calcium carbonate formation will be needed as the hydraulic conductivity increases. Strength could not be obtained in the result of giving the application solution (calcium chloride and urea) to the coarse-grained soil at high flow rates (0.400 mL/sec-0.666 mL/sec), while strength could be obtained at low flow rate (0.266 mL/sec). On fine grained soil, strength results at low flow rate (0.266 mL/sec) were not obtained as desired, but better results were obtained at higher flow rates (0.400 mL/sec-0.666 mL/sec) as the application time extended. In the result of our analysis, it has been determined that in cases where the soil hydraulic conductivity is high, it is necessary to accumulate calcium carbonate by decreasing the flow rate and the application must be performed by increasing the flow rate as the hydraulic conductivity decreases.

The test arrangement used in the studies is as follows. The culture medium or application solution given from the top accumulates in the lower vessel by passing through the soil matrix (by filtering). Then, this accumulated solution is given to the soil from the upper part by means of a peristaltic pump. When blocking starts as a result of calcium carbonate precipitation, the solutions overflowing from the upper part are delivered to the lower container through the pipes by accumulating in the upper container. The cycle continues in this way during the application. Therefore, it can be provided that both the culture medium (bacteria) and the application solution (calcium chloride and urea) reach all over the soil.

The most important elements that distinguish the invention from other patents in the present art are the use of a different bacteria and the application method specific to the soil. Since the soils have different clearance volumes, the clearance volumes must be considered to connect the grains together or to reduce hydraulic conductivity by filling among the grains. In the present invention, with the application method that will be prepared in accordance with the hydraulic conductivity of the soil, improvement can be provided in the applied soil both in a shorter time and by using chemicals more effectively. Increasing strength in the soil improvement is an indicator of how successful the soil improvement is. Higher strength values than the existing patents could be obtained by using a different bacteria and method in the invention.

Claims

1. A method for biological improvement of sand soils by using Viridibacillus arenosi bacteria, wherein the method comprises following process steps: i. Preparing the liquid growth culture, ii. inoculating the Viridibacillus arenosi bacteria to the liquid growth medium, iii. Shaking the liquid growth medium cultivated at the determined time and agitation speed, iv. Determining the hydraulic conductivity of the soil in which the application will be performed, v. Applying the culture medium to the soil at a determined flow rate according to the hydraulic conductivity of the soil, vi. Applying the urea and calcium chloride solution to the soil at the determined time and flow rate.

2. The method for biological improvement of sand soils according to claim 1 , wherein; the liquid growth medium includes Tryptic Soy Broth, Manganese Sulfate Monohydrate and Nickel Chloride Hexahydrate.

3. The method for biological improvement of sand soils according to claim 2, wherein; the liquid growth medium contains 30 g/L Tryptic Soy Broth, 20 g/L urea, 12 mg/L Manganese Sulfate Monohydrate and 24 mg/L Nickel Chloride Hexahydrate by weight.

4. The method for biological improvement of sand soils according to claim 1 , wherein; Bacterial culture is performed in the iii. in the treatment step, shaking the inoculated broth at 100 - 125 rpm for 40 - 50 hours.

5. The method for biological improvement of sand soils according to claim 4, wherein; Bacterial culture is performed in the iii. in the treatment step, shaking the inoculated broth at 120 rpm for 48 hours.

6. The method for biological improvement of sand soils according to claim 1 , wherein; the culture medium is applied to the soil for 15 to 30 hours at the flow rate determined according to the hydraulic conductivity of the soil in the v. process step.

7. The method for biological improvement of sand soils according to claim 6, wherein; the culture medium is applied to the soil for 24 hours at the flow rate determined according to the hydraulic conductivity of the soil in the v. process step.

8. The method for biological improvement of sand soils according to claim 1 , wherein; the average pH is between 7 and 9 during the cultivation of Viridibacillus arenosi bacteria to the liquid growth medium.

9. The method for biological improvement of sand soils according to claim 8, wherein; the average pH is 8.3 during the cultivation of Viridibacillus arenosi bacteria to the liquid growth medium.

10. The method for biological improvement of sand soils according to claim 1 , wherein; the growth temperature during shaking is 25±5^tC.

11. The method for biological improvement of sand soils according to claim 1 , wherein; the average medium temperature during the application performed on the soil in v. and vi. process steps are between 15-35Ό.

12. The method for biological improvement of sand soils according to claim 1 , wherein; the average pH of the culture medium taken from the shaker after 48 hours is between 7 and 9.

13. The method for biological improvement of sand soils according to claim 12, wherein; the average pH of the culture medium taken from the shaker after 48 hours is 8.3.

14. A sand soil improved by the method according to claim 1 to 13.

15. The improved sand soil according to claim 14, wherein; the relative density is between 25-50%.

16. The improved sand soil according to claim 15, wherein; the relative density is 35%.

17. The improved sand soil according to claim 14, wherein; it comprises culture medium of 0.5-2%, calcium chloride of 3-8%, urea of 3-6% and sand of 84-93.5%, by weight in average.

18. The improved sand soil according to claim 17, wherein; it comprises culture of 1%, calcium chloride of 5%, urea of 4% and sand of 90%, by weight in average.

19. The improved sand soil according to claim 14, wherein; it comprises 0.5-1 M calcium chloride and 1-2 M urea.

20. The improved sand soil according to claim 19, wherein; it comprises 0.75 M calcium chloride and 1.5 M urea.