Effects of Cationic Polypeptide on CaCO3 Crystallization and Sand

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Effects of cationic polypeptide on CaCO3 crystallization and sand solidification by microbial-induced carbonate precipitation Thiloththama Hiranya Kumari Nawarathna, Kazunori Nakashima, Masahiro Fujita, Momoko Takatsu, and Satoru Kawasaki ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/acssuschemeng.8b01658 • Publication Date (Web): 13 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018

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ACS Sustainable Chemistry & Engineering

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Effects of cationic polypeptide on CaCO3 crystallization and sand solidification by

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microbial-induced carbonate precipitation

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Thiloththama H. K. Nawarathna§, Kazunori Nakashima‡*, Masahiro Fujita§, Momoko

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Takatsu§, Satoru Kawasaki‡

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§

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University, Kita 13, Nishi 8, Kita-Ku, Sapporo, 060-8628, Japan

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‡

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Division of Sustainable Resources Engineering, Graduate School of Engineering, Hokkaido

Division of Sustainable Resources Engineering, Faculty of Engineering, Hokkaido

University, Kita 13, Nishi 8, Kita-Ku, Sapporo, 060-8628, Japan

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*Corresponding author (K. Nakashima)

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TEL./FAX: +81-11-706-6322

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E-mail: [email protected]

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1 ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

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The use of organic additives to improve microbial-induced carbonate precipitation (MICP) is

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a novel and innovative idea. This study is the first to address the effects of the cationic

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biopolymer poly-lysine (poly-Lys) on CaCO3 crystallization and sand solidification by MICP.

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CaCO3 was precipitated with and without poly-Lys by hydrolysis of urea by using ureolytic

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bacteria, Pararhodobacter sp., in the presence of CaCl2 under different experimental

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conditions. The morphologies and polymorphs of the oven-dried precipitates were

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investigated using scanning electron microscopy and X-ray diffraction. A larger amount of

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precipitate was obtained with poly-Lys than with the conventional MICP method. The curve

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for the relationship between the poly-Lys concentration and amount of precipitate was bell

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shaped. In the presence of poly-Lys, the morphology changed from rhombohedral crystals to

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twin spherical crystals. The effects of poly-Lys on sand solidification were also investigated

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by syringe solidification at different bacterial injection intervals with and without poly-Lys.

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The addition of poly-Lys gave strongly cemented sand specimens that were stronger than

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those obtained by the conventional method. The results confirm that poly-Lys addition is an

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effective and sustainable way to improve the MICP efficiency and production of green

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materials for engineering applications.

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KEYWORDS: Biocement, Biomineral, Calcium Carbonate, Morphology, Poly-Lysine


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INTRODUCTION

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The amount of suitable land available for construction is decreasing because of rapid

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population growth and urbanization. Unsuitable ground is therefore being used for building

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constructions and appropriate ground improvement techniques are needed to improve the

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properties of weak soils. The available traditional ground improvement techniques have

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various limitations and most of these methods are environmentally unfriendly. Recently,

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biological approaches have attracted attention as environmentally friendly ground

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improvement methods.

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One innovative idea is to use non-pathogenic microorganisms that are naturally present

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in the subsurface soil to improve the properties of uncemented soils.1 These microorganisms

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support to provide the cementing materials through enzymatic reactions. CaCO3 is a well-

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known cementing material and a common biomineral. The formation of artificial CaCO3 as a

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cementing material via biological processes is an ecofriendly method for treating uncemented

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soils. This concept is called microbial-induced carbonate precipitation (MICP) and this bio-

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cement can be introduced as a sustainable construction material.2 MICP is a bio-geochemical

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process in which CaCO3 precipitation in the soil matrix is achieved by urea hydrolysis using

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enzyme urease, which is produced by ureolytic bacteria. The overall process involves the

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biochemical reactions represented by equations 1–3.3-8 The precipitated CaCO3 acts as a

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binding material, which binds soil grains together at the particle–particle interfaces, leading

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to increased soil stiffness and strength.

