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Earth stabilisation via carbonate precipitation by plant-derived urease for

building applications

Article  in  Geomechanics for Energy and the Environment · December 2020

DOI: 10.1016/j.gete.2020.100230

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 1 Paper published in:

2 A. Cuccurullo, D. Gallipoli, A.W. Bruno, C. Augarde, P. Hughes, C. La Borderie (2021).

3 Earth stabilisation via carbonate precipitation by plant-derived urease for building

4 applications.

5 Geomechanics for Energy and the Environment, ??: 100230

6 https://doi.org/10.1016/j.gete.2020.100230

8 Earth stabilisation via carbonate precipitation by plant-

9 derived urease for building applications
10
11 A. Cuccurullo1, D. Gallipoli2, A. W. Bruno3, C. Augarde4, P. Hughes4, C. La Borderie1
12 1
Laboratoire SIAME, Université de Pau et des Pays de l’Adour, France
2
13 Dipartimento di Ingegneria Civile, Chimica e Ambientale, Università degli Studi di Genova, Italy
3
14 School of Engineering, Newcastle University, United Kingdom
4
15 Department of Engineering, Durham University, United Kingdom
16
17 ABSTRACT: Raw (unfired) earth represents a sustainable and efficient alternative to traditional construction
18 materials but its dissemination into building practice has been hindered by a relatively high vulnerability to water
19 erosion. In this respect, enzyme induced carbonate precipitation (EICP) can improve the durability of earth
20 materials without using traditional chemical binders such as cement and lime. EICP utilises the urease enzyme to
21 catalyse the hydrolysis of urea, which produces carbonate ions that react with the calcium ions dissolved in the
22 pore water, thus resulting in the precipitation of calcium carbonate. The calcium carbonate fills the soil voids and
23 binds particles together, which reduces water permeability and increases material strength. The plant-derived
24 urease enzyme is a hexameric protein produced by the metabolism of microbes but it is also found in the tissues
25 of many common plants. This work proposes a relatively low-cost and simple stabilisation technology that makes
26 use of crude urease enzyme extracted from soybeans. This technology is applied to the stabilisation of compacted
27 earth, whose properties are then assessed via unconfined compression, moisture buffering and durability tests. The
28 findings suggest a noticeable improvement of material strength and durability, though further investigation is
29 necessary to increase the competitiveness of EICP stabilisation against standard techniques using cement and lime.

30 Keywords: Soil stabilisation; Biocementation; Enzyme induced carbonate precipitation (EICP); Plant-derived
31 urease; Earth construction

32
33 1. INTRODUCTION

34 1.1. Context

35 Raw earth is a construction material consisting of a compacted mix of soil and water which is put in
36 place with the least possible transformation (Jaquin et al., 2009). Archaeological evidence from clay-
37 rich regions of ancient Mesopotamia (between the Euphrates and Tigris rivers) and Afghanistan
38 indicates that raw earth was used for the construction of buildings since 10000 BC. Earth is harmless to
39 humans, can be locally sourced and is therefore easily transported to the construction site. Earth is also
40 recyclable, inexhaustible and, when properly manufactured, offers high strength, excellent hygrothermal
41 properties and small embodied energy at very low costs. Therefore, raw earth materials can dramatically
42 reduce exploitation of natural resources not only during construction but also during the service life of
43 buildings by cutting down heating/air conditioning needs and limiting demolition waste (Gallipoli et al.,
44 2017).

45 Despite the above eco-friendly attributes, the dissemination of earth building into routine engineering
46 practice has been hindered by the high vulnerability of this construction technique to water erosion.
47 Earth rapidly absorb any free water which it meets (Walker, 2000; Jayasinghe and Kamaladasa, 2007)
48 exhibiting an increase of moisture content that generally follows a linear trend with the square root of
49 time (Lucas, 1918; Washburn, 1921). Moisture ingress reduces the strength and stiffness of raw earth
50 while producing structural damages caused by swelling of the clay fraction. For example, Bui et al.
51 (2009) reported erosion depths between 5 mm and 10 mm on the surface of 400 mm thick unstabilised
52 earth wallets exposed to a wet continental climate for twenty years. This also explains why well-
53 preserved earth buildings, which may date back hundreds of years, are relatively common in dry climates
54 but are very unusual in wet climates.

55 One of the main challenges of earth building therefore lies in the improvement of material durability by
56 means of environmental-friendly stabilisation methods that avoid energy intensive processes or
57 environmentally unfriendly binders such as cement or lime. Stabilisation by cement or lime also
58 undermines the hygroscopic properties of raw earth, thus reducing the hygro-thermal inertia of building
59 walls with a consequent increase of operational energy (Bui et al., 2014; Bruno, 2016). Alternative
60 chemical binders, such polymers or alkoxysilanes, have also been used to stabilise the superficial layer
61 of earth walls exposed to environmental actions. These techniques have, however, encountered limited
62 success as they do not counteract the swelling/shrinkage of clay minerals but only diminish the
63 symptoms of the wearing process, thus being ineffective in the long-term (Price et al., 2011).

64 1.2. Research objectives

65 This paper describes a biomediated earth stabilisation method that relies on the hydrolysis of urea
66 (ureolysis) to induce the precipitation of calcium carbonate with the consequent enhancement of material
67 properties (Stocks-Fischer et al., 1999; Barkay and Schaefer, 2001; DeJong et al., 2006; Whiffin et al.,
68 2007; De Muynck et al., 2010; Dilrukshi and Kawasaki, 2016). The application of microbial processes
69 to the catalysis of ureolytic reactions inside soils has captured the attention of many scientists across the
70 world (Ismail et al., 2002; Whiffin, 2004; Mitchell and Santamarina, 2005; De Jong et al., 2006, Van
71 Paassen et al., 2011; Ng et al., 2012). Past research has shown that ureolytic reactions are catalysed by
72 the urease enzyme, which is produced by the metabolic activity of microbes, either exogenous or
73 endogenous to the soil (Whiffin, 2004; DeJong et al., 2006; Whiffin et al., 2007; De Muynck et al.,
74 2010; van Paassen et al., 2010; Al Qabany and Soga, 2013; Cheng et al., 2013; Cheng et al., 2014; El
75 Mountassir et al., 2014; Martirena et al., 2014; Gomez et al., 2015; Choi et al., 2016; Terzis and Laloui,
76 2017; Terzis and Laloui, 2018). Based on these findings, researchers have developed microbially
77 induced carbonate precipitation (MICP) techniques, which have proven generally successful in
78 stabilising earth materials but have also displayed a number of shortcomings. The main shortcoming is
79 perhaps the unsuitability of MICP to the stabilisation of fine-grained or compacted materials (i.e., clays
80 and silts) due to the impossibility of microorganisms to migrate through very small pores (Morales et
81 al., 2015). For example, the Sporosarcina pasteurii bacteria has a size of the order of microns, which is
 82 larger than most pores inside fine-grained materials. Furthermore, microbial colonies remain alive inside
83 the soil long after the stabilisation reactions have occurred with potential negative impacts on the
84 surrounding ecosystem.

