Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e8, 2015 www.elsevier.com/locate/jbiosc
Effects of bentonite and yeast extract as nutrient on decrease in hydraulic conductivity of porous media due to CaCO3 precipitation induced by Sporosarcina pasteurii an Eryürük,1 Suyin Yang,2 Daisuke Suzuki,2 Iwao Sakaguchi,2 and Arata Katayama1, 2, * Kag 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 25 November 2014; accepted 30 January 2015 Available online xxx
The reduction mechanism of hydraulic conductivity was investigated in porous media treated with bentonite and CaCO3 precipitates induced by growing cells of Sporosarcina pasteurii (ATCC 11859). Bentonite, the bacterial cells, and a precipitation solution, composing of 0.5 M CaCl2 and 0.5 M urea with or without 2% weight/volume yeast extract allowing the bacterial growth were sequentially introduced into the continuous-flow columns containing glass beads between 0.05 and 3 mm in diameter. The treatments reduced the hydraulic conductivity of the columns from between 8.4 3 10L1 and 4.1 3 10L3 cm/s to between 9.9 3 10L4 and 2.1 3 10L6 cm/s as the lowest. With yeast extract, the conductivity continuously decreased during four days of the experiment, while became stable after two days without yeast extract. Introduction of the bacterial cells did not decrease the conductivity. The reduction in hydraulic conductivity was inversely correlated with the volume occupied by the depositions of bentonite and CaCO3 precipitates in column, showing the same efficiency but a larger effect of the CaCO3 precipitates with increasing volume by bacterial growth. The smaller glass beads resulted in larger volume of the depositions. Bentonite increased the deposition of CaCO3 precipitates. Analysis using the KozenyeCarman equation suggested that without yeast extract, bentonite and the CaCO3 precipitates formed aggregates with glass beads, thus increasing their diameter and consequently decreasing the pore size in the column. With yeast extract, in addition to the aggregates, the individual CaCO3 precipitates formed separately from the aggregates reduced the hydraulic conductivity. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Microbial clogging; Glass beads; Bentonite; Yeast extract; CaCO3; Hydraulic conductivity; Sporosarcina pasteurii; KozenyeCarman equation]
Landfill is the simplest, cheapest, and most cost-effective way of disposing of waste (1). However, groundwater, which is a resource of drinking water, can become contaminated due to leakage from landfills (2). Therefore, geosynthetic clay liners (3e5) were investigated to decrease the hydraulic conductivity of landfills to avoid movement of contaminated fluid from the landfill to groundwater. Although geosynthetic clay liners provide low hydraulic conductivity, several studies have indicated that the increase in the hydraulic conductivity of clay liners might be possible due to permeation of inorganic salt solutions (6e8) and seasonal drying, which causes shrinkage of the clay and the development of cracks (9). Hence, microbial clogging, which gives rise to insoluble inorganic and organic compounds such as microbially induced calcium carbonate (10,11), is considered as a maintenance technique instead of simple clay liners to keep the hydraulic conductivity of landfills at the required level. Microbially induced calcium carbonate is a common phenomenon in nature (12), and hydrolysis of urea is one of the methods to
* 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).
precipitate calcium carbonate in the presence of urea and calcium (13). Bacterial CaCO3 precipitation has been studied for different purposes, such as improving the mechanical properties of soil (14,15), repairing construction (16), decreasing the hydraulic conductivity of porous media (17e19), reducing leakage from channels (20), and immobilizing heavy metals by co-precipitation with CaCO3 precipitates (21e23). Our previous research demonstrated that the hydraulic conductivity of porous media was decreased by the CaCO3 precipitation induced by resting Sporosarcina pasteurii (S. pasteurii) (24). However, the hydraulic conductivity was decreased by only one order or less than two orders, achieving a hydraulic conductivity of 3.07 105 cm/s (24), which is not enough to maintain the liner for the landfill site, where lower than 106 cm/s of the hydraulic conductivity is required with 50 cm of the thickness (Ministry of Environment Japan, Ministerial Order No. 3, revised 21 February 2013, http://law.e-gov.go.jp/htmldata/S52/ S52F03102004001.html). Thus, it is important to examine the ureolytic microbial growth in the presence of various nutrients for further decreasing the hydraulic conductivity of porous media. Also, because the addition of nutrients increases the population of indigenous bacteria widely present in the environment (25), it would minimize engineering difficulties such as transportation of bacterial cells through the soil (26).
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.01.020
Please cite this article in press as: Eryürük, K., et al., Effects of bentonite and yeast extract as nutrient on decrease in hydraulic conductivity of porous media due to CaCO3 precipitation induced by Sporosarcin..., J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.020
2
ERYÜRÜK ET AL.
However, bentonite, which has a high specific surface area in addition to high swelling potential (27) and is a clay mineral, can be used to reduce the hydraulic conductivity of the soil in spite of the probability of crack occurrence in bentonite. Microbially induced carbonate precipitation may repair the possible cracks in bentonite to maintain the low hydraulic conductivity. Thus, it can be said that the combination of the usage of bentonite and microbially induced CaCO3 precipitation may have great potential to clog the soil pores to provide low hydraulic conductivity. It is essential to understand the mechanism of reduction in the hydraulic conductivity of porous media by the bacterial CaCO3 precipitation in combination with the bentonite treatment for the application of this technology. However, there are a few studies on the quantitative effect of bacterial CaCO3 precipitation on the hydraulic conductivity of porous media. Our previous research elucidated that the decrease in the hydraulic conductivity of porous media by the CaCO3 precipitation induced by resting S. pasteurii was successfully described by the modified KozenyeCarman equation (24). Further study should be conducted to evaluate the effect of the growth of bacteria inducing the CaCO3 precipitation in combination with bentonite application on the clogging of porous media. In this paper, a quantitative study was conducted to elucidate the reduction mechanism of hydraulic conductivity of porous media by using the continuous-flow columns packed with glass beads with different diameters as model porous media with different pore sizes, and by treating the columns with bentonite as clogging clay and the CaCO3 precipitates induced by S. pasteurii (ATCC 11859) with yeast extract as nutrient allowing the bacterial growth. MATERIALS AND METHODS Column clogging experiment Column clogging experiments were carried out using S. pasteurii ATCC 11859 to induce CaCO3 precipitation at 22 C, as previously reported in detail (24). The column with a 3-cm inner diameter and 10 cm in height was provided using 50-mL plastic syringes (Terumo SS-50ESZ, Terumo Corporation, Tokyo, Japan). Different sizes of glass beads (Potters-Ballotini Co. Ltd., Ibaraki, Japan), with average diameters of 0.05 mm, 0.25 mm, 1.0 mm, and 3.0 mm (abbreviated as GB0.05, GB0.25, GB1.0, and GB3.0, respectively), were used as porous media. The column had 25 mL of the pore volume. S. pasteurii ATCC 11859 was introduced into the column as 100 mL of suspension (four pore volumes of the column) with a cell density of 2.15 109 cells/mL at a flow rate of 2 mL/min using a peristaltic pump. A magnetic stirrer was used to ensure a homogenous bacterial suspension during 50 min of the introduction. 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 continuously introduced into the column with a flow rate of 3 mL/min for four days (totally 17,500 mL of the precipitation solution). The hydraulic conductivity of the column was measured using a manometer. The effect of bentonite on the reduction of the hydraulic conductivity of the column was examined by introducing 25 mL of the suspension made by 4.5 g of bentonite (5% weight/weight of the amount of glass beads) and distilled water with thorough mixing using a magnetic stirrer. The bentonite (Wako Pure Chemical Industries, Ltd., Osaka, Japan) contained a negligible amount of calcium (Supplementary Table S1), and the average particle size and the proportions of particles smaller than 0.45 mm in diameter were 5.0 mm and 0.12%, respectively. The bentonite suspension was introduced into the column at a flow rate of 2 mL/min using the peristaltic pump. Then, the bacterial suspension and the precipitation solution were introduced as described above, and the hydraulic conductivity was measured. The effect of yeast extract as a nutrient on the hydraulic conductivity was examined by introducing yeast extract (Becton, Dickinson and Company, NJ, USA) as a dissolved component of the precipitation solution at a concentration of 2% weight/ volume with an adjusted pH of 6.8. The precipitation solution was continuously introduced at a flow rate of 3 mL/min using the peristaltic pump as described above. The experiment was stopped before all the precipitation solution was introduced in the case that the leakage of the precipitation solution was observed from the tube connecting points of the column without the effluent solution because of the high pressure inside the column due to the low hydraulic conductivity, which was 2.1 106 cm/s. The conditions of the clogging treatments using S. pasteurii ATCC 11859, bentonite, the precipitation solution, and yeast extract are summarized in Table 1. The estimation of bacterial population in the column The number of bacterial cells in the column was estimated by multiplying the number of bacterial
J. BIOSCI. BIOENG., TABLE 1. Clogging treatment conditionsa. Condition
0b 1 2 3 4 5 6
S. pasteurii ATCC 11859
Bentonite
Precipitation solution
Yeast extract
c þ þ þ þ þ
þ þ þ
þ þ þ þ
þ þ
a Treatments with materials were carried out by introducing sequentially 25 mL of bentonite suspension (2 mL/min), 100 mL of cell suspension of S. pasteurii (2 mL/ min), and 17,500 mL of precipitation solution with or without yeast extract (3 mL/ min), in this order, to the columns. b Condition 0 denotes the condition before the treatments. c Symbols: þ, presence of material; , absence of material.
cells initially deposited with the growth proportion obtained in the batch culture, subtracting the number of bacterial cells eluted from the column. This estimation was carried out because the number of bacterial cells in the column cannot be counted by the direct microscopic method due to the formation of CaCO3 precipitation. The batch culture was provided by the introduction of 1 mL of 109 cell suspension into 20 mL of the sterilized precipitation solution, including 2% w/v yeast extract. Then, the cultures were incubated at 22 C, and an aliquot of the culture was taken at appropriate intervals to count the number of bacterial cells under the microscope (BX50WI, Olympus, Tokyo, Japan). During the microbial clogging experiments of the columns, samples were taken from the effluent after appropriate intervals, and the number of bacteria in the effluent was counted microscopically. The deposited amount of CaCO3 precipitation and bentonite The amount of CaCO3 precipitation in the columns of glass beads was determined as previously reported (24). The precipitated CaCO3 was dissolved into 0.1 N HNO3 and measured by inductively coupled plasma atomic emission spectroscopy (PerkinElmer Optima 3300DV, PerkinElmer, Waltham, MA, USA). The amount of CaCO3 precipitation in the columns was calculated using the Ca2þ concentration. The deposited amount of bentonite was obtained by subtracting the amount of bentonite eluted from the column. Densities of CaCO3 (2.71 g/cm3) and bentonite (0.80 g/cm3) were used to convert the amounts to the volumes of CaCO3 and bentonite. Size analysis of CaCO3 precipitates and bentonite One milliliter cell suspension, which contained 109 cells, was inoculated into 20 mL of the sterilized precipitation solution, including 2% w/v yeast extract using a sterile syringe, and incubated at 22 C. Then, samples were taken at appropriate intervals and placed on a glass slide to measure the size of the crystals in the CaCO3 precipitates using a microscope (Olympus BX50WI). Bentonite was suspended well in distilled water, and then the suspension was placed on a glass slide to measure the size of the bentonite particles under the microscope. The pore size distribution of the columns Matric potential was measured to evaluate the pore size distribution of the columns using a wide-range pF meter (DIK-3404, Daiki Co., Ltd., Saitama, Japan). The diameter of the pore (d, cm) in the column was estimated from the matric potential head (j, cm) by the following equation (28,29): d ¼ 0:3=j
(1)
The average pore size in the columns was calculated from the proportion of the individual size ranges of the pores.