CONH + H O H NCOO + NH 1 H NCOO + H O → HCO + NH 2 Ca + HCO + NH → CaCO + NH 3 57

Previous research has shown that MICP can increase the soil strength and stiffness, and

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maintain its permeability, with minimum disturbance to the soil.1,8-11 CaCO3 precipitation is 3 ACS Paragon Plus Environment


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governed by four major factors: calcium concentration, carbonate concentration, pH, and

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availability of nucleation sites.11 The MICP efficiency can be improved by adjusting these

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parameters with suitable chemicals, but adding chemicals to improve the efficiency can

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damage the environment. It is therefore better to use naturally formed environmentally

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friendly additives. The use of naturally formed organic additives to improve the MICP

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performance is therefore attractive. In nature, biominerals are mainly deposited in organic

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matrixes that consist mainly of macromolecules such as proteins or polysaccharides, which

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act as templates for mineral nucleation and growth.12-14 It has been proven that combinations

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of inorganic minerals and organic macromolecules provide good hybrid materials with well-

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controlled morphologies, structures, and unique properties.15-18 In the present study, we

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mimicked this concept to improve the efficiency of MICP by producing new bio-inspired

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materials.

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Previous studies have shown the effects of acidic polypeptides on CaCO3

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crystallization,19-22 but there are few reports of the use of basic polypeptides to control CaCO3

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crystallization.15,20 This study is the first to address the effects of a cationic polypeptide on

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CaCO3 crystallization and the MICP process by using the ureolytic bacteria Pararhodobacter

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sp. Most of the MICP related research works were based on the Sporosarcina pasteurii,

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which is Gram positive bacteria with high urease activity.11, 23 Similar to S. pasteurii, Gram

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negative Pararhodobacter sp has been successfully applied in sand solidification 4,10,24 due to

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its high urease activity and long term stability.6 Poly-lysine (poly-Lys) was used as the

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cationic polypeptide; at neutral pH its amino group side chains are positively charged.20

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EXPERIMENTAL SECTION

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Preparation of bacterial cell culture

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Ureolytic bacteria Pararhodobacter sp. isolated from beach sand in Sumuide, Nago,

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Okinawa, Japan was used in this study.25,26 Bacterial cells were pre-cultured in Zobell 2216E

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medium (polypeptone (Nihon Seiyaku Co., Ltd., Tokyo, Japan) 5.0 g/L, yeast extract (BD

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Biosciences Advanced Bioprocessing, Miami, FL, USA) 1.0 g/L, FePO4 (Junsei Chemical

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Co., Ltd., Tokyo, Japan) 0.1 g/L in artificial sea water, pH 7.6–7.8; 5 mL) by shaking at

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30 °C and 160 rpm for 24 h. The pre-culture (1 mL) was inoculated into fresh Zobell 2216E

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medium (100 mL). The mixture was kept in the shaking incubator under the same conditions

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as those used for pre-culturing for 48 h. The cells were collected by centrifugation of the

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bacterial culture. The cells were resuspended in sterilized water to adjust the cell

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concentration (OD600 = 1), which was determined by UV-visible spectroscopy (V-730,

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JASCO Corporation, Tokyo, Japan).

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Precipitation of CaCO3 by ureolytic bacteria

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CaCO3 precipitation was achieved by urea hydrolysis with bacteria in the presence of

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CaCl2. The bacteria were added to a substrate solution containing urea (0.3 mol/L; Wako

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Pure Chemical Industries Ltd., Tokyo, Japan) and CaCl2 (0.3 mol/L; Wako Pure Chemical

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Industries Ltd., Tokyo, Japan) in the presence or absence of poly-Lys (Wako Pure Chemical

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Industries Ltd., Tokyo, Japan). The reaction mixture (10 mL) was shaken at 30 °C and 160

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rpm for 24 h. The samples were centrifuged to separate the CaCO3 precipitate from the

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supernatant. The precipitates were dried in an oven at 100 °C for 24 h, and then the dry

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weights of the precipitates were determined. The reactions were performed at various

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bacterial concentrations (OD600 = 0.01–0.2) in the presence (10 mg/L) or absence of poly-Lys.



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The same reactions were conducted at various poly-Lys concentrations (0-50 mg/L) with a

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bacterial concentration of OD600 = 0.1. All experiments were done in triplicate.

