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Graphene oxide on microbially induced calcium carbonate precipitation
International Biodeterioration & Biodegradation 145 (2019) 104767
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Graphene oxide on microbially induced calcium carbonate precipitation ∗
T
Guowang Tang, Guihe Wang , Yuxiu An, Haonan Zhang School of Engineering and Technology, China University of Geosciences (Beijing), Beijing, 100083, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Bio mineralization Calcium carbonate Morphology Graphene oxide
The improvement of microbial induced calcium carbonate precipitate (MICP) using additives has attracted much attention due to its great influence on the quality of precipitate. In this paper, graphene oxide (GO) was used as an additive to induce CaCO3 precipitate. Using scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), the effects of GO on immobilization of bacteria, CaCO3 crystals and the consolidation of sand was studied. The addition of GO caused the rapid growth and the larger size of the CaCO3 crystals. Additionally, the morphology of the rhombohedral crystals was unchanged. The precipitation capability of CaCO3 during the MICP process was improved significantly. X-ray diffraction (XRD) noted that stable calcite was formed during the CaCO3 precipitate process with GO, compared to calcite and vaterite formed. More importantly, the unconfined compressive strength of the consolidated sand was significantly enhanced. These results show that GO has outstanding properties.
1. Introduction Traditional grouting methods, such as chemical grouting, cement consolidation and polymer grouting, have been developed and widely used; however, environmental pollution is a major limitation (van Paassen et al., 2010). Thus, a new grouting method, known as microbially induced calcium carbonate precipitation (MICP), has emerged (Amarakoon and Kawasaki, 2018; Cheng et al., 2013). MICP has attracted extensive attention due to its outstanding properties and potential engineering applications (Dopffel et al., 2018; Jimenez-Lopez et al., 2008; Kumari et al., 2014; van Paassen et al., 2010). At present, it is generally acknowledged that the reaction mechanism can be summarized in three equations (Eq. (1) - (3)) (Fujita et al., 2017; Khan et al., 2015; Nawarathna et al., 2018) these equations summarize the biochemical reactions of calcium carbonate precipitation when bacteria serve as nucleation sites.
urease CO(NH2)2 + H2 O → H2 NCO O− + NH+4
H2
NCOO−
+ H2 O→
HCO−3
Ca2 ++HCO32 − + NH3
+ NH3
bacterial
→
CaCO3 + NH+4
(1) (2) (3)
First, urea is hydrolyzed to carbamate and ammonium ions (Reaction (1)). Then, carbonate is spontaneously hydrolyzed to produce bicarbonate and second ammonia (Reaction (2)). The precipitation of CaCO3 is obtained in the presence of dissolved Ca2+ (Reaction (3)).
∗
Great application prospect for the MICP process have been exhibited in many fields. For instance, it has been applied in lessen wind erosion (Gu et al., 2018; Maleki et al., 2016); treatment of the environment (Kumari et al., 2014; Okwadha and Li, 2011; Wang et al., 2018); consolidation of sand (Martinez et al., 2013; Soon et al., 2013); repair of cracks in concrete structures (Vashisht et al., 2018; Vijay et al., 2017); and improvement of oil recovery (Nemati et al., 2005; Whiffin et al., 2007). MICP is also a common bio-mineralization process in nature; however, the laboratory research plays an important role. Current laboratory research mainly focuses on several different aspects. First, the effect of different bacteria, such as Bacillus subtilis sp, Bacillus sp (Li et al., 2018), Bacillus megaterium and Pararhodobacter sp, have been analyzed during the MICP process (Amarakoon and Kawasaki, 2018; Cardoso et al., 2018; Gat et al., 2017; Schwantes-Cezario et al., 2017b; Zhang et al., 2014). Second, many scholars have investigated the precipitation efficiency of CaCO3 crystals and sand consolidation during the MICP process and found that it is influenced by Ca2+ concentrations, urea concentrations, temperature, pH, incubation period, calcium sources and degree of saturation (Achal and Pan, 2014; Cheng et al., 2013; De Muynck et al., 2010; Kim et al., 2018; Omoregie et al., 2017). Finally, permeability, porosity, stiffness, shear strength, unconfined compressive strength (UCS), microstructure and shear wave velocity have been extensively studied. These parameters determine the effect of cementation (Amiri and Bundur, 2018; Cheng and Shahin, 2016; Martinez et al., 2013; Qian et al., 2010). Additives have improved soil mechanical properties by bio-
Corresponding author. E-mail address: [email protected] (G. Wang).
https://doi.org/10.1016/j.ibiod.2019.104767 Received 4 June 2019; Received in revised form 7 August 2019; Accepted 19 August 2019 0964-8305/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Formation mechanism of CaCO3 crystals in the presence of GO.
1643 cm−1, and a strong and wide OH group stretching vibration band appeared near 3436 cm−1 (Haghighi et al., 2013; Jin et al., 2018).
mineralizing CaCO3 precipitation catalyzed through bacterial urease. Several additives, including low water-soluble nacre proteins (Heinemann et al., 2011), poly-Lys (Nawarathna et al., 2018), fiber (Li et al., 2016), modified polymer (Wang et al., 2018) and activated carbon (Zhao et al., 2018), have been studied and show good performance improvement. However, few studies have been carried out to investigate the influence of graphene oxide (GO). This study evaluated the effects of GO as an additive during the MICP process. The reaction rate of Ca2+; the precipitated number of CaCO3 crystals; and the polymorph, morphology and microstructure of CaCO3 crystals at various bacterial concentrations were analyzed using the gravimetric method, X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) to assess change in the crystals.