85 Because of the above limitations, some authors have recently proposed the direct use of the urease
86 enzyme as an alternative to the cultivation of microbes inside the soil, which has led to a new category
87 of enzyme induced carbonate precipitation (EICP) techniques for the stabilisation of earth materials
88 (Nemati and Voordouw, 2003; Yasuhara et al., 2012; Neupane et al., 2013; Park et al., 2014; Nam et al.,
89 2015; Kavazanjian and Hamdan, 2015; Carmona et al., 2016; Dilrukshi and Kawasaki, 2016;
90 Kavazanjian et al., 2017). The molecule of the urease enzyme has a size of around 12 nm and can fit in
91 very small pores, thus overcoming the main disadvantage of MICP. Furthermore, unlike microbial
92 colonies, the urease enzyme has a relatively short life span with an activity that naturally degrades with
93 time, thus avoiding any significant impact on the surrounding ecosystem (Pettit et al., 1976). Traditional
94 EICP techniques make use of pure reagent-grade enzyme that is purchased from chemical suppliers at
95 relatively high cost. Conversely, the present work employs a crude plant-derived urease enzyme, which
96 is extracted from soybeans via a simple and cost-effective process that could be easily adopted by
97 building practitioners.

98 The paper starts with an overview of the key aspects that govern the kinetics of urea hydrolysis. This
99 provides the basis for optimising the catalyst action of the urease enzyme and, therefore, maximising
100 calcium carbonate precipitation. Here, the implicit assumption is that a larger precipitation of calcium
101 carbonate corresponds to a greater improvement of the mechanical characteristics of the earth material.
102 An optimised EICP method is then devised and applied to the stabilisation of compacted silty clay
103 samples, which are subjected to unconfined compression, moisture buffering and durability tests to
104 assess the improvement of the material hygro-mechanical properties.

105 2. ENZYME INDUCED CARBONATE PRECIPITATION

106 2.1. Chemical reactions leading to calcium carbonate precipitation

107 The hydrolysis of one mole of urea (CO(NH2)2) in water generates one mole of ammonia (NH3) and one
108 mole of carbamic acid (NH2COOH) (Equation 1) (Dilrukshi et Kawasaki, 2016). One mole of
109 NH2COOH is then hydrolysed into another mole of NH3 plus one mole of carbonic acid (H2CO3)
110 (Equation 2). The two moles of NH3 and one mole of H2CO3, obtained from the above reactions,
111 equilibrate in water to form one mole of carbonate ions (CO32-) and two moles of ammonium ions (NH4+)
112 (Equations 3 and 4). The above reactions also produces hydroxide ions (OH-), which in turn leads to an
113 increase of alkalinity.

114 Therefore, the overall result of the hydrolysis of one mole of CO(NH2)2 is the production of two moles
115 of NH4+ and one mole of CO32- (Equation 5). In the presence of calcium ions (Ca2+) under supersaturated
116 conditions, one mole of CO32- then precipitates into one mole of calcium carbonate (CaCO3) (Equation
117 6), which is the ultimate objective of the earth stabilisation method.

118 𝐶𝑂(𝑁𝐻2 )2 + 𝐻2 𝑂 → 𝑁𝐻2 𝐶𝑂𝑂𝐻 + 𝑁𝐻3 (1)

119
120 𝑁𝐻2 𝐶𝑂𝑂𝐻 + 𝐻2 𝑂 → 𝑁𝐻3 + 𝐻2 𝐶𝑂3 (2)
121
122 2𝑁𝐻3 + 2𝐻2 𝑂 → 2𝑁𝐻4+ + 2𝑂𝐻 − (3)
123
124 2𝑂𝐻 − + 𝐻2 𝐶𝑂3 → 𝐶𝑂32− + 2𝐻2 𝑂 (4)
125
126 𝐶𝑂(𝑁𝐻2 )2 + 2𝐻2 𝑂 → 2𝑁𝐻4+ + 𝐶𝑂32− (5)
127
128 𝐶𝑎2+ + 𝐶𝑂32− → 𝐶𝑎𝐶𝑂3 (6)
129 The above reactions indicate that precipitation of calcium carbonate is a rather straightforward chemical
130 process that is governed by the following factors: the source of Ca2+ ions, the concentration of urea and
131 Ca2+ ions (Hammes and Verstraete, 2002; Ng et al., 2012) and, finally, the pH and temperature of the
132 solution (Kile et al., 2000; Castanier et al., 1999). The next sections briefly describe the influence of
133 each one of these factors on the precipitation of calcium carbonate.