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X-ray diffraction and scanning electron microscopy analyses

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The polymorphs of the CaCO3 precipitate were identified by X-ray diffraction (XRD;

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MiniFlex™, Rigaku Co., Ltd., Tokyo, Japan) analysis of ground samples under Ni-filtered

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Cu 1.5406 Å radiation. The crystalline phases were identified with MATCH 3.4 software.

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Scanning electron microscopy (SEM; Miniscope TM3000, Hitachi, Tokyo, Japan) was used

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to investigate the morphologies of the precipitated CaCO3 crystals.

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Sand solidification in syringe

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Sand solidification in syringe was performed with or without poly-Lys to check the

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effects of the polymer on sand solidification under various conditions, as shown in Table 1.

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The method previously described by Danjo and Kawasaki was used for the experiments.24

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Mikawa sand (40 g, mean diameter D50 = 0.6 mm) was oven dried at 110 °C for 2 days

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and placed in a 35 mL syringe (mean diameter D50 = 2.5 cm and height 7 cm) in three layers.

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Each layer was subjected to 20 hammer blows. Then first poly-Lys (5 ml, 100 mg/L) was

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injected to the syringe and next bacterial solution (16 mL, OD600 = 1) was injected and kept

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it 5-10 min to allow fixation of the bacteria to sand particles. After that, drained out the

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solution from the outlet, leaving 2 mL of the solution above the surface. After that

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cementation solution (20 mL; 0.3 M urea, 0.3 M CaCl2, 0.02 M sodium hydrogen carbonate,

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0.2 M ammonium chloride, and 3 g/L nutrient broth (BD Biosciences Advanced

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Bioprocessing, Miami, FL, USA)) was injected into the syringe, and drained, leaving 2 mL of

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the solution above the surface to maintain wet conditions. All solutions were injected through


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gravity. Ammonium chloride was added to the cementation solution as a nitrogen source of

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the bacteria. Experiments were done in triplicate in an incubator at 30 °C.

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Every 3 days, the pH and Ca2+ concentration of the drainage were measured. The

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experiments were conducted with various bacterial injection intervals. In one set, bacteria

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were injected only once, on the first day. In another set, bacteria were injected twice, i.e., on

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the first day and again after 7 days. During the experiment poly-Lys was injected twice, on

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the first day and again after 7 days. After curing for 14 days, the unconfined compressive

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strength (UCS) of the samples were determined with a needle penetration device (SH70,

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Maruto Testing Machine Company, Tokyo, Japan). First, needle was injected in to the sand

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specimen and penetration force (N) and the penetration distance (mm) were recorded.

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Unconfined compressive strengths (UCS) of the samples were determined by using the

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calibration equation given in equation 4 which was provided by the manufacture. In the

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instrument manual it is mentioned that this calibration equation was developed by

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considering 114 natural rock samples and 50 improved soils with cements. log = 0.978 log% + 2.621 4

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x= Penetration gradient (N/mm)

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y= Unconfined Compressive Strength

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RESULTS AND DISCUSSION

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Effects of poly-Lys on CaCO3 precipitation

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Figure 1 shows the amounts of CaCO3 formed by ureolytic bacteria at different

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concentrations in the presence or absence of poly-Lys. In both cases, the amount of CaCO3

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precipitate increased with increasing bacterial concentration. The amount of precipitate

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formed is directly related to the urease activity of the relevant bacteria.27 The urease activity

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of Pararhodobacter sp. increases with increasing cell concentration.6 The increase in urease



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activity with increasing cell concentration leads to increased precipitation, and causes rapid

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nucleation and growth of CaCO3 at high bacterial cell concentrations. Previous reports have

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stated that the rate of CaCO3 precipitation with Sporosarcina pasteurii depends on the rate of

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urea hydrolysis and to increase the rate of urea hydrolysis the ureolytic biomass must be

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increased.8 A higher concentration of urease also increases the CaCO3 formation rate.14

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The amount of precipitate formed in the system with poly-Lys was higher than that

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formed in the absence of poly-Lys. The positive effect of poly-Lys on CaCO3 precipitation is

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clearer at low bacterial concentrations. A significant effect of poly-Lys was not detected at

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higher bacterial concentrations; this indicates that the bacterial activity may be the dominant

