Experimental Study on the Effective Production of Biocement for Soil Solidification and Wind Erosion Control

Abstract

Biocement can be achieved through the microbially induced carbonate precipitation (MICP) process. Such a method can potentially be utilized as an eco-friendly method for civil and environmental engineering applications such as soil ground improvement and wind erosion control of surface soil. In this method, one key step is the effective production of ureolytic bacteria. In previous laboratory and field studies, the cultivation and production of the bacteria used for the MICP were usually expensive and time-consuming. The purpose of this study was to optimize the cultivation method of the ureolytic bacteria (Sporosarcina pasteurii), and soil stabilization tests were conducted to verify the effectiveness of the cultured bacteria used to strengthen soil against the wind-induced erosion. Bacterial cultivation methods were studied by investigating the effects of different cultivation media and conditions. Testing variables included the types and concentrations of nitrogen sources (urea or NH₄Cl), pH values (7.5–9.5), cultivation conditions (batch or chemostat condition), and different carbon sources. It was found that, with the same amount of nitrogen source, the test with pure urea had the highest biomass yield, urease activity, and specific urease activity than the other tests with pure NH₄Cl or both NH₄Cl and urea. The use of urea as the nitrogen source in the media also led to an increase in pH, which was not found in the test with pure NH₄Cl. As for the factor of urea concentration, the tests with a higher urea concentration had a higher biomass yield, urease activity, and pH. The factor of pH values also played an important role. The test with an 8.5 initial pH value had a higher biomass yield, urease activity, and specific urease activity than the tests with 7.5 and 9.5 initial pH values. In the chemostat condition, the ureolytic bacteria could be effectively produced with urease activity up to 7 mmol/L/min, as compared with around 12 mmol/L/min activity in the batch condition. Thus, the optimum nitrogen source, pH value, and cultivation condition for the cultivation of Sporosarcina pasteurii was urea, 8.5, and batch condition, respectively. In addition, when soybean milk powder or milk powder was used as the carbon source, the urease activity was around 2.5 mmol/L/min, which is also high enough to be used for biocement.

1. Introduction

Bacteria are widespread in natural soil environments. Their activities can affect the chemical, physical, and mechanical properties of soils. The adoption of microbial methods to solve engineering problems has gained increasing attention in the last decade. One of the most attractive microbial processes is microbially induced calcite precipitation (MICP). In this process, calcium carbonate can be produced through the hydrolyzation of urea by ureolytic bacteria. This method can be used for stabilizing soil particles and improving the shear strength of soil. Such a method can potentially be utilized as an eco-friendly method for civil and environmental engineering applications. Soil grounds with weak strata may have engineering problems including excessive ground settlement, loss of stability, earthquake liquefaction, etc. Instead of using conventional techniques such as soil compaction or chemical stabilizations for ground treatments, the MICP method can be adopted as an eco-friendly solution to enhance the strength and mechanical properties of ground soils so that the ground can satisfy the criteria for buildings and structures [1,2,3]. Another application is the use of the MICP method to reduce the permeabilities of soils or the sealing of rock fractures for the remediation of leakage in reservoirs and river dikes, or the control of pollutant migration in contaminated grounds [4,5]. Soil erosion and fugitive dust pollution caused by wind is a serious environmental problem in arid regions. The MICP method can be applied to the surface of soil to stabilize soil and suppress fugitive dust [6,7,8,9,10]. The MICP method has also been tested as a sustainable solution to repair concrete structures [11]. In the MICP method based on the microbial ureolysis process, in the first step, urea is hydrolyzed into ammonium and carbonate catalyzed by ureolytic bacteria. If aqueous calcium is present in the system, CaCO₃ can precipitate in the second step. The reaction is as follows,

$${\mathrm{NH}}_2{-\mathrm{CO}-\mathrm{NH}}_2{+2\mathrm{H}}_2\mathrm{O}\stackrel{\mathrm{ureolytic} \mathrm{bacteria} \mathrm{or} \mathrm{urease}}{\to }{2\mathrm{NH}}_4{}^{+}{+\mathrm{CO}}_3{}^{2-}$$