134 2.1.1. Source of Ca2+ ions

135 Biomineralisation processes generally lead to the precipitation of different calcium carbonate phases,
136 including anhydrous polymorphs phases, such as calcite, aragonite and vaterite. In particular,
137 precipitation of calcium carbonate by mixing concentrated solutions of Ca2+ and CO32− ions involves the
138 following three consecutive steps: a) formation of amorphous calcium carbonate characterised by low
139 stability and high solubility, b) transformation of amorphous calcium carbonate into vaterite and c)
140 transformation of thermodynamically unstable vaterite into stable calcite (Wei et al., 2003; Shen et al.,
141 2006; Hua et al., 2007). Furthermore, different sources of Ca2+ ions induce precipitation of calcium
142 carbonate crystals with different shapes as revealed by electron microscopy. The rhombohedral shape,
143 which is induced by calcium chloride (CaCl2), is characteristic of calcite, i.e. the most stable form of
144 calcium carbonate (De Yoreo and Vekilov, 2003; Favre et al., 2009; Gorospe et al., 2013). Instead,
145 calcium acetate induces a lettuce like or lamellar shape that is characteristic of vaterite, a metastable
146 form of calcium carbonate. Finally, calcium lactate and calcium gluconate induce a more complex
147 growth of vaterite crystals with spherical shapes (Tai and Chen, 1998). Overall, calcium chloride
148 appears the best option to enhance urease activity and maximise the production of a stable form of
149 calcium carbonate (Achal and Pan, 2014).

150 2.1.2. Concentration of chemical reagents (urea and Ca2+ ions)

151 Nemati and Voordouw (2003) conducted one of the earliest investigations into the parameters that affect
152 the efficiency of EICP. Their objective was to enhance fossil fuels recovery by consolidating sand in oil
153 reservoirs and tracing contaminants in ground water systems. Nemati and Voordouw (2003) conducted
154 test-tube experiments to evaluate the influence of the amount of urease enzyme and the concentration
155 of reagents (i.e. urea, calcium chloride and skim milk powder as stabiliser) on the rate and quantity of
156 calcium carbonate precipitation. They concluded that, for a constant concentration of reagents, an
157 increase in the amount of urease enzyme produced a faster reaction. Conversely, a proportional increase
158 of reagents concentration resulted in a slower reaction, though the final quantity of precipitate increased
159 if sufficient time was allowed. Nemati and Voordouw (2003) concluded that, as the concentration of
160 reagents increases, longer curing times are needed for completely converting reactants into products.
161 Other studies (Yasuhara et al., 2012; Neupane et al., 2013; Simatupang and Okamura, 2017; Carmona
162 et al., 2017; Almajed et al., 2018; Chandra and Ravi, 2018) confirmed that, for a fixed amount of urease
163 enzyme, higher concentrations of urea and calcium chloride yield greater quantities of precipitate as
164 curing time increases. Nevertheless, if amount of urease enzyme and curing time are both fixed, there is
165 an optimal concentration of the reagents leading to the highest precipitation of calcium carbonate.

166 Urease activity also plays a key role in determining the optimal amount of enzyme, i.e. the amount that
167 maximises reaction rate while avoiding inhibition of catalysis at high reagents concentrations. Nemati
168 and Voordouw (2003) used purified urease with a very high activity of 26,100 Units/g, which explains
169 why only a very little amount of enzyme was necessary (0.01 – 0.1 g/L) to initiate the ureolytic reactions
170 without any inhibition of catalysis at all reagents concentration. Other studies, which employed enzymes
171 of different nature and activity, observed instead inhibition of catalysis at large reagents concentrations
172 with a consequent reduction of calcium carbonate precipitation.

173 2.1.3. pH

174 The pH of the reaction medium is a crucial abiotic factor that influences the activity of the urease enzyme
175 during hydrolysis of urea (Labana et al., 2005). Rohy et al. (2019) observed that an acidic reaction
176 medium delays the EICP reaction rate, which is also consistent with the results from Cheng et al. (2018)
177 about delayed MICP stabilisation. In particular, Rohy et al. (2009) investigated the compressive strength
178 of soil columns of 100 mm and 50 mm diameter where for each increment in the molarity of the reagents
179 (urea-CaCl2), a greater quantity of purified urease was used. The samples were allowed to cure for 3, 7
180 and 14 days at room temperature, before being oven dried at 105°C for 24 hours. Mechanical tests
181 outlined an increase of strength with increase in the reagents concentration from 1 mol/L to 3 mol/L
182 after 3 days of curing. Measurements of the pH before and after adding the free urease, showed a
183 decrease with the increase in the molarity of the reagents (pH of 5.69 and 4.46 for 1mol/L and 3mol/L
184 solutions, respectively). This initial decrease of the pH was shown to be due to the increase of the urea
185 concentration and the later addition of free urease led to decreased rate of carbonate precipitation.
186 Therefore, 3 mol/L solution samples were prepared and cured for 3, 7 and 14 days. Samples cured for
187 14 days recorded a compressive strength of 3 MPa, equal to an increase of 500% compared to the
188 strength of samples cured for 3 days. The increased strength pointed towards a decreased rate of
189 precipitation due to which 3 days were not enough for the precipitation reaction to stabilize.

190 2.1.4. Temperature

191 Neupane et al. (2015) investigated the effect of temperature on both EICP reaction rate and amount of
192 precipitate inside coarse sand columns of 5 cm diameter and 100 cm height. Their objective was to
193 reduce the reaction rate in order to achieve an uniform distribution of precipitate over large soil areas.
194 The sample columns were injected with two cementing solutions, containing fixed amounts of urea,
195 calcium chloride and purified urease enzyme at mixing temperatures of 5°C and 23.5°C, respectively.
196 The solution at temperature of 23.5°C generated very large amounts of calcium carbonate near the
197 injection point. Precipitation progressively reduced down to a depth of 60 cm, beyond which no
198 significant amount of precipitate was detected. On the contrary, the columns injected with cementing
199 solution at temperature of 5°C, exhibited a smaller but much more uniform precipitation of calcium
200 carbonate. This indicates that a decrease of temperature from 23.5°C to 5°C reduces the reaction rate
201 and induces smaller amounts of precipitation. These results are in agreement with those of Nemati and
202 Voordouw (2003), who found that the reactants conversion rate was 100% after 120 hours at
203 temperatures between 30°C and 50°C but it dropped to 70% after 300 hours at a temperature of 20°C.
204 Similarly, van Paassen (2009) found that the rate of ureolysis doubled approximately every 8°C as
205 temperature increased from 5°C to 70°C.