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factor rather than the effects of poly-Lys. Acidic polypeptides clearly affect CaCO3

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crystallization because the negative charge on the carboxylic group is strongly attracted to

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Ca2+ and in the presence of CO32−, a CaCO3 nucleus can be formed.19,21,22,28 The effects of

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basic polypeptides on CaCO3 crystallization are unclear and difficult to explain. One research

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group reported chemical precipitation of CaCO3 from Ca(NO3)2 and Na2CO3 in the presence

100 μm

2−

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of L-Lys. They suggested that the positive charge on L-Lys strongly attracts CO3

ions,

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resulting in CO32− ion enrichment and higher super saturation of CO32− ions in local regions.

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This is favorable for CaCO3 crystal nucleation and growth.21 The positive effect of poly-Lys

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on CaCO3 precipitation can be similarly explained. Most acidic polypeptides inhibit CaCO3

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nucleation and growth.12,21 However, the addition of poly-Lys does not inhibit nucleation and

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growth of CaCO3. The interactions between poly-Lys and calcite are purely electrostatic

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interactions between the positively charged polypeptide and the negatively charged calcite

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surface.20,28 Poly-Lys therefore has a positive effect on CaCO3 crystallization and can be used

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to improve the MICP efficiency.

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The crystal morphologies of the samples prepared at various bacterial concentrations

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were examined by SEM (Figure 2). Figure 2(a) shows that well-developed rhombohedral


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crystals were obtained at low bacterial concentrations. In contrast, at higher bacterial

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concentrations, individual rhombohedral crystals were not be observed and rhombohedral

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crystal agglomeration occurred. Another important point is that the crystal size decreased

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with increasing bacterial concentration. This may be because the number of nucleation sites

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increased with increasing bacterial concentration. Urease from Pararhodobacter sp. is not

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secreted into the extracellular medium but accumulates in or on the cell as a membrane

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enzyme.6 The bacterial cells can therefore act as nucleation sites for CaCO3 crystals. Because

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of the lack of nucleation sites at low bacterial concentrations, after the initial calcite

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precipitation on the nucleation sites, calcite crystals can grow continuously and produce

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larger crystals.29 However, at higher bacterial concentrations, each cell acts as a nucleation

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site and a large number of small crystals are produced, without growth of the individual

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crystals.

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Figure 3 shows the morphologies of the crystals in the presence of poly-Lys at various

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bacterial concentrations. In the presence of poly-Lys, the crystal morphology became

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ellipsoidal; this was especially clear at low bacterial concentrations. At higher bacterial

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concentrations, a combination of ellipsoidal and rhombohedral crystals was obtained.

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However, the XRD patterns in Figure 4 show that the main component of the obtained

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crystals, with and without poly-Lys, was calcite.

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Formation of larger number of smaller crystals in higher bacteria cell concentration

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would increase the efficiency of the MICP process in the solution due to produce of larger

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amount of CaCO3 precipitate.

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Effects of poly-Lys concentration on CaCO3 crystallization

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Figure 5 shows the amounts of precipitate formed at different concentrations of poly-

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Lys. A moderate bell-shaped relationship was observed between the amount of precipitate



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and the poly-Lys concentration. The amounts of precipitate formed at intermediate poly-Lys

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concentrations were higher than those formed at lower and higher concentrations. Visual

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observations indicated that when the poly-Lys concentration was high, the precipitate was a

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gel rather than a solid. Before oven drying, the volumes of precipitates formed at high poly-

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Lys concentrations were higher than those of the other samples, but the dry weights, i.e., after

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oven drying, were lower than those of the other samples. A similar relationship between the

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amount of precipitate and the poly-Lys concentration was previously reported by another

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research group.20 In their experiments, CaCO3 was precipitated by a chemical reaction

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between CaCl2 and Na2CO3 solutions in the presence of poly-Lys. They reported that

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increased calcite growth at low concentrations of poly-Lys and decreased growth at higher

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concentrations is typical of additives that are weakly, non-selectively bonded to the crystal

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surfaces.