2.3. Cementation media and the bacteria concentration The cementation medium (0.5 mol/L) was a mixture of 0.5 mol/L CaCl2 and 0.5 mol/L urea solutions. The concentration of cementation media was selected based on the Nawarathna et al. (2018) study. Bacterial suspensions of different concentrations (OD600 = 0.25, 0.5, 0.75 and 1.0) were prepared with bacteria and deionized water. 2.4. Precipitation experiments CaCO3 precipitation experiments were carried out at 25 °C in beakers containing 60 mL of bacterial suspension and 240 mL of the cementation medium. When the cementation medium concentrations were 0.5 mol/L, the precipitation experiments were carried out at various bacterial suspensions (OD600 = 0.25, 0.5, 0.75 and 1.0) without and with GO (10 mg/L). When bacterial concentrations equaled OD600 1.0, the same experiments were conducted at the different concentrations of GO (5, 10, 15 and 20 mg/L) with the cementation medium (0.5 mol/L). CaCO3 crystals on the wall of the beaker were obtained by filtering and then drying at 105 °C for 24 h.
2. Materials and methods 2.1. Microorganisms and growth conditions In this study, the Sporosarcina pasteurii (ATCC11859) strain was used for CaCO3 precipitation. This bacteria were used because they produce urease and have a capacity for crystallization of CaCO3 within the experimental system. The culture medium was prepared by mixing casein peptone (15 g/L), soy peptone (5 g/L), NaCl (5 g/L) and urea (20 g/L) into 1000 mL of deionized water. The final pH of the culture medium was approximately 7.3. The culture medium was sterilized at 121 °C for 30 min. Bacteria were incubated aerobically in the medium at 30 °C at a rotation rate of 120 rpm for 48 h. The culture was centrifuged at 5434 g at 4 °C for 8 min to pellet the bacteria. Bacterial pellets were resuspended in 1000 mL of deionized water and stored at 4 °C no longer than 48 h prior to use. The bacteria were diluted to an optical density at 600 nm (OD600) of 1.0. All chemical reagents, such as urea, calcium chloride (CaCl2), sodium chloride (NaCl), peptone from casein and peptone from soymeal were purchased from Sinopharm Chemical Reagent Co, Ltd (Shanghai, China).
2.5. Sands Sand from a river was used. Grading size distribution curves of sand are shown in Fig. S2. The average particle diameter of the sand (D50) is 127.26um. The D10, D30, and D60 are 22.26, 78.482 and 150.23 μm, respectively. The coefficient of uniformity (Cu) is 6.74 and the coefficient of gradation (Cc) is 1.84. 2.6. Consolidation experiments Consolidated sand samples were obtained using full contact flexible molds as described in Zhao et al. (2014). The size of the mold was 50 mm diameter and 100 mm height. The 300 g of sand and GO (0, 5, 10, 15, and 20 mg/l by total volume of cementing fluid) were uniformly mixed with 60 ml bacteria solution. The mold with the above sand mixture was submerged in the cementing fluid (0.5 mol/l) and reacted using a gaseous diffusion system shown in Fig. S3.
2.2. Characterization of GO GO, which was prepared from natural graphite powder (325 mesh, Qingdao Huatai Lubricant Sealing S&T Co. Ltd., Qingdao, China) by the modified Hummers' method (Wang et al., 2012), was obtained from Tsinghua University, china. The thickness (4–5 nm) and diameter (0.5–3.5 μ m) of GO was characterized by Atomic force microscope (AFM) under standard operating conditions shown in Fig. S1 (a). The oxygen-containing functional groups of GO were characterized by Fourier transform infrared spectroscopy (FT-IR, FTIR8400, SHIMADZU, Japan) shown in Fig. S1 (b). The characteristic absorption bands of C–O appeared at the wavelengths of 1045 cm−1 (Haghighi and Tabrizi, 2013; Jin et al., 2018). The band of the C]O group appeared at
2.7. Analytic methods A small amount of CaCO3 crystals was ground in a mortar. XRD measurements were performed using automatic XRD instruments (D8 Advance, Bruker-AXS, Germany). The scan rate was 0.15 s step−1 and the range of 2 θ was from 10° to 70°. The concentrations of Ca2+ were measured with a PHS-3C PH meter using a calcium ion electrode. The 2
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Fig. 2. SEM images of CaCO3 crystals in the presence of GO obtained in different time. 1min (a), 10min (b), 30 min (c), 60min (d), 90 min (e) 12 h (f), respectively. EDS patterns record of GO and CaCO3 crystals from the marked area A (g) and B (h).
morphology and microstructure of the crystals were analyzed by field emission scanning electron microscopy (FESEM, Zeiss Supra 55, Germany) after being sputter-coated with a thin layer of gold nanoparticles. In the meantime, energy dispersive spectroscopy (EDS, HORIBA, 7593-H) associated with the SEM were used to characterize the chemical composition.
During the MICP process, most oxygen-containing groups on the basal planes of GO are removed which negatively charged COO− groups remain at their edges; therefore, Ca2+ ions are preferentially adsorbed on the edges of GO when urea and CaCl2 are used. Negatively charged GO had a larger specific surface area and was encapsulated by Ca2+. Consequently, a large number of negatively charged bacteria (Bundeleva et al., 2011; De Muynck et al., 2010) were absorbed through the positively charged Ca2+ aggregates, which lead to accumulation and immobilization of the bacteria. Larger CaCO3 crystals were generated in the presence of sufficient Ca2+. To understand the mechanisms of the CaCO3 crystals growth in the presence of GO. Precipitates were collected at various stages as shown in Fig. 2a–f. In the first 1 min, it was found that CaCO3 crystals began to grow on graphene surface in the presence of bacteria when urea and
3. Results 3.1. Mechanism of GO on CaCO3 precipitation Fig. 1 shows that CO32 − was formed through hydrolysis of urea catalyzed by bacterial urease. Then, CO32 − reacted directly with Ca2+ cross-linked with GO in the presence of bacteria to precipitate CaCO3. 3
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Fig. 3. (A) The changes of Ca2+ concentration at different GO concentrations. (B) The concentration of Ca2+ at different bacterial concentrations (OD600) with and without GO (10 mg/L). (C) The total mass of CaCO3 precipitate at different GO concentrations. (D) The total mass of CaCO3 precipitate at different bacterial concentrations (OD600) with and without GO (10 mg/L).
