Biological cementation is a new process in which urea hydrolysis bacteria or free urease enzyme decompose urea and increases the pH of the environment and chemical interactions in the presence of calcium ions to form calcite. Nowadays, nano-calcite is widely used in engineering, such as increasing the strength of soil and concrete, as well as in medicine, such as drug delivery and cancer treatment. This study aimed to investigate the laboratory conditions for producing nano-calcite particles with appropriate quality, size and purity by Sporosarcina pasteurii enzyme extract for use in medical and engineering studies. This investigation aimed to make calcite by S. pasteurii enzyme extract and optimize influential factors in calcite production. For this purpose, the bacterium S. pasteurii was cultured in nutrient broth containing urea and nickel, and upon reaching the appropriate time, the cells were separated and washed. Then, their enzyme extract was prepared by sonication, and calcite precipitation was studied in different amounts of urea, calcium chloride, enzyme and temperature. The quality of produced calcite crystals and their ratio compared to other crystals were investigated by XRD and SEM analyses. According to the results of XRD analysis, it was found that in 0.5 M urea and 0.25 M calcium chloride, the highest amount of calcite is produced with 96%, and the least side products are produced. Examining the particle size histogram in the sample containing 0.5 M urea and 0.25 M calcium chloride revealed that the range of particles were between 50 and 100 nm. The nature and type of crystals were studied by electron microscopy, and EDX analysis showed the presence of calcium, oxygen, and carbon. According to the results, it was found that by the increase of the concentrations of urea and calcium, the range of particle size became larger. Also, the percentage of calcite produced in low urea and calcium chloride concentrations is higher than those in high concentrations.

Wind erosion and the phenomenon of Dust with all of its controlling methods is serious problem. This phenomenon lead environment degradation and fugitive dust storms. So, Study and use of the new methods to control this natural phenomenon is essential. In this study, the novel and environmental friendly method of soil biological stabilization was investigated with using an abundant bacterial species founding in nature and soil deposits. The scientific name of this bacterium is Sporosarcina Pasturii (PTCC 1645) and uses as the urease-positive bacterium. This bacterium produce urease enzyme which converts urea to ammonium and carbonate, resulting in the precipitation of calcite crystals that bridge the soil particles. In this study a mixture of cementation and bacterial-cell solutions uniformly sprayed onto the exposed top surfaces of the soils. The concentration of bacterial-cell solution was quantified in terms of its optical density at 600nm wavelength (OD600) which equal 1.5 (that is, approximately 1.5×108 bacterial cells·ml−1). The prepared equimolar urea–calcium chloride cementation solution included nutrient broth (3g.l-1), ammonium chloride (10g.l-1) and sodium bicarbonate (2.12g·l-1) prepared at 0.5M concentration. The mixture volume sprayed onto each specimen was equal to 1.5Vv (where Vv is the pore voids volume of the topmost 3-mm thick layer of the 20–mm deep loose sand tray-specimens). The bench scale experimental programme presented investigates the proposed technique’s effectiveness for stabilisation of two clean, angular to sub-angular medium silica sands and carbonate silty sands with different gradations (sand t60 and sand t90 with size ranges of 0.125–0·50 and 0.075–0.85mm, respectively and carbonate sand with size ranges of 0·001–0·85mm, and mean particle size (D50) values of 0.28, 0.24 and 0.20 mm, respectively), the time-dependent (retention time 3, 7, 14, 20 and 28 days) compressive strength development for the crustal sand layer following single- and double-MICP (with interval of 6 days) spray treatments, as well as wind tunnel experiments under the condition of wind velocity of 20 ms-1. The effect of dew formation on crustal compressive strength development with curing period and the efficiency of the MICP treatment for the outdoor environment compared to laboratory-controlled test conditions. A pocket penetrometer was used to determine the compressive strength of soils. Significant improvements in the Compressive strength of the treated soil samples were observed. The results show improving compressive strength with time. The highest compressive strength in the carbonate sand was obtained equals to 84 kPa. Silica sand with finer size distribution has shown more compressive strength than two other soils. Also the results showed that double-MICP spray treatments of the bacteria solution and cementation was more effective than single- MICP spray treatments in the compressive strength of soils, especially in the silica sand equals to 190% in a curing period of 28 days. Also, the cured MICP-treated crustal sand layer was stable to 20 m·s−1 winds that demonstrating the potential of biological stabilisation via the MICP process as an appropriate option for dealing with desertification and motion of sandy soil deposits.