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The SEM images in Figure 6 show that the crystal morphology changed greatly with

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changes in the poly-Lys concentration. At low poly-Lys concentrations, polyhedral crystals

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were obtained, as shown in the Figure 6(a). With increasing poly-Lys concentration, the

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morphology changed to peanut-like twin spheres, and at high poly-Lys concentrations, twin

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spheres were dominant, as shown in Figure 6(d). These morphology changes depend on the

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conformation of the poly-Lys chain. During urea hydrolysis, the pH of the medium increases,

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and under alkaline conditions poly-Lys has an α-helix confirmation.30 This α-helix

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confirmation leads to tangentially oriented growth on the sphere surface and vertically

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oriented growth on the equatorial zone, to form peanut-like twin spheres, as shown in Figure

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7.15 Twin spheres are probably formed because of crystal growth on both sides of the nuclear

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plate.

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Sand solidification in syringe

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Four sets of experiment (S1–S4) were performed with/without poly-Lys addition and

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with various bacterial injection times. Figure 8 shows the Ca2+ concentrations and pH values

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of the effluents every 3 days.

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Initially, a negligible amount of Ca2+ was detected in the effluents of all four samples.

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After 7 days, the effluent Ca2+ concentrations for S1 and S2 gradually increased with time,

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but the S3 and S4 effluent Ca2+ concentrations remained low for up to 12 days. Lower Ca2+

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concentrations in the effluent show that more Ca2+ was deposited as CaCO3 in the sand

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column, resulting in small amounts being washed out with the effluent. The initial low Ca2+

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concentrations in the effluents of all the samples provide evidence of initially high

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precipitation of CaCO3 in the sand column. However, the Ca2+ concentrations in the effluents

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in the systems without reinjection of bacteria (S1 and S2) increased gradually after 6 days,

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indicating insufficient consumption of Ca2+ ions and low precipitation of CaCO3. In contrast,

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the Ca2+ concentrations in the systems that were reinjected with bacteria (S3 and S4)

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remained low, indicating efficient CaCO3 precipitation in the column.

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The pH values for S1 and S2 decreased after 6 days, whereas the values for S3 and S4

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were almost constant up to 12 days and then began to decline. We found a close relationship

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between the Ca2+ concentration and effluent pH. In urea hydrolysis by urease, the pH of the

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solution increases because of ammonia formation. The pH of the effluent therefore remained

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high (alkaline) when the urease activity was high. However, urea hydrolysis rate decreased

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with time due to the deactivation of the bacteria. Bacterial cells itself could act as a

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nucleation site for CaCO3 deposition around the cell wall, leading to the gradual reduction of

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the bacterial urease activity.31 On the other hand, reinjection of the bacteria helped to

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maintain the active cell density hence could maintain high pH value. At high urease activity,

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CaCO3 is efficiently formed in the column, and the Ca2+ concentration in the effluent remains



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low. For Pararhodobacter sp., a significant loss of activity is seen under acidic (pH < 5) and

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alkaline (pH > 10) conditions, and the activity is maximum at around pH 8.6 To increase

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CaCO3 precipitation, it is therefore important to maintain the pH at around 7–8. The increase

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in the Ca2+ concentration and the decrease in the effluent pH are therefore attributed to

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decreased urease activity in the column. The urease activity was recovered by reinjection of

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the bacterial culture, to maintain a low Ca2+ concentration and high pH value throughout the

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test period.

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Solidified sand specimens after 14 days before and after UCS measurements are given

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in Figure 9. The estimated UCS values for the syringe solidification samples are shown

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graphically in Figure 10. All the sand specimens were strongly cemented, according to the

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classification system proposed by Shafii and Clough,

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values less than 1 MPa are classified as moderately cemented soil, strongly cemented soil is

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defined as having a UCS between 1 and 3 MPa, and samples with UCS values greater than 3

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MPa are classified as soft rock.

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in which cemented soils with UCS

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One interesting point is that the strength of the sand column decreased from top to

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bottom. This may be the result of high urease activity near the inlet, caused by accumulation

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of bacteria near the injection point. Pararhodobacter sp. are aerobic bacteria, therefore the

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oxygen concentration plays a significant role in the bacterial activity. Lack of oxygen in the

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bottom of the specimen would inhibit the bacterial activity, and the strength of the specimen

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would therefore decrease from top to bottom.4 It is important to achieve an even distribution

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of bacterial activity throughout the sand column to obtain a homogeneous sample. Control the

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rate of CaCO3 precipitation is also important. The injection rate of the cementation fluid

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plays a significant role in achieving a uniform distribution of bacteria through the sand

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column. At a low injection rate, immediate cementation can occur near the injection point.

