Accepted Manuscript

A decade of progress and turning points in the understanding of

bio-improved soils: A review

Dimitrios Terzis, Lyesse Laloui

PII: S2352-3808(18)30061-3
DOI: https://doi.org/10.1016/j.gete.2019.03.001
Reference: GETE 116

To appear in: Geomechanics for Energy and the Environment

Received date : 30 July 2018

Revised date : 8 January 2019
Accepted date : 4 March 2019

Please cite this article as: D. Terzis and L. Laloui, A decade of progress and turning points in the
understanding of bio-improved soils: A review, Geomechanics for Energy and the Environment
(2019), https://doi.org/10.1016/j.gete.2019.03.001

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 1 A decade of progress and turning points in the
2 understanding of bio-improved soils: A review
3 Dimitrios Terzis & Lyesse Laloui

4 Swiss Federal Institute of Technology, Lausanne, Switzerland (EPFL)

5 Abstract
6 Research and practice in the broader fields of civil and geotechnical engineering had long
7 ignored the presence of living microorganisms in the subsurface and the way it impacts
8 conventional practices. In the last 10 years, the term “microbial induced calcite precipitation”,
9 or that of “biogrouting” have gained momentum in the scientific literature. They are often
10 presented as the “next big thing” in geotechnical engineering applications that will solve
11 many kinds of problems, ranging from soil erosion to landslide risk mitigation and
12 liquefaction protection. Are the claimed benefits of the application of microorganisms in
13 conventional geotechnical problems real? The present review work aims to shape a complete
14 and comprehensive understanding of the progress reported in the field of bio-mediated soil
15 improvement. Specific focus is put on pivotal points in this decade-long path which is marked
16 by proof of fundamental concepts at multiple scales. Among the treated literature, reference is
17 made to over forty studies produced after 2016. As soil bio-reinforcement makes its steps
18 towards claiming a spot in mainstream geotechnical practice this review foresees to offer both
19 a look back on how far research has gone and a look forward by evaluating opportunities and
20 challenges which lie ahead.
21 Engineering nature-based and nature-inspired
22 solutions
23 Water, a constituent element of life found below the earth’s surface, attracted the interest of
24 researchers and engineers, as early as the fundamental theories of modern geotechnical
25 engineering were established. However, whilst eighty per cent of Earth’s total dry biomass is
26 found below its surface (Kallmeyer et al., 2012), the role of microorganisms in subterranean
27 engineering applications remained, until recently, relatively underexplored. Before moving
28 our focus to soil bio-improvement, it is pertinent to consider that some of these
29 microorganisms carry the oldest and most fascinating mechanisms and genetic sequences that
30 nature had millions of years of evolution to bring to perfection.

31 The major role of bacteria in applications has been addressed in various fields ranging from
32 agriculture, to enhance crop nutrition, or protect crops against pathogens (Amarger, 2002), to
33 electricity or light production, wastewater treatment and environmental remediation. Starting
34 from the aquatic environment, specific bacterial strains have been identified and attracted the
35 interest of researchers due to their ability to produce electricity. The first relevant ideas
36 appeared as early as 1911 (Potter, 1911). Decades later, progress has been reported and a
37 specific type of bacteria has been identified, such as strains of Shewanella Oneidensis, which
38 are able to conduct electrical current by using metals as intakes (Nealson & Hastings, 1979,
39 Lovley et al., 1987). Recent applications mobilize such strains as potential agents for
40 wastewater treatment (Kirchhofer et al., 2017) and electricity production (Hou et al., 2013).

41 However, many of the implemented technologies which mobilize unicellular organisms often
42 require genetic modifications to be done. Limitations thus emerge as far as their exploitation
43 is concerned since current regulations do not allow modified organisms to be released into the
44 environment. Their use remains within laboratories and their integration into real-world
45 problems is doubtful. New types of technology emerge to overcome such limitations. This
46 refers to technologies which do not require whole bacterial cells but only a specific protein or
47 enzyme found inside the cell walls, which can be isolated and extracted to execute genetic
48 codes and reaction sequences. These latter applications are often referred to as “cell-free” (Li
49 et al., 2014) technologies since they do not require whole cells but individual genetic
50 sequences and proteins found within the cell cluster or exerted in its surrounding.

51 Going beyond the direct use of microorganisms or their products in engineering processes, we
52 identify another trend at the crossroads of biomimicry and synthetic chemistry. Observations
53 of natural mechanisms have led to the development of new conceptual frameworks to address
54 engineering problems such as, for example, those referring to self-healing materials (Sordo
55 and Michaud, 2016, Koyama et al., 2016) inspired by wound healing processes in plants and
56 bones, or to development of performant fiber optics, based on spider silk (Thévenaz et al.,
57 2016). As another example, the unique architecture and properties of moth eye, which are
58 responsible for eliminating light reflection, gave birth to new concepts integrated in solar
59 panel cells (Gonzalez et al., 2015) for achieving increased performance and efficiency in solar
60 energy production.

61 Moving from the aquatic environment and the fields of material sciences and biomimicry to
62 the earth’s subsurface, one can observe that during the past decade, innovative solutions that
63 incorporate biological agents and mobilize natural processes emerge. Amongst such novel
64 approaches and applications are those that reduce heavy metals and radioactive pollutants,
65 such as uranium, from soils via bioremediation (Stylo et al., 2015, Hossain et al., 2017),
66 produce self-healing concrete (Ramachandran et al., 2001, Jonkers et al., 2010, Başaran
67 Bundur et al., 2017) for crack repair, and trace fractures in oil and gas reservoir formations
68 with recent a field application in Switzerland (Kittilä et al., 2016). In the same context,
69 knowledge has been evolving in the past years around a technique applied to enhance oil
70 extraction from trapped pores within reservoirs via the use of microbes. This technique is
71 commonly referred to as Microbial Enhanced Oil Recovery (MEOR) and has shown signs of
72 economic efficiency for the secondary and tertiary phases of oil recovery from reservoirs
73 (Lazar et al., 2007).

74 Bio-mediated and bio-inspired geo-mechanics opened new horizons for innovative

75 applications which provide unconventional solutions in geo-engineering problems (De Jong et
76 al., 2013). Some of these applications remain in a rather hypothetical sphere, such as
77 conceptual designs of systems of foundations and anchors inspired from tree roots, or
78 excavation systems inspired by the digging process observed in ant communities (DeJong et
79 al., 2014, Frost et al., 2017). Others target more practical applications such as the production
 80 of construction elements out of earth materials. This is the case of bio-bricks, a term used
81 (Achal et al., 2009, Bernardi et al., 2014) to describe bricks manufactured by mobilizing
82 Microbial Induced Calcite Precipitation (MICP) in prefabricated moulds. Such approaches for
83 the manufacturing of construction elements out of geo-materials triggered the curiosity of
84 researchers who extend these concepts in the framework of future deep-space human missions
85 where the inherent capacity of microorganisms to withstand extreme environmental
86 conditions can be used for building purposes (Roedel et al., 2014, Rothschild, 2016, Miranda
87 et al., 2017).

88 Another example relates to the presence of gas bubbles or the provision of gas bubble
89 generation in saturated sand which is considered to substantially mitigate liquefaction risks
90 (Eseller-Bayat, 2009, Viand and Eseller-Bayat, 2017). To this purpose, microbiological gas
91 production (Rebata‐Landa and Santamarina, 2006, Vilar-Sanz et al., 2013) is mobilized as a
92 means of desaturation via production of biogenic gas bubbles to achieve an overall increase in
93 liquefaction resistance.

