Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e6, 2014 www.elsevier.com/locate/jbiosc
Reducing hydraulic conductivity of porous media using CaCO3 precipitation induced by Sporosarcina pasteurii an Eryürük,1 Suyin Yang,2 Daisuke Suzuki,2 Iwao Sakaguchi,2 Tetsuji Akatsuka,2 Takayuki Tsuchiya,1 Kag and Arata Katayama1, 2, * Graduate School of Engineering, Department of Civil Engineering, Nagoya University, Nagoya 464-8601, Japan1 and EcoTopia Science Institute, Division of Environmental Research, Nagoya University, Nagoya 464-8603, Japan2 Received 21 April 2014; accepted 18 August 2014 Available online xxx
The effect on hydraulic conductivity in porous media of CaCO3 precipitation induced by Sporosarcina pasteurii (ATCC 11859) was investigated using continuous-flow columns containing glass beads between 0.01 mm and 3 mm in diameter. Resting S. pasteurii cells and a precipitation solution composed of 0.5 M CaCl2 and 0.5 M urea were introduced into the columns, and it was shown that the subsequent formation of CaCO3 precipitation reduced hydraulic conductivity from between 8.38 3 10L1 and 3.27 3 10L4 cm/s to between 3.70 3 10L1 and 3.07 3 10L5 cm/s. The bacterial cells themselves did not decrease the hydraulic conductivity. The amount of precipitation was proportional with the bacterial number in the column. The specific CaCO3 precipitation rate of the resting cells was estimated as 4.0 ± 0.1 3 10L3 mg CaCO3/cell. Larger amounts of CaCO3 precipitation were deposited in columns packed with small glass beads than in those packed with large glass beads, resulting in a greater reduction in the hydraulic conductivity of the columns containing small glass beads. Analysis using the KozenyeCarman equation suggested that the effect of microbially induced CaCO3 precipitation on hydraulic conductivity was not due to the formation of individual CaCO3 crystals but instead that the precipitate aggregated with the glass beads, thus increasing their diameter and consequently decreasing the pore size in the column. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Microbial clogging; Glass beads; CaCO3; Hydraulic conductivity; Sporosarcina pasteurii; KozenyeCarman equation]
Contamination of groundwater, which influences drinking water resources due to leakage from landfills, is a wide spread problem (1). It is desirable for the hydraulic conductivity of landfill clay liners with 50 cm of thickness and lower than 106 cm/s in order to prevent migration of contaminated fluid from the landfill to groundwater (Ministry of Environment Japan, ministerial order No. 3, revised 21st February 2013, http://law.e-gov.go.jp/htmldata/ S52/S52F03102004001.html). Clay liners, whose essential material is bentonite, have been investigated to decrease the hydraulic conductivity of landfills (2e4). However, the long-term durability of landfill liners is still arguable. Landfill waste degradation is a long-term process, and stabilization of waste to inert state continues for 20e30 years after the landfill completion (5). Several studies have indicated the permeation of inorganic salt solutions (6e8) and seasonal drying, which leads shrinkage of the clay and development of cracks (9), can cause the increase in hydraulic conductivity of clay liners. Thus, clay liners may not provide confinement well in the long-term. It is important to develop maintenance techniques instead of simple clay liners. Microbial
clogging, which results in production of insoluble organic and inorganic compounds by microbes (10,11), and occurs via many pathways, such as photosynthesis, urea hydrolysis, and sulfate reduction (12), is considered as one such maintenance method used for hazardous waste confinement. Bacterial and enzymatic CaCO3 precipitation has been successfully used to decrease the permeability of porous media (13e15). Sporosarcina pasteurii (formerly known as Bacillus pasteurii) has been used by many researchers to induce CaCO3 precipitation (16e18) because of the following advantages: S. pasteurii is not repressed by ammonium, it is classified as nonpathogenic, and it has high urease activity (19) and a high tolerance to salt (high electrical conductivity) and high pH. S. pasteurii produces urease, which is the enzyme that hydrolyzes urea to ammonia and carbon dioxide. The production of ammonia causes the pH of the surroundings to increase, which promotes CaCO3 precipitation in calcium-rich environments. The reaction steps and overall reaction are as follows (20): urease
NH2 CONH2 þ H2 O / 2NH3 þ CO2 2NH3 þ 2H2 O/2NHþ 4 þ 2OH
* Corresponding author at: EcoTopia Science Institute, Nagoya University, Chikusa, Nagoya 464-8603, Japan. Tel.: þ81 52 789 5856; fax: þ81 52 789 5857. E-mail address: [email protected] (A. Katayama).