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Increases the flow rate beyond the rate of urea consumption, and precipitation will lead to a


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uniform distribution of chemicals along the pathway.8 However, this area has not yet been

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well studied and it is difficult to give an exact explanation, therefore further investigation is

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needed.

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In this work, sand solidification was strengthened by reinjection of bacteria after 7 days.

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Reinjection of bacteria after 7 days effectively maintained a high urea hydrolysis rate, which

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led to higher CaCO3 precipitation and solidified sand with high UCS values. A number of

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other factors can cause a decrease in urease activity over long time periods, e.g.,

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encapsulation of bacteria as a result of precipitation or being trapped inside pores, a reduction

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in chemical transport (nutrients) through pore spaces because of precipitation, and space

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limitations caused by saturation of pore fluids and accumulation of metabolic wastes.27,29,33

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Bacterial cell concentration also significantly affect the strength of the sample. As shown in

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the Figure 3, a larger number of small crystals produced by higher cell concentration can

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create favorable bridge between sand particles by filling the pore spaces more efficiently than

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a small amount of larger crystals which were produced by lower cell concentration.

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The most important finding of this research is the positive effect of poly-Lys on sand

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solidification compared with the conventional method. Poly-Lys addition gave strongly

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cemented samples with better mechanical strength. The cell adhesion properties of poly-Lys

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could be one of the reasons for its positive effect on sand solidification. In sand solidification,

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the pore spaces between the sand particles should be filled effectively to achieve good

298

strength. Bridge formation between sand grains is also important for preventing movement of

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sand grains and improving the strength and stiffness of the specimen.34 SiO2 is the main

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constituent of sand and it has a negative charge because of dissociated silanol groups (–SiO−).

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Most bacteria have negatively charged cell surfaces, therefore there is a repulsive force

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between sand particles and bacterial cells. Positively charged poly-Lys could act as a binder

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and attach bacterial cells to sand particles, which would promote efficient cementation. The



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ionic strength of the solution and the salinity of the bacterial suspension also affect adsorption

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of bacteria on sand particles. The collision efficiency increases with increasing ionic strength

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of the solution, and adsorption of bacteria can be increased by increasing the salinity of the

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bacterial suspension.34-36 The addition of poly-Lys helps to increase the ionic strength of the

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solution and improves adsorption of bacteria on sand particles, therefore better cementation is

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achieved.

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And also as shown in the Figure 3 and Figure 6, ellipsoidal shaped crystals formed in the

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presence of the poly-Lys could fill the pore spaces more efficiently and hence better

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cementation could be achieved.

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The SEM images in Figure 11 show that in the absence of poly-Lys, cementation

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mainly occurs at the contact points between sand particles. This is the result of low

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adsorption of bacteria on the sand surface. The carbonate crystals formed in the absence of

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poly-Lys all have similar sizes and shapes, as shown in Figure 11(b). Figure 11(c) and (d)

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show SEM images of sand specimens solidified in the presence of poly-Lys. The sand

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particles are fully covered with carbonate and strong bonds are formed bridge between sand

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particles. These SEM images indicate that poly-Lys acts as a binder and facilitates bacterial

320

cell adsorption on the sand particles. The irregular shapes and sizes of the crystals also assist

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good cementation.

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According to the experimental results, the addition of poly-Lys was found to improve

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the efficiency of the MICP process. Therefore, naturally available cationic polypeptides/

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polysaccharides such as lysozyme and chitosan can be used for real field applications to

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improve the efficiency more sustainably in place of poly-Lys, supporting the concept of green

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engineering.

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329 330 331 332 333 334 335 336

AUTHOR INFORMATION

337

Corresponding Author

338

Kazunori Nakashima*

339

*Phone: +81-11-706-6322. Fax: +81-11-706-6322.

340

E-mail: [email protected]

341

Notes

342

The authors declare no competing financial interest.