94 Such innovative concepts have an important effect on the greater geo-engineering field both
95 from a research and industrial point of view. Firstly, they defy established practices by
96 bringing into surface alternative mechanisms, with some of them mobilizing processes which
97 are found in nature to serve as examples of efficiency. Nature itself is the perfect example of
98 achieving maximum performance at minimum energy needs. The authors believe that
99 emerging technologies, which have at their core biological processes, do push forward the
100 limits of thinking which inevitably will lead to progress, regardless of the individual fate of
101 each individual mechanism discussed above.

102 Soil bio-improvement

103 Bio-mediated urea hydrolysis and CaCO3 mineralization
104 The overview on the crossroads between biology and geo-engineering applications is herein
105 narrowed down to the specific case of bio-mineralization. Such mechanism is found in the
106 core of some of the aforementioned applications like those reported by Achal et al. (2009),
107 Jonkers et al. (2010), Bernardi et al. (2014) and Stylo et al. (2015). It is pertinent to take into
108 consideration the following clarification, regarding the use of the terms bio-mineralization
109 and bio-mediated mineralization. Bio-mineralization stands for an organism’s capacity to
110 produce and grow mineral structures within its body as part of bones or tissues. The bio-
111 mineralized element is composed mainly of calcium and phosphate with calcium carbonate
112 commonly found in corals or shells of marine organisms. Bio-mediated mineralization
113 characterizes nucleation of particles which do not grow within the organism’s body, or on its
114 tissues, but rather in the surrounding environment. The mineralized elements are considered
115 as a byproduct of the organism’s biological processes, or are due to changes in surrounding
116 environmental conditions, such as pH or concentration of chemical species.

117 Urea (CH4N2O) hydrolysis, or ureolysis, is such a chemical reaction which generates
118 favorable conditions which lead to precipitation of solid mineral calcium carbonate. Ureolysis
119 occurs in natural environments without the presence of organisms or it can be catalyzed as a
120 result of the metabolic activity of species due to the presence of the urease enzyme. CaCO3
121 nuclei are considered indirect products of urea hydrolysis, which precipitate in carbonate-rich
122 solutions under increased pH and under the presence of a calcium source. More precisely,
123 urea hydrolysis refers to the decomposition of one mole of urea into two moles of ammonium
124 and one mole of carbonate anion as shown in Equation 1.

125 𝐶𝐶𝐶𝐶4 𝑁𝑁2 𝑂𝑂 + 2𝐻𝐻2 𝑂𝑂 → 2𝑁𝑁𝑁𝑁4+ + 𝐶𝐶𝐶𝐶32− (Equation 1)

126 Catalysis by the urease enzyme is found to lead to overall faster completion times (Hausinger,
127 2013) up to a factor of 1014. The release of ammonium into the environment, due to urea
128 hydrolysis, induces pH increase, which is considered favorable for precipitation of CaCO3
129 under the presence of carbonate ions and the introduction of a calcium source into the system
130 (Equation 2).

131 𝐶𝐶𝐶𝐶2+ + 𝐶𝐶𝐶𝐶32− → 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶3 ↓ (Equation 2)

132 This mechanism of calcium carbonate mineralization is considered of utmost ecological and
133 geological importance. Biological mechanisms which trigger and govern this process are
134 associated with the reduction of carbon dioxide (CO2) in the atmosphere and its
135 mineralization to calcium carbonate (CaCO3). Such is the case of cyanobacteria (Kamennaya
136 et al., 2012) or plants which are found to capture CO2 from the atmosphere and precipitate it
137 around their roots into CaCO3 crystals. Canavalia ensiformis (jack bean) for example is
138 considered such an ureolytic plant which has been studied for its capacity to precipitate
139 CaCO3 (Park et al., 2014, Nam et al., 2015).

140 CaCO3 is found to precipitate in three main polymorphs: vaterite, aragonite and the much
141 more stable calcite (Wei et al., 2015). These CaCO3 phases exhibit different shapes,
142 geometries and solubility, with vaterite being spherical and highly soluble and calcite cubic
143 and less soluble (Plummer and Busenberg, 1982, Zhou et al., 2010). Environmental conditions
144 such as temperature, pH and pressure are considered to affect the precipitating polymorph as
145 well as transitions to metastable phases.

146 An aspect of the mechanism of soil bio-improvement, with controversial interpretations found
147 in the literature, refers to the role which microorganisms hold in the precipitated CaCO3
148 polymorph. Whilst the mechanism of MICP is widely appreciated as bio-mediated
149 precipitation, some results indicate that there exists dependency, at least for the initial
150 polymorph which precipitates, on the microbial agent responsible for urea hydrolysis. A study
151 performed by Wei et al. (2015) compares CaCO3 precipitation kinetics and morphologies
152 induced by the ureolytic Sporosarcina, Bacillus and Brevundimonas species. The precipitated
153 CaCO3 polymorphs are found to depend both on environmental conditions (Teng et al., 1998,
154 Davis et al., 2000) and to preferable polymorph precipitation by the adopted ureolytic gene
155 (Cañveras et al., 2001, Dupraz and Visscher, 2005).

156 The debate over the suitability of ureolysis-driven calcite precipitation as soil strengthening
157 technique is intensifying with respect to ammonium, a side-product of urea hydrolysis.
158 Despite the belief that ammonium can be captured and recycled through loops of recirculating
159 reactants, concerns over residual ammonium have led to the development of alternative
160 concepts. Among them we find denitrification using calcium acetate and calcium nitrate
161 (Pham et al. 2016, Hamdan et al. 2017). Despite successful applications based on
162 denitrification being reported, low reaction rates are believed to represent a major challenge,
163 if not the bottleneck of the process, which remains to be overcome towards application of
164 denitrification in realistic timeframes in conventional construction and geotechnical problems.

165 An alternative concept to improve efficiency of future in-situ applications based on calcite
166 mineralization relates to bio-stimulation of native calcifying species (Gomez et al. 2016,
167 Ohan, 2018). This strategy bypasses the direct introduction of ureolytic bacteria into the
168 ground by providing necessary nutrients and conditions for native species to grow and induce
169 precipitation of calcium carbonate bonds. An advantage of bio-stimulation is the reduced risk
170 of causing unwanted clogging of pores due to accumulation of bacterial cells, since these
171 latter are not injected directly into the ground, but grow gradually and homogenously due to
172 nutrient-rich environments. However, preliminary quality control analyses are required in the
173 area of foreseen applications to identify native species and to adapt accordingly stimulation
174 strategies which target the favoured growth and metabolic activity of calcifying strains against
175 all other native species. A recent study by Gat et al. (2016) deals with the effect of MICP on
176 the indigenous bacterial population composition of coastal sand samples collected from a
177 semi-arid environment in Israel, which yielded low initial ureolytic potential. The study
178 introduces organic carbon to biostimulate indigenous, ureolytic species and provides with
179 evidence of nitrification during the period following application of MICP, where ammonia
180 oxidation occurs.

181 Microbially Induced Calcite Precipitation (MICP)

182 To date, the term cementation has been associated with the introduction of artificially
183 manufactured cementitious mixtures and industrial fluids in the subsurface. MICP induces a
184 peculiar type of soil cementation with calcium carbonate acting as cementing element. The
185 reactive mechanism at its core is urea hydrolysis with the catalyzing activity of the urease
186 enzyme (Equation 1), which is found in several bacterial strains (Whiffin, 2004). Fujita et al.
187 (2000) firstly reported on the correlation between the rate of calcium carbonate precipitation
188 and that of urea hydrolysis. Among urease-bearing species, Sporosarcina pasteurii (Yoon et
189 al., 2001), a bacterium isolated from soils, is found to yield the highest urea hydrolysis rates.