RESULTS Changes in hydraulic conductivity by the column clogging treatments The hydraulic conductivity of the columns packed with different average diameters of glass beads (0.05e3 mm) was measured after the treatments with different conditions, as shown in Fig. 1. The hydraulic conductivity of the initial conditions of the columns ranged from 8.4 101 to 4.1 103 cm/s. Introduction of only cell suspension (condition 1) did not change the hydraulic conductivity. Introduction of only bentonite suspension (condition 2) did not decrease so much in the hydraulic conductivity, as shown by the range of 6.2 101 to 9.9 104 cm/s, although the extent of the decrease was larger in the column packed with small glass beads. The decrease was attributed to the larger amount of deposition of bentonite in the column (Supplementary Table S2). Our previous research (24)
Please cite this article in press as: Eryürük, K., et al., Effects of bentonite and yeast extract as nutrient on decrease in hydraulic conductivity of porous media due to CaCO3 precipitation induced by Sporosarcin..., J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.020
VOL. xx, 2015
BENTONITE AND YEAST EXTRACT ENHANCE MICROBIAL CLOGGING
3
FIG. 1. Hydraulic conductivity of columns packed with different sizes of glass beads (open circle) before (condition 0) and after application of condition 1 (plus sign), condition 2 (closed bar), condition 3 by Eryürük et al. (24) (asterisk), condition 4 (closed triangle), condition 5 (cross sign), and condition 6 (closed circle). Details of the conditions are summarized in Table 1.
showed that introduction of the precipitation solution in the presence of resting cells (condition 3) decreased the hydraulic conductivity to the range of 3.7 101 to 4.1 104 cm/s (Fig. 1). Combined introduction of bentonite suspension, cell suspension, and the precipitation solution (condition 4) decreased the hydraulic conductivity of the column in the range of 3.9 102 to 3.1 105 cm/s. The difference in the hydraulic conductivity between conditions 3 and 4 was larger than that between conditions 1 and 2. This suggested that bentonite promoted the deposition of microbial CaCO3 precipitates. The hydraulic conductivity decreased to the range of 2.3 103 to 3.7 106 cm/s for the combined introduction of cell suspension and precipitation solution including yeast extract (condition 5), and to the range of 9.9 104 to 2.1 106 cm/s for the combined introduction of bentonite suspension, cell suspension, and the precipitation solution including yeast extract (condition 6). Conditions 5 and 6 containing yeast extract as a nutrient allowed the growth of S. pasteurii ATCC 11859 and resulted in the lower hydraulic conductivity. Condition 6 achieved the lowest hydraulic conductivity in the column with GB0.05, which showed the leakage of the precipitation solution from the tube connecting points of the column in the experiment with reaching the limit for the measurement of hydraulic conductivity, 2.1 106 cm/s. Correspondence between numbers of bacterial cells deposited in the column and the decrease in hydraulic conductivity In the presence of yeast extract in precipitation solution, the hydraulic conductivity continued to decrease during four days of the clogging experiment (Fig. 2A and Supplementary Fig. S1), which corresponded to 700 pore volumes, while without yeast extract, the hydraulic conductivity became stable after 2 days of infiltration, regardless of the different sizes of glass beads, which corresponded to 350 pore volumes. Growth of S. pasteurii ATCC 11859 in the batch culture with yeast extract did not stop within four days (Fig. 2B and Supplementary Fig. S1). Although the numbers of eluted cells in the clogging experiment decreased with time in all the columns, the numbers differed between the columns depending on the glass bead sizes. Under the conditions 3 and 4 without yeast extract, the estimated numbers of the deposited bacterial cells were larger in the columns with smaller glass beads (Supplementary Table S3), corresponding to the larger reduction of the hydraulic conductivity. Because the numbers of the eluted cells were much smaller than those of the deposited bacterial cells, the number of the deposited cells in the column was maintained at an almost constant level during 4 days of the
FIG. 2. (A) Time course changes in hydraulic conductivity in column packed with GB0.05. Symbols: plus sign, condition 1; closed bar, condition 2; asterisk, condition 3; closed triangle, condition 4; cross sign, condition 5; and closed circle, condition 6. (B) For GB0.05, the number of cells in the solution (open circle), the number of cells in effluent (closed square), and the estimated number of cells in the column (cross sign).
experiment. It was considered that the urease activity disappeared in 2 days without yeast extract. Under the conditions 5 and 6 with yeast extract allowing the bacterial growth, the numbers of the deposited cells after 4 days were estimated to be approximately the same in all the columns, regardless of the different sizes of glass beads (Supplementary Table S3). The correspondence between the number of the bacterial cells and the decrease in hydraulic conductivity suggested that the bacterial growth supported by yeast extract resulted in the continuous decrease in hydraulic conductivity due to the maintenance of the urease activity of the bacterium inducing the CaCO3 precipitation for the entire experimental period. It should be noted that the significant difference in the number of deposited bacterial cells (P < 0.05) was observed between conditions 3 and 4 (Supplementary Table S3). This suggested that bentonite promoted the deposition of bacterial cells in the column. The relation between reduction of hydraulic conductivity and the occupied volume of deposited CaCO3 precipitates and bentonite Fig. 3 shows the relation between the relative reduction rate of hydraulic conductivity and the volume occupied with both the CaCO3 precipitates and bentonite (Fig. 3A), only the CaCO3 precipitation (Fig. 3B), or only the bentonite (Fig. 3C). The amount of calcium in bentonite was negligibly low (Supplementary Table S2) comparing with the amount of CaCO3 precipitates in the columns, therefore, the amount of CaCO3 precipitates estimated from calcium amount was little affected by the deposited bentonite. The relative reduction rate of hydraulic conductivity showed a significant correlation with either the volume occupied with both the CaCO3 precipitates and bentonite or with the only CaCO3 precipitation but not with bentonite. In addition, the effect of bentonite was limited at less than a oneorder decrease in hydraulic conductivity, as shown by the results of condition 1 in Fig. 3A. These results suggested that the reduction in hydraulic conductivity was dependent mainly on the
Please cite this article in press as: Eryürük, K., et al., Effects of bentonite and yeast extract as nutrient on decrease in hydraulic conductivity of porous media due to CaCO3 precipitation induced by Sporosarcin..., J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.020
4
ERYÜRÜK ET AL.
J. BIOSCI. BIOENG., did not have a unique particle size, and the particle size of the bentonite was calculated as 5.0 mm on average.
FIG. 3. Relationship between the proportion of the hydraulic conductivity of the treated column to the original hydraulic conductivity and (A) the occupied volume of CaCO3 precipitated and bentonite in the column, (B) the occupied volume of CaCO3 precipitated in the column, and (C) the occupied volume of bentonite in the column. Symbols: closed bar, condition 2; asterisk, condition 3; closed triangle, condition 4; cross sign, condition 5; and closed circle, condition 6. Relative reduction rate 1 (at 100%) is equal to condition 0.