the Ca2+ the concentration in the presence of GO decreased faster than that in the absence of GO. In the beginning, When the bacterial concentration was OD600 0.5, 0.75 and 1.0, the Ca2+ concentration decreased more rapidly in the presence of GO than in the absence of GO. The above result shows that GO was conducive to the Ca2+ reaction. Although Ca2+ in the absence of GO was not completely deposited, Ca2+ in the presence of GO was completely deposited after 36 h. The total mass of the CaCO3 precipitate increased by at least 53% in presence of GO is shown in Fig. 3 (C). Fig. 3 (D) shows that total quality of the CaCO3 precipitate catalyzed by bacterial urease at different bacterial concentrations with (10 mg/L) and without GO. The CaCO3 precipitate increased with increasing bacterial concentrations without GO. When the bacterial concentrations were OD600 0.25, 0.5, 0.75 and 1.0, the CaCO3 precipitate increased by at least 171%, 73%, 33% and 26%, respectively, in the presence of GO (10 mg/L) when compared to without GO. The total mass of the CaCO3 precipitate reached its maximum at the bacterial concentration of 0.5 in the presence of GO because Ca2+ was completely deposited.
CaCl2 were used (Fig. 2 (a)). At 10 min, the characteristic spherical of vaterite became more and more evident, suggesting that vaterite gradually formed. However, at 90 min almost no characteristic spherical corresponding to vaterite phases appeared, according to FESEM results (Fig. 2b–d). The crystals were rhombohedral when the deposition time was 60 min. In the first 90 min, with the increasing time of deposition, GO around crystals is becoming less and less. By 12 h, the shape of crystals gradually became more regular, and Go was not detected as shown in Fig. 2f, which mean GO is encapsulated in calcium carbonate as nucleation sites. This result is consistent with the results shown in Fig. 1. EDS was used to further characterize effect of GO in the CaCO3 crystals growth. EDS indicates that the atomic ratios of C, O and Ca for sit A and sit B are 68: 32: 0 and 63: 23: 12, respectively (Fig. 2(g and h)). The result certifies that Ca2+ ions can be adsorbed by GO; bacteria can be adsorbed by Ca2+ ions and the CaCO3 crystals growth can be influenced by GO during the MCIP process. 3.2. Influence of GO on CaCO3 precipitation
3.3. Influence of GO on the polymorph and morphology of CaCO3 crystals at various bacterial concentrations
Calcium carbonate precipitation is influenced by GO, with the concentration of GO possibly playing a key role. Changes in the Ca2+ concentration at different GO concentrations are shown in Fig. 3 (A). The concentration of Ca2+ in the presence of GO decreased faster than that in the absence of GO. When the concentration of GO was 10, 15 and 20 mg/L, the change in the Ca2+ concentration was basically the same. The effect of GO became more apparent at different bacterial concentrations (Fig. 3 (B)). The reaction rate of Ca2+ increased with increasing bacterial concentrations. When the bacterial concentration was OD600 0.25, the change in the Ca2+ concentration was basically the same with and without GO during the first 24 h. However, after 24 h,
The morphologies of CaCO3 crystals prepared at different bacterial concentrations without GO were invested by SEM (Fig. 4 (a, b, c and d)). The size of the CaCO3 crystals were in the range of 7–22 μm, 18–48 μm, 17–38 μm and 24–113 μm, respectively. Regular rhombohedra crystals were obtained at low bacterial concentrations. When the bacterial concentration was OD600 0.25, vaterite was produced (Fig. 4a). When the bacterial concentration was OD600 1.0, the agglomeration phenomenon of CaCO3 crystals occurred. An important 4
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The morphologies of the CaCO3 crystals prepared at different bacterial concentrations with 10 mg/L GO were also invested by SEM (Fig. 4 (a1, b1, c1 and d1)). The size of the CaCO3 crystals were in the range of 24–53 μm, 17–67 μm, 38–72 μm and 28–113 μm, respectively. Larger CaCO3 crystals were obtained in the presence of GO than in the absence GO at the various bacterial concentrations. This is because the bacteria were gathered together through the adsorption of Ca2+ on the edges of GO. The morphology of the rhombohedral crystals, which is the most advantageous morphology for sand consolidation, was unchanged. Another important phenomenon is that the size of the crystals decreased with increasing bacterial concentrations in the presence of 10 mg/L GO. This is because GO can act as a nucleation site (Wang et al., 2012). Because adequate Ca2+ existed, CaCO3 crystals could continuously grow, thereby generating larger CaCO3 crystals. However, there was not enough Ca2+ at higher concentration of nucleation sites; thus, a larger number of small CaCO3 crystals were generated without the growth of individual crystals. Calcite was obtained at the various bacterial concentration, regardless of GO addition. A small amount of vaterite was obtained without addition of GO in Fig. S4. This result is consistent with the results shown in Fig. 4 (a). Thus, we suggest that more stable crystals were obtained in the presence of GO. At the same time, a number of researchers have concluded that calcite catalyzed by urease of bacteria is more conducive to the consolidation of sand (Amiri and Bundur, 2018; Zhao et al., 2014). The morphology of CaCO3 crystals at different GO concentrations are shown in Fig. 5. The size of the CaCO3 crystals decreased with increasing concentrations of GO. An important reason for this is that the number of nucleation sites increased. rhombohedral crystals are the main morphology at the low GO concentration. When the GO concentration was 20 mg/L, sphere crystals were obtained. 3.4. Influence of GO on sand consolidation Consolidated sand samples from day 6 at different GO concentrations are shown in Fig. 6. Better integrity and compactness were observed with GO than without GO. The result is same with other literature (Schwantes-Cezario et al., 2017a). The unconfined compressive strength tests are shown in Fig. 7. One important point is that the unconfined compressive strength of the consolidated sand was obviously improved at most by about 100% in the presence of 15 mg/L GO. SME images show that a small amount of CaCO3 precipitate was adsorbed on the surface of the sand in the absence of GO (Fig. S5 (a and b)). However, a larger number of CaCO3 precipitate absorbed on the surface of the sand in the presence of GO (Fig. S5 (c and d)). This result is consistent with the results shown in Fig. 3 (c). Thus, we consider that the unconfined compressive strength increased with increasing the number of CaCO3 precipitate. This maybe reason why the consolidation strength of sand particles is higher in the presence of than in the absence of GO shown in Fig. 7. Al Qabany and Soga, 2013b also suggest that the unconfined compressive strength increased with the amount of CaCO3 precipitation.