(1)

$${\mathrm{Ca}}^{2+}{+\mathrm{CO}}_3{}^{2-}\to {\mathrm{CaCO}}_3\downarrow$$

(2)

To use the MICP method in practice, one key step is the cultivation of ureolytic bacteria. The cultivation of ureolytic bacteria can be achieved either in the laboratory or in-situ. In laboratory conditions, bacterial cultivation is 10–60% of the total cost of the method [12]. Several research studies have concentrated on the optimization of bacterial cultivation methods in order to improve the biomass yield and reduce the cost, only from the aspect of the carbon source and nitrogen source, respectively [13,14,15,16,17,18]. The bacterial cultivation methods can be optimized by varying the cultivation medium compositions or cultivation conditions. As for the nitrogen sources, several substances containing ammoniacal nitrogen can be use in the cultivation media including urea, ammonium chloride, and ammonium sulphate [19,20,21]. As for the carbon sources, yeast extract and nutrient broth are used in most research studies [19,20,21]. Some cheap materials or waste materials have also been tested as carbon sources for bacterial cultivation, including chicken manure effluent, waste from diary and brewery industries, corn steep liquor, and food-grade yeast extract [13,15,17,18]. Although batch conditions have been adopted in most of the MICP studies for the production of ureolytic bacteria, it has been found that ureolytic bacteria could also be cultivated in an open and chemostat condition using the enrichment culture technique [14].

In this study, several influencing factors such as nitrogen sources, carbon sources, pH values, and cultivation conditions were considered comprehensively to optimize the cultivation method of the ureolytic bacteria (Sporosarcina pasteurii) used in biocement for the stabilization of soil against wind-induced erosion. The types and concentrations of nitrogen sources were varied to search the optimum cultivation medium. Furthermore, pH is an important factor affecting the growth of ureolytic bacteria. A slight change in the pH value can greatly affect the growth rate and activity of ureolytic bacteria [14]. So, the influence of pH on bacterial cultivation was studied. The chemostat culture technique was also experimented for the production of Sporosarcina pasteurii. In addition to laboratory-grade reagents, some inexpensive commercial materials, including milk powder and soybean milk powder, were tested for bacterial cultivation. Last, soil surface stabilization tests were carried out to evaluate the MICP treatment effects against wind-induced erosion.

2. Materials and Methods

2.1. Bacterial Strain

Sporosarcina pasteurii (CGMCC1.3687 from China General Microbiological Culture Collection Center) was used in this study. It is the same species as DSM 33 from the DSMZ Culture Center. A freeze-dried strain was firstly cultivated aseptically and aerobically on a solid medium consisting of 20 g/L yeast extract, 10 g/L NH₄Cl, 24 mg/L NiCl₂·6H₂O, 12 mg/L MnSO₄·H₂O, 20 g/L tryptic soy agar, and NaOH for the adjustment of pH to 8.5. The bacterial strain on the solid medium was then moved to a liquid medium. The liquid medium had the same composition as the solid medium except the tryptic soy agar. The liquid culture was incubated aerobically at 35 °C and 100 rpm shaking condition for 24 h. The harvested bacteria suspension had around 1.3 OD600-based biomass concentration and 7 mmol/L/min urease activity. The harvested bacteria were stored at 4 °C before being used in the experiments.

2.2. Biomass and Activity Determinations

The biomass was determined using an optical density method. The optical density at 600 nm (OD600) was measured using a Shanghai Qinghua 721 spectrophotometer. According to Cheng and Cord-Ruwisch [14], if the values of OD600 were greater than 0.8, the bacterial suspension should be diluted to obtain a value less than 0.8, and the actual OD600 values should take the dilution factor into account.