190 Moreover, due to their wall electronegativity, S. pasteurii cells attach on soil grains where
191 they induce urea hydrolysis and release CO32- in their micro-environment. There, precipitation
192 of CaCO3 occurs under the presence of a calcium source. This process of bacterial attachment
193 on soil grains to ultimately induce the growth of calcite cubic particles is presented in a
194 schematic approach in Figure 1. Apart from this schematic representation, insight into the real
195 geometries of calcite bonds is provided in Figure 2 (Terzis et al., 2016) where cubic particles
196 of CaCO3 are captured via Scanning Electron Microscopy (SEM). Hierarchical expansion of
197 the bigger particle surfaces is observed, with planes found to reproduce those of individual
198 calcite crystals observed in their vicinity. Figure 3 (Venuleo et al., 2016) presents an assembly
199 of soil grains bound by calcite particles, all exhibiting similar sizes and geometries, captured
200 via SEM.

201
202 Figure 1 Schematic representation of S. pasteurii cells (black) attaching on soil grains (brown dodecahedra) and inducing the
203 formation and growth of calcite cubic particles (white); system of grains (top) and porous assembly of soil grains (bottom)
204 (Terzis, 2018)
205
206 Figure 2 Particles of CaCO3 captured via SEM revealing hierarchical expansion of planes (arrows) for bigger bond particles,
207 which reproduce the geometries of neighbouring single crystals (circles); black arrows indicate traces of bacteria
208 encapsulated on the crystals’ contact plane with sand grains (Terzis et al., 2016)

209 Depending on the desired final CaCO3 content, the quick overall reaction times of MICP can
210 range from several hours to a few days, thus rendering the technique a feasible solution for
211 modern engineering applications. Moreover, another advantage of MICP is the reduced
212 energy requirement for in-situ applications, which implies reduced overall application costs.
213 This is attributed to the low viscosity and increased workability of the employed bacterial and
214 reactive solutions. Lower pressures are required for their propagation via infiltration through
215 granular media, instead of mixing highly pressurized fluids with soils. MICP is thus widely
216 appreciated as a non-intrusive, permeation mechanism which leaves the initial soil state intact
217 during deposition and growth of calcite bonds upon introduction of reactive species. The need
218 of extensive injection repetitions is avoided since MICP offers the ability to generate flow
219 networks to circulate bacteria and reactants and thus improve larger soil volumes through few
220 infiltration steps. A further sign of the economic efficiency of the process lies in recirculating
221 the utilized water during infiltration of the targeted volume. This allows, on one hand,
222 reducing the volume of water needed for dissolving reactant elements and, on the other hand,
223 performing quality control tests on the effluent solutions and capturing produced NH4+, a by-
224 product which is widely used as fertilizer in agricultural applications.
225
226 Figure 3 Calcite particles (arrows) binding sand grains (Venuleo et al., 2016)

227 To fully understand the overall performance of MICP, one should account for the
228 improvement in the obtained mechanical properties of the bio-treated geo-material, in relation
229 to the mass of products used to achieve the targeted bond content. MICP is reported to yield
230 cohesion values in the range of hundreds of kPa (Terzis et al., 2016) and values of
231 compressive strength in the absence of confinement above 10 MPa (van Paassen, 2009). More
232 importantly, the desired level of improvement can be determined and controlled according to
233 the nature of the foreseen geo-engineering application. For example, MICP can be designed to
234 endow the targeted material with cohesion values in the range of 50-60 kPa to increase its
235 resistance against liquefaction. Alternatively, MICP can be implemented for producing
236 materials with strength and stiffness values close to those of typical concrete, which allows
237 considering earth materials for structural purposes. The technique’s greatest advantages are its
238 adaptability and flexibility in the design of the final soil improvement solutions for tailoring,
239 at will, desired material properties in a given area of interest.

240 The technique has been studied in a series of frameworks which require different levels in
241 calcite bond contents. Below, a summary of problems considered suitable for implementing
242 solutions based on MICP is presented with reference to relevant works:

243 i. Improve soil mechanical properties for securing the necessary bearing capacity
244 (van Paassen et al,. 2010, Gomez et al., 2016)
245 ii. Bio-clogging; a term used to describe the clogging of pores for mitigating
246 leakages in reservoir formations or the construction of underground barriers
247 (Ivanov and Chu, 2008, Cunningham et al., 2011, Ebigbo et al., 2012, Minto et
248 al., 2016)
249 iii. Slope stabilization (Cheng, 2012, DeJong et al., 2015)
250 iv. Mitigating liquefaction risk (Montoya et al., 2013, Han et al., 2016)
251 v. Fugitive dust control (Gomez et al., 2015)
252 vi. Production of masonry and bio-bricks (Achal et al., 2009, Dosier, 2015)
253 vii. Erosion protection (Jiang and Soga, 2017), targeting slopes and river banks
254 (Anbu et al., 2016)
255 viii. Vegetation applications related to soil erosion (Gomez et al., 2013)
256 ix. Stabilization of tunnelling walls (Fauriel, 2012)
257 x. Increasing soil thermal conductivity (Venuleo et al., 2016)
258 xi. Land reclamation (Lian et al., 2018)

259 State of the art

260 The characterization of the behavioural characteristics of the bio-improved geo-material at
261 laboratory scale is treated here in detail. Interest is further put on studies which up-scale
262 MICP to evaluate overall feasibility of the bio-cementation mechanism towards providing
263 solutions to real-world problems. Next, we dedicate a section to studies developed around
264 multi-physical modelling of the complex biological, chemical and hydraulic phenomena
265 coupled in MICP. Except for notions of multiphysical modelling, the review extends to
266 incorporate studies which address the behaviour of bio-improved soil in constitutive
267 modelling frameworks. Finally, works developed around the numerical study of the bio-
268 improved geo-material are presented with a focus on the use of the Discrete Element Method
269 (DEM).

270 The bio-improved geo-material at laboratory scale

271 Specimen preparation processes are reported covering various set-ups and applied conditions
272 to ultimately produce bio-improved specimens. The overall challenge in each case is no other
273 than the homogeneity in the samples’ calcite distribution. Typically, specimens are produced
274 in cylindrical moulds using peristaltic pumps to introduce reactive elements either via batch
275 (stop-flow) or continuous flow. One peculiar preparation method was conducted by Chou et
276 al. (2011) who placed sand samples in permeable moulds within a reservoir and applied
277 continuous agitation for recirculating dissolved elements through the permeable cloth.
278 Another approach is reported by Keykha et al. (2014) who used electrodes for distributing
279 reactants, i.e. dissolved ions of calcium and urea, throughout sand columns by generating an
280 electric field to drive carbonate dianions (CO32-) and calcium cations (Ca2+) across the length
281 of the columns. Moreover, a different approach suggests circulation of bacteria and chemical
282 reactants within specimens placed in triaxial cells, under confinement, to ultimately obtain
283 samples at the desired dimensions, which can be subsequently subjected to mechanical
284 loading (Lin et al., 2015). The advantage of this approach is that complex sample coring is
285 avoided. This technique allows testing lower bond contents as reported in the work by
286 Montoya and DeJong (2015). Finally, medium scale (0.5 m diameter) tanks are used by
287 Cheng (2012) as a means of obtaining larger bio-cemented volumes to core samples for
288 mechanical testing. Overall, homogenous calcite distribution at laboratory scale would imply
289 reliable experimental data sets, as far as the mechanical response is concerned, and would
290 facilitate comparison between results produced in various studies. Detecting calcite
291 distribution remains a challenge with various approaches suggested towards accurate and
292 representative measurements (Terzis et al., 2016, Nafisi and Montoya, 2018) at the scale of
293 conventional geotechnical testing.