Fig. 4. SEM images of CaCO3 crystals at different bacterial concentrations without GO at D600 = 0.25 (a), D600 = 0.50 (b), D600 = 0.75 (c), D600 = 1.0 (d) and with GO (10 mg/L) at D600 = 0.25 (a1), D600 = 0.50 (b1), D600 = 0.75 (c1), D600 = 1.0 (d1).
4. Discussions The experimental investigation on effect of GO on MICP using Sporosarcina pasteurii revealed that GO can be successfully utilized in order to accelerate the deposition of Ca2+, increase the total mass of CaCO3 and improve the unconfined compressive strength of consolidated sand samples. Fig. 7 demonstrates optimal concentration of GO found for the unconfined compressive strength of the consolidated sand sample in this study. The increase in the total amount of calcium carbonate precipitation was crucial because the amount of calcium carbonate precipitation was proportional to the unconfined compressive strength of consolidated sand (Al Qabany et al., 2013a; Whiffin et al., 2007). The increase of the rate of Ca2+ ions was also very important, which was of great significance for controlling the reaction
Fig. 5. SEM images of CaCO3 crystals catalyzed by bacteria (OD600 = 1.0) at GO concentrations of 5 mg/L (a), 10 mg/L (b), 15 mg/L (c) and 20 mg/L (d).
characteristic of this phenomenon is that the size of the crystals increases with increasing bacterial concentrations, which may be because there is enough Ca2+ involved in the reaction. 5
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Fig. 6. Consolidated sand samples with different GO concentrations.
may be flushed out during injection after CaCO3 precipitation (Al Qabany et al., 2013a). GO is a structural analogue of graphene containing O-containing functional groups (Liu et al., 2019). Go exhibits aromatic structure, and functional groups are mainly found at the edges (Mauter and Menachem, 2008). In the literature, because of its sharp edges, GO may have adverse effects on human body and the environment (Du et al., 2017). However, in this study, GO was used as the nucleus of CaCO3 crystals growth, and was not detected at the end of the experiment. Therefore, adverse effects can be avoided. We believe that GO did not produce any toxicity in this study. 5. Conclusions This paper investigated the effects of GO during the MICP process. It has been revealed that GO can accelerate the deposition of Ca2+, increase the total mass of CaCO3 and improve the unconfined compressive strength of consolidated sand samples. The SEM and XRD analyses demonstrate that larger and more stable calcite can be obtained with than without GO. At the same time, SEM analysis of consolidated sand sample indicates that more CaCO3 crystals were adsorbed on the surface of sand particles with than without GO. The unconfined compressive strength of the consolidated sand was obviously improved at most by about 100% in the presence of 15 mg/L GO.
Fig. 7. The unconfined compressive strength of the consolidated sand at different GO concentrations.
time of MICP progress. Many literatures have also studied how the rate of Ca2+ ions reaction can be increased (Bains et al., 2015; Cuthbert et al., 2012; Gat et al., 2014). Theoretically, bacteria can decompose 0.4 mol of urea and induce precipitate of 0.4 mol of CaCO3. However, this was not observed experimentally at the various bacterial concentrations (OD600 = 0.25, 0.5, 0.75 and 1.0) because the hydrolysis rate of urea generally depends on the rate of bacterial urease activity rather than the amount of minerals in the media (Bachmeier et al., 2002). When GO was added, urea was completely decomposed and Ca2+ ions were completely reacted to form CaCO3 precipitate, which may be because the presence of negatively charged GO might create additional nucleation sites by attracting positively charged Ca2+ ions and trigger further precipitation of CaCO3 (Wang et al., 2012). Meanwhile, literature reported the activity of bacteria could be enhanced in the presence of GO (Wang et al., 2013), which may be the reason why reaction rate of Ca2+ ions was increased. The effect of GO on size of crystals can be attributed to adsorption for bacteria, because of adsorption, several bacteria acting as a nucleus at the same time can lead to larger crystal growth. The relationship between bacterial OD and GO concentration were shown in Fig. S6. The bacterial OD was lowest in the presence of 15 mg/l GO, which showed that the adsorption effect was the best with 15 mg/l GO and proved why the unconfined compressive strength was the highest with 15 mg/l GO. The above result revealed that the size of the crystals can be changed by using GO during the MICP process. This point was important because the size of the CaCO3 crystals may affect the effect of bio-cementation (Choi et al., 2016). In general, the unconfined compressive strength of consolidated sand should be enhanced with increasing concentrations of GO. However, when concentration of GO was 20 mg/L, the unconfined compressive strength of the consolidated sand sample decreased. This may be because sphere crystals appeared in Fig. 5 (d)). Sphere crystals
Conflicts of interest The authors declare that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgement This work was financially supported by Study on Mechanism of Seepage Grouting in Unsaturated Fine Sand Layer under Vacuum Negative Pressure [grant number 3-2-2019-025]. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ibiod.2019.104767. References Achal, V., Pan, X.L., 2014. Influence of calcium sources on microbially induced calcium carbonate precipitation by Bacillus sp CR2. Appl. Biochem. Biotechnol. 173, 307–317. Al Qabany, A., Soga, K., 2013a. Effect of chemical treatment used in MICP on engineering properties of cemented soils. Geotechnique 63, 331–339. Al Qabany, A., Soga, K., 2013b. Effect of chemical treatment used in MICP on engineering properties of cemented soils. Geotechnique 63, 331–339. Amarakoon, G., Kawasaki, S., 2018. Factors affecting sand solidification using MICP with pararhodobacter sp. Mater. Trans. 59, 72–81. Amiri, A., Bundur, Z.B., 2018. Use of corn-steep liquor as an alternative carbon source for biomineralization in cement-based materials and its impact on performance. Constr.