One of the most common methods of soil improvement is to use additives in order to improve strength properties and permeability of the soil. Cements or chemicals are usually used as binders for soil particles, which lead to increase the soil shear strength and reduce its hydraulic conductivity (i. e. permeability). Nevertheless, these materials are not suitable for soil improvement in the long term because they require significant natural resources. The use of cement and chemicals for soil improvement is expensive and time-consuming. Management of renewable natural resources (microorganisms and their products) could lead to solve geotechnical and environmental problems and achieve great economic benefits in the building industry. In addition, the application of microbial biotechnology in the building industry make easier some of the existing methods of construction. Using the latest microbial biotechnology, a new type of building materials, namely biocement, has been produced as an alternative to cement or chemicals. Biocementation is the improvement of strength and stiffness of rock and soil by using microbial activity and their products. The process of the formation of precipitates or biocement in the presence of microorganisms is called microbialy induced calcium precipitation (MICP). Biocement can be used in solid and liquid states. In the liquid state, biogrout can flow like water with very low viscosity. Therefore, compared to cement and chemicals, it will be transmitted into the soil, more easily. Naturally, biocement is formed in the presence of microorganisms in ambient temperature and thus, it requires less energy. Because of the abundance of microorganisms in the nature and easy to reproduce with low cost, this type of cement is sustainable. The Microorganisms that are suitable for the production of biocement are usually non-pathogenic and environment friendly. In addition, unlike cement, soil can be improved without disturbance of ground and the environment; since microorganisms can penetrate into the soil and grow in it. This dissertation aims to realize the effect of ground condition on the MICP process in non-cohesive soils. Since this method is still in the laboratory stage, for being used in practical projects, it is required to carry out laboratory experiments, including relative density and particle size distribution, to evaluate the performance of this method in different ground conditions. For this purpose, it was used from Sandy soil with different silt contents of 0%, 5%, 10%, 15% and 20% in two states of Loose (Dr = 40%) and dense (Dr = 100%) conditions in this research. The high urease activity and non-pathogenic bacteria S. Pasteurii was also used in the MICP process. In order to consider the soil conditions on the efficiency of this type of improvement method, uniaxial compressive test parameters and precipitated calcium carbonate content were investigated. According to the results, increasing of silt content from 0% to 20%, leads to reductions of 40% and 46% in precipitated calcium carbonate content, increases of 57% and 41% in the uniaxial strength and increases of 79% and 71% in the elasticity modulus of the samples in two loose and dense conditions, respectively. It seems that these changes were resulted from shrinking of the empty space and increasing of the contact area between the soil particles.

Dust events are among the serious environmental challenges in some countries. Sustainable solutions can be applied to tackle this problem by considering soil as a living ecosystem. Biocementation based on the production of carbonates by heterotrophic bacteria is one of the favorable methods for suppressing the dust from wind erosion, because this type of bacteria produces calcium carbonate (main product) as well as water and carbon dioxide (by-products). In the present research, bacterial species of Bacillus amyloliquefaciens was sampled for analysis. First, bacteria were cultivated to reach a predetermined concentration. Next, bacterial cells and nutrients in the form of solution were sprayed on the soil surface. Then, samples were tested in a closed-circuit wind tunnel. Three main groups of samples were tested: (1) without sand bombardment and undisturbed soil surface, (2) with sand bombardment and undisturbed soil surface, and (3) without sand bombardment and with disturbed soil surface. The results show that the implemented method for soil stabilization is e cient. Moreover, based on the results of the second group of tests, curing duration, amount of water, temperature-water interaction, and waterbacterial cells interaction were found to be of considerable signi cance.

Introduction: The present study was focused on the statistical optimization of growth parameters for enhancing the Microbially Induced Calcite Precipitation (MICP) using ureolytic yeast strain. Materials and Methods: Thirteen yeast strains were tested for the synthesis of urease enzyme by phenol-hypochlorite assay and were further evaluated for calcite precipitation test. The growth parameters were optimized using the best ureolytic strain by Box-Behnken Design (BBD) and the extracted MICP was characterized through instrumental analysis. Results: Among thirteen yeast strains, Candida tropicalis NN4, Spathospora sp. NN04, Wickerhamomyces anomalus VIT-NN01 and Candida dubliniensis NN03 showed positive results for the synthesis of urease enzyme. Spathospora sp. was found to be the most potent strain for MICP. A significant enhancement in MICP by Spathospora sp. was observed under optimized conditions viz. A-urea concentration (80. 0 g/L), B-calcium chloride (45. 0 g/L), C-pH (9. 0) and D-inoculum dosage (8%, v/v). The actual value (34. 4± 0. 12 g/L) was in agreement with predicted value (34. 7± 0. 01g/L) with the R 2 value (0. 9900), confirming the validity of the model. The FTIR of MICP confirmed the fundamental bands of CO3 stretching and bending vibrations, observed at 1394. 23 and 874. 85 cm-1. The Scanning Electron Microscope (SEM) images of biomotar revealed aggregated polymorphs of MICP interconnected with yeast mycelium and spores. The Energy Dispersive X-Ray Spectrometer (EDX) analysis indicated the presence of calcite in the biomotar. A remarkable improvement in the compressive strength (28 to 44 MPa) and morphological changes were observed in biocement mortar as compared to cement mortar. Conclusions: This result is the first report on the implementation of ureolytic Spathospora towards the application of Biocementation through MICP using BBD.