343 344

ACKNOWLEDGEMENTS

345

This work was partly supported by JSPS KAKENHI Grant Number JP15K18273 and

346

JP16H04404

347



348

Page 16 of 35

REFERENCES

349

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MICP with pararhodobacter sp. Materials Transactions. 2017, 59 (1), 71-81; DOI

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ureolysis by Sporosarcina pasteurii. Journal of Applied Microbiology. 2015, 118,

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1321–1332; DOI 10.1111/jam.12804.

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Supersaturation on Spontaneous Precipitation of Calcium Carbonate in the Presence

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of Charged Poly-L-Amino Acids. Journal of Colloidal and Interface Science. 2009,

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(34) Harkes, M. P.; Paassen, L. A. V.; Booster, J. L.; Whiffin, V. S.; Loosdrechta, M. C.

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M. V. Fixation and distribution of bacterial activity in sand to induce carbonate

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precipitation for ground reinforcement. Ecological Engineering. 2010, 36 (2), 112-

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459


Figure captions

460 461

Figure 1. Amounts of precipitate formed at various bacterial concentrations (OD600) with or

462

without addition of poly-Lys. Error bars show the standard deviation of the values in three

463

independent experiments.

464

465

Figure 2. SEM images of CaCO3 precipitates at various bacterial concentrations without

466

poly-Lys: (a) OD600 = 0.01, (b) OD600 = 0.05, (c) OD600 = 0.1, and (d) OD600 = 0.2.

467 468

Figure 3. SEM images of CaCO3 precipitates at various bacterial concentrations with poly-

469

Lys (10 mg/L): (a) OD600 = 0.01, (b) OD600 = 0.05, (c) OD600 = 0.1, and (d) OD600 = 0.2.

470 471

Figure 4. XRD patterns of CaCO3 precipitates (a) without poly-Lys and (b) with poly-Lys.

472 473

Figure 5. Variations in amount of carbonate precipitated with changes in poly-Lys

474

concentration. Error bars show the standard deviation of the values in three independent

475

experiments.

476 477

Figure 6. SEM images of CaCO3 precipitated by bacteria (OD600 = 0.1) at different poly-Lys

478

concentrations: (a) 1 mg/L, (b) 10 mg/L, (c) 30 mg/L, and (d) 50 mg/L.

479 480

Figure 7. Formation of twin-sphere crystals in presence of poly-Lys.



Page 22 of 35

481 482

Figure 8. Time courses of (a) Ca2+ concentration and (b) pH of effluent in sand solidification

483

tests.

484 485

Figure 9. Solidified sand specimens after 14 days (a) before UCS measurements and (b) after

486

UCS measurements.

487 488

Figure 10. Estimated UCS values of solidified sand specimens. Error bars show the standard

489

deviation of the values in three independent experiments.

490 491

Figure 11. SEM images of cemented sand samples: without poly-Lys (a,b) and with poly-Lys

492

(c,d).

493


Page 23 of 35

494 0.3

495

0.25

496 497 498 499

Precipitate (g)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.2 0.15 Without poly-Lys

0.1 0.05

With poly-Lys

0

500

0

0.05 0.1 0.15 0.2 0.25 Bacteria Concentration (OD600)

501 502

Figure 1. Amounts of precipitate formed at various bacterial concentrations (OD600)

503

with or without addition of poly-Lys. Error bars show the standard deviation of the

504

values in three independent experiments.



(b) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 35

505 506

(a)

(b)

507 508 509 510 511 100 µm

100 µm

512 513

(d)

(c)

514 515 516 517 518 519

100 µm

100 µm

520 521 522

Figure 2. SEM images of CaCO3 precipitates at various bacterial concentrations without poly-Lys: (a) OD600 = 0.01, (b) OD600 = 0.05, (c) OD600 = 0.1, and (d) OD600

523

= 0.2.


Page 25 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


524 525

(b)

(a)

526 527 528 529 530 531

100 µm

100 µm

532 (c)

(d)

533 534 535 536 537 538

100 µm

100 µm

539 540 541

Figure 3. SEM images of CaCO3 precipitates at various bacterial concentrations with poly-Lys (10 mg/L): (a) OD600 = 0.01, (b) OD600 = 0.05, (c) OD600 = 0.1, and (d) OD600 =

542

0.2.