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Graphene oxide on microbially induced calcium carbonate precipitation ∗
T
Guowang Tang, Guihe Wang , Yuxiu An, Haonan Zhang School of Engineering and Technology, China University of Geosciences (Beijing), Beijing, 100083, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Bio mineralization Calcium carbonate Morphology Graphene oxide
The improvement of microbial induced calcium carbonate precipitate (MICP) using additives has attracted much attention due to its great influence on the quality of precipitate. In this paper, graphene oxide (GO) was used as an additive to induce CaCO3 precipitate. Using scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), the effects of GO on immobilization of bacteria, CaCO3 crystals and the consolidation of sand was studied. The addition of GO caused the rapid growth and the larger size of the CaCO3 crystals. Additionally, the morphology of the rhombohedral crystals was unchanged. The precipitation capability of CaCO3 during the MICP process was improved significantly. X-ray diffraction (XRD) noted that stable calcite was formed during the CaCO3 precipitate process with GO, compared to calcite and vaterite formed. More importantly, the unconfined compressive strength of the consolidated sand was significantly enhanced. These results show that GO has outstanding properties.
1. Introduction Traditional grouting methods, such as chemical grouting, cement consolidation and polymer grouting, have been developed and widely used; however, environmental pollution is a major limitation (van Paassen et al., 2010). Thus, a new grouting method, known as microbially induced calcium carbonate precipitation (MICP), has emerged (Amarakoon and Kawasaki, 2018; Cheng et al., 2013). MICP has attracted extensive attention due to its outstanding properties and potential engineering applications (Dopffel et al., 2018; Jimenez-Lopez et al., 2008; Kumari et al., 2014; van Paassen et al., 2010). At present, it is generally acknowledged that the reaction mechanism can be summarized in three equations (Eq. (1) - (3)) (Fujita et al., 2017; Khan et al., 2015; Nawarathna et al., 2018) these equations summarize the biochemical reactions of calcium carbonate precipitation when bacteria serve as nucleation sites.
urease CO(NH2)2 + H2 O → H2 NCO O− + NH+4
H2
NCOO−
+ H2 O→
HCO−3
Ca2 ++HCO32 − + NH3
+ NH3
bacterial
→
CaCO3 + NH+4
(1) (2) (3)
First, urea is hydrolyzed to carbamate and ammonium ions (Reaction (1)). Then, carbonate is spontaneously hydrolyzed to produce bicarbonate and second ammonia (Reaction (2)). The precipitation of CaCO3 is obtained in the presence of dissolved Ca2+ (Reaction (3)).
∗
Great application prospect for the MICP process have been exhibited in many fields. For instance, it has been applied in lessen wind erosion (Gu et al., 2018; Maleki et al., 2016); treatment of the environment (Kumari et al., 2014; Okwadha and Li, 2011; Wang et al., 2018); consolidation of sand (Martinez et al., 2013; Soon et al., 2013); repair of cracks in concrete structures (Vashisht et al., 2018; Vijay et al., 2017); and improvement of oil recovery (Nemati et al., 2005; Whiffin et al., 2007). MICP is also a common bio-mineralization process in nature; however, the laboratory research plays an important role. Current laboratory research mainly focuses on several different aspects. First, the effect of different bacteria, such as Bacillus subtilis sp, Bacillus sp (Li et al., 2018), Bacillus megaterium and Pararhodobacter sp, have been analyzed during the MICP process (Amarakoon and Kawasaki, 2018; Cardoso et al., 2018; Gat et al., 2017; Schwantes-Cezario et al., 2017b; Zhang et al., 2014). Second, many scholars have investigated the precipitation efficiency of CaCO3 crystals and sand consolidation during the MICP process and found that it is influenced by Ca2+ concentrations, urea concentrations, temperature, pH, incubation period, calcium sources and degree of saturation (Achal and Pan, 2014; Cheng et al., 2013; De Muynck et al., 2010; Kim et al., 2018; Omoregie et al., 2017). Finally, permeability, porosity, stiffness, shear strength, unconfined compressive strength (UCS), microstructure and shear wave velocity have been extensively studied. These parameters determine the effect of cementation (Amiri and Bundur, 2018; Cheng and Shahin, 2016; Martinez et al., 2013; Qian et al., 2010). Additives have improved soil mechanical properties by bio-
Corresponding author. E-mail address: [email protected] (G. Wang).
https://doi.org/10.1016/j.ibiod.2019.104767 Received 4 June 2019; Received in revised form 7 August 2019; Accepted 19 August 2019 0964-8305/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Formation mechanism of CaCO3 crystals in the presence of GO.
1643 cm−1, and a strong and wide OH group stretching vibration band appeared near 3436 cm−1 (Haghighi et al., 2013; Jin et al., 2018).
mineralizing CaCO3 precipitation catalyzed through bacterial urease. Several additives, including low water-soluble nacre proteins (Heinemann et al., 2011), poly-Lys (Nawarathna et al., 2018), fiber (Li et al., 2016), modified polymer (Wang et al., 2018) and activated carbon (Zhao et al., 2018), have been studied and show good performance improvement. However, few studies have been carried out to investigate the influence of graphene oxide (GO). This study evaluated the effects of GO as an additive during the MICP process. The reaction rate of Ca2+; the precipitated number of CaCO3 crystals; and the polymorph, morphology and microstructure of CaCO3 crystals at various bacterial concentrations were analyzed using the gravimetric method, X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) to assess change in the crystals.