294 A principal difference between MICP and traditional cementation mechanisms for soils is that
295 soil structure remains intact throughout treatment by infiltration. Therefore, the initial packing
296 of grains holds a governing role on the formation of the final lattice of bonds. The effect of
297 initial dry density on the obtained mechanical response was treated in the work by Chou et al.
298 (2011) for low calcite contents that yielded peak deviator stresses in the range below 50 kPa.
299 Van Paassen (2009) reports values of unconfined compressive strength (UCS) between 1-12
300 MPa. He concludes that soils with lower initial relative density require increased mass of
301 bond contents to reach similar strength with respect to the same material which was treated
302 via MICP under initially higher relative densities. A hypothesis is that calcite bonds
303 precipitate preferably around the contact points of grains. Thus, denser initial states
304 correspond to larger number of contact points among particles, as shown schematically in
305 Figure 4. Attention should be made on the way the calcite bond content is expressed. This can
306 be done in terms of volumetric fraction, for example, of the total soil volume or with respect
307 to the fraction of the solid phase. It is easily understood that looser initial states correspond to
308 increased fraction of pores per given soil volume thus bond contents expressed in terms of
309 fraction of the solid phase can be considerably higher with respect to denser initial states.In
310 this direction, Mahawish et al. (2018a) established a correlation between strength and stiffness
311 of coarse sand with the increase in the amount of deposited CaCO3, initial relative density and
312 dry density.

313
314 Figure 4 Schematic representation of contact points and bond (grey) repartition for minimum (left) and maximum (right)
315 relative dry densities; white lines represent intergranular contacts and idealized intergranular force transmission

316 MICP is widely treated as a cementation mechanism targeting shallow depths and thus most
317 available results report the mechanical behaviour of treated soils in terms of UCS. Fewer
318 results are available for triaxial drained tests under confinement or for one-dimensional
319 oedometric compression. In this direction, some relevant work is reported by Feng and
320 Montoya (2014, 2015), Fauriel (2012) and Terzis and Laloui (2017). Additionally, while the
321 material is considered to yield tensile strength, few attempts have been made towards
322 quantifying this parameter. Li et al. (2017) report results on the effect of the presence of fibres
323 on the overall tensile strength of sand stabilized via MICP and van Paassen (2009) presents
324 results of Brazilian tensile strength (BTS) tests, with values reaching up to 500 kPa.

325 As far as the permeability of the material is concerned, results suggest (Whiffin et al., 2007,
326 Cheng et al., 2013, Zamani and Montoya, 2016) that the hydraulic conductivity reduces up to
327 one order of magnitude. In addition, Kirkland et al. (2017) performed low-field nuclear
328 magnetic resonance to detect changes in porosity. They conclude that the final porosity is in
329 the range of 85% of the initial value, which was measured prior to application of MICP, with
330 the rest porous space occupied by calcite. Contrary, clogging of pores refers to the intentional
331 calcification around the vicinity of an injection source or within fissures in rock formations.
332 Such attempt is reported by Cunningham et al. (2011) for ultimately sealing fractures and
333 mitigating leakages in well-bores.

334 A confusing point in the literature relates to the characterization of the level of cementation
335 within produced samples. Terms such as light or heavy cementation appear in several studies
336 and are associated with bond contents as low as 3%. However, it is commonly appreciated
337 that contents below 3% fail to endow materials with the necessary mechanical integrity under
338 unconfined conditions for laboratory experimentation. For example, van Paassen (2009)
339 reports the highest range of CaCO3 content found in literature between 12-24%. These latter
340 results allowed establishing a trend between the precipitated mass and the expected range of
341 strength. Montoya and DeJong (2015) showed that calcite contents as low as 1% lead to
342 increase in shear strength, under undrained conditions, with respect to the untreated material
343 at loose state. In this same work, the achieved range of calcite content (1.3 %) is characterized
344 by authors as moderate cementation. It should be noted that tests, in this case, were ran at an
345 initial confinement of 100 kPa, followed by increase in the mean effective pressure due to
346 excess pore water pressure generation. Thus, understanding the specific conditions under
347 which samples are tested is important towards evaluating the overall calcite content, and
348 therefore its classification within the low-moderate-heavy cementation spectrum. Overall,
349 results available in the literature refer to a variety of CaCO3 contents, ranging from 0.6 and
350 1.2 % (DeJong et al. 2006) to 25 % (van Paassen, 2009). Contents that might appear very low,
351 such as contents lower than 2 %, can indeed endow the desired, improved properties to geo-
352 materials, given the role of confinement in problems of geo-technical interest.

353 Common agreement is found among researchers as far as the effect of treatment conditions on
354 the obtained fabric and mechanical properties of MICP-treated soils is concerned. More
355 precisely, among such parameters reported in the literature are environmental conditions,
356 concentrations of biological and chemical elements, the speed and direction of the imposed
357 reactive flow field. Because of varying treatment conditions the geometries and overall spatial
358 distribution of CaCO3 bonds are found to be affected. More precisely, Al Qabany and Soga
359 (2013) reported increasing UCS for increasing dry density of MICP-cemented specimens.
360 Results obtained in this latter work attempt to highlight the role of chemical factors in the
361 geometrical and spatial distribution of crystals and associate this role with the obtained
362 engineering properties of the material. Somewhat surprisingly, for the larger crystals observed
363 after injection of 1 mol/litre (M) of CaCl2, no increase in UCS was reported for any calcite
364 content. Indeed, these samples were reported not to exhibit homogenous cementation and the
365 cemented volume was limited to the vicinity of the injection point. Such evidence suggests
366 that there exists a series of factors, determined based on the decision-making around the
367 provided treatment, which reflects to distinctive geometries and distribution of the bond
368 lattice. These geometries lead to varying post-treatment mechanical response, regardless of
369 the base material chosen for treatment. More studies (Whiffin et al., 2007; Al Qabany et al.,
370 2011; Martinez et al., 2013) have attempted to further capture the effect of treatment
371 conditions on the overall MICP efficiency by providing with qualitative descriptions of
372 observed precipitate behaviours. The effect of environmental factors was investigated on the
373 bio-cementation of coarse sand by Mahawish et al. (2018b) who found that the optimal
374 conditions for the bio-cementation of their adopted base material is reactant concentration of
375 1 mol/L at 20oC.

376 Cheng et al. (2013) introduced a simple way for performing injections by allowing reactants
377 to flow into columns through infiltration from the surface. Results showed significant
378 variation of the obtained UCS values for samples of the same calcite content. Calcite was
379 found to be more efficiently distributed and lead to increased UCS for conditions where soil is
380 partially saturated with reactant solutions during treatment. This is attributed to liquid menisci
381 that form around the grains and maintain reactants -and thus precipitated nuclei- around the
382 crucial grain-to-grain contact points. This was the first study to demonstrate that CaCO3
383 crystals, which were formed under lower degrees of saturation, exhibited more efficient
384 distribution around the areas of desired crystal deposition, i.e. contact points among grains.
385 Such distinctive particle deposition is considered to contribute to increased strength of the
386 cemented samples for lower calcite contents. A schematic representation of this hypothesis is
387 presented in Figure 5.

388
389 Figure 5 Schematic representation of bonds precipitating within the liquid phase for 100% saturation conditions (left) and
390 within liquid menisci at lower degree of saturation (right)

391 No further results are available in the literature regarding the effect of the degree of saturation
392 during MICP-treatment on the post-treatment mechanical response. This might be attributed
393 to the complex control of the degree of saturation in granular materials with poor water
394 retention capacity. However, the finding itself, based on the hypothesis of more efficient bond
395 distribution yielded due to liquid menisci, serves as another example of the importance of
396 controlling external factors which hold strong effect on the obtained mechanical response.
397 Results obtained from UCS tests in the same study by Cheng et al. (2013) show that for
398 partially saturated conditions (Sr=20%) same level of strength (in the range of 3 MPa) is
399 reached at only one third of the total bond content required for specimens which were MICP-
400 treated under fully saturated conditions.

