Investigating the Factors Affecting the Properties of Coral Sand Treated with Microbially Induced Calcite Precipitation

Microbial-induced carbonate precipitation (MICP) can be used to cement coral sand to improve its engineering properties to protect coastal structures. In this study, a series of laboratory tests were conducted to test the effect of the MICP method by using an ureolytic bacterium (Sporosarcina pasteurii). In order to determine the activity of bacteria, the growth properties of the microbial strain were observed under different culture conditions (different pH and temperature). )e effect of partial size distribution and nutrient concentration on the soil permeability and unconfined compressive strength was then examined in coral sand.)e results showed that the pH had less effect on the bacteria growth compared to temperature.)e bacteria can growth well at pH over 8 and temperature higher than 20°C. )e well-degraded soil has higher unconfined compressive strength (1.91–2.61 MPa) than poor-degraded soil (1.31MPa). )e similar trend was also found in permeability reduction. )e unconfined compressive strength increased as the biocement solution concentration increased to 1mol/L and then decreased at 1.5mol/L.


Introduction
Coral sand is widely distributed on coral reefs and seashores in South China Sea.In coastal engineering, there are many buildings and breakwaters or other structures constructed on coral sand, or using coral sand as backfill materials for road embankment or airport runways.However, the coral sands consist of shells and corals with high void ratio.e strength is rather low compared to silica sand and can be easily crushed under load.So it is necessary to improve its strength before it is used as foundation or backfill materials.ere are many traditional technologies such as pile driving, grouting, and vibrocompaction, which have been successfully applied to other soils.However, these methods are ineffective when applied to calcareous sand due to its high carbonate contents and low strength.For the grouting method, it may also cause pollution to the marine environment.Other methods may cause the breakdown of coral sand [1,2].
In recent years, a novel ground improving method was proposed to minimize the environmental problems by microbial-induced carbonate precipitation (MICP [3,4]).
e MICP technique involves two biochemical reactions: (1) the hydrolysis of urea by the urease enzyme produced by bacteria and (2) the precipitation of calcite with the presence of Ca 2+ .With the production of calcite, the soil properties were changed.
It has been well studied by the researchers to optimize the treatment in the laboratory or field experiments on silicon sand [7].Harkes et al. reported that the unconfined compression strength (UCS) varied from 0.2 to 20 MPa according to the amount of calcite precipitation [15].Others also found similar results that the UCS ranges from 1 to 12 MPa for silicon sand [16].Meanwhile, the MICP effect quite relied on the experimental conditions, such as the pH, temperatures, and chemical species concentrations [6,8,17].
e change of the conditions can influence both the activity of the bacteria and the chemical reaction rate and then affect the precipitation of calcite.It is difficult to develop an injection approach that can generate homogeneous distributed calcite in the soil.Previous studies have reported nonhomogeneous distribution of calcite in soil especially in long distance injection.In order to achieve a homogeneous calcite fill in the soil, they have tried to slow down the injection flow or inject with some fixation solution with bacteria.But these works did not completely solve the problem, more detailed researches are still required to get a better strategy.
Although soil improvement using MICP has shown a great promise in silicon sand, a few studies have been reported to apply the MICP effect on coral sand [13,18,19].Based on the previous studies of the silicon sand, this study aimed at determining the efficiency of MICP treatment to improve the properties of coral sand.First, the growth characteristics of the bacteria were measured under various culture conditions.en, a series of tests were conducted to explore the effect of particle size distribution and cementation solutions on the efficacy of MICP.Soil permeability and UCS tests were conducted on the biocemented soil columns.

Bacteria Cultivation.
e test bacteria Sporosarcina pasteurii (ATCC 11859) was used in this study.e bacterial strain was cultured in the liquid medium under various conditions (pH and temperature) and found the optimal growth conditions.e cell density was quantified by measuring the absorbance of the suspension using a spectrophotometer at 600 nm wavelength (OD 600 ).e bacteria were first grown on the plate media and incubated at 30 °C.
e cultivation solution ingredients are listed in Table 1.After the plate growth, the bacteria were harvested and inoculated in the liquid media to grow for 24 h at 180 rpm with an aeration of 1 : 2.5 (200 mL of the media in a 500 mL flask) to an optical density of 600 nm (OD 600 ) of 1-1.3. is OD 600 value can ensure the bacteria had high urease activity during the experiment.
e following two factors were analyzed to investigate the growth condition of the bacterial strain: (1) the pH of the liquid culture media ranged from 8 to 11 and (2) the culturing temperature varied from 5 to 35 °C.