CO2 þ OH /HCO 3
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.08.009
Please cite this article in press as: Eryürük, K., et al., Reducing hydraulic conductivity of porous media using CaCO3 precipitation induced by Sporosarcina pasteurii, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.08.009
2
ERYÜRÜK ET AL.
FIG. 1. Setup of column clogging procedure.
J. BIOSCI. BIOENG.,
FIG. 2. Hydraulic conductivity of columns packed with different sizes of glass beads (open square), before and after clogging treatment (closed circle), in comparison with the previous results for the hydraulic conductivity of glass beads reported by Hasegawa et al. (25) (open diamond), Taniguchi and Sakura (26) (plus sign), and Cho and Kumano (27) (asterisk).
CaCl2 /Ca2þ þ 2Cl Ca2þ þ HCO 3 þ OH /CaCO3 þ H2 O
Overall reaction: NH2 CONH2 þ CaCl2 þ 2H2 O/2NHþ 4þ 2Cl þ CaCO3 Hence, microbial clogging is expected to decrease the hydraulic conductivity of porous media. However, the effect of CaCO3 precipitation on reducing hydraulic conductivity has not been extensively studied, except for using in sealing cracks (21), for increasing the resistance of cementitious materials (22) and for achieving the co-precipitation of Sr (23). It is essential to understand quantitatively the effect of bacterial clogging on the hydraulic conductivity of porous media for the application of this technology. Thus, in this study, the bacterial clogging experiments were conducted to elucidate quantitatively how hydraulic conductivity of porous media is decreased by the microbially-induced CaCO3 precipitation, by using the columns packed with glass beads with different diameters and the resting cells of S. pasteurii. MATERIALS AND METHODS Microorganism and preparation of culture S. pasteurii (ATCC 11859) was used to induce CaCO3 precipitation during experiments. Tris-YE, containing 130 mM Tris buffer (pH 9.0), 10 g/L (NH4)2SO4, and 20 g/L yeast extract, was used as the culture medium, to which 2% agar was added to obtain a solid medium for the stock culture. Each reagent was autoclaved at 121 C for 15 min separately before use. Resting cells of S. pasteurii were provided to obtain the quantitative relation between microbial population and the effect on the hydraulic conductivity. To prepare the cells for the experiments, one loopful of S. pasteurii was aseptically inoculated into 1 L Tris-YE medium, and the culture was incubated overnight at 30 C under shaking conditions (120 rpm). The bacterial cells were then precipitated by centrifugation at 10,000 g for 10 min and washed twice with distilled water. Finally, the bacterial cells were resuspended in 100 mL distilled water to achieve an optical density of 2.25 at 600 nm (OD600; 2.15 109 cells/mL) as measured with a spectrophotometer (Hitachi U-1900 Spectrophotometer, Tokyo, Japan). Column clogging treatment The experiments were conducted using 50 mL plastic syringes (Terumo SS-50ESZ, Terumo Corporation, Tokyo, Japan) as plastic columns that had a 3 cm inner diameter and were 10 cm in height (Fig. 1). Different sizes of glass beads (Potters-Ballotini Co. Ltd., Ibaraki, Japan), with average diameters of 0.01 mm, 0.05 mm, 0.25 mm, 0.50 mm, 1.0 mm, 2.0 mm, and 3.0 mm (abbreviated as GB0.01, GB0.05, GB0.25, GB0.50, GB1.0, GB2.0, and GB3.0, respectively), were used as porous media in the experiments. Before use, the glass beads were washed with an acidic solution (0.1 N HCl) and subsequently rinsed with distilled water until a neutral pH was obtained. The columns were packed with 90 g of the washed glass beads under saturated conditions. The cell suspension (4 pore volumes of glass beads, 100 mL) was introduced into the column with a flow rate of 2 mL/min using a peristaltic pump at 22 C. A magnetic stirrer was used to ensure a homogenous bacterial suspension. During the