206 Nevertheless, increasing temperature to very high levels may lead to a progressive denaturation of the
207 urease enzyme. For example, Illeová et al. (2003) demonstrated that enzyme activity was completely
208 lost after 40 min of exposure to a temperature of 87.5°C. Dhami et al. (2014) found that the urease
209 enzyme was stable at 35°C but its activity decreased by almost 47 % when temperature increased to
210 55°C. Wu et al. (2017) investigated urea hydrolysis in the absence of a calcium source and observed
211 decreasing rates of ammonium production at temperatures above 30°C with no ammonium production
212 at 50°C.

213 3. MATERIALS AND METHODS

214
215 3.1. Bouisset soil

216 The soil used in the present work was provided by the brickwork factory Bouisset from the region of
217 Toulouse (France). It is one of the five soils supplied by different brickwork factories in France, which
218 were tested in a separate project (TERCRUSO, 2013). The project TERCRUSO (2013) showed that the
219 Bouisset soil possesses similar hygro-mechanical characteristics but better water durability properties
220 than the other four soils, which is the reason why it was selected in the present study.

221 Figure 1 shows the grain size distribution of the Bouisset soil, which was determined as the average of
222 three independent measurements by means of wet sieving and sedimentation tests in compliance with
223 the norms XP P94-041 (AFNOR, 1995) and NF P 94-057 (AFNOR, 1992). Figure 1 also presents the
224 lower and upper limits of the recommended grading ranges for the manufacture of compressed earth
225 bricks according to AFNOR (2001); CRATerre-EAG (1998) and MOPT (1992).
226
227 Figure 1: Grain size distribution of the Bouisset soil in relation to existing recommendations for the manufacture
228 of compressed earth bricks by AFNOR (2001); CRATerre-EAG (1998) and MOPT (1992).

229 Inspection of Figure 1 indicates that the Bouisset soil exhibits a well graded grain size distribution that
230 is slightly finer than the limit prescribed by current recommendations for compressed earth bricks. The
231 specific gravity of the solid particles, Gs is reported in Table 1 and was measured as the average of four
232 pycnometer tests performed according to the norm NF P 94-054 (AFNOR, 1991). The plasticity
233 properties of the fine fraction (i.e. the fraction smaller than 0.400 mm) were measured according to the
234 norm NF P94-051 (AFNOR, 1993). In particular, the Atterberg limits, i.e. the liquid limit wL, plastic
235 limit wP and plasticity index IP, were all determined as the average of four independent measurements
236 and are given in Table 1.

237 Figure 2 shows the plasticity characteristics of the Bouisset soil with reference to the Casagrande chart,
238 which indicates that the material can be classified as a low plasticity clay. Note also that the Bouisset
239 soil falls inside the recommended plasticity region for the manufacture of compressed bricks according
240 to AFNOR (2001); CRATerre–EAG (1998) and Houben and Guillaud (1994). The hydro-mechanical
241 behaviour of earth materials is significantly influenced by the mineralogy of the clay fraction. Previous
242 studies of the Bouisset soil (TERCRUSO, 2013) have indicated a predominantly kaolinitic clay fraction,
243 which indicates a small tendency to swell/shrink upon wetting/drying and, therefore, makes this soil
244 suitable for earth construction. Another measure of the soil sensitivity to water is the clay activity, A,
245 which is defined as the ratio between the plasticity index and the percentage of clay fraction (i.e. the
246 percentage of the soil that is smaller than 0.002 mm). For the Bouisset soil, the activity has been
247 established to be 0.30, which classifies this material as low active (Skempton, 1953).
248 Table 1: Main properties of Bouisset soil (after Cuccurullo et al., 2019a).

Grain size distribution

Gravel content (> 2 mm, %) 0
Sand content (≤ 2 mm, %) 31
Silt content (≤ 63 μm, %) 35
Clay content (≤ 2 μm, %) 34
Specific gravity, Gs 2.65
Atterberg limits
Plastic limit, wP (%) 18.7
Liquid limit, wL (%) 29.0
Plasticity index, IP(%) 10.3
Clay activity, A 0.30
249
250 Figure 2: Plasticity properties of the Bouisset soil in relation to existing recommendations for the manufacture of
251 compressed earth bricks by Houben and Guillaud (1994) and AFNOR (2001); CRATerre-EAG (1998) (after
252 Bruno, 2016).

253 3.2. Plant-derived urease enzyme

254 The urease enzyme was the first nickel metalloenzyme ever purified and crystallised from jack beans
255 (canavalia ensiformis), an achievement that yielded the Nobel Prize in Chemistry to James B. Summer
256 in 1946. Since then, different types of nickel dependent ureases have been isolated from bacteria, fungi
257 and plants.

258 Chemical suppliers commercialise pure reagent-grade urease enzyme, which is effective for catalysing
259 the hydrolysis of urea, but it is also very expensive and, therefore, not suitable for routine construction.
260 Because of this, the present work has explored an alternative and cost-effective EICP stabilisation
261 method that is based on the use of crude urease enzyme extracted from soybeans. Soybeans have been
262 either centrifuged into a juice or crushed into a fine powder to provide two alternative sources of the
263 urease enzyme, which have been tested to assess their respective activities.

264 3.2.1. Soybeans liquid extract

265 The procedure for obtaining the liquid urease extract consisted in soaking dry soybeans into distilled
266 water (in a ratio of 1g:10 ml) for 24 hours before centrifuging both beans and water in a 700-Watt
267 blender for 1 minute (Figure 3). This resulted in the extraction of a semi-transparent grey-yellow juice,
268 which was collected in a glass while the crushed beans were retained in the blender. To increase enzyme
269 concentration, the crushed beans were recovered from the blender and centrifuged two more times to
270 extract additional liquid that was mixed with the previously collected juice.

271
272 Figure 3: Procedure for obtaining soybeans liquid extract: soybeans are soaked in water (a), soaked soybeans are
273 centrifuged in a blender (b), soybeans juice is collected (c).

274
275 3.2.2. Fine soybeans powder

276 An alternative procedure was also followed to obtain the urease enzyme in the form of fine soybeans
277 powder. Soybeans were crushed by using an electrical grinder and passed through a sieve to collect the
278 finest particles with dimensions smaller than 0.400 mm (Figure 4). This cut-off size was chosen to boost
279 the fine fraction of the earth mix and to avoid that material imperfections, caused by the presence of
280 large inclusions, may increase the vulnerability of the stabilised earth to water erosion.