543 544

(a) 120000

545

C

100000

546

80000

Intensity

547 548

60000 40000

549 20000

550

C

C

C C

C

C

C

0 10

551

20

30

40 50 2Ɵ (Deg)

552

60

70

80

70

80

553 (b)

554

100000 C

80000

555 Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 35

556 557

60000 40000

558 20000

559 10

561

563

C C

C C

0

560

562

C

C

20

30

40 50 2Ɵ (Deg)

60

C

Figure 4. XRD patterns of CaCO3 precipitates (a) without poly-Lys and (b) with polyLys.

564


Page 27 of 35

565 566 0.3

567

0.25

568 569 570

Precipitate (g)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.2 0.15 0.1

571

0.05

572

0 0

5

573

10 15 20 25 30 35 40 45 50 55 Poly-Lys concentration (mg/L)

574 575

Figure 5. Variations in amount of carbonate precipitated with changes in poly-Lys

576

concentration. Error bars show the standard deviation of the values in three

577

independent experiments.



578

(a) (a)

Page 28 of 35

(b)

579 580 581 582 583

100 µm

100 µm

584

(c)

(d)

585 586 587 588 589 590

100 µm

100 µm

591

Figure 6. SEM images of CaCO3 precipitated by bacteria (OD600 = 0.1) at different 592

poly-Lys concentrations: (a) 1 mg/L, (b) 10 mg/L, (c) 30 mg/L, and (d) 50 mg/L. 593 594


Page 29 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


595 596 597 598 599 600 601 602 603 604 605 606

(c)

607 608 609

Figure 7. Formation of twin-sphere crystals in presence of poly-Lys.

610



611

18000

(a)

16000

Ca2+ Concentration (ppm)

612 613 614 615 616 617

14000 Inlet

12000 10000

S1

8000

S2

6000

S3

4000

S4

2000 0

618

0

2

4

619

6 8 10 Time (Days)

12

14

16

620 621

8.5

(b)

622

8

623 624

7.5

S1

pH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 35

S2

7

625

S3

626

6.5

627

6

S4

0

628

2

4

6

8

10

12

14

16

Time (Days)

629 630

Figure 8. Time courses of (a) Ca2+ concentration and (b) pH of effluent in sand

631

solidification tests. (Arrows indicate the time of bacteria reinjection to the sand

632

specimens.)

633


Page 31 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649

Figure 9. Solidified sand specimens after 14 days (a) before UCS measurements 650 651

and (b) after UCS measurements.

652



653 6

654

Bacteria injection once

Bacteria injection twice

655 5

656 657 658 659 660 661

Estimated UCS (MPa)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 35

4

3

2

662 663

1

664 665 666 667

0 Bot

Mid S1

Top

Bot

Mid

Top

Bot

S2

Mid S3

Top

Bot

Mid

Top

S4

668 669 670

Figure 10. Estimated UCS values of solidified sand specimens. Error bars show the standard deviation of the values in three independent experiments.

671 672

S1 and S3 (open): without addition of poly-Lys,

673

S2 and S4 (closed): with addition of poly-Lys 674


Page 33 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


675 676

(a)

(b)

677 678 679 680 681 100 µm

682 683

(c)

100 µm

(d)

684 685 686 687 688 100 µm

689

100 µm

690 691

Figure 11. SEM images of cemented sand samples: without poly-Lys (a,b) and with

692

poly-Lys (c,d). 693 694



Page 34 of 35

Table 1. Experimental conditions for sand solidification

695 696

poly-Lys

bacteria injection

S1

-

once b

699

S2

100 mg/L a

once b

700

S3

-

twice c

S4

100 mg/L a

twice c

697 698

701 702 703

a

Initial concentration of the poly-Lys solution applied to the column in solidification test.

704

b

Injection of bacteria culture only at the beginning of solidification test.

705

c

Injection of bacteria culture at the beginning and after 7 days of solidification test.

706


Page 35 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

707


For Table of Contents Use Only

708 709 710 711 712 713 714 715 716

Synopsis

717

Addition of poly-lysine is more sustainable way to improve the efficiency of bio-cementation

718

by bacteria.


Effects of Cationic Polypeptide on CaCO3 Crystallization and Sand

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