401 Considering the above, there exists a series of external factors that need to be meticulously
402 chosen and controlled in order to achieve efficient application of MICP. However, the role of
403 intrinsic properties of the base material subjected to MICP on the properties of the calcite
404 lattice remains to be understood. While most studies refer to sandy soils treated under MICP,
405 less is known about the effect of grain size distribution or that of fine content of the base
406 material, with most recent the contribution from Zamani and Montoya (2018) on the
407 undrained monotonic shear response of MICP-treated silty sands. Along similar lines, Nafisi
408 and Montoya (2018) proposed a framework which accounts for the particle size effect on bio-
409 cementation of sands. Gomez and DeJong (2017) compared various types of sands with fine
410 contents up to 13% subjected to MICP while Cardoso et al. (2018) investigated the role of
411 clay contents reaching 28% on the MICP process. All the above works highlight the critical
412 role the base materials’ intrinsic properties hold in the resulted mechanical response and bio-
413 improved fabric. In a similar attempt to provide with quantified data on the evolution of the
414 mechanical properties and of the micro-structure of bio-improved soils, Terzis and Laloui
415 (2017, 2018) compare the behaviour of two types of sand of different grain size distribution
416 which were subjected to MICP under identical external treatment conditions. Samples yielded
417 overall calcite contents between 7-8%. Results are summarized in Figures 6 and 7, and reveal
418 that medium-grained sand yields considerably higher peak strength under drained
419 conventional triaxial compression and under unconfined compressive strength tests, despite
420 similar response of the two materials in their untreated state (Figure 6).
421
422 Figure 6 Mechanical response for untreated (dashed) and MICP-treated (solid) fine- (grey) and medium-grained (black) sand;
423 (top) deviatoric stress versus axial strain; (bottom) normalized specific volume versus axial strain

424
425 Figure 7 Unconfined compressive strength for fine- and medium-grained MICP-treated specimens (a); evolution of the
426 Young’s modulus for fine- and medium-grained bio-improved sand with respect to increasing bond content (b)

427 Therefore, it becomes clear that the average calcite content cannot serve as the sole indicator
428 of the expected, improved mechanical behaviour of the bio-treated state. This is critical
429 towards interpreting literature results of bio-treated soils, such as those summarized in Table
430 1, which were produced under different set-ups, targeting different calcite ranges and using
431 various quantities to capture and describe the improved mechanical response.

432 More precisely, Table 1 summarizes a series of key findings reported in the literature in
433 representative studies during the last decade. This summary illustrates the range of calcite
434 contents achieved and the variety of conditions tested to characterize mechanical and physical
435 properties of bio-improved soils.
436 Table 1 – Overview of key findings reported in the literature on mechanical and physical properties of bio-improved soils

CaCO3 (%) Stiffness

Source Strength other remarks
range reported quantities
 van Paassen et 1300-13000
0.7-12 MPa c’=280-540 kPa 100 m3 experiment
al. 12-27 MPa
(UCS and triaxial) φ’= 39-58o (fine sand)
(2010) (Eur)
up to 2067 kPa
4.5-11.5 (coarse) (UCS coarse)
Cheng (2012) - - 2m column injection
0-27.5 (fine) up to 3700 kPa
(UCS fine)
k-value** in the
Cheng et al. 150-2300 kPa 20-180 MPa MICP under various
1-14 same order of
(2013) (UCS) (E) degrees of saturation
magnitude
one order of
Same base
Al Qabany & up to 3000 kPa magnitude
0-8 - material/different
Soga (2013) (UCS) reduction in k-
treatment conditions
value
Feng & Montoya 1.4/3/5.3 280-800 kPa c’=5-59 kPa Injections directly in
-
(2015) loose/moderate/heavy * (qmax, σ’3=100 kPa) φ’= 33-41o triaxial cell
up to 250 MPa two types of sand
400 kPa
Lin et al. (2015) 1-2.5 (Ei, tangent - adopted 20/30 and
(σ’3=100 kPa)
(Janbu, 1968)) 50/70
up to1350 MPa qmax=1437/1870
comparison with
Duraisamy (small-strain kPa for σ’3=50/500
0.26-9.34 50-950 kPa (UCS) gypsum cemented
(2016) shear modulus kPa and
samples
Gmax) CaCO3=4.2%
one order of
Jiang and Soga magnitude application for erosion
0-1.4 - -
(2017) reduction in k- control in gravels
value (10-4 m/s)
two types of sand
Terzis and Laloui up to 11.3 MPa E up to 1950 quantified
3-10% adopted: D50=0.19
(2018) (UCS) MPa microscopic data
mm and D50=0.39 mm
437 - Results not available

438 * Characterization of the level of cementation

439 ** Hydraulic conductivity

440 From the studies presented in Table 1, specific focus is put on strength and stiffness, cohesion
441 and friction properties as well as on hydraulic conductivity (Cheng et al. 2013, Al Qabany &
442 Soga 2013, Jiang and Soga, 2017). Comparison is also done with other cemented materials,
443 such as gypsum-cemented sands (Duraisamy, 2016). For calcite contents above 3%, UCS
444 values are commonly reported, since samples maintain their integrity without the application
445 of confining pressure. Contents up to 27% (van Paaseen et al, 2010) endow sands with
446 properties that resemble those of building materials. However, as previously discussed in this
447 paper, MICP is herein studied from a geotechnical perspective and therefore the role of
448 confinement needs to be understood in relation with the targeted calcite content. Contents as
449 low as 2% can therefore be sufficient to improve mechanical properties, to satisfy safety
450 factors and to meet the required bearing capacity limits.

451 The role of confinement is further understood in Figure 6, which presents the response of two
452 different base materials under three confining pressures (30, 100, 200 kPa). Maximum peak
453 deviatoric stress reaches shy of 2800 kPa for medium-graned sand under 200 kPa of
454 confinement. Duraisamy (2016) reports experimental data on triaxial drained tests for MICP-
455 treated sand of calcite contents up to 7.3%, with peak deviatoric stress reaching 2600 kPa
456 under 50 kPa of confinement. It should be noted that this latter work adopts sand of a mean
457 diameter (D50) equal to 0.39 mm as base material. Similar sand is adopted by Terzis and
458 Laloui (2017) which yields peak deviatoric stress equal to 2500 kPa under 30 kPa of
459 confinement (figure 6). Both studies conclude that the effect of bio-cementation is more
460 pronounced for lower values of confinement. As another example of this key finding, Table 1
461 reports a range of 1437-1870 kPa for the peak deviatoric stress yielded by samples of 4.2%
462 calcite content under confinements of 50 kPa and 500 kPa respectively.

463
464 Figure 8 (adapted and enriched with respect to El Mountassir et al. 2018) UCS evolution with respect to CaCO3 content

465 A comprehensive summary of six studies presenting experimental results on the UCS of bio-
466 improved sands was presented in the review work by El Mountassir et al. (2018). Figure 8 is
467 enriched with recent data from Mahawish et al. (2018c) on coarse-graned bio-cemented sand..
468 A distinctive trend is established by Terzis and Laloui (2018) as far as the evolution of UCS
469 of medium-granied sand (D50=0.39 mm) is concerned, compared to fine sand (D50=0.19 mm),
470 which is found to validate the trend of the rest of the literature works.

471 Recent literature studies report results from undrained triaxial shear tests. Ciu et al. (2017)
472 performed tests for bio-improved samples which reached 11.87 % in calcite content. Results
473 reveal a peak deviatoric stress of 2750 kPa for the maximum calcite content achieved (11.87
474 %) and an effective cohesion which reaches just below 450 kPa.

