Effect of cell density on decrease in hydraulic conductivity by microbial calcite precipitation – AMB Express

Inoculum preparation, growth conditions and measurement of OD

The strain used in this study was Sporosarcina pasteurii [American Type Culture Collection (ATCC) 11859] to induce calcium carbonate precipitation during experiments. The medium (Tris-YE) for cultures was prepared containing Tris buffer (130 mM,pH 9.0), ammonium sulphate (10 g/L) and yeast extract (20 g/L). For stock culture, solid medium was prepared adding 2% agar per liter of liquid medium. The components of media were autoclaved separately at 121 ℃ for 15 min and mixed before usage (Eryürük et al. 2015a).

To observe the relation between cell density and the hydraulic conductivity, resting cells of Sporosarcina pasteurii were provided by inoculation of cells in Tris-YE medium overnight at 30 ℃ with 120 rpm shaking. Then the cells were harvested using centrifugation at 10,000*g for 10 min and rinsed two times using distilled water (Eryürük et al. 2015a, b). Ultimately, the cells were suspended in 100 mL distilled water obtaining the optical densities (OD) of 0.15, 0.75, and 2.25 (abbreviated as OD₆₀₀ 0.15, 0.75, and 2.25) at 600 nm. The OD values were measured using a spectrophotometer (Hitachi U-1900 Spectrophotometer, Tokyo, Japan). The aliquots, which were diluted, were taken from cell suspension to count the number of bacterial cells under the microscope (Olympus BX50WI, Tokyo, Japan). The optical densities of 2.25, 0.75, and 0.15 at 600 nm corresponded to 2.15 * 10⁹ cells/mL, 8.10 * 10⁸ cells/mL, 5.89 * 10⁸ cells/mL, respectively.

Experimental setup and conditions

50 mL-volume of plastic syringes, whose inner diameter was 3 cm inner diameter and height was 10 cm (Fig. 1), were used as columns in the experiments. 90 g of glass beads in average diameter of 0.25 mm, 0.50 mm, 1.0 mm, 2.0 mm, and 3.0 mm were used as the porous media and saturated conditions were provided for each experiment. Before conducting each experiment, an acidic solution (0.1 N HCl) was used to wash the glass beads and then the glass beads were rinsed with distilled water until a neutral pH was achieved.

Experimental setup revised from Eryürük et al. 2015a

All experiments were carried out at 22 ℃ as constant temperature and a peristaltic pump controlled the down flow influent rate. First, Sprosarcina pasteurii was added into the column as 100 mL of cell suspension (four pore volumes of the packed glass beads) with 2 mL/min of flow rate. Homogeneous cell suspension during introduction of cell suspension was provided using magnetic stirrer. The effluent from the columns was collected to count the number of cells, that flowed out the column, under the microscope (Olympus BX50WI, Tokyo, Japan) and the number of cells deposited in the columns was estimated by subtracting the number of cells eluted from the number of cells introduced to the columns. Then, a 0.22 μm membrane filter was employed to sterilize the precipitation solution which consisted of 500 mM CaCl₂ and 500 mM urea and had pH 6.8. After that, flow rate of 3 mL/min was used to introduce the precipitation solution (700 pore volumes of the packed glass beads) into the column. The hydraulic conductivity of the column was measured using a manometer. (Eryürük et al. 2015a, b). The experiments were continued by introducing the precipitation solution until no change was measured in the hydraulic conductivity of the columns. All experiments were performed in triplicate.

Measurement of CaCO₃ precipitated in the experiments

The porous media material, which was glass beads, was taken into a beaker at the end of the experiments. The glass beads were rinsed two times using distilled water to remove excess Ca⁺⁺. Then, 0.1 N HNO₃ was added into the beaker to dissolve CaCO₃ formed by Sporosarcina pasteurii. After that, inductively coupled plasma atomic emission spectroscopy (PerkinElmer Optima 3300DV, PerkinElmer, Waltham, Massachusetts, USA) was employed to determine Ca⁺⁺ concentration of the samples which were obtained from the beaker. Finally, detected Ca⁺⁺ concentration was used to calculate the amount of CaCO₃ precipitated (Eryürük et al. 2015a).

Measurement of pore size distribution before and after experiments and calculation of occupied volume of CaCO₃

Matric potential was measured with a wide range pF meter (DIK-3404, Daiki Co., Ltd., Saitama, Japan) to estimate the pore size distribution of the glass beads. The columns were first weighed, and then their bottoms were covered with Advantec No. 6 filter paper (Advantec MFS, Inc., CA, USA). The columns, as well as the ceramic filters of the pF meter, were then saturated with distilled water. The columns were weighed again after 24 h. The ceramic filters and columns were then placed in the pF meter. The pF values 1.3, 1.6, 2.0, 2.5, 3.0, 3.5, and 3.8 were applied by pressure to determine pore sizes of 75–150 μm, 31–75 μm, 10–30 μm, 4–9 μm, 1–3 μm, 0.45–1.00 μm, and < 0.45 μm, respectively. For pF values 1.3–3.0, air was the pressure supplier; for pF values 3.5 and 3.8, nitrogen gas was the pressure supplier. The columns were subjected to pressure for seven days in order to achieve equilibrium. The columns were then reweighed. The pore size distribution of glass beads was then calculated using weight differences between samples after different pF values were applied. The proportion of the individual size ranges of the pores was used to calculate the average pore size in the columns.

The occupied volume of CaCO₃ was also calculated using specific gravity of CaCO₃, which is 2.7 g/cm³, and the amount of CaCO₃ precipitation after treatment.

Analysis of hydraulic conductivity using modified Kozeny-Carman equation

Modified Kozeny-Carman equation (Eryürük et al. 2015a) was used to interpret quantitative relation between the microbial CaCO₃ precipitation and the reduction of the hydraulic conductivity.

where K_Ca+B denotes hydraulic conductivity with bacterial clogging, f denotes the shape factor, ρ denotes the density of liquid, g denotes gravity, n denotes porosity, r_Ca denotes the specific CaCO₃ precipitation rate, B denotes the number of cells deposited, V denotes the volume of column, µ denotes the dynamic viscosity of the liquid, and D denotes the average diameter of glass beads.