CaCO3 precipitation. The amount of CaCO3 precipitates was larger in the columns packed with smaller glass beads under the same treatment conditions. The amount of CaCO3 precipitates in the columns with the deposit of bentonite was larger than that in the columns without bentonite. Deposited bentonite was considered to increase the deposition of the CaCO3 precipitation in the column, and indirectly promote the reduction of the hydraulic conductivity. Particle sizes of the CaCO3 precipitates and bentonite Fig. 4AeF show the microscopic observation of the formation of crystals of the CaCO3 precipitates in the static culture of the bacterium with yeast extract. The precipitation reaction started just 1 min after the cells were inoculated and mixed with the precipitation solution (Fig. 4A), and the CaCO3 precipitates grew to a maximum of approximately 10 mm in diameter in a few minutes. After a couple of hours (Fig. 4B), the number of 10-mm-diameter precipitates increased and began to form clusters, which were aggregated or individually larger than 10 mm (Fig. 4CeF). At the same time, precipitates smaller than 15 mm in diameter were also maintained (with 10 mm as the average diameter). After 4 days, the aggregates or individual particles with 25 mm as the average diameter became approximately the same in number as that of the precipitates with 10 mm as the average diameter (Supplementary Table S4). In the culture without yeast extract, the precipitates did not make the aggregates, and the average diameter was 10 mm, as previously reported (24). Fig. 4G and H confirmed that bentonite
Porosity and average pore size in columns before and after the clogging experiments Changes in pore size distribution were estimated from the water retention curve of the column packed with glass beads after the treatments. The larger deposition of the CaCO3 precipitates and/or bentonite in the columns with the smaller glass beads resulted in a larger extent of the decrease in porosity and estimated pore size. The largest reduction in pore size was observed in the introduction of bentonite suspension, cell suspension, and precipitation solution including yeast extract (condition 6). Before the clogging experiments (condition 0), the pore sizes were 49.7 mm, 66.1 mm, 77.6 mm, and 96.8 mm for the columns packed with GB0.05, GB0.25, GB1.0, and GB3.0, respectively. The pore size did not change in condition 1. Except condition 1, the pore sizes were reduced in response to the treatment; in condition 2, to 40.0 mm, 60.4 mm, 70.9 mm, and 88.4 mm for the columns packed with GB0.05, GB0.25, GB1.0, and GB3.0, respectively; in condition 3, to 33.0 mm, 52.0 mm, 64.1 mm, and 80.3 mm for the columns packed with GB0.05, GB0.25, GB1.0, and GB3.0, respectively; in condition 4, to 23.0 mm, 50.9 mm, 58.6 mm, and 70.3 mm for the columns packed with GB0.05, GB0.25, GB1.0, and GB3.0, respectively; in condition 5, to 20.1 mm, 32.6 mm, 52.4 mm, and 60.4 mm for the columns packed with GB0.05, GB0.25, GB1.0, and GB3.0, respectively; and in condition 6, to 16.1 mm, 28.9 mm, 49.3 mm, and 55.9 mm for the columns packed with GB0.05, GB0.25, GB1.0, and GB3.0, respectively. The pore volume of the columns also decreased in response to the treatment; in condition 2, from 25 cm3 (the same volume for all the columns) to 20.6 cm3, 22.5 cm3, 23.1 cm3, and 24.4 cm3 for the columns packed with GB0.05, GB0.25, GB1.0, and GB3.0, respectively; in condition 3, to 20.0 cm3, 21.3 cm3, 22.5 cm3, and 23.8 cm3 for columns with GB0.05, GB0.25, GB1.0, and GB3.0, respectively; in condition 4, to 13.1 cm3, 15.0 cm3, 17.5 cm3, and 19.4 cm3 for columns with GB0.05, GB0.25, GB1.0, and GB3.0, respectively; in condition 5, to 7.5 cm3, 10.0 cm3, 11.9 cm3, and 13.1 cm3 for columns with GB0.05, GB0.25, GB1.0, and GB3.0, respectively; and in condition 6, to 5.7 cm3, 7.5 cm3, 9.4 cm3, and 10.6 cm3 for columns with GB0.05, GB0.25, GB1.0, and GB3.0, respectively. The extent in the decrease in pore volume corresponded to that in porosity (Supplementary Table S5). DISCUSSION The hydraulic conductivity of the column with GB0.05 treated under condition 6 decreased to 2.1 106 cm/s after 4 days of infiltration of the precipitation solution with yeast extract (condition 6). This was the lowest hydraulic conductivity that was able to be measured in the experimental setup. Although the decrease in hydraulic conductivity was faster after 2 days, the continuous decrease in the hydraulic conductivity was still observed after 4 days in the columns treated with Condition 6. This suggested that the combination of bentonite and the precipitation solution with yeast extract would cause a further decrease in the hydraulic conductivity from the growth of ureolytic S. pasteurii (ATCC 11859) even after 4 days and would achieve the hydraulic conductivity required for the clay lining for the landfill, which should be lower than 106 cm/s of hydraulic conductivity with 50 cm of thickness (Ministry of Environment Japan, Ministerial Order No. 3, revised 21 February 2013, http://law.e-gov.go.jp/htmldata/S52/ S52F03102004001.html). Bentonite and the CaCO3 precipitates affected the hydraulic conductivity of the column to a similar extent, as shown in Fig. 3A. The efficiency in decreasing the hydraulic conductivity was 13.6%/ cm3 of bentonite as the relative reduction rate and 10.3%/cm3 of the CaCO3 precipitates, respectively. However, the effect of the CaCO3
Please cite this article in press as: Eryürük, K., et al., Effects of bentonite and yeast extract as nutrient on decrease in hydraulic conductivity of porous media due to CaCO3 precipitation induced by Sporosarcin..., J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.020
VOL. xx, 2015
BENTONITE AND YEAST EXTRACT ENHANCE MICROBIAL CLOGGING
5
FIG. 4. Photographs of the CaCO3 precipitates induced by S. pasteurii ATCC 11859 in a liquid static culture containing yeast extract after (A) 1 min, (B) 360 min, (C) 1440 min, (D) 2880 min, (E) 4320 min, and (F) 5760 min of incubation, and photographs of bentonite (G) and (H). Closed scale bars, 10 mm; open scale bars, 50 mm.
precipitates was larger because of the larger deposition volume in the column. Without yeast extract in the precipitation solution (conditions 3 and 4), the largest deposited volume of the CaCO3 precipitates was observed in the column with GB0.05 as 14 cm3. With the supply of yeast extract in the precipitation solution, the larger deposition of the CaCO3 precipitates was observed up to 24 cm3 in the column with GB0.05 (conditions 5 and 6). Continuous urease activity with yeast extract resulted in larger volumes of the CaCO3 precipitates, which clogged the pores of the column with glass beads and provided very low hydraulic conductivity. The larger volume of deposited bentonite and the CaCO3 precipitates were observed in the columns with smaller glass beads under the same treatment conditions. The specific CaCO3 precipitation rate of cells was calculated as 4.0 0.1 103 mg CaCO3/cell (condition 3) (24) and 7.0 0.1 103 mg CaCO3/cell (condition 4) under nonbacterial growth conditions and 1.8 0.1 103 mg CaCO3/cell
(condition 5) and 2.1 0.1 103 mg CaCO3/cell (condition 6) under bacterial growth conditions. Under the bacterial growth conditions, the larger amount of CaCO3 precipitates would cause less availability of CaCl2, urea and yeast extract in the precipitation solution for bacteria that had been grown inside the aggregates of the CaCO3 precipitates, and the less availability would lead the smaller value of specific CaCO3 precipitation rate. The deposited amount of bentonite was limited so that the clogging effect of bentonite only was small (condition 2). However, the deposited bentonite increased the depositing efficiency of the cells of S. pasteurii (ATCC 11859) and the CaCO3 precipitates induced, as shown by the difference between conditions 3 and 4 and between conditions 5 and 6. The deposition of bentonite resulted in the larger deposit number of the bacterial cells (Supplementary Table S3), the larger deposit amount of the CaCO3 precipitates and the larger decrease in hydraulic conductivity
Please cite this article in press as: Eryürük, K., et al., Effects of bentonite and yeast extract as nutrient on decrease in hydraulic conductivity of porous media due to CaCO3 precipitation induced by Sporosarcin..., J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.020
6
ERYÜRÜK ET AL.
J. BIOSCI. BIOENG.,
FIG. 5. Comparison of measured and estimated values of hydraulic conductivity using the modified KozenyeCarman equation: (A) condition 2; (B) condition 4; (C) condition 5; and (D) condition 6. In the estimation, the value of the shape factor (f) was set at 0.2534, which was determined by Eryürük et al. (24). Values of other parameters used are described in the text, except for the specific surface area (S). The values of S in conditions 2 and 4 (A, B) were estimated either by assuming that the glass beads, bentonite, bacterial cells, and CaCO3 precipitates formed one spherical particle with a diameter consisting of the total of the diameters of the individual particles (closed circle) or by assuming that the individual particles were separately present in the column (closed square). Another assumption was made for conditions 5 and 6 (C, D) in which none of the cell detached from the glass beads (closed circle), 20% of the cells detached from the glass beads and formed 20% of the total number of the CaCO3 precipitates as separately present in the pore solution with yeast extract (cross sign), 50% of the cells detached from the glass beads and formed 50% of the total number of the CaCO3 precipitates as separately present in the pore solution with yeast extract (closed triangle), and 100% of the cells detached from the glass beads and formed 100% of the total number of the CaCO3 precipitates as separately present in the pore solution with yeast extract (closed square). The symbols of 1.E e x (x ¼ 1e10) at axes mean 1.0 10x.