The rate of electrical conductivity (EC) change was used to determine the urease activity based on the method given in Whiffin et al. [22]. With the catalyzation of ureolytic bacteria, urea was hydrolyzed to ammonium and carbonate, leading to an increase in EC. For the preparation of a testing sample, 3 mL bacterial suspension was added into 27 mL of the testing solution containing 1.11 mol/L urea. In a standard testing condition (1 mol/L urea and 20 °C), 1 mS/cm/min EC increased at a rate that corresponded to a 11 mmol/L/min urea hydrolyzing rate or urease activity. During the test, the relative EC change in mS/cm/min was recorded over 5 min. As the measurement of the urease activity required the addition of 27 mL of urea testing solution into 3 mL bacterial suspension liquid, the actual urease activity values should take the 10-time dilution factor into consideration.

2.3. Sample Preparation and Microbial Treatment

In order to comprehensively study the influence of multiple factors on bacterial culture, a total of 12 bacterial growth tests were conducted with different nitrogen source types, concentrations, and initial pH values. In each test sample, the cultivation medium had 150 mL volume and consisted of 20 g/L yeast extract, 24 mg/L NiCl₂·6H₂O, and 12 mg/L MnSO₄·H₂O, as well as different types and concentrations of nitrogen sources. The nitrogen source types, concentrations, and initial pH values in the test samples are given in

Table 1. Parameters of bacterial growth tests with different nitrogen source types, concentrations, and pH.

Samples	NH4Cl (g/L)	Urea (g/L)	Initial pH
AU1	10	/	7.5
AU2	/	10	7.5
AU3	10	/	8.5
AU4	6.67	3.33	8.5
AU5	3.33	6.67	8.5
AU6	/	10	8.5
AU7	10	/	9.5
AU8	/	10	9.5
U1	/	3.33	8.5
U2	/	6.67	8.5
U3	/	10	8.5
U4	/	13.33	8.5

Note: The cultivation conditions of “AU” and “U” are the batch condition and chemostat condition, respectively.

. The cultivation media were autoclaved under 121 °C for 30 min before inoculation. After the autoclave process, the cultivation media were negative. Urea could decompose at a high temperature. So, urea solutions were sterilized using 0.22 micrometer syringe filters before adding into the autoclaved cultivation media. The cultivation media were inoculated with 1% volume of the bacteria suspension, which was obtained as described in Section 2.1. The liquid cultures of the cultivation tests were incubated aerobically at 35 °C and 100 rpm shaking condition for 24 h. During cultivation, the urase activities, OD600 values, and pH values were measured every 3 h.

2.4. Chemostat Cultivation

The chemostat cultivation tests were carried out to explore whether Sporosarcina pasteurii could be produced in a continuous condition. A chemostat is a bioreactor to which fresh medium is continuously added, at the same time, culture liquid containing left over nutrients, metabolic end products, and microorganisms is continuously removed at the same rate to keep the culture volume constant. The chemostat cultivation is a cultivation method in which microorganisms can be continuously harvested. The cultivation medium was the same as Test AU6 in Table 1, which was found to be the optimum cultivation medium based on the results of the batch cultivation tests. A schematic of the chemostat bioreactor is shown in Figure 1. The bioreactor had 500 mL operation volume. The temperature and the pH in the reactor could be adjusted using a feedback control system. Cultivation medium and harvested bacteria were pumped into or out of the bioreactor at constant rates using peristaltic pumps. Clean sterilized air was injected into the bioreactor to ensure the aerobic condition. After inoculation, the bacteria were first cultivated in the batch condition for 24 h. After 24 h, fresh cultivation medium was continuously provided at a rate of 0.65 mL/min, and the bacteria were harvested at the same rate. The hydraulic retention time was around 13 h. Temperature was controlled at 35 °C. During the tests, the urase activities, OD600 values, and pH values were measured. Tubes and sensors installed in the bioreactor were sterilized to ensure the aseptic condition. Nutrients added were autoclaved, and air pumped into the bioreactor was filtered.