Soil Column Preparation.
e sands used in this study were collected from Nansha Island.e sands were crushed down and sieved through 5 mm sieve to be used in the experiments.
ree different particle size distributions (PSDs) were prepared by mixing different size sands.In Figure 1, soil #1 and #2 are classified as well graded, and #3 is poorly graded.e mean particle size of soil #1 is fine, and #3 is coarse.e summary of the properties (porosity e, dry density ρ d , coefficient of uniformity C u , and coefficient of curvature C c ) is presented in Table 2. e relative density of soil was around 2.7-2.85g/cm 3 .
e soil columns were prepared in a PVC column with 50 mm in diameter and 120 mm in height.A mesh and filter paper were placed at the bottom of the column to minimize the loss of soil particles during the test.

MICP Treatment.
A peristaltic pump was used to inject the bacteria and cementation solutions to the soil column from the bottom to the top and then let it drain from the top to the bottom.e flow rate was set to 1 mL/min.Each sample was flushed by 1.5 pore volumes of bacteria-0.5 M CaCl 2 solution first.e bacteria-CaCl 2 solution was kept within the sample for 6 h to allow the bacteria to attach to the soil surface.en, 1.5 pore volume cementation solutions (urea and CaCl 2 ) were pumped to the soil sample and kept for 12 h.e bacteria and cementation solution were injected four times as described before.
To determine the effect of various conditions on the UCS of the specimen, the following conditions were considered: the concentrations of urea and CaCl 2 used were 0.5 mol/L, 1.0 mol/L, and 1.5 mol/L.Each sample was prepared in triplicates.

Bacteria Growth Conditions Tests.
e growth curve of the microbial strain in different culture conditions (pH and temperature) was obtained to investigate the effects of various conditions on bacterial growth.

e Effect of pH.
e cell densities were reflected by the value of OD 600 .OD 600 obtained at different pH values from 8 to 11 is shown in Figure 2. e results showed that the OD 600 can reach to above 1 after about 8 h.e data presented showed that there is only a slight difference between pH over 9 especially at the stable stage of the bacteria growth curve.It means that these bacteria prefer alkaline environment and can be used in the environmental pH at 9-11.For the biochemical reaction, the calcium carbonate precipitated when pH is above 8.3 and increased up to 9, and the pH tends to lower back to neutral afterwards [10,20].As the coral sand mainly exists in seawater, the pH is always larger than 7.So it is possible to apply these bacteria to coral sand from the aspect of the pH value.e bacteria densities increased with the increase in temperature.As shown in Figure 3, at low temperatures (5-10 °C), the nal OD 600 was less than 1.0, which was rather low to apply in soil.e best temperature for the bacteria growth is 30-35 °C.It has also been reported by Whi n [21] and van Paassen [16] that the urease activity increased with temperature up to 60 °C.
From the aspect of temperature, the bacteria are not applicable to deep seabed but can be used in the o shore area.Or indigenous bacteria which can produce urease enzyme may be used to precipitate calcite in deep seabed.
e temperature had a more signi cant in uence on the bacteria growth ability compared to the pH value (comparing Figures 2 and 3).So it is more important to control the temperature when applying the MICP technique in engineering applications.us, the bacteria used in the following soil column experiments were grown at 30 °C with pH at 9.

Permeability.
e soil permeability was measured during the MICP process after each injection of cementation solution.