introduction of the bacteria, samples were taken from the effluent to count the number of bacteria eluted from the column and thus calculate the number of bacteria that had been deposited into the column. The introduction of the bacterial suspension was complete after 50 min. Then, the precipitation solution (0.5 M CaCl2 and 0.5 M urea; pH 6.8) was sterilized through a 0.22 mm membrane filter and introduced into the column with a flow rate of 3 mL/min. The precipitate solution was continuously introduced until the hydraulic conductivity of the column became stable. Effect of bacterial population size on reduction in hydraulic conductivity To examine the effect of bacterial population size on reduction in hydraulic conductivity, experiments were conducted with cell suspensions at various densities (OD600 0.15, OD600 0.75, and OD600 2.25), using the same procedure as described above. One milliliter cell suspension, which Size analysis of CaCO3 precipitate contained 109 cells, was injected into 20 mL sterilized precipitation solution using a syringe. Then, samples were taken at appropriate intervals and placed on a glass slide to measure the size of the crystals in the CaCO3 precipitate using a microscope (Olympus BX50WI, Tokyo, Japan). Determination of the amount of CaCO3 precipitated into the columns After the column clogging treatment, the glass beads were removed from the columns, placed into a beaker, and mixed with 0.1 N HNO3 to dissolve the precipitated CaCO3. Then, samples were taken to detect their Ca2þ concentration using inductively coupled plasma atomic emission spectroscopy (PerkinElmer Optima 3300DV, PerkinElmer, Waltham, MA, USA). After the Ca2þ concentration was measured, the amount of CaCO3 that had precipitated into the columns was calculated. Pore size distribution of glass beads before and after column clogging To estimate the pore size distribution of the glass beads, matric potential was measured using a wide range pF meter (DIK-3404, Daiki Co., Ltd., Saitama, Japan). First, the columns were weighed; then, the bottoms of the columns were covered with Advantec No. 6 filter paper (Advantec MFS, Inc., CA, USA). Next, the columns were saturated with distilled water, as were the ceramic filters of the pF meter. After 24 h, the columns were weighed again. Next, the ceramic filters and the columns were put into the pF meter. Then, pressure in the pF meter was adjusted to pF values 1.3, 1.6, 2.0, 2.5, 3.0, 3.5, and 3.8, to identify pore sizes of 75e150 mm, 31e75 mm, 10e30 mm, 4e9 mm, 1e3 mm, 0.45e1.00 mm, and
Reducing hydraulic conductivity of porous media using CaCO3 precipitation induced by Sporosarcina pasteurii an Eryürük,1 Suyin Yang,2 Daisuke Suzuki,2 Iwao Sakaguchi,2 Tetsuji Akatsuka,2 Takayuki Tsuchiya,1 Kag and Arata Katayama1, 2, * Graduate School of Engineering, Department of Civil Engineering, Nagoya University, Nagoya 464-8601, Japan1 and EcoTopia Science Institute, Division of Environmental Research, Nagoya University, Nagoya 464-8603, Japan2 Received 21 April 2014; accepted 18 August 2014 Available online xxx
The effect on hydraulic conductivity in porous media of CaCO3 precipitation induced by Sporosarcina pasteurii (ATCC 11859) was investigated using continuous-flow columns containing glass beads between 0.01 mm and 3 mm in diameter. Resting S. pasteurii cells and a precipitation solution composed of 0.5 M CaCl2 and 0.5 M urea were introduced into the columns, and it was shown that the subsequent formation of CaCO3 precipitation reduced hydraulic conductivity from between 8.38 3 10L1 and 3.27 3 10L4 cm/s to between 3.70 3 10L1 and 3.07 3 10L5 cm/s. The bacterial cells themselves did not decrease the hydraulic conductivity. The amount of precipitation was proportional with the bacterial number in the column. The specific CaCO3 precipitation rate of the resting cells was estimated as 4.0 ± 0.1 3 10L3 mg CaCO3/cell. Larger amounts of CaCO3 precipitation were deposited in columns packed with small glass beads than in those packed with large glass beads, resulting in a greater reduction in the hydraulic conductivity of the columns containing small glass beads. Analysis using the KozenyeCarman equation suggested that the effect of microbially induced CaCO3 precipitation on hydraulic conductivity was not due to the formation of individual CaCO3 crystals but instead that the precipitate aggregated with the glass beads, thus increasing their diameter and consequently decreasing the pore size in the column. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Microbial clogging; Glass beads; CaCO3; Hydraulic conductivity; Sporosarcina pasteurii; KozenyeCarman equation]
Contamination of groundwater, which influences drinking water resources due to leakage from landfills, is a wide spread problem (1). It is desirable for the hydraulic conductivity of landfill clay liners with 50 cm of thickness and lower than 106 cm/s in order to prevent migration of contaminated fluid from the landfill to groundwater (Ministry of Environment Japan, ministerial order No. 3, revised 21st February 2013, http://law.e-gov.go.jp/htmldata/ S52/S52F03102004001.html). Clay liners, whose essential material is bentonite, have been investigated to decrease the hydraulic conductivity of landfills (2e4). However, the long-term durability of landfill liners is still arguable. Landfill waste degradation is a long-term process, and stabilization of waste to inert state continues for 20e30 years after the landfill completion (5). Several studies have indicated the permeation of inorganic salt solutions (6e8) and seasonal drying, which leads shrinkage of the clay and development of cracks (9), can cause the increase in hydraulic conductivity of clay liners. Thus, clay liners may not provide confinement well in the long-term. It is important to develop maintenance techniques instead of simple clay liners. Microbial
clogging, which results in production of insoluble organic and inorganic compounds by microbes (10,11), and occurs via many pathways, such as photosynthesis, urea hydrolysis, and sulfate reduction (12), is considered as one such maintenance method used for hazardous waste confinement. Bacterial and enzymatic CaCO3 precipitation has been successfully used to decrease the permeability of porous media (13e15). Sporosarcina pasteurii (formerly known as Bacillus pasteurii) has been used by many researchers to induce CaCO3 precipitation (16e18) because of the following advantages: S. pasteurii is not repressed by ammonium, it is classified as nonpathogenic, and it has high urease activity (19) and a high tolerance to salt (high electrical conductivity) and high pH. S. pasteurii produces urease, which is the enzyme that hydrolyzes urea to ammonia and carbon dioxide. The production of ammonia causes the pH of the surroundings to increase, which promotes CaCO3 precipitation in calcium-rich environments. The reaction steps and overall reaction are as follows (20): urease
NH2 CONH2 þ H2 O / 2NH3 þ CO2 2NH3 þ 2H2 O/2NHþ 4 þ 2OH
* Corresponding author at: EcoTopia Science Institute, Nagoya University, Chikusa, Nagoya 464-8603, Japan. Tel.: þ81 52 789 5856; fax: þ81 52 789 5857. E-mail address: [email protected] (A. Katayama).
CO2 þ OH /HCO 3
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.08.009
Please cite this article in press as: Eryürük, K., et al., Reducing hydraulic conductivity of porous media using CaCO3 precipitation induced by Sporosarcina pasteurii, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.08.009
2
ERYÜRÜK ET AL.
FIG. 1. Setup of column clogging procedure.
J. BIOSCI. BIOENG.,
FIG. 2. Hydraulic conductivity of columns packed with different sizes of glass beads (open square), before and after clogging treatment (closed circle), in comparison with the previous results for the hydraulic conductivity of glass beads reported by Hasegawa et al. (25) (open diamond), Taniguchi and Sakura (26) (plus sign), and Cho and Kumano (27) (asterisk).