2.5. Alternative Carbon Sources

A list of the carbon sources used in the test samples is given in

Table 2. Parameters of bacterial growth tests with different carbon sources.

Substrates (g/L)	Urea (g/L)	Initial pH	Protein Content (g/100 g)
Yeast extract (20)	10	8.5	68.1
Industrial-grade peptone (30)	10	8.5	59.1
Soybean milk powder (60)	10	8.5	16.8
Milk powder (60)	10	8.5	18.1

, including yeast extract, industrial-grade peptone, soybean milk powder, and milk powder. The other components in the media were the same as Test AU6, as described in Section 2.3.

2.6. Soil Stabilization Test

One of the promising applications of the MICP method is the stabilization of soil surfaces for erosion control and the fugitive dust suppression [6,7]. In this study, sandy soils received the treatment with various amounts of the bacteria. The treatment liquid was prepared by mixing the bacterial suspensions and the urea–CaCl₂ solution together at a 1:1 volume ratio. The OD600 biomass concentrations ranged from 0.3–1.8, and the urease activities ranged from 0.9 to 14.7 mmol/L/min in the different test samples. The urea–CaCl₂ solution contained 0.2 mol/L equimolar urea and CaCl₂. The sandy soil used in the tests was Fujian Sand, which is a Chinese standard sand with an angular particle shape and more than 96% quartz content. The mean size of the soil particles was 0.5 mm. The specific gravity, uniformity coefficient, coefficient of curvature, maximum void ratio, and minimum void ratio of the soil were 2.65, 4.22, 0.53, 0.704, and 0.368, respectively. Each soil sample had 3 kg sandy soil and was prepared in steel trays with 18 × 26 cm² surface areas, as shown in Figure 2a. The soil was compacted to achieve a dry density of 1.28 g/cm³. The treatment liquid was sprayed onto the surfaces of the soils with 4 L per square-meter surface area. The soil samples were cured at 20 °C for 3 days. The treatment effects were evaluated using a wind tunnel, as shown in Figure 2b. The wind speed in the wind erosion tests was 14 m/s (Grade 7 wind according to Beaufort wind scale) and the wind lasted 10 min in each test. The surface strengths of the soil samples were also measured using a flat-bottom penetrometer with a 6 mm diameter.

3. Results and Discussion

3.1. Effect of Nitrogen Sources

In previous studies, urea or ammonia salts were used as the nitrogen sources for the cultivation of Sporosarcina pasteurii and other ureolytic species. In the experiments, urea, NH₄Cl, and the combination of both were tested. The results are presented in Figure 3. There were four tests with a total of 10 g urea and/or NH₄Cl, as can be seen in Table 1. As shown in the figure, Test AU6 with purely 10 g urea and no NH₄Cl had the highest OD600 and urease activity. The OD600 increased to 2.6 at 21 h, which was about 50% higher than the other three tests. The urease activity in Test AU6 increased to the peak value of 13.5 mmol/L/min at 6 h, followed by a gradual decrease afterwards. In the other three tests, the urease activities gradually increased in the tests. The specific urease activity in Test AU6 also increased to a peak value of 9.8 mmol/L/min/OD at 6 h and decreased after 6 h. In the other three tests, the specific urease activities increased within the first 3 h and remained roughly constant afterwards until the end of the tests. The pH values in the three tests with urea additions (AU4, AU5, and AU6) increased in the first 3 h, remained almost constant from 3 h to 12 h, and gradually decreased after 12 h. The pH value in Test AU3 (pure NH₄Cl) had a gradual decreasing trend in the tested time range.

The effect of the urea concentrations on the bacterial growth were investigated. The results are presented in Figure 4. The test samples had a range of urea concentrations from 3.3 to 13.3 g/L. With the increase in urea concentration, both OD600 and urease activity increased greatly in the tested condition. Test U3 with 10 g/L urea had the highest specific urease activity, which wa 12.5 mmol/L/min/OD at 9 h.