2.3. Cementation media and the bacteria concentration The cementation medium (0.5 mol/L) was a mixture of 0.5 mol/L CaCl2 and 0.5 mol/L urea solutions. The concentration of cementation media was selected based on the Nawarathna et al. (2018) study. Bacterial suspensions of different concentrations (OD600 = 0.25, 0.5, 0.75 and 1.0) were prepared with bacteria and deionized water. 2.4. Precipitation experiments CaCO3 precipitation experiments were carried out at 25 °C in beakers containing 60 mL of bacterial suspension and 240 mL of the cementation medium. When the cementation medium concentrations were 0.5 mol/L, the precipitation experiments were carried out at various bacterial suspensions (OD600 = 0.25, 0.5, 0.75 and 1.0) without and with GO (10 mg/L). When bacterial concentrations equaled OD600 1.0, the same experiments were conducted at the different concentrations of GO (5, 10, 15 and 20 mg/L) with the cementation medium (0.5 mol/L). CaCO3 crystals on the wall of the beaker were obtained by filtering and then drying at 105 °C for 24 h.
2. Materials and methods 2.1. Microorganisms and growth conditions In this study, the Sporosarcina pasteurii (ATCC11859) strain was used for CaCO3 precipitation. This bacteria were used because they produce urease and have a capacity for crystallization of CaCO3 within the experimental system. The culture medium was prepared by mixing casein peptone (15 g/L), soy peptone (5 g/L), NaCl (5 g/L) and urea (20 g/L) into 1000 mL of deionized water. The final pH of the culture medium was approximately 7.3. The culture medium was sterilized at 121 °C for 30 min. Bacteria were incubated aerobically in the medium at 30 °C at a rotation rate of 120 rpm for 48 h. The culture was centrifuged at 5434 g at 4 °C for 8 min to pellet the bacteria. Bacterial pellets were resuspended in 1000 mL of deionized water and stored at 4 °C no longer than 48 h prior to use. The bacteria were diluted to an optical density at 600 nm (OD600) of 1.0. All chemical reagents, such as urea, calcium chloride (CaCl2), sodium chloride (NaCl), peptone from casein and peptone from soymeal were purchased from Sinopharm Chemical Reagent Co, Ltd (Shanghai, China).
2.5. Sands Sand from a river was used. Grading size distribution curves of sand are shown in Fig. S2. The average particle diameter of the sand (D50) is 127.26um. The D10, D30, and D60 are 22.26, 78.482 and 150.23 μm, respectively. The coefficient of uniformity (Cu) is 6.74 and the coefficient of gradation (Cc) is 1.84. 2.6. Consolidation experiments Consolidated sand samples were obtained using full contact flexible molds as described in Zhao et al. (2014). The size of the mold was 50 mm diameter and 100 mm height. The 300 g of sand and GO (0, 5, 10, 15, and 20 mg/l by total volume of cementing fluid) were uniformly mixed with 60 ml bacteria solution. The mold with the above sand mixture was submerged in the cementing fluid (0.5 mol/l) and reacted using a gaseous diffusion system shown in Fig. S3.
2.2. Characterization of GO GO, which was prepared from natural graphite powder (325 mesh, Qingdao Huatai Lubricant Sealing S&T Co. Ltd., Qingdao, China) by the modified Hummers' method (Wang et al., 2012), was obtained from Tsinghua University, china. The thickness (4–5 nm) and diameter (0.5–3.5 μ m) of GO was characterized by Atomic force microscope (AFM) under standard operating conditions shown in Fig. S1 (a). The oxygen-containing functional groups of GO were characterized by Fourier transform infrared spectroscopy (FT-IR, FTIR8400, SHIMADZU, Japan) shown in Fig. S1 (b). The characteristic absorption bands of C–O appeared at the wavelengths of 1045 cm−1 (Haghighi and Tabrizi, 2013; Jin et al., 2018). The band of the C]O group appeared at
2.7. Analytic methods A small amount of CaCO3 crystals was ground in a mortar. XRD measurements were performed using automatic XRD instruments (D8 Advance, Bruker-AXS, Germany). The scan rate was 0.15 s step−1 and the range of 2 θ was from 10° to 70°. The concentrations of Ca2+ were measured with a PHS-3C PH meter using a calcium ion electrode. The 2
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Fig. 2. SEM images of CaCO3 crystals in the presence of GO obtained in different time. 1min (a), 10min (b), 30 min (c), 60min (d), 90 min (e) 12 h (f), respectively. EDS patterns record of GO and CaCO3 crystals from the marked area A (g) and B (h).
morphology and microstructure of the crystals were analyzed by field emission scanning electron microscopy (FESEM, Zeiss Supra 55, Germany) after being sputter-coated with a thin layer of gold nanoparticles. In the meantime, energy dispersive spectroscopy (EDS, HORIBA, 7593-H) associated with the SEM were used to characterize the chemical composition.