281

282 Figure 4: Sieving of the fine fraction (smaller than 0.400 mm) of crushed soybeans.

283 3.3. Chemical reagents

284 The chemical reagents used in the proposed EICP stabilisation method are urea and calcium chloride
285 (Figure 5). Urea is a colourless and odourless organic material, which serves an important role in the
286 metabolism of nitrogen compound and is the main nitrogen containing substance of the urine of
287 mammals. It is also highly soluble in water and practically non-toxic. The hydrolysis of urea exhibits a
288 high carbonate conversion rate compared to other calcium carbonate precipitation processes (Whiffin et
289 al., 2007; Harkes et al., 2010). Calcium chloride is instead a salt that provides an excellent source of
290 calcium ions because of its high hygroscopicity and solubility in water. The water solubility of calcium
291 chloride at room temperature is up to 100 times greater than that of other calcium salts such as calcium
292 hydroxide or calcium nitrate, which makes calcium chloride a very effective reagent to generate high
293 concentrations of calcium ions. This is an important aspect to consider during EICP stabilisation because
294 larger concentrations of calcium ions correspond to higher calcium carbonate precipitation (Park et al.,
295 2014). The urea and calcium chloride used in the present work were supplied by Labbox and their
296 properties are synthetically described in Table 2.
297

298
299 Figure 5: Urea (CO(NH2)2) powder and flakes of calcium chloride (CaCl2).

300 Table 2: Properties of the urea (CO(NH2)2) and calcium chloride (CaCl2) used in the present work.
Chemical
Labbox reference MW (g/mol) Appearance Assay (%)
formula
Urea CO(NH2)2 UREA-00P-1K0 60.06 White crystals, powder >99.6 %
Calcium
chloride CaCl2 CACH-A0P-1K0 110.98 White pearls >94 %
301

302 3.4. Preliminary test-tubes experiments

303 3.4.1. Liquid soybeans extract

304 This section briefly describes previous research undertaken by the same authors of this study
305 (Cuccurullo et al., 2019b), which is propaedeutic to the extensions presented in this paper. Such research
306 investigated the influence of reagents concentration on the efficiency of the EICP reactions inside test-
307 tubes containing liquid soybeans extract.

308 Twelve test-tubes with a capacity of 40 ml were filled with the fresh liquid soybeans extract that was
309 produced as described in Section 3.2.1. The liquid soybeans extract in each test-tube was mixed with
310 twelve different equimolar concentrations of urea and calcium chloride, namely 0.010 mol/L, 0.025
311 mol/L, 0.050 mol/L, 0.10 mol/L, 0.50 mol/L, 1.00 mol/L, 1.50 mol/L, 2.00 mol/L, 2.50 mol/L, 3.00
312 mol/L, 3.50 mol/L and 4.00 mol/L. The urea was added to the fresh liquid extract, shortly after
313 centrifugation of the soybeans, while the calcium chloride was added after 24 hours. This allowed the
314 evaluation of the enzymatic activity of the soybeans extract via the measurement of electrical
315 conductivity at 1 and 24 hours after the addition of urea. Figure 6 compares the values of electrical
316 conductivity measured at these two times inside the test-tubes containing different concentrations of
317 urea. The maximum value of electrical conductivity changes from about 27000 µS/cm after 1 hour, in
318 correspondence of an urea concentration of 0.50 mol/L, to about 100000 µS/cm after 24 hours, in
319 correspondence of an urea concentration of 2.00 mol/L. After 24 hours, equimolar concentrations of
320 calcium chloride were introduced in each test-tube, which induced the rapid precipitation of calcium
321 carbonate at the bottom of the test-tubes as visually observed. An equilibrium condition was reached
322 after about 72 hours from the addition calcium chloride, with no more material being deposited at the
323 bottom of the test-tubes (Figure 7). The material inside each test-tube was then filtrated and weighted,
324 which showed that the amount of precipitate was largest for a concentration of reagents between 1.50
325 mol/L and 2.50 mol/L. This concentration level is also consistent with the electrical conductivity values
326 measured in Figure 6 and with the visual observation of precipitate inside test-tubes in Figure 7.

327
328 Figure 6: Measurements of electrical conductivity 1 and 24 hours after adding urea to the liquid soybeans extract
329 (urea concentration varying from 0.010 mol/L to 4 mol/L) (after Cuccurullo et al., 2019b).
330

331 Figure 7: Visual inspection of test-tubes 72 hours after adding calcium chloride (equimolar urea and calcium
332 chloride concentrations varying from 0.010 mol/L to 4 mol/L).

333 3.4.2. Fine soybeans powder

334 The previous test-tube investigation was repeated on the fine soybeans powder to find, also in this case,
335 the optimum equimolar concentrations of urea and calcium chloride that maximise precipitation of
336 calcium carbonate. Twelve test-tubes were filled with 40 ml of distilled water which was mixed with 4
337 g of the fine soybeans powder produced as described in Section 3.2.2. This amount of soybeans powder
338 was chosen for consistency with the liquid extract, which was produced by centrifuging dry soybeans
339 and distilled water in the proportion of 1g:10 ml (see Section 3.2.1).

340 Similar to the previous test, urea was added to each test-tube in twelve different concentrations of 0.010
341 mol/L, 0.025 mol/L, 0.050 mol/L, 0.10 mol/L, 0.50 mol/L, 1.00 mol/L, 1.50 mol/L, 2.00 mol/L, 2.50
342 mol/L, 3.00 mol/L, 3.50 mol/L and 4.00 mol/L. Figure 8 shows the measurements of electrical
343 conductivity after 1 and 24 hours from the addition of urea to the test-tubes. As before, the enzymatic
344 activity appears to increase significantly over time with a peak of about 125000 µS/cm after 24 hours,
345 in correspondence of a concentration of 2.50 mol/L. Figure 8 also includes the previous measurements
346 of electric conductivity on the liquid soybeans extract and indicates that the fine powder leads to higher
347 levels of electrical conductivity after 24 hours while a negligible difference between the two cases is
348 observed after 1 hour. The higher enzymatic activity of the fine powder compared to the liquid extract
349 is particularly evident after 24 hours when higher concentrations of urea are employed.