CaCl2 /Ca2þ þ 2Cl Ca2þ þ HCO 3 þ OH /CaCO3 þ H2 O
Overall reaction: NH2 CONH2 þ CaCl2 þ 2H2 O/2NHþ 4þ 2Cl þ CaCO3 Hence, microbial clogging is expected to decrease the hydraulic conductivity of porous media. However, the effect of CaCO3 precipitation on reducing hydraulic conductivity has not been extensively studied, except for using in sealing cracks (21), for increasing the resistance of cementitious materials (22) and for achieving the co-precipitation of Sr (23). It is essential to understand quantitatively the effect of bacterial clogging on the hydraulic conductivity of porous media for the application of this technology. Thus, in this study, the bacterial clogging experiments were conducted to elucidate quantitatively how hydraulic conductivity of porous media is decreased by the microbially-induced CaCO3 precipitation, by using the columns packed with glass beads with different diameters and the resting cells of S. pasteurii. MATERIALS AND METHODS Microorganism and preparation of culture S. pasteurii (ATCC 11859) was used to induce CaCO3 precipitation during experiments. Tris-YE, containing 130 mM Tris buffer (pH 9.0), 10 g/L (NH4)2SO4, and 20 g/L yeast extract, was used as the culture medium, to which 2% agar was added to obtain a solid medium for the stock culture. Each reagent was autoclaved at 121 C for 15 min separately before use. Resting cells of S. pasteurii were provided to obtain the quantitative relation between microbial population and the effect on the hydraulic conductivity. To prepare the cells for the experiments, one loopful of S. pasteurii was aseptically inoculated into 1 L Tris-YE medium, and the culture was incubated overnight at 30 C under shaking conditions (120 rpm). The bacterial cells were then precipitated by centrifugation at 10,000 g for 10 min and washed twice with distilled water. Finally, the bacterial cells were resuspended in 100 mL distilled water to achieve an optical density of 2.25 at 600 nm (OD600; 2.15 109 cells/mL) as measured with a spectrophotometer (Hitachi U-1900 Spectrophotometer, Tokyo, Japan). Column clogging treatment The experiments were conducted using 50 mL plastic syringes (Terumo SS-50ESZ, Terumo Corporation, Tokyo, Japan) as plastic columns that had a 3 cm inner diameter and were 10 cm in height (Fig. 1). Different sizes of glass beads (Potters-Ballotini Co. Ltd., Ibaraki, Japan), with average diameters of 0.01 mm, 0.05 mm, 0.25 mm, 0.50 mm, 1.0 mm, 2.0 mm, and 3.0 mm (abbreviated as GB0.01, GB0.05, GB0.25, GB0.50, GB1.0, GB2.0, and GB3.0, respectively), were used as porous media in the experiments. Before use, the glass beads were washed with an acidic solution (0.1 N HCl) and subsequently rinsed with distilled water until a neutral pH was obtained. The columns were packed with 90 g of the washed glass beads under saturated conditions. The cell suspension (4 pore volumes of glass beads, 100 mL) was introduced into the column with a flow rate of 2 mL/min using a peristaltic pump at 22 C. A magnetic stirrer was used to ensure a homogenous bacterial suspension. During the
introduction of the bacteria, samples were taken from the effluent to count the number of bacteria eluted from the column and thus calculate the number of bacteria that had been deposited into the column. The introduction of the bacterial suspension was complete after 50 min. Then, the precipitation solution (0.5 M CaCl2 and 0.5 M urea; pH 6.8) was sterilized through a 0.22 mm membrane filter and introduced into the column with a flow rate of 3 mL/min. The precipitate solution was continuously introduced until the hydraulic conductivity of the column became stable. Effect of bacterial population size on reduction in hydraulic conductivity To examine the effect of bacterial population size on reduction in hydraulic conductivity, experiments were conducted with cell suspensions at various densities (OD600 0.15, OD600 0.75, and OD600 2.25), using the same procedure as described above. One milliliter cell suspension, which Size analysis of CaCO3 precipitate contained 109 cells, was injected into 20 mL sterilized precipitation solution using a syringe. Then, samples were taken at appropriate intervals and placed on a glass slide to measure the size of the crystals in the CaCO3 precipitate using a microscope (Olympus BX50WI, Tokyo, Japan). Determination of the amount of CaCO3 precipitated into the columns After the column clogging treatment, the glass beads were removed from the columns, placed into a beaker, and mixed with 0.1 N HNO3 to dissolve the precipitated CaCO3. Then, samples were taken to detect their Ca2þ concentration using inductively coupled plasma atomic emission spectroscopy (PerkinElmer Optima 3300DV, PerkinElmer, Waltham, MA, USA). After the Ca2þ concentration was measured, the amount of CaCO3 that had precipitated into the columns was calculated. Pore size distribution of glass beads before and after column clogging To estimate the pore size distribution of the glass beads, matric potential was measured using a wide range pF meter (DIK-3404, Daiki Co., Ltd., Saitama, Japan). First, the columns were weighed; then, the bottoms of the columns were covered with Advantec No. 6 filter paper (Advantec MFS, Inc., CA, USA). Next, the columns were saturated with distilled water, as were the ceramic filters of the pF meter. After 24 h, the columns were weighed again. Next, the ceramic filters and the columns were put into the pF meter. Then, pressure in the pF meter was adjusted to pF values 1.3, 1.6, 2.0, 2.5, 3.0, 3.5, and 3.8, to identify pore sizes of 75e150 mm, 31e75 mm, 10e30 mm, 4e9 mm, 1e3 mm, 0.45e1.00 mm, and