475 Moving beyond conventional geo-technical testing, we notice that the bio-improved geo-
476 material offers research challenges related to micro-mechanical testing. Montoya and Feng
477 (2015) investigated particle crushing between calcite grains in contact and bond-breakage
478 evolution during shearing to determine whether MICP bonds experience cohesive or adhesive
479 failure. They performed surface energy measurements and studied particle-bond-particle
480 deformation patterns coupled with electron microscopy to conclude that the failure
481 mechanism is rather of cohesive nature. Microscale experimentations donnot limit to the study
482 of mechanics. Transport phenomena can capture real-time data and reveal critical information
483 on the involved mechanisms which result to the formation of precipitates. In such an attempt,
484 Singh et al. (2015) provided with a micromodel of a porous medium to study the precipitation
485 evolution at the micro-scale using the bacterial strain Pseudomonas stutzeri (Lalucat et al.,
486 2006). The above approaches, bring the research focus to the smallest scale possible, studying
487 particle-to-particle or cell-to-particle phenomena and can serve towards robust upscaling of
488 microscopic phenomena which were treated, until recently, through a series of hypotheses.

489 The challenge of up-scaling

490 Several field-scale applications have been reported that target the bio-cementation of larger
491 volumes of fine-grained sandy soils (van Paassen et al., 2010), or stabilization of sands to
492 mitigate fugitive dust (Gomez et al., 2015, 2016, Hamdan and Kavazanjian, 2016) and apply
493 MICP to field experiments in gravels (van Paassen, 2011). All these studies provided crucial
494 findings related to the overall feasibility of MICP at large experimental scale, such as the
495 distribution of calcite content over a large soil volume, and further revealed several challenges
496 for the application of an efficient and reproducible soil improvement process that was based
497 on MICP.

498 More precisely, van Paassen (2009) performs MICP treatment in 1 m3 and 100 m3
499 experiments. The resulted volume allowed for coring samples which are considered to exhibit
500 homogenous calcite distribution at laboratory scale. Results reveal UCS values above 10 MPa
501 and values of Young’s modulus up to 13 GPa. This latter test was duplicated under similar
502 conditions and with the same base material by Filet et al. (2012) with results for UCS
503 remaining in the range around 500 kPa. At a subsequent attempt, van Paassen (2011) applies
504 MICP in field conditions, to an area with gravels, targeting the stabilization of the soil
505 substrate prior to excavation and installation of a pipeline. This application confirmed the
506 presence of mineralized CaCO3; however, no bio-cemented samples were further obtained for
507 testing. Gomez et al. (2015) performed MICP via surface percolation in an in-situ application
508 in Californian desert (USA), where the goal was to cement the upper crust of the targeted
509 area, for ultimately providing evidence towards the efficiency of MICP for mitigating fugitive
510 dust. Results show that a cemented crust was formed of about 2 cm in depth. Focusing on the
511 impact of environmental factors towards upscaling MICP, Mortensen et al. (2011) reported
512 that their biological treatment shows robust efficiency over various soil types, which were
513 adopted as base materials, and water compositions referring to varying concentrations of
514 ammonium chloride and salinity levels. Such attempts suggest that a crucial step prior to
515 upscaling is considering the properties of the in-situ base material and perform feasibility
516 studies under laboratory conditions. Moreover, continuous monitoring of the reaction process
517 is necessary, as a means of obtaining real-time data sets corresponding to key factors related
518 to the evolution of precipitation and MICP efficiency. Such real-time quality control can take
519 place via collection and analysis of effluent volumes or via geophysical methods to monitor
520 density change due to bio-cementation.

521 These works at the larger experimental scale further reveal the need of a new approach, as far
522 as the utilization of urease and the conception of a reproducible application mechanism are
523 concerned. A common reference between all these attempts is the need to cultivate bacteria
524 cells at the site of application. Thus, bioreactors need to be built, with the provision of
525 agitation systems for incubating bacterial cultures which often becomes a complicated task. In
526 this direction, a cell-free approach of MICP has been studied in Terzis and Laloui (2018). The
527 main finding is that urea hydrolysis and calcite precipitation persist in a cell-free environment,
528 after complete breakdown of rehydrated bacteria that were prepared in dry-powder form. The
529 use of freeze-dried cells as inoculum is considered to facilitate in-situ application of MICP
530 and contribute to overall reproducible soil bio-improvement applications and subsequent
531 quality control. Similar finding regarding the persistence of urease, which maintains its
532 ureolytic activity in the absence of viable cells, were reported by Mortensen et al. (2011). In
533 this study cell lysis occurred due to anoxic conditions and the enzyme was believed to keep
534 hydrolysing urea, for an uknwon time, till its ultimate degradation.

535 Multi-physical modelling

536 The processes involved in MICP offer researchers with challenges that are related to multi-
537 physical modelling. How far can bacteria travel from a given injection source? What is their
538 activity during continuous recirculation of reactants and what is the final distribution of
539 calcite along the imposed flow path?

540 Researchers have attempted to provide answers towards addressing these uncertainties by
541 bringing notions of reactive-transport processes together and addressing involved couplings
542 with the use of analytical methods. This is possible by accounting for phenomena such as
543 attachment of bacterial cells, their rate of growth, decay and their efficiency to hydrolyse urea,
544 among others. Such progress unifies the contributing factors of MICP and allows for
545 predictive modelling as far as the final mass and distribution of calcite within porous media
546 are concerned. At a second stage, the precipitated mass of calcite can be introduced as a
547 parameter to mechanical constitutive modelling to finally predict the behavioural
548 characteristics of the material. Fauriel and Laloui (2012) developed a numerical tool based on
549 Michael-Mentis kinetics for the reaction process. The model was implemented for two
550 configurations, one that simulates the application of MICP under a system of existing
551 buildings and a second which simulates stabilization of the walls of a tunnelling excavation
552 (Fauriel, 2012). Relevant parameters have been studied in numerical simulations by Dupraz et
553 al. (2009) among which variations in pH and calcite precipitation. Akimana et al. (2016)
554 coupled reactive modelling with X-Ray computed tomography as a means of calibrating their
555 numerical transport model and estimating the radial precipitation of CaCO3, extending this
556 way the predictive modelling of MICP in the three-dimensional space. Recently, Nassar et al.
557 (2018) presented a complete modelling approach which uses zero fitting parameters to
558 simulate and predict bio-stimulation in a 1.7 m diameter tank. Results showed good
559 agreement with the actual experiment which was ran in the tank, where 3 injection wells were
560 employed.

561 It becomes clear that such modelling attempts require calibration with experimentally
562 measured parameters for a robust prediction of the final mass of calcite and its spatial
563 distribution. Such an attempt was provided by Lauchnor et al. (2015) on whole cell kinetics of
564 urease hydrolysis using S. pasteurii. This is possible only in the case of large-scale tests,
565 provided that the experimental set-up is equipped with the necessary monitoring systems.
566 Martinez et al. (2014) provide a similar framework for MICP, where theoretical prediction of
567 reactive transport and kinetics of ureolysis are addressed, including mass of precipitated
568 CaCO3 and urease activity. More precisely, obtained parameters were calibrated against
569 experimentally measured values in a 0.5 m column test. Results revealed good agreement with
570 theoretically predicted parameters. However, authors in this latter work agree that complexity
571 in predictions will increase when parameters such as bacterial growth or reactive transport in
572 the 3D domain enter the problem. Similar conclusions are derived in the work by Barkouki et
573 al. (2011) where a bio-geo-chemical model is developed to capture precipitation kinetics and
574 transport phenomena for the case of batch treatments (stop-flow as mentioned by the authors)
575 and continuous flow in MICP induced by S. pasteurii. In this latter work, the imposed flow
576 regime and time allowed between batches for precipitation to occur are found to affect the
577 homogeneity in calcite distribution.