The process of biomineralization as a method of biological soil improvement refers to a set of biochemical reactions in which the sediment formed by bacteria causes the grains of soil to graft and thus improve its properties. In each of the previous researches in the field of biomineralization, one of the methods of mixing, adsorption and injection have been used to add cementation solution to the soil and so far, no comparison has been made on these methods. What is very important in the biomineralization process as a soil remediation method and in fact, one of the most important challenges of this method is the uniform penetration of the Biocementation solution and, consequently the proportional distribution of sediments formed in the soil matrix. In the present study, after optimizing the precipitation conditions, the effect of the sample-making method on the biological improvement of Kermanshah clay fine-grained soil with two bacteria Bacillus megaterium and Lysinibacillus boronitolerans was studied. The results show that the method of sample making (in terms of adding Biocementation solutions) has a significant effect on the improvement of the compressive strength of samples. The maximum improvement of soil compressive strength (up to 2.68) occurred for Bacillus megaterium in the adsorption method. In general, the addition of solution to the soil by adsorption method has been more effective in the proper placement of sediment between soil grains than the mixing and injection method. It has been more effective and in fact the solutions have been allowed to move between the soil grains to the corners where the grains join, thus resulting in the formation of effective sediment at the grains joint. Also, biological stabilization of the consolidation test sample reduced the soil void ratio change from 0.584 to 0.354 and the compression index from 0.077 to 0.038. The addition of Biocementation solutions to the sample of atterberg limits test sample reduced the plasticity index from 26 to 19.

Introduction: In many areas of the world, the mechanical properties of soils for utilization of land are not sufficient. For improvement of these lands, soil stabilization such as compacting, installation of nails, elders of piles, mixing soil with lime or cement before or during constructions on the surface or inside of the ground can be useful. Microbially induced carbonate precipitation (MICP), due to its versatility and stable performance, has been recently attracted the attention of many researchers in the field of the geotechnical engineering around the world. MICP is a biological technique that is naturally caused to create a cementation agent, which is known as calcium carbonate or calcite by controlling the metabolism of bacteria. Although there are many biological processes that can be lead to MICP, but the using of urea hydrolysis by bacteria is commonly used more. In this method, aerobic bacteria with the enriched urease enzymes inject into the soil. Hydrolysis of urea occurs when the bacteria speeds up the hydrolysis reaction to produce ammonium and carbonate ions. In the presence of soluble calcium ions, carbonate ions are precipitated and formed the calcium carbonate crystals. When these crystals are formed on a grain of soil or like a bridge between them, they prevent the movement of grains and thus improve the mechanical and geotechnical properties of the soil. Material and methods: In the present study, the effect of increasing fines on the improvement of Anzali sandy soil, and soil resistance parameters for improving the clean sand and its mixtures with a fine grained cohesive soil and a fine grained cohesionless soil separately in a percentage weight of 30 by MICP and using a small scale of direct shear test (6×6) have been investigated. In the present study the sandy soil was collected from the coast of Bandar Anzali Free Zone and for the preparation of samples of clayey sand and silty sand, Kaolinite clay soils and Firouzkooh broken silt were used, respectively. Anzali sand is poorly graded and had a rounded corner with an average particle size of 0. 2 mm, somewhat, sharpening cores are also found in its granulation. In addition, its fine grained content is very small (less than 1%). The Kaolinite clay is also labeled with a liquid limit of 40, a plastic limit of 25, and a plasticity index of 15 as an inorganic clay (CL). The used microorganism in this study is urease positive Sporosarcina pasteurii, which is maintained with the number of PTCC1645 at the Center Collective of Industrial Microorganisms of Iran Scientific and Research Organization. The bacterium was cultured in a culture medium containing 20 g/l yeast extract and 10 g/l ammonium chloride at pH 9 under aerobic conditions in incubator shaker machine at 150 rpm and temperature of 30 ° C. The organism was grown to late exponential/early stationary phase and stored at 4 ° C before injection in samples. A solution of calcium chloride and urea with a molar ratio of one is also used as a cementation solution. With the direct shear test (6cm×6cm) as a benchmarking of the shear strength in the before and after improvement steps, molds fitted with a shear box made of the galvanized sheet with a thickness of 0. 