350
351 Figure 8: Comparison of electrical conductivity measurements 1 and 24 hours after adding urea to test-tubes
352 (urea concentration varying from 0.010 mol/L to 4 mol/L).

353
354 The subsequent addition of equimolar concentrations of calcium chloride to the test-tubes led to the
355 rapid formation of a sticky yellow semi-liquid paste (Figure 9). Unlike the case of the liquid soybeans
356 extract, the relatively high consistency of this paste did not allow collection of the precipitated calcium
357 carbonate by means of filtration (see Section 3.4.1.).

358
359 Figure 9: Formation of a semi-liquid paste after addition of calcium chloride to test-tubes containing distilled
360 water, soybeans fine powder and urea (urea/calcium chloride concentration of 2.50 mol/L).

361 4. EXPERIMENTAL PROGRAM ON STABILISED EARTH

362 4.1. Samples manufacture

363 Cylindrical earth samples with a diameter of 50 mm and a height of 100 mm were manufactured as
364 follows: (i) the dry earth was passed through a 2 mm sieve to remove the largest particles; (ii) the sieved
365 material was carefully mixed with an amount of cementing solution (described later) equal to the Proctor
366 optimum water content of the Bouisset soil, that is 12.4 %; (iii) the moist soil was statically compacted
367 in 10 layers inside a cylindrical mould to attain the Proctor optimum dry density of the Bouisset soil,
368 that is 1.97 g/cm3 (the earth was compacted immediately after mixing it with the cementing solution,
369 without allowing any equalisation time, to prevent the premature precipitation of calcium carbonate
370 crystals); (iv) the surface of each compacted layer was lightly scarified before applying the subsequent
371 layer; (v) the final samples were equalised under controlled humidity (40 ± 5 %) and temperature (20 ±
372 5 ºC) until the mass changed less than 0.1 % over at least one week.

373 Three different stabilisation methods were selected for the subsequent campaign of laboratory tests. For
374 two of these methods, the source of urease enzyme was provided by either the liquid soybeans extract
375 or the suspension of fine soybeans powder in distilled water. In the third method, the previous two
376 sources of urease enzyme were combined into a suspension of fine soybeans powder in liquid soybeans
377 extract. In all cases, the cementing solution was completed by adding urea and calcium chloride in
378 equimolar concentrations of 2.50 mol/L. This concentration was chosen based on the previous test-tube
379 experiments (see section 3.4), which suggested a maximum carbonate precipitation for this amount of
380 reagents.

381 The three different stabilisation methods are summarised in Table 3 using the following codes:

382 - stabilisation using liquid soybeans extract is referred as SC;

383 - stabilisation using fine soybeans powder suspended in distilled water is referred as SP;
384 - stabilisation using fine soybeans powder suspended in liquid soybeans extract is referred as SCP.
385
386

387

388

389

390
391 Table 3: Composition of cementing solution for SC, SP and SCP stabilisation methods.

Reagents (2.50 mol/L

Stabilisation Liquid soybeans Fine soybeans
Distilled water urea/calcium
method ID extract powder
chloride)

SC × - - ×

SP - × × ×

SCP × × - ×
392
393 For the SP and SCP stabilisation methods, the amount of soybeans powder was equal to 1/12 of the dry
394 sample mass. This relatively little amount of soybeans powder was chosen after some preliminary tests
395 to limit the growth of mould on the sample surface during exposure to water. Finally, as a term of
396 comparison, a set of control (unstabilised) samples made of dry earth and distilled water were prepared
397 according to the same manufacturing procedure.

398 4.2. Mechanical properties

399 After equalisation at a relative humidity of 40 ± 5 % and a temperature of 20 ± 5 ºC, the samples were
400 subjected to unconfined compression tests with a constant axial displacement rate of 0.001 mm/s. Three
401 samples were tested for each stabilisation method to confirm the repeatability of measurements. The
402 peak compressive strength and Young’s modulus were calculated as the average of these three tests. The
403 Young’s modulus was simply measured as the slope of the tangent to the stress-strain curve over the
404 initial stress range where the material response is reasonably linear (Figure 10).

405

406 Figure 10: Stress-strain curve from unconfined compression tests and measurement of the Young’s modulus as
407 the slope of the initial tangent to the stress-strain curve.

408 Figure 11 shows the average peak compressive strength of all stabilised and unstabilised samples
409 together with the corresponding standard deviation bars. Inspection of Figure 11 indicates that
410 compressive strength varies depending on the chosen stabilisation method with the lowest, intermediate
411 and highest levels recorded for the SC, SP and SCP stabilised samples, respectively. Interestingly, only
412 SCP stabilisation, which is a combination of the SC and SP methods, generates a level of strength which
413 is higher than that of the unstabilised samples. Instead, the use of the liquid soybeans extract as a source
414 of urease enzyme (SC stabilisation) markedly undermines the mechanical properties of the material
415 resulting in a compressive strength that is twofold lower than that of the unstabilised samples. This result
416 is in apparent contradictions with the observations made in test-tube experiments (Cuccurullo et al.,
417 2019b), where precipitation of carbonate crystals was observed in a solution of liquid soybeans extract,
418 urea and calcium chloride. This is an important aspect that should be investigated in detail during future
419 research. To consider will be the presence of proteins, other than the urease enzyme, which may affect
420 the mechanical performance of the earth material. The visual examination of SP and SCP stabilised
421 samples also highlighted the formation of a crystallised white covering on the external material surface.
422 This observation provides further evidence of the greater efficiency of the fine soybeans powder as a
423 source of urease enzyme, either alone or in combination with the liquid soybeans extract.

424 Figure 12 shows the average measurements of Young’s modulus of all stabilised and unstabilised
425 samples together with corresponding standard deviation bars. Like compressive strength, the Young’s
426 modulus varies depending on the chosen stabilisation method with the highest, intermediate and lowest
427 levels of stiffness recorded for the SCP, SP and SC stabilised samples, respectively. In this case, all
428 three stabilisation methods generate lower values of Young’s modulus compared to the unstabilised
429 material.