e E ect of Sand Particle Distribution.
ree di erent SPDs of coral sand were used in the experiments.e concentration of the cementation solution in these tests was 1 mol/L.Figure 4 shows the cemented samples after the MICP process of three soil types.
e loose sand was cemented together by the MICP.However, the cementation e ect was not that good as there are still some pores at the surface, and the bonding between particles was weak.ere were more calcite precipitated at the top of the column and less at the middle and bottom.
e permeability of three soils during the MICP process is shown in Figure 5. ere was a signi cant reduction in soil #2 and a slight reduction in soil #1.For soil #3, the permeability almost remained the same before the MICP process.at was because soil #3 had a high initial porosity and larger particle size.e bacteria were di cult to attach  Advances in Civil Engineering to the soil particles and can be washed o by the injection ow even at very low ow rate.Meanwhile, even there were calcite precipitated between the soil particles, the pore space was not completely occupied to block the ow of water.For low porosity and ner sand particles, the soil pore space can easily be blocked by the calcite and then a ect the biocement e ect.To reach a better cementation e ect, the initial pore space cannot be too large or too small.Previous studies had reported that the optimal grain size for the biocementation process is between 50 and 400 μm for sand [22].e MICP cannot take place in very ne sand, and larger amount of nutrients were required in coarser sand.However, there was less report about the e ect of PSD.

e E ect of Solution Concentrations.
Figure 6 shows that the cemented samples after the MICP process at three di erent solution concentrations of 0.5, 1, and 1.5 mol/L.e soil used in these experiments was soil #2. e biocement e ect of 1 mol/L and 1.5 mol/L was better than 0.5 mol/L as illustrated in Figure 6.
Figure 7 shows the permeability of soil #2 cemented at three di erent solution concentrations.
e permeability was reduced after the biocement process.When the solution concentrations were low at 0.5 mol/L, the calcite amount in soil was low.e permeability only changed slightly.For 1.0 and 1.5 mol/L, the permeability change almost had the same trend.e higher the solution concentration injected, the better the MICP e ect.

e E ect of Sand Particle Distribution.
e soil was taken out from the column and dried for 7 days after the permeability test for the uncon ned compressive strength test.Table 3 shows the UCS strength of three soils during the test.Uncon ned compressive strengths were not obtained for every sample due to the poor cementation e ect of some samples.Soil 2# was cemented the best compared to 1# and 3#.ere were visible voids or de ections in soil 3# as the pores were larger.e UCS value was larger in soil 2# to about 2.61 MPa and 1.31 MPa for soil 3#.During the experiments, the same amounts of nutrients were injected to the column, but soil #1 and #3 had larger pore volumes than soil #2.

e E ect of Cementation Solution Concentration.
To investigate the e ect of cementation solution concentration on MICP-treated coral sand, the sand column was prepared under three di erent cementation solution concentrations using soil #2. e soil was selected because of its best MICP e ciency as described before.Table 4 shows the UCS strength of soil biocemented with three solution concentrations.
e biocement e ect of 1 mol/L and  Advances in Civil Engineering 1.5 mol/L was better than 0.5 mol/L.Although the amount of calcite increased as the concentrations increased, the distribution pattern at the pore scale was also a ected by the concentration.At higher concentration (1.5 mol/L), there were more calcite deposited at the inlet, while at lower concentration (mol/L), calcite distribution was more homogeneous.

Conclusions
MICP is a complex biochemical process which has been used to improve coral sand properties.Identi cation of di erent factors enables the control of MICP in geotechnical engineering.Understanding how di erent treatments and sand properties could a ect MICP is very important.is study describes the in uence of pH and temperature on bacteria growth, soil particle size distribution, and solution concentrations on soil permeability and strength.Based on the experimental data, the following conclusions were drawn: (1) e pH values within 8-11 had little e ect on the growth of bacteria.(2) e temperature had greater e ect on the bacteria activity: at low temperature, the bacteria did not have high enough density for the MICP application.e best temperature is over 30-35 °C.(3) e permeability of biocemented coral sand was reduced after the MICP process.However, the wellgraded sand and medium porosity sand has a larger reduction in the permeability.(4) e results of UCS showed that the SPD and solution concentrations have an obvious e ect on the MICPtreated coral sand.For well-cemented coral sand, the UCS can reach up to 2.6 MPa.
e MICP process was very complex.e results reported in this paper will be employed to further investigate the use of MICP on improving the soil strength.

Figure 2 :Figure 3 :Figure 1 :
Figure 2: e OD 600 value of the bacteria solution at di erent pH values.

Table 1 :
Summary of the microbial-induced carbonate precipitation recipe.

Table 2 :
Initial porosity and parameters for soils.

Table 3 :
Soil UCS after the biocement process for 3 soil types.

Table 4 :
Soil UCS after the biocement process for 3 soil types.