3.2. Effects of the Initial pH Value

The effects of pH values on bacterial growth are investigated here. There were two series of tests with NH₄Cl (Figure 5) and urea (Figure 6) as the nitrogen sources, respectively. In each series of tests, there were three test samples with an initial pH value of 7.5, 8.5, and 9.5, respectively. In both series of tests, the most favorable pH value wsa 8.5 in terms of the biomass yields and the urease activities. In the series of tests with urea, Test AU6 showed a maximum urease activity of 13.5 mmol/L/min at 6 h, which was much higher than Tests AU2 and AU8 with urea as the nitrogen sources and Tests AU1, AU3, and AU7 with NH₄Cl as the nitrogen sources. Among the three tests with NH₄Cl as the nitrogen sources, the urease activity, and the growth rate and biomass yield of Test AU3 (pH = 8.5) were higher than the other two tests. As for the pH variations, Tests AU2 (pH = 7.5) and AU6 (pH = 8.5) with urea as the nitrogen source showed a fast increase in pH to around 9.2 in the first 3 h, and a gradual decrease afterwards. In Test AU8 (pH = 9.5) with urea and the other three tests with NH₄Cl, the pH values showed a gradual decrease throughout the tests.

3.3. Chemostat Cultivation

Chemostat cultivation tests were carried out to investigate the performance of bacterial cultivation using the chemostat bioreactor. From the results of the batch cultivation tests presented in Section 3.1 and 3.2, it can be seen that the optimum medium composition for the cultivation of Sporosarcina pasteurii was 20 g/L yeast extract, 10 g/L urea, 24 mg/L NiCl₂·6H₂O, 12 mg/L MnSO₄·H₂O, and pH = 8.5. This optimum cultivation medium was also used in the chemostat cultivation test. The results are presented in Figure 7. The bacteria were cultivated in a batch condition in the first 24 h and the cultivation condition was switched to the chemostat condition with 13 h hydraulic retention time. From 24 h to 90 h, the pH in the bioreactor was controlled at 8.5. During this phase, both the OD600 and urease activity remained roughly constant. OD600 was around 1.3, and the urease activity was around 5 mmol/L/min. From 90 h, the pH was no longer controlled. The pH value drifted to around 9.3 at 102 h and remained roughly around this value until the end of the test. During this phase, OD600 showed a fast increase to around 2.0 and decreased back to around 1.3.

3.4. Alternative Carbon Sources

The use of alternative carbon sources including waste and mass production materials can greatly reduce the cost of bacterial cultivation. The urease activities using different carbon sources are presented in Figure 8. The urease activity using industrial-grade peptone was 5.6 mmol/L/min. This level of activity was high enough to be used for MICP applications. It can also be seen that the urease activities at 12 h using soybean milk powder or milk powder were around 2.5 mmol/L/min. This value was much lower than those using yeast extract and industrial-grade peptone. However, the 2.5 mmol/L/min activity was enough for soil treatment, as compared with previous studies [5,22,23,24]. These alternative carbon sources could also potentially be used in the chemostat cultivation condition to simplify the cultivation procedure.

3.5. Soil Stabilization Tests

The results of the soil stabilization and wind erosion tests are presented in Figure 9 and Figure 10. It can be seen that the treatment effect improved with the increase in bacterial activity. The soil mass losses against the urease activities are presented in Figure 9. The percentage soil mass loss was 13% when the bacterial activity was 0.9 mmol/L/min. At this condition, and soil sample was completely destroyed by wind forces. When the bacterial activity increased to around 2.0 or higher, the soil samples were intact and the percentage of soil losses was minimal. The surface strength and CaCO₃ content also increased with the bacterial activity, as shown in Figure 10. Such results also indicate that a bacterial suspension with 2 mmol/L/min activity or higher was sufficient for the soil surface stabilization and wind erosion control. It can also be seen that the bacteria cultivated using the alternative carbon sources (soybean milk powder and milk powder as shown in Figure 8) had enough activities to be used for the soil treatment.