578 As an example of the increased complexity of the actual phenomena involved in MICP, we
579 consider that as calcification progresses, bacterial cells get trapped in the precipitated nuclei
580 and left deprived of oxygen and nutrients. Thus, they do not contribute to further hydrolysis.
581 Moreover, the presence of gas bubbles alters considerably flow regimes. As discussed in the
582 work by Cheng et al (2013), partially saturated conditions can induce more efficient
583 distribution of calcite particles altering the relationship between calcite content and
584 mechanical response. The existence of a design approach which quantifies parameters such as
585 hydrolysis rates, decay of bacteria, as well as crucial environmental conditions would allow
586 predictive modelling to take place on a more representative basis with respect to the physical
587 mechanisms of MICP which have been captured and reported experimentally.

588 Constitutive modelling

589 The bio-improved geo-material’s peculiar structure would classify a potential attempt to
590 address its behaviour in a modelling framework in the grey area between rock and soil
591 mechanics. The fabric of bio-improved soils does not resemble that of naturally cemented
592 soils or artificially cemented soils yielded upon application of jet grouting, for example. This
593 is due to distinctive geometries of the binding element, i.e. CaCO3, and varying trends in bond
594 sizes and spatial distributions reproduced according to applied treatment conditions.

595 Fauriel (2012) implemented a theoretical model to capture the performance of bio-improved
596 soils by extending the constitutive concept that was developed for unsaturated aggregated
597 soils by Koliji et al. (2010) to account for the effects of bonding and density change. The main
598 features of this proposed framework are the incorporation of a hardening variable, to account
599 for the effects of the apparent oveconsolidation due to the density change upon precipitation
600 and the incorporation of a destructuration parameter to govern the post-yield softening
601 behaviour of the geo-material. However, the physical significance of these parameters
602 remains unclear and their conception and calibration are based on a series of back-analysis
603 prediction of experimental data available in the literature. This model, however, did set the
604 basis for addressing the behaviour of MICP-treated soils within the critical-state concept. The
605 main features reproduced are the elastic response upon initial yielding, post-yield softening
606 and a residual condition which represents the response of the completely destructured state.
607 As widely reported by researchers, bio-improved materials yield brittle failure in the small-
608 strain region. Though, less is known about the underlying mechanisms and the contribution of
609 dilatancy in the peak strength.

610 Cheng et al. (2013) in their work on MICP under partially saturated conditions implement a
611 mechanistic model on a simplified geometry to account for the volumetric fraction of bonds
612 yielded during application of MICP at various degrees of saturation. A statistical analysis
613 captures the evolution of bond diameters and the way they affect changes in porosity.
614 Therefore, physical insight is provided offering tangible evidence on the geometry of the
615 lattice of bonds. This work sets the basis for expressing the evolution of microstructural
616 quantities with respect to the evolution of the calcite bond content. Despite its simplified
617 approach based on idealistic geometries of grains, it reflects the principle of bond growth
618 upon continuous deposition of precipitated nuclei.

619 Nweke and Pestana (2018) introduced a strength correlation which takes into account
620 nonlinearities of failure envelopes observed in bio-cemented sands. In the suggested
621 approach, the strength of the bio-improved material is expressed as a function of post-MICP
622 density, applied confinement and by accounting for the material’s intrinsic mineralogy.

623 Similar materials which exhibit bonded structures could inspire further progress in modelling
624 the behaviour of MICP-treated soils. For example, gas-hydrate bearing soils (Uchida et al.,
625 2012, Gai and Sánchez, 2016, Sánchez et al., 2017) are studied by incorporating formulations
626 to account for the different hydrate particle habits, as a means of expressing structural
627 characteristics in the adopted modelling approaches based on the critical state concept. This
628 approach was extended to capture the case of bio-imporved soils in a recent work by Gai and
629 Sánchez (2018) which incorporates a critical state yield surface, sub-loading concepts and
630 also accounts fot bond degradation. Efforts to model the behaviour of microbially-cemented
631 soils resulted in a modelling formulation proposed by Gajo et al. (2019) which incorporates
632 micro-scale inspired parameters. More precisely, cross-scale functions are introduced to
633 express a relationship between the evolving microscopic variables and the resulting
634 macroscopic material behaviour. These parameters refer to the reactive surface area, cross-
635 sectional area and the number of bonds for materials which experience dissolution or
636 precipitation. This model was successfully validated against experimental data provided by
637 Montoya and DeJong (2015). The same experimental data series were used by Gai and
638 Sánchez (2018) to validate their proposed model.

639 Numerical modelling

640 Numerical simulations, especially through implementation of Discrete Element Method
641 (DEM) codes, have facilitated significantly the study of the behaviour of packings of grains
642 with bonds (Shen et al., 2016), as well as the statistical analysis of microscopic quantities,
643 such as size of grains, number of contacts and orientations (Jiang et al., 2003, Altuhafi et al.,
644 2016, Druckrey et al., 2016), all found to affect the overall mechanical response of granular
645 materials. The main advantage of such analyses lies in the ability to reproduce different
646 packings and geometries of grains and bonds and attribute strength and stiffness
647 characteristics to different phases of the solid skeleton. Except for the peak strength further,
648 focus by researchers has been put on the post-yield behaviour. Such relevant work is reported
649 by Jiang et al. (2014) who performed 2D DEM numerical analyses focusing on the
650 degradation evolution in cemented geo-materials by providing, in addition, with microscopic
651 interpretation of the underlying mechanisms.

652 For the specific case of bio-improved soils, except for multi-physical modelling where
653 coupled bio-chemical and transport phenomena are addressed in a unified framework, a series
654 of works treating their behavioural characteristics is reported using numerical tools (Evans et
655 al. 2014, Montoya & Feng 2015, Yang et al. 2016, Feng et al. 2017). As concluded by Evans
656 et al. (2014) the principle of parallel bonds, widely applicable in DEM simulations of granular
657 materials, is not considered representative for the case of bio-improved soils. This happens
658 due to the vanishing of individual particles when their strength limit is reached, which implies
659 that mass conservation is not respected. For larger cementitious particles, which better reflects
660 the case of bio-improved soils, Evans et al. (2014) provide with a more robust design of the
661 3D packing geometry. They introduce cementitious bonds in chain formations and conclude
662 that parameter calibration needs to be done, with the sizes of bonds considered as crucial
663 parameter governing the overall response. Recently, Yang et al. (2016) treat the case of very
664 low calcite contents in the range of 0.6 % in DEM simulations. The main finding relates to
665 understanding the contribution of inter-particle friction and increasing shear resistance to the
666 dilation and re-arrangement of the packing structure in an attempt to interpret the macro-scale
667 response with respect to particle-scale phenomena. Finally, notions of contact stiffness,
668 contact mechanics and inter-particle friction (Yang et al., 2012) are incorporated in such
669 approaches to ultimately express the improved structure and understand the chain force
670 distribution within the solid matrix, a term commonly confronted in DEM simulations.

671 Insight into the micro-scale

672 While new understanding around the geometries and spatial distribution of the CaCO3 crystals
673 within the new improved structure has been reported, relevant works which became available
674 until 2016 have provided only with qualitative description of microstructural properties. What
675 we know so-far about calcite crystalline bonds is that they develop their structures following
676 hierarchical plane expansion, like the ones shown in Figure 2, but how large they eventually
677 grow remains undetermined. A hypothesis adopted by the authors is that increased contact
678 area between bonds and grains results into lower intergranular stresses. Among the questions
679 that remain to be answered is how these bonds increase their diameters and how bond sizes
680 vary with respect to the intrinsic properties of the base material and the final overall calcite
681 content. Relevant work towards addressing these uncertainties was provided recently in
682 Dadda et al. (2017, 2018) and Terzis and Laloui (2018). In this latter work, a workflow is
683 presented which incorporates X-Ray micro-computed tomography and 3D volume
684 reconstruction to capture and analyse the critical contact area and a series of micro-scale
685 parameters.