(Fig. 3A). It should be noted that the specific CaCO3 precipitation rate of the cells was larger under the conditions with bentonite (conditions 4 and 6) than the corresponding conditions without bentonite (conditions 3 and 5), suggesting the enhanced formation of the CaCO3 precipitate. Thus, bentonite indirectly enhanced the clogging. In the previous paper (24) in which the clogging treatment of condition 3 was carried out, a modified KozenyeCarman equation showed the good agreement by assuming that the glass beads, bacterial cells, and CaCO3 precipitates became one particle but not
KCaþB
r B sV B sV B ¼ f rg n Ca B Ben Ben V V V
3 ,
2 8 < 4m :
porosity, rCa denotes the specific CaCO3 precipitation rate, B denotes the number of bacterial cells deposited (Supplementary Table S3), V denotes the volume of the column, m denotes the dynamic viscosity of the liquid, and D denotes the average diameter of the glass beads. To identify the effects of bentonite and yeast extract on the decrease in hydraulic conductivity, Eq. 2 was further modified by introducing the specific microbial volume (sVB), specific volume of bentonite (sVBen), and amount of bentonite deposited (BBen):
6
6B ðr
p
3 Ca þ sVB þ sVBen Þ þ D
by assuming that they were separate particles. The modified KozenyeCarman equation, which estimates hydraulic conductivity based on the specific surface area and the porosity of the porous medium, was as follows: KCaþB ¼ f rgðn rCa B=VÞ3
1=3 2 ð1 n þ rCa B=VÞ2 6rCa B=p þ D3 m 6
r denotes the density of liquid, g denotes gravity, n denotes
(2)
where KCaþB denotes the hydraulic conductivity of the porous medium with microbial clogging, f denotes the shape factor,
92 =
1=3 ;
r B sV B sV B 1 n þ Ca þ B þ Ben Ben V V V
2
3 5
(3)
Here, the density of the precipitation solution including yeast extract (r) was measured at 22 C as 1072 kg/m3. The value of g used was 9.80665 m/s2. The dynamic viscosity of the precipitation solution including yeast extract (m) has previously been shown to be 0.0140 kg/ms (30). The value of fd0.2532din the columns was the same as the previous study (24). The bacterial number in the column was estimated at 4.5 1010 cells under bacterial growth conditions after 4 days, and it was estimated to be approximately the same for all the columns packed with different sizes of glass beads (Supplementary Table S3). In conditions 2 and 4, as in the previous paper (24) with condition 3, Eq. 3 showed the good
Please cite this article in press as: Eryürük, K., et al., Effects of bentonite and yeast extract as nutrient on decrease in hydraulic conductivity of porous media due to CaCO3 precipitation induced by Sporosarcin..., J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.020
VOL. xx, 2015
BENTONITE AND YEAST EXTRACT ENHANCE MICROBIAL CLOGGING
agreement by assuming that the bentonite, glass beads, bacterial cells, and CaCO3 precipitates became one particle (Fig. 5A and B). Therefore, the bentonite, bacterial cells, and CaCO3 precipitates would attach to the surface of the glass beads in the column; increase the glass bead sizes; decrease the porosity of the column; and decrease the hydraulic conductivity in conditions 2, 3, and 4. In conditions 5 and 6 with bacterial growth, good agreement was obtained by assuming that 50% of the cells were attached to the glass beads to form CaCO3 precipitates attached on the glass beads, and 50% of the cells detached from the glass beads to form CaCO3 precipitates as separate and individual particles in the column pores. The separate CaCO3 precipitates may attach on the glass beads but all the surface of the precipitates was considered as effective at decreasing the hydraulic conductivity. All of the bentonite, which remained in the column, was considered as attached on the glass beads to form the aggregates. When considering that 0%, 80%, and 100% of the cells were attached to the glass beads to form the aggregates with the CaCO3 precipitates, meaning that 100%, 20%, and 0% of the cells detached from the glass beads to form the separate CaCO3 precipitates, respectively, the calculated and measured hydraulic conductivity did not agree well (Fig. 5C and D). The large and small CaCO3 precipitates were observed in the static culture using the precipitation solution with yeast extract, and the ratio of the number of large and small precipitates was approximately 1:1. This observation corresponded well to the results of the analysis from the KozenyeCarman equation, which found that 50% of the total number of the CaCO3 precipitates formed large particles by attaching to the glass beads, and 50% of the total number of the CaCO3 precipitates separately formed small particles. The growth of the bacterial population using yeast extract led to the formation of the separate CaCO3 precipitates in addition to the attached CaCO3 precipitates on the glass beads. Correspondingly, the decrease in the porosity caused a decrease in the hydraulic conductivity of the column. In summary, the effects of bentonite and yeast extract on the clogging of porous media due to CaCO3 precipitation induced by S. pasteurii ATCC11859 under the growing conditions were quantitatively evaluated using columns packed with glass beads. Analysis using the modified KozenyeCarman equation indicated that the mechanism underlying the reduction in hydraulic conductivity was not only the aggregation of the glass beads with the bentonite and the CaCO3 precipitates, which increased the effective size of glass beads, but also the formation of individual CaCO3 precipitates separated from the glass beads in the column pore in the presence of yeast extract allowing the bacterial growth. Although the addition of only bentonite suspension did not change the hydraulic conductivity significantly, bentonite promoted the deposition of the CaCO3 precipitates in the column. Bacterial growth using nutrient had a larger effect on decreasing the hydraulic conductivity by the CaCO3 precipitates. The combination of bentonite suspension, cell suspension, and the precipitation solution including yeast extract resulted in the highest decrease in hydraulic conductivity. The lowest hydraulic conductivity achieved in this study was 106 cm/s. Microbially induced CaCO3 precipitation could be applied as a technology to landfills to decrease hydraulic conductivity by providing CaCl2, urea, and a nutrient to enhance ureolytic bacteria in situ. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2015.01.020.
ACKNOWLEDGMENTS This work was partly supported by the Environmental Technology Development Fund in the Ministry of the Environment, Japan (ZB-1205) and the Environmental Biological Remediation and
7
Conservation Project C-1 (2013e2014) at the EcoTopia Science Institute, Nagoya University, Japan.
References 1. Allen, A.: Containment landfills: the myth of sustainability, Eng. Geol., 60, 3e19 (2001). 2. Röling, W. F. M., van Breukelen, B. M., Braster, M., Lin, B., and van Verseveld, H. W.: Relationship between microbial community structure and hydrochemistry in a landfill leachate-polluted aquifer, Appl. Environ. Microbiol., 67, 4619e4629 (2001). 3. Gleason, M. H., Daniel, D. E., and Eykholt, G. R.: Calcium and sodium bentonite for hydraulic containment applications, J. Geotech. Geoenviron., 123, 438e445 (1997). 4. Benson, C. H., Daniel, D. E., and Boutwell, G. P.: Field performance of compacted clay liners, J. Geotech. Geoenviron. Eng., 125, 390e403 (1999). 5. Benson, C. H., Thorstad, P. A., Jo, H.-Y., and Rock, S. A.: Hydraulic performance of geosynthetic clay liners in a landfill final cover, J. Geotech. Geoenviron. Eng., 133, 814e827 (2007). 6. Petrov, R. J. and Rowe, R. K.: Geosynthetic clay liner (GCL) e chemical compatibility by hydraulic conductivity testing and factors impacting its performance, Can. Geotech. J., 34, 863e885 (1997). 7. Egloffstein, T. A.: Natural bentonitesdInfluence of the ion exchange and partial desiccation on permeability and self-heating capacity of bentonites used in GCLs, Geotext. Geomembranes, 19, 427e444 (2001). 8. Kolstad, D. C., Benson, C. H., and Edil, T. B.: Hydraulic conductivity and swell of nonprehydrated geosynthetic clay liners permeated with multispecies inorganic solutions, J. Geotech. Geoenviron. Eng., 130, 1236e1249 (2004). 9. Daniel, E. D. and Wu, Y.-K.: Compacted clay liners and covers for arid sites, J. Geotech. Eng., 119, 223e237 (1993). 10. Stocks-Fischer, S., Galinat, J. K., and Bang, S. S.: Microbial precipitation of CaCO3, Soil Biol. Biochem., 31, 1563e1571 (1999). 11. Chou, C.-W., Seagren, E. A., Aydilek, A. H., and Lai, M.: Biocalcification of sand through ureolysis, J. Geotech. Geoenviron. Eng., 137, 1179e1189 (2011). 12. Hammes, F. and Verstraete, W.: Key roles of pH and calcium metabolism in microbial carbonate precipitation, Rev. Environ. Sci. Biotechnol., 1, 3e7 (2002). 13. Okwadha, G. D. O. and Li, J.: Optimum conditions for microbial carbonate precipitation, Chemosphere, 81, 1143e1148 (2010). 14. Harkes, M. P., Van Paassen, L. A., Booster, J. L., Whiffin, V. S., and Van Loosdrecht, M. C. M.: Fixation and distribution of bacterial activity in sand to induce carbonate precipitation for ground reinforcement, Ecol. Eng., 36, 112e117 (2010). 15. Whiffin, V. S., Van Paassen, L. A., and Harkes, M. P.: Microbial carbonate precipitation as a soil improvement technique, Geomicrobiol. J., 24, 417e423 (2007). 16. De Muynck, W., Cox, K., De Belie, N., and Verstraete, W.: Bacterial carbonate precipitation as an alternative surface treatment for concrete, Constr. Build. Mater., 22, 875e885 (2008). 17. Nemati, M. and Voordouw, G.: Modification of porous media permeability, using calcium carbonate produced enzymatically in situ, Enzyme Microb. Technol., 33, 635e642 (2003). 18. Yasuhara, H., Neupane, D., Hayashi, K., and Okamura, M.: Experiments and predictions of physical properties of sand cemented enzymatically-induced carbonate precipitation, Soil Found., 52, 539e549 (2012). 19. Chu, J., Stabnikov, V., and Ivanov, V.: Microbially induced calcium carbonate precipitation on surface or in the bulk of soil, Geomicrobiol. J., 29, 544e549 (2012). 20. Gollapudi, U. K., Knutson, C. L., Bang, S. S., and Islam, M. R.: A new method for controlling leaching through permeable channels, Chemosphere, 30, 695e705 (1995). 21. Mitchell, A. C. and Ferris, F. G.: The coprecipitation of Sr into calcite precipitates induced by bacterial ureolysis in artificial groundwater: temperature and kinetic dependence, Geochim. Cosmochim. Acta, 69, 4199e4210 (2005). 22. Fujita, Y., Ferris, F. G., Lawson, R. D., Colwell, F. S., and Smith, R. W.: Calcium carbonate precipitation by ureolytic subsurface bacteria, Geomicrobiol. J., 17, 305e318 (2000). 23. Fujita, Y., Redden, G. D., Ingram, J. C., Cortez, M. M., Ferris, F. G., and Smith, R. W.: Strontium incorporation into calcite generated by bacterial ureolysis, Geochim. Cosmochim. Acta, 68, 3261e3270 (2004). 24. Eryürük, K., Yang, S. Y., Suzuki, D., Sakaguchi, I., Akatsuka, T., Tsuchiya, T., and Katayama, A.: Reducing hydraulic conductivity of porous media using CaCO3 precipitation induced by Sporosarcina pasteurii, J. Biosci. Bioeng., 119, 331e336 (2015). 25. Mobley, H. L. and Hausinger, R. P.: Microbial ureases: significance, regulation, and molecular characterization, Microbiol. Rev., 53, 85e108 (1989). 26. Mortensen, B. M., Haber, M. J., DeJong, J. T., Caslake, L. F., and Nelson, D. C.: Effects of environmental factors on microbial induced calcium carbonate precipitation, J. Appl. Microbiol., 111, 338e349 (2011).
Please cite this article in press as: Eryürük, K., et al., Effects of bentonite and yeast extract as nutrient on decrease in hydraulic conductivity of porous media due to CaCO3 precipitation induced by Sporosarcin..., J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.020
8
ERYÜRÜK ET AL.
27. Cerato, A. B. and Lutenegger, A. J.: Determination of surface area of finegrained soils by the ethylene glycol monoethyl ether (EGME) method, Geotech. Test J., 25, 1e7 (2002). 28. Lal, R. and Shukla, M. K.: Principles of soil physics, pp. 313e314. Marcel Dekker, Inc., New York (2004).
J. BIOSCI. BIOENG., 29. Fang, H.-Y.: Introduction to environmental geotechnology, pp. 194. CRC Press LLC, Boca Raton, Florida (1997). 30. Goel, A.: States of matter, pp. 21e23. Discovery Publishing House, New Delhi (2006).