4. Discussions

4.1. Urea or Ammonium Salt as Nitrogen Source

It can be clearly seen in the experimental results that bacterial cultivation using urea had a much higher biomass yield and urease activity than that using NH₄Cl. The growth of bacteria in the urea medium was associated with the increase in pH, which did not occur in the purely NH₄Cl medium. The increase in pH is thought to be due to ionic equilibrium in the urea hydrolysis process, which produces more OH^- than H⁺ [25]. The consistency of the higher activity and increase in pH could be because of the increase in protonmotive force (PMF) [26,27,28]. PMF is an electrochemical or chemiosmotic gradient of protons that is formed by active proton pumping and it is modulated by secondary ion movements. In bacterial cells, it is PMF that drives the entry of protons and adenosine-triphosphate (ATP) synthesis via membrane-bound adenosine triphosphatase (ATPase). The PMF comprises the chemical gradient of protons (ΔpH) and the transmembrane electrical potential (ΔΨ). ΔpH (alkaline inside) contributes to PMF. Thus, with urea in the cultivation medium, the hydrolysis of urea and the simultaneous pH increase contributes to PMF and in turn ATP synthesis. In addition, the produced intracellular ammonium ion, unlike freely permeable ammonia, can diffuse only slowly through the membrane, resulting in an ion gradient and transmembrane electrical potential (ΔΨ) that drives ATP synthesis [14,29]. However, with NH₄Cl in the medium, the contribution of urea hydrolysis to the PMF and ATP synthesis is absent, which may cause a lower urease activity compared with bacterial growth with urea.

4.2. Effect of pH

In the batch cultivation condition, it was found that the optimum initial pH was around 8.5 (Figure 6). Such a result is consistent with previous studies, which have shown that Sporosarcina pasteurii has relatively high urease activities in the 8.5–9 pH range [30,31]. By comparing the performance of urea and ammonium chloride nitrogen sources at different initial pH values, it was also found that when urea was the nitrogen source, the urease activity of ureolytic bacteria would reach its peak value after 6 to 12 h of culture under any initial pH conditions, and the storage was relatively stable. When ammonium chloride was the nitrogen source, the urease activity of ureolytic bacteria increased linearly with time at all initial pH conditions. In addition, the urease activity of the urea hydrolyzing bacteria cultured at an optimal pH for 21 h with ammonium chloride as the nitrogen source was lower than that of the ureolytic bacteria cultured at a non-optimal pH for 6 h with urea as the nitrogen source, indicating that the pH adaptation range of ureolytic bacteria was wider when urea was selected as the nitrogen source than ammonium chloride as the nitrogen source. In addition, selecting urea as the nitrogen source for the culture of ureolytic bacteria could greatly shorten the culture time and improve the activity of the target bacteria.

The main reason for the above results is that the pH of the microbial growth environment, as one of the important factors affecting microbial life activities, does not only affect the biological activity of microorganisms by changing the charge of macromolecular substances in microorganisms, but also reduces the absorption and utilization of nutrients by microorganisms by changing the charge of microbial cell membrane [32]. During the culture of ureolytic bacteria, the pH in the culture medium was measured every 3 h, as shown in Figure 5d and Figure 6d. It was observed that when ureolytic bacteria were cultured under the condition that the nitrogen source was ammonia chloride, the pH of the culture medium gradually decreased during the growth process due to the acidic substances produced by bacterial metabolism. When urea was selected as the nitrogen source, the hydrolysis of urea balanced the acidic substances produced by bacterial metabolism, resulting in a rise in pH of the culture medium. At the same time, with the continuous consumption of urea in the culture medium and the increasing amount of acidic substances produced by bacterial metabolism, the pH of AU2, AU6, and AU8 samples was maintained between 9.0–9.5. Because the hydrolysis of urea can balance the acidic substances produced by bacterial metabolism and slow down the decrease in the pH of the culture medium, the pH adaptation range of ureolytic bacteria was wider when urea was selected as the nitrogen source than when ammonium chloride was selected as the nitrogen source.