430

431 Figure 11: Compressive strength measured from unconfined compression tests on unstabilised, SC stabilised, SP
432 stabilised and SCP stabilised samples.

433
434 Figure 12: Young’s modulus measured from unconfined compression tests on unstabilised, SC stabilised, SP
435 stabilised and SCP stabilised samples.

436 4.3. Hygroscopic properties

437 The hygroscopic behaviour of the stabilised earth was investigated via the measurement of the moisture
438 buffering value (MBV). Samples were exposed to step cycles of relative humidity, between 53 % and
439 75 %, under a constant temperature of 23 °C inside a climatic chamber (CLIMATS Type EX2221HA).
440 Each of these two humidity levels was maintained for twelve hours while the sample mass was recorded
441 every two hours as prescribed by the norm ISO 24353 (2008) for the characterisation of the hygro-
442 thermal behaviour of building materials exposed to cyclic variations of humidity over a daily time. Each
443 sample was placed in the upright position inside an aluminium foil pan so that only the top and lateral
444 surfaces were directly exposed to the atmosphere inside the climatic chamber. The total area of the
445 exposed surface was, therefore, higher than the minimum value of 0.010 m2 required by the norm ISO
446 24353 (2008).

447 Before the start of the tests, all samples were equalised at a temperature of 23 °C and a relative humidity
448 of 53 %. Equalisation was assumed to be complete when the mass of the sample changed less than 0.1
449 % over at least one week, which took generally a period of two weeks. After equalisation, the samples
450 were exposed to the cyclic changes of relative humidity as previously described. Distinct MBVs were
451 calculated during the moisture uptake and moisture release stages of each cycle according to the
452 following equation:

453 𝑀𝐵𝑉 = 𝛥𝑚 𝑆 𝛥%𝑅𝐻 (7)

454 where ∆m is the absolute value of the sample mass variation (in grams) at end of each relative humidity
455 stage, S is the exposed surface (in square meters) and ∆%RH is the imposed change of relative humidity
456 (in percentage). The values of ∆m measured at the end of a moisture uptake stage (i.e. at the end of the
457 exposure to the high humidity level) provide the “MBV uptake” while the values of ∆m measured at the
458 end of a moisture release stage (i.e. at the end of the exposure to the low humidity level) provide the
459 “MBV release”. To take into account the change of sample dimensions caused by swelling, the exposed
460 surface was calculated from the average of three height measurements and three diameter measurements
461 taken at the beginning of the test (i.e. at T = 23 °C and RH = 53 %) and at the end of each moisture
462 uptake stage (i.e. at T = 23 °C and RH = 75 %). This assures that small variations of sample dimensions,
463 owed to swelling upon wetting at high humidity, are duly considered. To confirm the repeatability of
464 measurements, three replicate samples were tested, with the relevant MBVs calculated as the average of
465 these three measurements.

466 Figure 13 shows the variation of both MBV uptake and MBV release over consecutive humidity cycles
467 for all stabilised and unstabilised samples (solid markers indicate the MBV uptake while hollow markers
468 indicate the MBV release). As expected, the MBVs of the uptake and release stages tend to converge
469 towards the same steady state value as the number of cycles increases. In general, five cycles were
470 sufficient to attain steady state conditions for all materials.

471

472 Figure 13: MBVs measured during the uptake and release stages of subsequent humidity cycles on unstabilised,
473 SC stabilised, SP stabilised and SCP stabilised samples. Solid markers indicate the MBV uptake while hollow
474 markers indicate the MBV release.

475 The final MBVs of the different materials were calculated as the averages of the uptake and release
476 values of the last three stable cycles (Table 4). A stable cycle is defined as a humidity cycle where the
477 uptake and release values are approximately equal, thus indicating the achievement of steady state
478 conditions. Inspection of Table 4 indicates that the unstabilised earth exhibits a moisture buffering
479 capacity that is greater than that of the SP and SCP stabilised earth, which incorporates the fine soybeans
480 powder as a source of urease enzyme. An explanation of this result is proposed later when the results of
481 water immersion tests are presented. Yet, even for the SP and SCP stabilised earth, the MBV value
482 remains larger than 2, which indicates good hygroscopic properties.

483 Interestingly, the highest moisture buffering capacity is recorded for the SC stabilised earth, which
484 incorporates the liquid soybeans extract as a source of urease enzyme. In this case, a very high MBV
485 close to 4 is attained, which is even greater than that of the unstabilised earth. A possible explanation of
486 this result might be found in the modification of the pore size distribution of the earth due to the
487 precipitation of calcium carbonate inside the small voids penetrated by the liquid soybeans extract. This
488 precipitation of calcium carbonate may increase the fraction of nanopores compared to the unstabilised
489 earth, thus increasing the material capacity of absorbing/releasing vapour under changing ambient
490 humidity. Therefore, SC stabilisation might not be very effective in increasing the stiffness and strength
491 of the earth but offers a way to improve the material hygroscopic properties.
492 Table 4: MBVs of unstabilised, SC stabilised, SP stabilised and SCP stabilised samples.