6 mm and it consists of two main parts, the body, in the middle of which an exhaust pipe was embedded in the injector waste fluid. At the bottom of the samples, a layer of filter paper was placed in order to prevent soil washes, and then all samples with a thickness of 2 cm, with a relative density of 30% at the same weight and height were pressed. In the upper part of the samples, a layer of filter paper is similarly used to prevent the discontinuity of soil particles when injected biological materials are used. Biological solutions are injected from the top to the specimens and allowed to penetrate under the influence of gravitational and capillary forces in the sample and discharge the inhaled fluid from the exhaust pipe. The criterion for determining the volume of the solution to inject into each sample is the pure volume (PV) of soil. The preparation process of the samples was initiated by injection of a PV water unit, followed by a two-layer mixture of bacterial suspensions and cementation solutions, each with a volume of one PV, and then for biological reactions, 24 hours to the sample at laboratory temperature (25 ± 2) is given. After the time of incubation, the solution of cementation is injected into the sample for a period of three days and every 24 hours. The processing time of samples is also considered 28 days. In this study, optical density (OD) was selected as a benchmark for estimating the concentration of bacterial cells in the culture medium, and in all stages of development, and precisely before injection of bacteria suspension into soil samples, it was measured by a spectrophotometer device at 600 nm (OD600) wavelength, which was obtained for all bacterial suspensions in the range of 1. 7 to 2 before the injection. To determine the activity of urea bacteria, 1 ml of bacterial suspension was added to nine milliliters of 1. 11 molar urea solution, and by immersing the electrode of the electrical conductivity in the solution, its conductivity was recorded for 5 minutes at 20 ± 2 ° C. The rate of urea activity in the pre-treatment stage for all specimens was in the range of 0. 8 to 1. 23 mS min-1. In order to evaluate the shear strength parameters of soil samples, before and after the improvement operations, a direct shear test was used based on the ASTM D3080 standard. This test was performed for all samples under stresses of 50, 100 and 150 kPa in undrained conditions at a loading speed of 1 mm/min up to a strain of 15%. Also, samples of soil with a moisture content of 7% and a relative density of 30% (as already mentioned) have been restored. SEM analysis was carried out to determine the distribution of sediment between soil particles and EDX analysis in order to identify carbonate calcium sediment formation elements in improved soil samples, by scanning electron microscopy on Anzali sandy soil samples in before and after improvement conditions. Conclusions: The effect of the increasing cohesive and cohesionless fines on the bio-treated process of sandy soil is the main subject of this research. For this purpose, three samples of clean sand, sand containing 30% clay and sand mixture with 30% silt in a relative density of 30% were treated with MICP method and their shear strength parameters were evaluated by direct shear test after 28 days of processing. Using the direct shear test and analyses of SEM and EDX data, the results are represented as below: 1. The microbial sediment of carbonate calcium has greatly improved the resistance properties of all three soil samples. 2. A sample of clayey sand, in spite of a higher improvement compared to the other samples with an average shear strength of 113. 7% in comparison to to its untreated state, it has the lowest shear strength among the three improved samples. 3. Increasing the clay content of 30% increases the soil voids. On the other hand, it reduces the friction angle and shear strength of the soil in the pre-treated state and also facilitates easier movement of the bacteria between the pores in the soil. More favorable distribution of sediment calcium carbonate was occurred and, as a result, increased adhesion between soil particles. 4. The increase of cohesionless fine particles creates more bonding points between sand particles and, therefore, calcium carbonate crystals form shorter distances between the soil bridges. As a result, with the end of the improvement process, the shear strength parameters of the sandy soil containing 30% of the silt compared to the clean sand have a higher value. 5. SEM images of the clean sand in both before and after improvement show that the calcium carbonate precipitation occurred with a uniform and thin layer that surrounds sand grains and another part of the sediments formed in the joint of grains. 6. Cube-shaped crystalline sediments confirm that the sediment formed in the soil is a stable type of calcite and that the relative increase in the friction angle of the improvement samples can be attributed to solid particles and multifaceted sediments. Also, the elements of carbon, oxygen, and calcium, which are the main components for the formation of calcium carbonate deposits, have been found in the EDX analysis of improvement sand samples.