4.3. Chemostat Cultivation

In the chemostat condition, after removing the pH control = 8.5 in the third step, the pH drifted to around 9.3, accompanying a considerable increase in the urease activity, and the activity of urease also increased by about 40% compared with the first step. The pH value remained around 9.3 in the third step, which was different from the change in pH in Figure 8d. This is because the pumping system of the chemostat bioreactor constantly draws out the bacterial liquid in the chemostatic bioreactor and injects fresh culture liquid, providing a continuous flow of urea. In the first step of the batch cultivation condition, the environmental pH abruptly increased to 9.5 during the sample preparation procedures. Such a pH shock may impair or slow down PMF and ATP synthesis [28]. However, in the chemostat condition, the urea hydrolysis led to a gradual pH increase, creating a pH gradient that may contribute to ATP synthesis [27]. The urease activity increased to around 7 mmol/L/min in the third stage of the chemostatic condition. In comparison, the activity was around 12 mmol/L/min with the same cultivation medium in the batch condition (shown in Figure 3).

4.4. Large-Scale Cultivation Methods

The MICP method based on the microbial ureolysis process could potentially be used for construction applications. So, the production of large amounts of the bacteria are required. For the cultivation of ureolytic bacteria, both batch and continuous (chemostat) cultivation methods can be used. The chemostat cultivation method is suitable for factory productions because of the automatic continuous feeding of the nutrients and the continuous harvesting of the bacteria. It has been found in this study, as well as by Cheng and Cord-Ruwisch, that chemostat cultivation can produce ureolytic bacteria reliably [11]. At the construction site, the batch cultivation method was easier to carry out. In a field trial of desert soil solidification carried out by the authors’ team, we used a two-step cultivation strategy. That is, small amounts of bacteria were cultivated in a sterile condition in the laboratory, and these small amounts of bacteria were used as inoculums for large-scale non-sterile batch cultivation at the construction site. The bacteria obtained in this way had ureolytic activities ranging 4–7 mmol/L/min, which is more than enough for the soil treatment. However, the weakness of this study is that it does not compare the strengthening effects of bacteria cultured by the different cultivation methods.

5. Conclusions

In this study, efforts were made to obtain an effective cultivation method for ureolytic bacteria for MICP applications. Bacterial cultivation methods were studied by investigating the effects of different cultivation media and conditions. Testing variables included the types and concentrations of nitrogen sources (urea or NH₄Cl), pH values (7.5–9.5), cultivation conditions (batch or chemostat condition), and different carbon sources. The following conclusions could be made,

• In the tests with different nitrogen source types, the cultivation medium with pure urea had the highest biomass yield, urease activity, and specific urease activity compared with the other tests with pure NH₄Cl or both NH₄Cl and urea. The use of urea as the nitrogen source in the media also led to an increase in pH, which was not found in the test with pure NH₄Cl;
• In the tests with different urea concentrations, the tests with a higher urea concentration had a higher biomass yield, urease activity, and pH value;
• In the tests with different pH values. the test with 8.5 initial pH value had a higher biomass yield, urease activity, and specific urease activity than the tests with 7.5 and 9.5 initial pH values;
• In the chemostat condition with continuous feeding into and harvesting from the bioreactor, the ureolytic bacteria could be steadily produced with OD600 of around 1.3 and urease activity of around 7 mmol/L/min;
• When using soybean milk powder or milk powder as the carbon sources, the urease activity was around 2.5 mmol/L/min. Although it was lower than that using the laboratory-grade or industrial-grade carbon sources, this level of urease activity was high enough to be used for MICP soil cementation, as demonstrated by the soil stabilization test.