686 To understand the importance of such findings one should consider the role of fabric
687 anisotropy in sands. Particle sizes, shape and orientation influence the behavioural
688 characteristics of natural sands and are all related to sample preparation. Relevant works
689 explore these effects for various types of sand (Altuhafi et al., 2016, Druckrey et al., 2016).
690 However, an approach to quantify microscale parameters of the MICP-improved soil fabric
691 remains to be determined. This task seems challenging because of the fabric anisotropy
692 induced by the applied bio-treatment conditions to the porous material.

693 Towards mainstream engineering applications

694 Are we ever going to witness a mainstream geo-engineering application based completely on
695 bio-mediated processes? This question is continuously involved in the branch of geotechnical
696 engineering studied in the present review. To offer tangible evidence, and thus treat this
697 question on a less philosophical and more pragmatic approach, there is need to provide with a
698 robust description of the processes involved in the problem. Processes which start from the
699 moment calcifying bacterial cells flow within the porous medium and mineral deposition
700 occurs, until the formation of the final structure and subsequent deformation under
701 mechanical loading. Most importantly, answering the above question requires
702 multidisciplinary approach and cooperation among experts who treat problems of varying
703 nature.

704 Common agreement is established among researchers suggesting that current knowledge
705 produced around MICP at laboratory scale provides with the necessary proof-of-concept and
706 establishes sufficient know-how to set the basis for performing the ultimate passing towards
707 large-scale, real applications. Attempts carried-out so far refer to various injection set-ups and
708 flow schemes and to different geo-technical problems. Rich data series reveal, on one hand,
709 the strong potential that lies in MICP and, on the other hand, limitations and challenges that
710 remain to be addressed.

711 To successfully implement MICP in real-field problems a series of uncertainties needs to be

712 overcome. Among these, we prioritize the type of biological agent which will be used as a
713 source of bio-cementation, its state and readiness-to-use. This agent should allow: (i)
714 performing quality controls in advance, during preparation of the bio-cementation process and
715 application in-situ; (ii) reducing the necessary on-site equipment to facilitate overall MICP in
716 reasonable timeframes and costs and (iii) establishing a mechanism which is easy-to-replicate
717 and to adapt to different set-ups instead of requiring major and costly redesigning.

718 Conclusions
719 We presented an overview of the crossroads between biology and engineering by highlighting
720 example applications where biological or bio-inspired materials and methods have been
721 integrated in practical solutions in various engineering fields. This illustrates the broader
722 principle which guides the motivation behind the present work. This is no other than the
723 fundamental, yet complex, idea of getting inspired from nature, to ultimately provide answers
724 to engineering problems by coupling technical innovation and economic efficiency with
725 environmental sustainability.
726 By focusing on soil bio-improvement we identify uncertainties involved in the sample
727 preparation process which reflect highly variable mechanical response. This explains the lack
728 of a unified constitutive framework dedicated to the bio-improved material, despite recent
729 attempts which have provided with validation between experimental data series and numerical
730 simulations. We conclude that without a robust description of the mechanical properties of the
731 engineered calcite bonds, without an understanding of evolving fabric parameters in relation
732 with the treatment process or the base material’s intrinsic properties, such modelling attempts
733 will inevitably use back-analysis to fit parameters or highly depend on a series of hypothesis.

734 To summarize, this review provided groundwork which can inspire the conception and
735 development of tools to design, implement and control bio-mediated soil improvement
736 applications. In light of the studied literature, we classify below three priorities to be
737 incorporated in the design of future applications based on MICP: (i) complete correlation of
738 the material foreseen to be improved with the chosen bio-treatment strategy, (ii) standardized
739 and reproducible treatment methods where strategies to introduce bio-cementation agents and
740 treat by-products are meticulously designed and monitored and finally, (iii) a design method
741 which targets and achieves the mass of calcite bonds to ultimately meet desired mechanical
742 properties.

743 Acknowledgements
744 The authors express their sincere thanks to the Swiss National Science Foundation (SNSF)
745 (grant 200021_140246) and Swiss Federal Commission for Scholarships for Foreign Students
746 (Swiss Government Excellence Scholarship ESKAS-Nr: 2014·0276) for their financial
747 support. Additionally the authors would like to acknowledge the support of the Lombardi
748 Foundation (Switzerland).

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772 Construction and Building Materials, Volume 55, pp. 462-469.

773 Cañveras, S. Sanchez-Moral, V. Sloer, C. Saiz-Jimenez, J., 2001. Microorganisms and microbially induced fabrics in cave walls.
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775 Cardoso, R., Pires, I., Duarte, S. O., & Monteiro, G. A. (2018). Effects of clay's chemical interactions on biocementation.
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781 Cheng, L., Shahin, M.A. and Mujah, D., 2016. Influence of key environmental conditions on microbially induced cementation
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817 Dupraz, S., Parmentier, M., Ménez, B. & Guyot, F., 2009. Experimental and numerical modeling of bacterially induced pH
818 increase and calcite precipitation in saline aquifers. Chemical Geology, Volume 265, pp. 44-53.

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829 Fauriel, S., 2012. Multiphysical Modelling of Soils with a Focus on Microbially Induced Calcite Precipitation.

830 Fauriel, S. & Laloui, L., 2012. A bio-chemo-hydro-mechanical model for microbially induced calcite precipitation in soils.
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832 Feng, K. and Montoya, B.M., 2014. Behavior of Bio-Mediated Soil in k0 Loading. In New Frontiers in Geotechnical
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850 Gat, D., Ronen, Z. and Tsesarsky, M., 2016. Soil bacteria population dynamics following stimulation for ureolytic microbial-
851 induced CaCO3 precipitation. Environmental Science & Technology, 50(2), pp.616-624.

852 Gomez, M.G., DeJong, J.T., Martinez, B.C., Hunt, C.E., deVlaming, L.A., Major, D.W. and Dworatzek, S.M., 2013. Bio-
853 mediated soil improvement field study to stabilize mine sands. Proceedings of GeoMontreal 2013.

854 Gomez, M.G., Martinez, B.C., DeJong, J.T., Hunt, C.E., deVlaming, L.A., Major, D.W. and Dworatzek, S.M., 2015. Field-scale
855 bio-cementation tests to improve sands. Proceedings of the Institution of Civil Engineers-Ground Improvement, 168(3),
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857 Gomez, M.G., Anderson, C.M., Graddy, C.M., DeJong, J.T., Nelson, D.C. and Ginn, T.R., 2016. Large-Scale Comparison of
858 Bioaugmentation and Biostimulation Approaches for Biocementation of Sands. Journal of Geotechnical and
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866 Géotechnique, Volume 66, pp. 546-555.

867 Hamdan, N., Kavazanjian Jr, E., Rittmann, B. E., & Karatas, I. 2017. Carbonate mineral precipitation for soil improvement
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880 Jiang, N.J. and Soga, K., 2016. The applicability of microbially induced calcite precipitation (MICP) for internal erosion
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882 Jiang, M., Zhang, F. & Sun, Y., 2014. An evaluation on the degradation evolutions in three constitutive models for bonded
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1032
 Among the treated literature, reference is made to over forty studies produced after
2016.
 As soil bio-reinforcement makes its steps towards claiming a spot in mainstream
geotechnical practice this work foresees to offer not only a look back on how far
research has gone but also an attempt to look forward by evaluating opportunities and
challenges which lie ahead.
 The authors believe that emerging technologies, which mobilize biological processes,
do push forward the limits of thinking which inevitably will lead to progress,
regardless of the individual fate of each of the mechanisms discussed above.
 6 sub-domains in the main body of this review study on soil bio-improvement
condense several intellectual turning points which have pushed forward the
understanding of involved processes as well as their reproducibility and
standardization.