During the MICP process, most oxygen-containing groups on the basal planes of GO are removed which negatively charged COO− groups remain at their edges; therefore, Ca2+ ions are preferentially adsorbed on the edges of GO when urea and CaCl2 are used. Negatively charged GO had a larger specific surface area and was encapsulated by Ca2+. Consequently, a large number of negatively charged bacteria (Bundeleva et al., 2011; De Muynck et al., 2010) were absorbed through the positively charged Ca2+ aggregates, which lead to accumulation and immobilization of the bacteria. Larger CaCO3 crystals were generated in the presence of sufficient Ca2+. To understand the mechanisms of the CaCO3 crystals growth in the presence of GO. Precipitates were collected at various stages as shown in Fig. 2a–f. In the first 1 min, it was found that CaCO3 crystals began to grow on graphene surface in the presence of bacteria when urea and
3. Results 3.1. Mechanism of GO on CaCO3 precipitation Fig. 1 shows that CO32 − was formed through hydrolysis of urea catalyzed by bacterial urease. Then, CO32 − reacted directly with Ca2+ cross-linked with GO in the presence of bacteria to precipitate CaCO3. 3
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Fig. 3. (A) The changes of Ca2+ concentration at different GO concentrations. (B) The concentration of Ca2+ at different bacterial concentrations (OD600) with and without GO (10 mg/L). (C) The total mass of CaCO3 precipitate at different GO concentrations. (D) The total mass of CaCO3 precipitate at different bacterial concentrations (OD600) with and without GO (10 mg/L).
the Ca2+ the concentration in the presence of GO decreased faster than that in the absence of GO. In the beginning, When the bacterial concentration was OD600 0.5, 0.75 and 1.0, the Ca2+ concentration decreased more rapidly in the presence of GO than in the absence of GO. The above result shows that GO was conducive to the Ca2+ reaction. Although Ca2+ in the absence of GO was not completely deposited, Ca2+ in the presence of GO was completely deposited after 36 h. The total mass of the CaCO3 precipitate increased by at least 53% in presence of GO is shown in Fig. 3 (C). Fig. 3 (D) shows that total quality of the CaCO3 precipitate catalyzed by bacterial urease at different bacterial concentrations with (10 mg/L) and without GO. The CaCO3 precipitate increased with increasing bacterial concentrations without GO. When the bacterial concentrations were OD600 0.25, 0.5, 0.75 and 1.0, the CaCO3 precipitate increased by at least 171%, 73%, 33% and 26%, respectively, in the presence of GO (10 mg/L) when compared to without GO. The total mass of the CaCO3 precipitate reached its maximum at the bacterial concentration of 0.5 in the presence of GO because Ca2+ was completely deposited.
CaCl2 were used (Fig. 2 (a)). At 10 min, the characteristic spherical of vaterite became more and more evident, suggesting that vaterite gradually formed. However, at 90 min almost no characteristic spherical corresponding to vaterite phases appeared, according to FESEM results (Fig. 2b–d). The crystals were rhombohedral when the deposition time was 60 min. In the first 90 min, with the increasing time of deposition, GO around crystals is becoming less and less. By 12 h, the shape of crystals gradually became more regular, and Go was not detected as shown in Fig. 2f, which mean GO is encapsulated in calcium carbonate as nucleation sites. This result is consistent with the results shown in Fig. 1. EDS was used to further characterize effect of GO in the CaCO3 crystals growth. EDS indicates that the atomic ratios of C, O and Ca for sit A and sit B are 68: 32: 0 and 63: 23: 12, respectively (Fig. 2(g and h)). The result certifies that Ca2+ ions can be adsorbed by GO; bacteria can be adsorbed by Ca2+ ions and the CaCO3 crystals growth can be influenced by GO during the MCIP process. 3.2. Influence of GO on CaCO3 precipitation
3.3. Influence of GO on the polymorph and morphology of CaCO3 crystals at various bacterial concentrations
Calcium carbonate precipitation is influenced by GO, with the concentration of GO possibly playing a key role. Changes in the Ca2+ concentration at different GO concentrations are shown in Fig. 3 (A). The concentration of Ca2+ in the presence of GO decreased faster than that in the absence of GO. When the concentration of GO was 10, 15 and 20 mg/L, the change in the Ca2+ concentration was basically the same. The effect of GO became more apparent at different bacterial concentrations (Fig. 3 (B)). The reaction rate of Ca2+ increased with increasing bacterial concentrations. When the bacterial concentration was OD600 0.25, the change in the Ca2+ concentration was basically the same with and without GO during the first 24 h. However, after 24 h,
The morphologies of CaCO3 crystals prepared at different bacterial concentrations without GO were invested by SEM (Fig. 4 (a, b, c and d)). The size of the CaCO3 crystals were in the range of 7–22 μm, 18–48 μm, 17–38 μm and 24–113 μm, respectively. Regular rhombohedra crystals were obtained at low bacterial concentrations. When the bacterial concentration was OD600 0.25, vaterite was produced (Fig. 4a). When the bacterial concentration was OD600 1.0, the agglomeration phenomenon of CaCO3 crystals occurred. An important 4
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The morphologies of the CaCO3 crystals prepared at different bacterial concentrations with 10 mg/L GO were also invested by SEM (Fig. 4 (a1, b1, c1 and d1)). The size of the CaCO3 crystals were in the range of 24–53 μm, 17–67 μm, 38–72 μm and 28–113 μm, respectively. Larger CaCO3 crystals were obtained in the presence of GO than in the absence GO at the various bacterial concentrations. This is because the bacteria were gathered together through the adsorption of Ca2+ on the edges of GO. The morphology of the rhombohedral crystals, which is the most advantageous morphology for sand consolidation, was unchanged. Another important phenomenon is that the size of the crystals decreased with increasing bacterial concentrations in the presence of 10 mg/L GO. This is because GO can act as a nucleation site (Wang et al., 2012). Because adequate Ca2+ existed, CaCO3 crystals could continuously grow, thereby generating larger CaCO3 crystals. However, there was not enough Ca2+ at higher concentration of nucleation sites; thus, a larger number of small CaCO3 crystals were generated without the growth of individual crystals. Calcite was obtained at the various bacterial concentration, regardless of GO addition. A small amount of vaterite was obtained without addition of GO in Fig. S4. This result is consistent with the results shown in Fig. 4 (a). Thus, we suggest that more stable crystals were obtained in the presence of GO. At the same time, a number of researchers have concluded that calcite catalyzed by urease of bacteria is more conducive to the consolidation of sand (Amiri and Bundur, 2018; Zhao et al., 2014). The morphology of CaCO3 crystals at different GO concentrations are shown in Fig. 5. The size of the CaCO3 crystals decreased with increasing concentrations of GO. An important reason for this is that the number of nucleation sites increased. rhombohedral crystals are the main morphology at the low GO concentration. When the GO concentration was 20 mg/L, sphere crystals were obtained. 3.4. Influence of GO on sand consolidation Consolidated sand samples from day 6 at different GO concentrations are shown in Fig. 6. Better integrity and compactness were observed with GO than without GO. The result is same with other literature (Schwantes-Cezario et al., 2017a). The unconfined compressive strength tests are shown in Fig. 7. One important point is that the unconfined compressive strength of the consolidated sand was obviously improved at most by about 100% in the presence of 15 mg/L GO. SME images show that a small amount of CaCO3 precipitate was adsorbed on the surface of the sand in the absence of GO (Fig. S5 (a and b)). However, a larger number of CaCO3 precipitate absorbed on the surface of the sand in the presence of GO (Fig. S5 (c and d)). This result is consistent with the results shown in Fig. 3 (c). Thus, we consider that the unconfined compressive strength increased with increasing the number of CaCO3 precipitate. This maybe reason why the consolidation strength of sand particles is higher in the presence of than in the absence of GO shown in Fig. 7. Al Qabany and Soga, 2013b also suggest that the unconfined compressive strength increased with the amount of CaCO3 precipitation.