Sample ID MBV [g/m2%RH]

Unstabilised soil 2.91

SC 3.79

SP 2.03

SCP 2.07
493
494 4.4. Durability properties
495 Durability against water erosion was assessed by means of immersion tests according to the norm DIN
496 18945 (2013). After equalisation to the laboratory atmosphere (corresponding to a temperature of 20 ±
497 5 °C and a relative humidity of 40 ± 5 %), the samples were weighed to record their initial mass 𝑚𝑖 and
498 then immersed in water for ten minutes. They were subsequently removed from water and equalised
499 again to the laboratory atmosphere to attain the same moisture content as before immersion. After
500 equalisation, the final mass mf was recorded and introduced, together with the initial mass mi, in the
501 following equation to calculate the percentage mass loss %Δm :
𝑚𝑖 −𝑚𝑓
502 %𝛥𝑚 = 𝑚𝑖
× 100 (8)

503 Each test was performed in three replicates to confirm the repeatability of the experimental procedure.
504 The mass loss was about 42% for the unstabilised samples but reduced to 13% for the SC stabilised
505 samples. An even better result was obtained by SP and SCP stabilisation, when only 1% of the initial
506 mass was lost. Figure 14 compares the pictures of the different samples before and after immersion
507 confirming that a significant improvement of durability is achieved by means of enzymatic stabilisation,
508 especially when the fine soybeans powder is used as a source of urease enzyme. Interestingly, the
509 combination of liquid soybeans extract and soybeans powder as a source of urease enzyme improves the
510 erosion resistance of the material to levels that are acceptable for routine building applications. The
511 lower mass loss experienced by the stabilised samples may be explained by the formation of calcium
512 carbonate bonds between earth particles and by the partial occlusion of voids with a consequent
513 reduction of permeability to water. Figure 15 compares the images captured by a 4K digital KEYENCE
514 microscope on the surface of SC and SP stabilised samples. The growth of organic ramifications on the
515 surface of SP stabilised earth may be responsible for the occlusion of voids, which explains the
516 hydrophobic behaviour and relatively low moisture buffering capacity of this material (see Section 4.3).

517

518 Figure 14: Reference sample before immersion (a); unstabilised sample after immersion exhibiting a mass loss of
519 42 % (b); SC stabilised sample after immersion exhibiting a mass loss of 13 % (c); SP stabilised sample after
520 immersion exhibiting a mass loss of 1 % (d); SCP stabilised sample after immersion exhibiting a mass loss of 1
521 % (e).

522

523 Figure 15: Images captured by a 4K digital KEYENCE microscope on the surface of SC stabilised samples (a)
524 and SP stabilised samples (b). Note the organic ramifications on the surface of the SP stabilised sample.

525 4.5. X-Ray Diffraction (XRD) analysis

526 Five grams of the unstabilised, SC stabilised and SP stabilised material were ground into a fine powder
527 and air dried to the laboratory environment before performing a X-Ray Diffraction (XRD) analysis.
528 Results from these XRD tests revealed the presence of kaolinite, microcline intermediate, muscovite,
529 quartz and calcium carbonate in the form of calcite (Figures 16, 17 and 18). Calcite was identified in all
530 samples, both unstabilised and stabilised. An approximate XRD quantitative analysis was performed
531 based on the construction of diffraction peak mathematic function model by means of polynomial whole
532 pattern fitting to the spectral lines using computer software (Rietveld refinement). This indicated an
533 amount of calcite of about 2 % for the unstabilised samples compared to about 8 % for the SP stabilised
534 samples.
535
536 Figure 16: XRD analysis on unstabilised sample.
537
538 Figure 17: XRD analysis performed on SC stabilised sample.
539
540 Figure 18: XRD analysis on SP stabilised sample.

541 5. CONCLUSIONS

542 This paper has presented an experimental study of the key factors that influence the kinetics of urea
543 hydrolysis and the consequent precipitation of calcium carbonate. The objective is to optimise enzyme
544 induced carbonate precipitation (EICP) techniques for the stabilisation of soils and, more specifically,
545 for the improvement of the hygro-mechanical and durability properties of compacted earth in building
546 applications. The main outcomes of this work are summarised as follows:
547 - Earth materials have been stabilised by exploiting the urease enzyme that is present in soybeans.
548 Both a liquid soybeans extract and a fine soybeans powder provide a good source of urease
549 enzyme to be used as a catalyst of the hydrolysis of urea. This leads to the production of
550 carbonate ions and the precipitation of calcium carbonate as a binding phase. Measurements of
551 electrical conductivity have highlighted that the urease enzyme activity increases when the fine
552 soybeans powder is used instead of the liquid soybeans extract. This increase of activity is
553 particularly noticeable in the presence of high urea concentrations.
554 - Results of unconfined compression tests on stabilised earth samples indicate that material
555 strength changes depending on the chosen stabilisation method with the highest levels of
556 strength obtained when fine soybeans powder and liquid soybeans extract are used together as
557 a source of urease enzyme. Interestingly, if the fine soybeans powder and liquid soybeans extract
558 are used alone, strength levels drop to values that are lower than those of the unstabilised
559 material.
560 - Unstabilised earth has an excellent capacity to buffer moisture and hence exhibits a high hygro-
561 thermal inertia. This capacity is not significantly changed by the stabilisation methods tested in
562 the present work. The moisture buffering capacity of the material tends to reduce slightly when
563 the fine soybeans powder is employed as a source of urease enzyme while it tends to increase
564 when the liquid soybeans extract is used.
565 - The water durability of both unstabilised and stabilised earth has been assessed by immersion
566 tests according to the norm DIN 18945 (2013). During water immersion, the unstabilised
567 samples loose about 42 % of their initial mass while the samples stabilised with a combination
568 of liquid soybeans extract and fine soybeans powder have exhibited a mass loss of only 1 %.
569 This negligible mass loss makes this stabilised material adequate for exposure to natural
570 weathering according to the norm DIN 18945 (2013). Digital microscope images have showed
571 the presence of organic ramifications on the surface of the earth stabilised with the fine soybeans
572 powder. This may explain the hydrophobic behaviour and smaller moisture buffering capacity
573 of this material compared to the unstabilised one.
574 - Results from X-Ray Diffraction (XRD) analyses of both stabilised and unstabilised earth has
575 revealed the presence of minerals like kaolinite, microcline intermediate, muscovite, quartz and
576 calcium carbonate in the form of calcite. An approximate quantitative interpretation of these
577 results detected an amount of calcite of about 2 % in the unstabilised samples, which increased
578 to about 8 % in the samples stabilised with the fine soybeans powder.

579 6. ACKNOWLEDGEMENTS

580 The authors wish to acknowledge the support of the European Commission via the Marie Skłodowska-
581 Curie Innovative Training Networks (ITN-ETN) project TERRE 'Training Engineers and Researchers
582 to Rethink geotechnical Engineering for a low carbon future' (H2020-MSCA-ITN-2015-675762).

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