Please cite this article in press as: Eryürük, K., et al., Effects of bentonite and yeast extract as nutrient on decrease in hydraulic conductivity of porous media due to CaCO3 precipitation induced by Sporosarcin..., J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.020
Effects of bentonite and yeast extract as nutrient on decrease in hydraulic conductivity of porous media due to CaCO3 precipitation induced by Sporosarcina pasteurii an Eryürük,1 Suyin Yang,2 Daisuke Suzuki,2 Iwao Sakaguchi,2 and Arata Katayama1, 2, * Kag 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 25 November 2014; accepted 30 January 2015 Available online xxx
The reduction mechanism of hydraulic conductivity was investigated in porous media treated with bentonite and CaCO3 precipitates induced by growing cells of Sporosarcina pasteurii (ATCC 11859). Bentonite, the bacterial cells, and a precipitation solution, composing of 0.5 M CaCl2 and 0.5 M urea with or without 2% weight/volume yeast extract allowing the bacterial growth were sequentially introduced into the continuous-flow columns containing glass beads between 0.05 and 3 mm in diameter. The treatments reduced the hydraulic conductivity of the columns from between 8.4 3 10L1 and 4.1 3 10L3 cm/s to between 9.9 3 10L4 and 2.1 3 10L6 cm/s as the lowest. With yeast extract, the conductivity continuously decreased during four days of the experiment, while became stable after two days without yeast extract. Introduction of the bacterial cells did not decrease the conductivity. The reduction in hydraulic conductivity was inversely correlated with the volume occupied by the depositions of bentonite and CaCO3 precipitates in column, showing the same efficiency but a larger effect of the CaCO3 precipitates with increasing volume by bacterial growth. The smaller glass beads resulted in larger volume of the depositions. Bentonite increased the deposition of CaCO3 precipitates. Analysis using the KozenyeCarman equation suggested that without yeast extract, bentonite and the CaCO3 precipitates formed aggregates with glass beads, thus increasing their diameter and consequently decreasing the pore size in the column. With yeast extract, in addition to the aggregates, the individual CaCO3 precipitates formed separately from the aggregates reduced the hydraulic conductivity. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Microbial clogging; Glass beads; Bentonite; Yeast extract; CaCO3; Hydraulic conductivity; Sporosarcina pasteurii; KozenyeCarman equation]
Landfill is the simplest, cheapest, and most cost-effective way of disposing of waste (1). However, groundwater, which is a resource of drinking water, can become contaminated due to leakage from landfills (2). Therefore, geosynthetic clay liners (3e5) were investigated to decrease the hydraulic conductivity of landfills to avoid movement of contaminated fluid from the landfill to groundwater. Although geosynthetic clay liners provide low hydraulic conductivity, several studies have indicated that the increase in the hydraulic conductivity of clay liners might be possible due to permeation of inorganic salt solutions (6e8) and seasonal drying, which causes shrinkage of the clay and the development of cracks (9). Hence, microbial clogging, which gives rise to insoluble inorganic and organic compounds such as microbially induced calcium carbonate (10,11), is considered as a maintenance technique instead of simple clay liners to keep the hydraulic conductivity of landfills at the required level. Microbially induced calcium carbonate is a common phenomenon in nature (12), and hydrolysis of urea is one of the methods to
* 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).
precipitate calcium carbonate in the presence of urea and calcium (13). Bacterial CaCO3 precipitation has been studied for different purposes, such as improving the mechanical properties of soil (14,15), repairing construction (16), decreasing the hydraulic conductivity of porous media (17e19), reducing leakage from channels (20), and immobilizing heavy metals by co-precipitation with CaCO3 precipitates (21e23). Our previous research demonstrated that the hydraulic conductivity of porous media was decreased by the CaCO3 precipitation induced by resting Sporosarcina pasteurii (S. pasteurii) (24). However, the hydraulic conductivity was decreased by only one order or less than two orders, achieving a hydraulic conductivity of 3.07 105 cm/s (24), which is not enough to maintain the liner for the landfill site, where lower than 106 cm/s of the hydraulic conductivity is required with 50 cm of the thickness (Ministry of Environment Japan, Ministerial Order No. 3, revised 21 February 2013, http://law.e-gov.go.jp/htmldata/S52/ S52F03102004001.html). Thus, it is important to examine the ureolytic microbial growth in the presence of various nutrients for further decreasing the hydraulic conductivity of porous media. Also, because the addition of nutrients increases the population of indigenous bacteria widely present in the environment (25), it would minimize engineering difficulties such as transportation of bacterial cells through the soil (26).
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.01.020
Please cite this article in press as: Eryürük, K., et al., Effects of bentonite and yeast extract as nutrient on decrease in hydraulic conductivity of porous media due to CaCO3 precipitation induced by Sporosarcin..., J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.020
2
ERYÜRÜK ET AL.
However, bentonite, which has a high specific surface area in addition to high swelling potential (27) and is a clay mineral, can be used to reduce the hydraulic conductivity of the soil in spite of the probability of crack occurrence in bentonite. Microbially induced carbonate precipitation may repair the possible cracks in bentonite to maintain the low hydraulic conductivity. Thus, it can be said that the combination of the usage of bentonite and microbially induced CaCO3 precipitation may have great potential to clog the soil pores to provide low hydraulic conductivity. It is essential to understand the mechanism of reduction in the hydraulic conductivity of porous media by the bacterial CaCO3 precipitation in combination with the bentonite treatment for the application of this technology. However, there are a few studies on the quantitative effect of bacterial CaCO3 precipitation on the hydraulic conductivity of porous media. Our previous research elucidated that the decrease in the hydraulic conductivity of porous media by the CaCO3 precipitation induced by resting S. pasteurii was successfully described by the modified KozenyeCarman equation (24). Further study should be conducted to evaluate the effect of the growth of bacteria inducing the CaCO3 precipitation in combination with bentonite application on the clogging of porous media. In this paper, a quantitative study was conducted to elucidate the reduction mechanism of hydraulic conductivity of porous media by using the continuous-flow columns packed with glass beads with different diameters as model porous media with different pore sizes, and by treating the columns with bentonite as clogging clay and the CaCO3 precipitates induced by S. pasteurii (ATCC 11859) with yeast extract as nutrient allowing the bacterial growth. MATERIALS AND METHODS Column clogging experiment Column clogging experiments were carried out using S. pasteurii ATCC 11859 to induce CaCO3 precipitation at 22 C, as previously reported in detail (24). The column with a 3-cm inner diameter and 10 cm in height was provided using 50-mL plastic syringes (Terumo SS-50ESZ, Terumo Corporation, Tokyo, Japan). Different sizes of glass beads (Potters-Ballotini Co. Ltd., Ibaraki, Japan), with average diameters of 0.05 mm, 0.25 mm, 1.0 mm, and 3.0 mm (abbreviated as GB0.05, GB0.25, GB1.0, and GB3.0, respectively), were used as porous media. The column had 25 mL of the pore volume. S. pasteurii ATCC 11859 was introduced into the column as 100 mL of suspension (four pore volumes of the column) with a cell density of 2.15 109 cells/mL at a flow rate of 2 mL/min using a peristaltic pump. A magnetic stirrer was used to ensure a homogenous bacterial suspension during 50 min of the introduction. 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 continuously introduced into the column with a flow rate of 3 mL/min for four days (totally 17,500 mL of the precipitation solution). The hydraulic conductivity of the column was measured using a manometer. The effect of bentonite on the reduction of the hydraulic conductivity of the column was examined by introducing 25 mL of the suspension made by 4.5 g of bentonite (5% weight/weight of the amount of glass beads) and distilled water with thorough mixing using a magnetic stirrer. The bentonite (Wako Pure Chemical Industries, Ltd., Osaka, Japan) contained a negligible amount of calcium (Supplementary Table S1), and the average particle size and the proportions of particles smaller than 0.45 mm in diameter were 5.0 mm and 0.12%, respectively. The bentonite suspension was introduced into the column at a flow rate of 2 mL/min using the peristaltic pump. Then, the bacterial suspension and the precipitation solution were introduced as described above, and the hydraulic conductivity was measured. The effect of yeast extract as a nutrient on the hydraulic conductivity was examined by introducing yeast extract (Becton, Dickinson and Company, NJ, USA) as a dissolved component of the precipitation solution at a concentration of 2% weight/ volume with an adjusted pH of 6.8. The precipitation solution was continuously introduced at a flow rate of 3 mL/min using the peristaltic pump as described above. The experiment was stopped before all the precipitation solution was introduced in the case that the leakage of the precipitation solution was observed from the tube connecting points of the column without the effluent solution because of the high pressure inside the column due to the low hydraulic conductivity, which was 2.1 106 cm/s. The conditions of the clogging treatments using S. pasteurii ATCC 11859, bentonite, the precipitation solution, and yeast extract are summarized in Table 1. The estimation of bacterial population in the column The number of bacterial cells in the column was estimated by multiplying the number of bacterial
J. BIOSCI. BIOENG., TABLE 1. Clogging treatment conditionsa. Condition
0b 1 2 3 4 5 6
S. pasteurii ATCC 11859
Bentonite
Precipitation solution
Yeast extract
c þ þ þ þ þ
þ þ þ
þ þ þ þ
þ þ
a Treatments with materials were carried out by introducing sequentially 25 mL of bentonite suspension (2 mL/min), 100 mL of cell suspension of S. pasteurii (2 mL/ min), and 17,500 mL of precipitation solution with or without yeast extract (3 mL/ min), in this order, to the columns. b Condition 0 denotes the condition before the treatments. c Symbols: þ, presence of material; , absence of material.
cells initially deposited with the growth proportion obtained in the batch culture, subtracting the number of bacterial cells eluted from the column. This estimation was carried out because the number of bacterial cells in the column cannot be counted by the direct microscopic method due to the formation of CaCO3 precipitation. The batch culture was provided by the introduction of 1 mL of 109 cell suspension into 20 mL of the sterilized precipitation solution, including 2% w/v yeast extract. Then, the cultures were incubated at 22 C, and an aliquot of the culture was taken at appropriate intervals to count the number of bacterial cells under the microscope (BX50WI, Olympus, Tokyo, Japan). During the microbial clogging experiments of the columns, samples were taken from the effluent after appropriate intervals, and the number of bacteria in the effluent was counted microscopically. The deposited amount of CaCO3 precipitation and bentonite The amount of CaCO3 precipitation in the columns of glass beads was determined as previously reported (24). The precipitated CaCO3 was dissolved into 0.1 N HNO3 and measured by inductively coupled plasma atomic emission spectroscopy (PerkinElmer Optima 3300DV, PerkinElmer, Waltham, MA, USA). The amount of CaCO3 precipitation in the columns was calculated using the Ca2þ concentration. The deposited amount of bentonite was obtained by subtracting the amount of bentonite eluted from the column. Densities of CaCO3 (2.71 g/cm3) and bentonite (0.80 g/cm3) were used to convert the amounts to the volumes of CaCO3 and bentonite. Size analysis of CaCO3 precipitates and bentonite One milliliter cell suspension, which contained 109 cells, was inoculated into 20 mL of the sterilized precipitation solution, including 2% w/v yeast extract using a sterile syringe, and incubated at 22 C. Then, samples were taken at appropriate intervals and placed on a glass slide to measure the size of the crystals in the CaCO3 precipitates using a microscope (Olympus BX50WI). Bentonite was suspended well in distilled water, and then the suspension was placed on a glass slide to measure the size of the bentonite particles under the microscope. The pore size distribution of the columns Matric potential was measured to evaluate the pore size distribution of the columns using a wide-range pF meter (DIK-3404, Daiki Co., Ltd., Saitama, Japan). The diameter of the pore (d, cm) in the column was estimated from the matric potential head (j, cm) by the following equation (28,29): d ¼ 0:3=j
(1)
The average pore size in the columns was calculated from the proportion of the individual size ranges of the pores.