Fig. 4. SEM images of CaCO3 crystals at different bacterial concentrations without GO at D600 = 0.25 (a), D600 = 0.50 (b), D600 = 0.75 (c), D600 = 1.0 (d) and with GO (10 mg/L) at D600 = 0.25 (a1), D600 = 0.50 (b1), D600 = 0.75 (c1), D600 = 1.0 (d1).
4. Discussions The experimental investigation on effect of GO on MICP using Sporosarcina pasteurii revealed that GO can be successfully utilized in order to accelerate the deposition of Ca2+, increase the total mass of CaCO3 and improve the unconfined compressive strength of consolidated sand samples. Fig. 7 demonstrates optimal concentration of GO found for the unconfined compressive strength of the consolidated sand sample in this study. The increase in the total amount of calcium carbonate precipitation was crucial because the amount of calcium carbonate precipitation was proportional to the unconfined compressive strength of consolidated sand (Al Qabany et al., 2013a; Whiffin et al., 2007). The increase of the rate of Ca2+ ions was also very important, which was of great significance for controlling the reaction
Fig. 5. SEM images of CaCO3 crystals catalyzed by bacteria (OD600 = 1.0) at GO concentrations of 5 mg/L (a), 10 mg/L (b), 15 mg/L (c) and 20 mg/L (d).
characteristic of this phenomenon is that the size of the crystals increases with increasing bacterial concentrations, which may be because there is enough Ca2+ involved in the reaction. 5
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Fig. 6. Consolidated sand samples with different GO concentrations.
may be flushed out during injection after CaCO3 precipitation (Al Qabany et al., 2013a). GO is a structural analogue of graphene containing O-containing functional groups (Liu et al., 2019). Go exhibits aromatic structure, and functional groups are mainly found at the edges (Mauter and Menachem, 2008). In the literature, because of its sharp edges, GO may have adverse effects on human body and the environment (Du et al., 2017). However, in this study, GO was used as the nucleus of CaCO3 crystals growth, and was not detected at the end of the experiment. Therefore, adverse effects can be avoided. We believe that GO did not produce any toxicity in this study. 5. Conclusions This paper investigated the effects of GO during the MICP process. It has been revealed that GO can accelerate the deposition of Ca2+, increase the total mass of CaCO3 and improve the unconfined compressive strength of consolidated sand samples. The SEM and XRD analyses demonstrate that larger and more stable calcite can be obtained with than without GO. At the same time, SEM analysis of consolidated sand sample indicates that more CaCO3 crystals were adsorbed on the surface of sand particles with than without GO. The unconfined compressive strength of the consolidated sand was obviously improved at most by about 100% in the presence of 15 mg/L GO.
Fig. 7. The unconfined compressive strength of the consolidated sand at different GO concentrations.
time of MICP progress. Many literatures have also studied how the rate of Ca2+ ions reaction can be increased (Bains et al., 2015; Cuthbert et al., 2012; Gat et al., 2014). Theoretically, bacteria can decompose 0.4 mol of urea and induce precipitate of 0.4 mol of CaCO3. However, this was not observed experimentally at the various bacterial concentrations (OD600 = 0.25, 0.5, 0.75 and 1.0) because the hydrolysis rate of urea generally depends on the rate of bacterial urease activity rather than the amount of minerals in the media (Bachmeier et al., 2002). When GO was added, urea was completely decomposed and Ca2+ ions were completely reacted to form CaCO3 precipitate, which may be because the presence of negatively charged GO might create additional nucleation sites by attracting positively charged Ca2+ ions and trigger further precipitation of CaCO3 (Wang et al., 2012). Meanwhile, literature reported the activity of bacteria could be enhanced in the presence of GO (Wang et al., 2013), which may be the reason why reaction rate of Ca2+ ions was increased. The effect of GO on size of crystals can be attributed to adsorption for bacteria, because of adsorption, several bacteria acting as a nucleus at the same time can lead to larger crystal growth. The relationship between bacterial OD and GO concentration were shown in Fig. S6. The bacterial OD was lowest in the presence of 15 mg/l GO, which showed that the adsorption effect was the best with 15 mg/l GO and proved why the unconfined compressive strength was the highest with 15 mg/l GO. The above result revealed that the size of the crystals can be changed by using GO during the MICP process. This point was important because the size of the CaCO3 crystals may affect the effect of bio-cementation (Choi et al., 2016). In general, the unconfined compressive strength of consolidated sand should be enhanced with increasing concentrations of GO. However, when concentration of GO was 20 mg/L, the unconfined compressive strength of the consolidated sand sample decreased. This may be because sphere crystals appeared in Fig. 5 (d)). Sphere crystals
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