RESULTS Changes in hydraulic conductivity by the column clogging treatments The hydraulic conductivity of the columns packed with different average diameters of glass beads (0.05e3 mm) was measured after the treatments with different conditions, as shown in Fig. 1. The hydraulic conductivity of the initial conditions of the columns ranged from 8.4 101 to 4.1 103 cm/s. Introduction of only cell suspension (condition 1) did not change the hydraulic conductivity. Introduction of only bentonite suspension (condition 2) did not decrease so much in the hydraulic conductivity, as shown by the range of 6.2 101 to 9.9 104 cm/s, although the extent of the decrease was larger in the column packed with small glass beads. The decrease was attributed to the larger amount of deposition of bentonite in the column (Supplementary Table S2). Our previous research (24)
Please cite this article in press as: Eryürük, K., et al., Effects of bentonite and yeast extract as nutrient on decrease in hydraulic conductivity of porous media due to CaCO3 precipitation induced by Sporosarcin..., J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.020
VOL. xx, 2015
BENTONITE AND YEAST EXTRACT ENHANCE MICROBIAL CLOGGING
3
FIG. 1. Hydraulic conductivity of columns packed with different sizes of glass beads (open circle) before (condition 0) and after application of condition 1 (plus sign), condition 2 (closed bar), condition 3 by Eryürük et al. (24) (asterisk), condition 4 (closed triangle), condition 5 (cross sign), and condition 6 (closed circle). Details of the conditions are summarized in Table 1.
showed that introduction of the precipitation solution in the presence of resting cells (condition 3) decreased the hydraulic conductivity to the range of 3.7 101 to 4.1 104 cm/s (Fig. 1). Combined introduction of bentonite suspension, cell suspension, and the precipitation solution (condition 4) decreased the hydraulic conductivity of the column in the range of 3.9 102 to 3.1 105 cm/s. The difference in the hydraulic conductivity between conditions 3 and 4 was larger than that between conditions 1 and 2. This suggested that bentonite promoted the deposition of microbial CaCO3 precipitates. The hydraulic conductivity decreased to the range of 2.3 103 to 3.7 106 cm/s for the combined introduction of cell suspension and precipitation solution including yeast extract (condition 5), and to the range of 9.9 104 to 2.1 106 cm/s for the combined introduction of bentonite suspension, cell suspension, and the precipitation solution including yeast extract (condition 6). Conditions 5 and 6 containing yeast extract as a nutrient allowed the growth of S. pasteurii ATCC 11859 and resulted in the lower hydraulic conductivity. Condition 6 achieved the lowest hydraulic conductivity in the column with GB0.05, which showed the leakage of the precipitation solution from the tube connecting points of the column in the experiment with reaching the limit for the measurement of hydraulic conductivity, 2.1 106 cm/s. Correspondence between numbers of bacterial cells deposited in the column and the decrease in hydraulic conductivity In the presence of yeast extract in precipitation solution, the hydraulic conductivity continued to decrease during four days of the clogging experiment (Fig. 2A and Supplementary Fig. S1), which corresponded to 700 pore volumes, while without yeast extract, the hydraulic conductivity became stable after 2 days of infiltration, regardless of the different sizes of glass beads, which corresponded to 350 pore volumes. Growth of S. pasteurii ATCC 11859 in the batch culture with yeast extract did not stop within four days (Fig. 2B and Supplementary Fig. S1). Although the numbers of eluted cells in the clogging experiment decreased with time in all the columns, the numbers differed between the columns depending on the glass bead sizes. Under the conditions 3 and 4 without yeast extract, the estimated numbers of the deposited bacterial cells were larger in the columns with smaller glass beads (Supplementary Table S3), corresponding to the larger reduction of the hydraulic conductivity. Because the numbers of the eluted cells were much smaller than those of the deposited bacterial cells, the number of the deposited cells in the column was maintained at an almost constant level during 4 days of the
FIG. 2. (A) Time course changes in hydraulic conductivity in column packed with GB0.05. Symbols: plus sign, condition 1; closed bar, condition 2; asterisk, condition 3; closed triangle, condition 4; cross sign, condition 5; and closed circle, condition 6. (B) For GB0.05, the number of cells in the solution (open circle), the number of cells in effluent (closed square), and the estimated number of cells in the column (cross sign).
experiment. It was considered that the urease activity disappeared in 2 days without yeast extract. Under the conditions 5 and 6 with yeast extract allowing the bacterial growth, the numbers of the deposited cells after 4 days were estimated to be approximately the same in all the columns, regardless of the different sizes of glass beads (Supplementary Table S3). The correspondence between the number of the bacterial cells and the decrease in hydraulic conductivity suggested that the bacterial growth supported by yeast extract resulted in the continuous decrease in hydraulic conductivity due to the maintenance of the urease activity of the bacterium inducing the CaCO3 precipitation for the entire experimental period. It should be noted that the significant difference in the number of deposited bacterial cells (P < 0.05) was observed between conditions 3 and 4 (Supplementary Table S3). This suggested that bentonite promoted the deposition of bacterial cells in the column. The relation between reduction of hydraulic conductivity and the occupied volume of deposited CaCO3 precipitates and bentonite Fig. 3 shows the relation between the relative reduction rate of hydraulic conductivity and the volume occupied with both the CaCO3 precipitates and bentonite (Fig. 3A), only the CaCO3 precipitation (Fig. 3B), or only the bentonite (Fig. 3C). The amount of calcium in bentonite was negligibly low (Supplementary Table S2) comparing with the amount of CaCO3 precipitates in the columns, therefore, the amount of CaCO3 precipitates estimated from calcium amount was little affected by the deposited bentonite. The relative reduction rate of hydraulic conductivity showed a significant correlation with either the volume occupied with both the CaCO3 precipitates and bentonite or with the only CaCO3 precipitation but not with bentonite. In addition, the effect of bentonite was limited at less than a oneorder decrease in hydraulic conductivity, as shown by the results of condition 1 in Fig. 3A. These results suggested that the reduction in hydraulic conductivity was dependent mainly on the
Please cite this article in press as: Eryürük, K., et al., Effects of bentonite and yeast extract as nutrient on decrease in hydraulic conductivity of porous media due to CaCO3 precipitation induced by Sporosarcin..., J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.020
4
ERYÜRÜK ET AL.
J. BIOSCI. BIOENG., did not have a unique particle size, and the particle size of the bentonite was calculated as 5.0 mm on average.
FIG. 3. Relationship between the proportion of the hydraulic conductivity of the treated column to the original hydraulic conductivity and (A) the occupied volume of CaCO3 precipitated and bentonite in the column, (B) the occupied volume of CaCO3 precipitated in the column, and (C) the occupied volume of bentonite in the column. Symbols: closed bar, condition 2; asterisk, condition 3; closed triangle, condition 4; cross sign, condition 5; and closed circle, condition 6. Relative reduction rate 1 (at 100%) is equal to condition 0.
CaCO3 precipitation. The amount of CaCO3 precipitates was larger in the columns packed with smaller glass beads under the same treatment conditions. The amount of CaCO3 precipitates in the columns with the deposit of bentonite was larger than that in the columns without bentonite. Deposited bentonite was considered to increase the deposition of the CaCO3 precipitation in the column, and indirectly promote the reduction of the hydraulic conductivity. Particle sizes of the CaCO3 precipitates and bentonite Fig. 4AeF show the microscopic observation of the formation of crystals of the CaCO3 precipitates in the static culture of the bacterium with yeast extract. The precipitation reaction started just 1 min after the cells were inoculated and mixed with the precipitation solution (Fig. 4A), and the CaCO3 precipitates grew to a maximum of approximately 10 mm in diameter in a few minutes. After a couple of hours (Fig. 4B), the number of 10-mm-diameter precipitates increased and began to form clusters, which were aggregated or individually larger than 10 mm (Fig. 4CeF). At the same time, precipitates smaller than 15 mm in diameter were also maintained (with 10 mm as the average diameter). After 4 days, the aggregates or individual particles with 25 mm as the average diameter became approximately the same in number as that of the precipitates with 10 mm as the average diameter (Supplementary Table S4). In the culture without yeast extract, the precipitates did not make the aggregates, and the average diameter was 10 mm, as previously reported (24). Fig. 4G and H confirmed that bentonite
Porosity and average pore size in columns before and after the clogging experiments Changes in pore size distribution were estimated from the water retention curve of the column packed with glass beads after the treatments. The larger deposition of the CaCO3 precipitates and/or bentonite in the columns with the smaller glass beads resulted in a larger extent of the decrease in porosity and estimated pore size. The largest reduction in pore size was observed in the introduction of bentonite suspension, cell suspension, and precipitation solution including yeast extract (condition 6). Before the clogging experiments (condition 0), the pore sizes were 49.7 mm, 66.1 mm, 77.6 mm, and 96.8 mm for the columns packed with GB0.05, GB0.25, GB1.0, and GB3.0, respectively. The pore size did not change in condition 1. Except condition 1, the pore sizes were reduced in response to the treatment; in condition 2, to 40.0 mm, 60.4 mm, 70.9 mm, and 88.4 mm for the columns packed with GB0.05, GB0.25, GB1.0, and GB3.0, respectively; in condition 3, to 33.0 mm, 52.0 mm, 64.1 mm, and 80.3 mm for the columns packed with GB0.05, GB0.25, GB1.0, and GB3.0, respectively; in condition 4, to 23.0 mm, 50.9 mm, 58.6 mm, and 70.3 mm for the columns packed with GB0.05, GB0.25, GB1.0, and GB3.0, respectively; in condition 5, to 20.1 mm, 32.6 mm, 52.4 mm, and 60.4 mm for the columns packed with GB0.05, GB0.25, GB1.0, and GB3.0, respectively; and in condition 6, to 16.1 mm, 28.9 mm, 49.3 mm, and 55.9 mm for the columns packed with GB0.05, GB0.25, GB1.0, and GB3.0, respectively. The pore volume of the columns also decreased in response to the treatment; in condition 2, from 25 cm3 (the same volume for all the columns) to 20.6 cm3, 22.5 cm3, 23.1 cm3, and 24.4 cm3 for the columns packed with GB0.05, GB0.25, GB1.0, and GB3.0, respectively; in condition 3, to 20.0 cm3, 21.3 cm3, 22.5 cm3, and 23.8 cm3 for columns with GB0.05, GB0.25, GB1.0, and GB3.0, respectively; in condition 4, to 13.1 cm3, 15.0 cm3, 17.5 cm3, and 19.4 cm3 for columns with GB0.05, GB0.25, GB1.0, and GB3.0, respectively; in condition 5, to 7.5 cm3, 10.0 cm3, 11.9 cm3, and 13.1 cm3 for columns with GB0.05, GB0.25, GB1.0, and GB3.0, respectively; and in condition 6, to 5.7 cm3, 7.5 cm3, 9.4 cm3, and 10.6 cm3 for columns with GB0.05, GB0.25, GB1.0, and GB3.0, respectively. The extent in the decrease in pore volume corresponded to that in porosity (Supplementary Table S5). DISCUSSION The hydraulic conductivity of the column with GB0.05 treated under condition 6 decreased to 2.1 106 cm/s after 4 days of infiltration of the precipitation solution with yeast extract (condition 6). This was the lowest hydraulic conductivity that was able to be measured in the experimental setup. Although the decrease in hydraulic conductivity was faster after 2 days, the continuous decrease in the hydraulic conductivity was still observed after 4 days in the columns treated with Condition 6. This suggested that the combination of bentonite and the precipitation solution with yeast extract would cause a further decrease in the hydraulic conductivity from the growth of ureolytic S. pasteurii (ATCC 11859) even after 4 days and would achieve the hydraulic conductivity required for the clay lining for the landfill, which should be lower than 106 cm/s of hydraulic conductivity with 50 cm of thickness (Ministry of Environment Japan, Ministerial Order No. 3, revised 21 February 2013, http://law.e-gov.go.jp/htmldata/S52/ S52F03102004001.html). Bentonite and the CaCO3 precipitates affected the hydraulic conductivity of the column to a similar extent, as shown in Fig. 3A. The efficiency in decreasing the hydraulic conductivity was 13.6%/ cm3 of bentonite as the relative reduction rate and 10.3%/cm3 of the CaCO3 precipitates, respectively. However, the effect of the CaCO3
Please cite this article in press as: Eryürük, K., et al., Effects of bentonite and yeast extract as nutrient on decrease in hydraulic conductivity of porous media due to CaCO3 precipitation induced by Sporosarcin..., J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.020
VOL. xx, 2015
BENTONITE AND YEAST EXTRACT ENHANCE MICROBIAL CLOGGING
5
FIG. 4. Photographs of the CaCO3 precipitates induced by S. pasteurii ATCC 11859 in a liquid static culture containing yeast extract after (A) 1 min, (B) 360 min, (C) 1440 min, (D) 2880 min, (E) 4320 min, and (F) 5760 min of incubation, and photographs of bentonite (G) and (H). Closed scale bars, 10 mm; open scale bars, 50 mm.
precipitates was larger because of the larger deposition volume in the column. Without yeast extract in the precipitation solution (conditions 3 and 4), the largest deposited volume of the CaCO3 precipitates was observed in the column with GB0.05 as 14 cm3. With the supply of yeast extract in the precipitation solution, the larger deposition of the CaCO3 precipitates was observed up to 24 cm3 in the column with GB0.05 (conditions 5 and 6). Continuous urease activity with yeast extract resulted in larger volumes of the CaCO3 precipitates, which clogged the pores of the column with glass beads and provided very low hydraulic conductivity. The larger volume of deposited bentonite and the CaCO3 precipitates were observed in the columns with smaller glass beads under the same treatment conditions. The specific CaCO3 precipitation rate of cells was calculated as 4.0 0.1 103 mg CaCO3/cell (condition 3) (24) and 7.0 0.1 103 mg CaCO3/cell (condition 4) under nonbacterial growth conditions and 1.8 0.1 103 mg CaCO3/cell
(condition 5) and 2.1 0.1 103 mg CaCO3/cell (condition 6) under bacterial growth conditions. Under the bacterial growth conditions, the larger amount of CaCO3 precipitates would cause less availability of CaCl2, urea and yeast extract in the precipitation solution for bacteria that had been grown inside the aggregates of the CaCO3 precipitates, and the less availability would lead the smaller value of specific CaCO3 precipitation rate. The deposited amount of bentonite was limited so that the clogging effect of bentonite only was small (condition 2). However, the deposited bentonite increased the depositing efficiency of the cells of S. pasteurii (ATCC 11859) and the CaCO3 precipitates induced, as shown by the difference between conditions 3 and 4 and between conditions 5 and 6. The deposition of bentonite resulted in the larger deposit number of the bacterial cells (Supplementary Table S3), the larger deposit amount of the CaCO3 precipitates and the larger decrease in hydraulic conductivity
Please cite this article in press as: Eryürük, K., et al., Effects of bentonite and yeast extract as nutrient on decrease in hydraulic conductivity of porous media due to CaCO3 precipitation induced by Sporosarcin..., J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.020
6
ERYÜRÜK ET AL.
J. BIOSCI. BIOENG.,
FIG. 5. Comparison of measured and estimated values of hydraulic conductivity using the modified KozenyeCarman equation: (A) condition 2; (B) condition 4; (C) condition 5; and (D) condition 6. In the estimation, the value of the shape factor (f) was set at 0.2534, which was determined by Eryürük et al. (24). Values of other parameters used are described in the text, except for the specific surface area (S). The values of S in conditions 2 and 4 (A, B) were estimated either by assuming that the glass beads, bentonite, bacterial cells, and CaCO3 precipitates formed one spherical particle with a diameter consisting of the total of the diameters of the individual particles (closed circle) or by assuming that the individual particles were separately present in the column (closed square). Another assumption was made for conditions 5 and 6 (C, D) in which none of the cell detached from the glass beads (closed circle), 20% of the cells detached from the glass beads and formed 20% of the total number of the CaCO3 precipitates as separately present in the pore solution with yeast extract (cross sign), 50% of the cells detached from the glass beads and formed 50% of the total number of the CaCO3 precipitates as separately present in the pore solution with yeast extract (closed triangle), and 100% of the cells detached from the glass beads and formed 100% of the total number of the CaCO3 precipitates as separately present in the pore solution with yeast extract (closed square). The symbols of 1.E e x (x ¼ 1e10) at axes mean 1.0 10x.
(Fig. 3A). It should be noted that the specific CaCO3 precipitation rate of the cells was larger under the conditions with bentonite (conditions 4 and 6) than the corresponding conditions without bentonite (conditions 3 and 5), suggesting the enhanced formation of the CaCO3 precipitate. Thus, bentonite indirectly enhanced the clogging. In the previous paper (24) in which the clogging treatment of condition 3 was carried out, a modified KozenyeCarman equation showed the good agreement by assuming that the glass beads, bacterial cells, and CaCO3 precipitates became one particle but not
KCaþB
r B sV B sV B ¼ f rg n Ca B Ben Ben V V V
3 ,
2 8 < 4m :
porosity, rCa denotes the specific CaCO3 precipitation rate, B denotes the number of bacterial cells deposited (Supplementary Table S3), V denotes the volume of the column, m denotes the dynamic viscosity of the liquid, and D denotes the average diameter of the glass beads. To identify the effects of bentonite and yeast extract on the decrease in hydraulic conductivity, Eq. 2 was further modified by introducing the specific microbial volume (sVB), specific volume of bentonite (sVBen), and amount of bentonite deposited (BBen):
6
6B ðr
p
3 Ca þ sVB þ sVBen Þ þ D
by assuming that they were separate particles. The modified KozenyeCarman equation, which estimates hydraulic conductivity based on the specific surface area and the porosity of the porous medium, was as follows: KCaþB ¼ f rgðn rCa B=VÞ3
1=3 2 ð1 n þ rCa B=VÞ2 6rCa B=p þ D3 m 6
r denotes the density of liquid, g denotes gravity, n denotes
(2)
where KCaþB denotes the hydraulic conductivity of the porous medium with microbial clogging, f denotes the shape factor,
92 =
1=3 ;
r B sV B sV B 1 n þ Ca þ B þ Ben Ben V V V
2
3 5
(3)
Here, the density of the precipitation solution including yeast extract (r) was measured at 22 C as 1072 kg/m3. The value of g used was 9.80665 m/s2. The dynamic viscosity of the precipitation solution including yeast extract (m) has previously been shown to be 0.0140 kg/ms (30). The value of fd0.2532din the columns was the same as the previous study (24). The bacterial number in the column was estimated at 4.5 1010 cells under bacterial growth conditions after 4 days, and it was estimated to be approximately the same for all the columns packed with different sizes of glass beads (Supplementary Table S3). In conditions 2 and 4, as in the previous paper (24) with condition 3, Eq. 3 showed the good
Please cite this article in press as: Eryürük, K., et al., Effects of bentonite and yeast extract as nutrient on decrease in hydraulic conductivity of porous media due to CaCO3 precipitation induced by Sporosarcin..., J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.020
VOL. xx, 2015
BENTONITE AND YEAST EXTRACT ENHANCE MICROBIAL CLOGGING
agreement by assuming that the bentonite, glass beads, bacterial cells, and CaCO3 precipitates became one particle (Fig. 5A and B). Therefore, the bentonite, bacterial cells, and CaCO3 precipitates would attach to the surface of the glass beads in the column; increase the glass bead sizes; decrease the porosity of the column; and decrease the hydraulic conductivity in conditions 2, 3, and 4. In conditions 5 and 6 with bacterial growth, good agreement was obtained by assuming that 50% of the cells were attached to the glass beads to form CaCO3 precipitates attached on the glass beads, and 50% of the cells detached from the glass beads to form CaCO3 precipitates as separate and individual particles in the column pores. The separate CaCO3 precipitates may attach on the glass beads but all the surface of the precipitates was considered as effective at decreasing the hydraulic conductivity. All of the bentonite, which remained in the column, was considered as attached on the glass beads to form the aggregates. When considering that 0%, 80%, and 100% of the cells were attached to the glass beads to form the aggregates with the CaCO3 precipitates, meaning that 100%, 20%, and 0% of the cells detached from the glass beads to form the separate CaCO3 precipitates, respectively, the calculated and measured hydraulic conductivity did not agree well (Fig. 5C and D). The large and small CaCO3 precipitates were observed in the static culture using the precipitation solution with yeast extract, and the ratio of the number of large and small precipitates was approximately 1:1. This observation corresponded well to the results of the analysis from the KozenyeCarman equation, which found that 50% of the total number of the CaCO3 precipitates formed large particles by attaching to the glass beads, and 50% of the total number of the CaCO3 precipitates separately formed small particles. The growth of the bacterial population using yeast extract led to the formation of the separate CaCO3 precipitates in addition to the attached CaCO3 precipitates on the glass beads. Correspondingly, the decrease in the porosity caused a decrease in the hydraulic conductivity of the column. In summary, the effects of bentonite and yeast extract on the clogging of porous media due to CaCO3 precipitation induced by S. pasteurii ATCC11859 under the growing conditions were quantitatively evaluated using columns packed with glass beads. Analysis using the modified KozenyeCarman equation indicated that the mechanism underlying the reduction in hydraulic conductivity was not only the aggregation of the glass beads with the bentonite and the CaCO3 precipitates, which increased the effective size of glass beads, but also the formation of individual CaCO3 precipitates separated from the glass beads in the column pore in the presence of yeast extract allowing the bacterial growth. Although the addition of only bentonite suspension did not change the hydraulic conductivity significantly, bentonite promoted the deposition of the CaCO3 precipitates in the column. Bacterial growth using nutrient had a larger effect on decreasing the hydraulic conductivity by the CaCO3 precipitates. The combination of bentonite suspension, cell suspension, and the precipitation solution including yeast extract resulted in the highest decrease in hydraulic conductivity. The lowest hydraulic conductivity achieved in this study was 106 cm/s. Microbially induced CaCO3 precipitation could be applied as a technology to landfills to decrease hydraulic conductivity by providing CaCl2, urea, and a nutrient to enhance ureolytic bacteria in situ. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2015.01.020.
ACKNOWLEDGMENTS This work was partly supported by the Environmental Technology Development Fund in the Ministry of the Environment, Japan (ZB-1205) and the Environmental Biological Remediation and
7
Conservation Project C-1 (2013e2014) at the EcoTopia Science Institute, Nagoya University, Japan.
References 1. Allen, A.: Containment landfills: the myth of sustainability, Eng. Geol., 60, 3e19 (2001). 2. Röling, W. F. M., van Breukelen, B. M., Braster, M., Lin, B., and van Verseveld, H. W.: Relationship between microbial community structure and hydrochemistry in a landfill leachate-polluted aquifer, Appl. Environ. Microbiol., 67, 4619e4629 (2001). 3. Gleason, M. H., Daniel, D. E., and Eykholt, G. R.: Calcium and sodium bentonite for hydraulic containment applications, J. Geotech. Geoenviron., 123, 438e445 (1997). 4. Benson, C. H., Daniel, D. E., and Boutwell, G. P.: Field performance of compacted clay liners, J. Geotech. Geoenviron. Eng., 125, 390e403 (1999). 5. Benson, C. H., Thorstad, P. A., Jo, H.-Y., and Rock, S. A.: Hydraulic performance of geosynthetic clay liners in a landfill final cover, J. Geotech. Geoenviron. Eng., 133, 814e827 (2007). 6. Petrov, R. J. and Rowe, R. K.: Geosynthetic clay liner (GCL) e chemical compatibility by hydraulic conductivity testing and factors impacting its performance, Can. Geotech. J., 34, 863e885 (1997). 7. Egloffstein, T. A.: Natural bentonitesdInfluence of the ion exchange and partial desiccation on permeability and self-heating capacity of bentonites used in GCLs, Geotext. Geomembranes, 19, 427e444 (2001). 8. Kolstad, D. C., Benson, C. H., and Edil, T. B.: Hydraulic conductivity and swell of nonprehydrated geosynthetic clay liners permeated with multispecies inorganic solutions, J. Geotech. Geoenviron. Eng., 130, 1236e1249 (2004). 9. Daniel, E. D. and Wu, Y.-K.: Compacted clay liners and covers for arid sites, J. Geotech. Eng., 119, 223e237 (1993). 10. Stocks-Fischer, S., Galinat, J. K., and Bang, S. S.: Microbial precipitation of CaCO3, Soil Biol. Biochem., 31, 1563e1571 (1999). 11. Chou, C.-W., Seagren, E. A., Aydilek, A. H., and Lai, M.: Biocalcification of sand through ureolysis, J. Geotech. Geoenviron. Eng., 137, 1179e1189 (2011). 12. Hammes, F. and Verstraete, W.: Key roles of pH and calcium metabolism in microbial carbonate precipitation, Rev. Environ. Sci. Biotechnol., 1, 3e7 (2002). 13. Okwadha, G. D. O. and Li, J.: Optimum conditions for microbial carbonate precipitation, Chemosphere, 81, 1143e1148 (2010). 14. Harkes, M. P., Van Paassen, L. A., Booster, J. L., Whiffin, V. S., and Van Loosdrecht, M. C. M.: Fixation and distribution of bacterial activity in sand to induce carbonate precipitation for ground reinforcement, Ecol. Eng., 36, 112e117 (2010). 15. Whiffin, V. S., Van Paassen, L. A., and Harkes, M. P.: Microbial carbonate precipitation as a soil improvement technique, Geomicrobiol. J., 24, 417e423 (2007). 16. De Muynck, W., Cox, K., De Belie, N., and Verstraete, W.: Bacterial carbonate precipitation as an alternative surface treatment for concrete, Constr. Build. Mater., 22, 875e885 (2008). 17. Nemati, M. and Voordouw, G.: Modification of porous media permeability, using calcium carbonate produced enzymatically in situ, Enzyme Microb. Technol., 33, 635e642 (2003). 18. Yasuhara, H., Neupane, D., Hayashi, K., and Okamura, M.: Experiments and predictions of physical properties of sand cemented enzymatically-induced carbonate precipitation, Soil Found., 52, 539e549 (2012). 19. Chu, J., Stabnikov, V., and Ivanov, V.: Microbially induced calcium carbonate precipitation on surface or in the bulk of soil, Geomicrobiol. J., 29, 544e549 (2012). 20. Gollapudi, U. K., Knutson, C. L., Bang, S. S., and Islam, M. R.: A new method for controlling leaching through permeable channels, Chemosphere, 30, 695e705 (1995). 21. Mitchell, A. C. and Ferris, F. G.: The coprecipitation of Sr into calcite precipitates induced by bacterial ureolysis in artificial groundwater: temperature and kinetic dependence, Geochim. Cosmochim. Acta, 69, 4199e4210 (2005). 22. Fujita, Y., Ferris, F. G., Lawson, R. D., Colwell, F. S., and Smith, R. W.: Calcium carbonate precipitation by ureolytic subsurface bacteria, Geomicrobiol. J., 17, 305e318 (2000). 23. Fujita, Y., Redden, G. D., Ingram, J. C., Cortez, M. M., Ferris, F. G., and Smith, R. W.: Strontium incorporation into calcite generated by bacterial ureolysis, Geochim. Cosmochim. Acta, 68, 3261e3270 (2004). 24. Eryürük, K., Yang, S. Y., Suzuki, D., Sakaguchi, I., Akatsuka, T., Tsuchiya, T., and Katayama, A.: Reducing hydraulic conductivity of porous media using CaCO3 precipitation induced by Sporosarcina pasteurii, J. Biosci. Bioeng., 119, 331e336 (2015). 25. Mobley, H. L. and Hausinger, R. P.: Microbial ureases: significance, regulation, and molecular characterization, Microbiol. Rev., 53, 85e108 (1989). 26. Mortensen, B. M., Haber, M. J., DeJong, J. T., Caslake, L. F., and Nelson, D. C.: Effects of environmental factors on microbial induced calcium carbonate precipitation, J. Appl. Microbiol., 111, 338e349 (2011).
Please cite this article in press as: Eryürük, K., et al., Effects of bentonite and yeast extract as nutrient on decrease in hydraulic conductivity of porous media due to CaCO3 precipitation induced by Sporosarcin..., J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.020
8
ERYÜRÜK ET AL.
27. Cerato, A. B. and Lutenegger, A. J.: Determination of surface area of finegrained soils by the ethylene glycol monoethyl ether (EGME) method, Geotech. Test J., 25, 1e7 (2002). 28. Lal, R. and Shukla, M. K.: Principles of soil physics, pp. 313e314. Marcel Dekker, Inc., New York (2004).
J. BIOSCI. BIOENG., 29. Fang, H.-Y.: Introduction to environmental geotechnology, pp. 194. CRC Press LLC, Boca Raton, Florida (1997). 30. Goel, A.: States of matter, pp. 21e23. Discovery Publishing House, New Delhi (2006).
Please cite this article in press as: Eryürük, K., et al., Effects of bentonite and yeast extract as nutrient on decrease in hydraulic conductivity of porous media due to CaCO3 precipitation induced by Sporosarcin..., J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.020
