Dissertations / Theses on the topic 'Microbially Induced Calcite Precipitation'

The Australian landscape has a large number of naturally cemented structures, which provide inspiration for a sustainable cementing material which does not produce carbon dioxide during the manufacturing phase. Structures such as corals, beach rocks and stromatolites are cemented through the process of Microbially Induced Calcium Carbonate Precipitation, (MICP). This thesis reports on the potential for MICP as a replacement or augmentation to chemical binders in geomaterials and evaluates the sustainability of MICP using Life Cycle Analysis.

Microbial Induced Calcite Precipitation (MICP) is an attractive alternative for a variety geotechnical ground improvement practices commonly used today and has a variety of potential applications. This research focuses primarily on its use as a soil stabilization technique using the bacteria Sporosarcina Pasteurii and a single injection point percolation method adapted from previous research in granular soils. This method, and most published data, show an inherent variability in both physical and engineering properties due to the distribution of precipitated calcite within the specimen. The focus of this research is on the quantification of the variability in shear strength parameters induced by MICP treatment in sand. Also, on the initial development of a new treatment method which aims to reduce this inherent variability and offer a more feasible option for field applications. The MICP treated soil columns were sampled at constant intervals from the injection point and then subject to direct shear testing (DST) and calcite distribution analysis. This analysis reiterates previously documented reduction in cementation as distance from injection point increases. The reduction in cementation results in reduced shear strength parameter improvements. This research also concluded a minimum of two percent mass of calcite per total mass of treated soil for significant strength improvements.

Microbially induced calcite precipitation (MICP) has been used for a number of years as a technique for the improvement of various geological materials. MICP has been used in a limited capacity in organic rich soils with varying degrees of success. Investigators hypothesized that microbially-induced cementation could be improved in organic soils by using a surfactant. Varying amounts of Sodium Dodecyl Sulfate (SDS) were added to soils of varying organic content and a mixing procedure was used to treat these soils via MICP. Treated specimens were tested for unconfined compressive strength (UCS). Results appeared to show direct relationships between SDS content and treated specimen strength although significant variability was present in the data. In addition, results also indicated that while addition of SDS during MICP treatment strengthens soil, the strengthening is likely from the formation of a calcium dodecyl sulfate (CDS) complex in which the CDS surrounds the soil in a matrix, and formation of MICP-induced calcite has very little to do with overall soil performance. As such, a new method for stabilizing loose soils dubbed ‘Surfactant-induced soil stabilization’ (SISS) was further explored by treating additional soil specimens. Samples treated using this technique showed increases in strength when compared to untreated specimens. In addition, preliminary data indicated that SISS treated specimens were insoluble. The SISS technique presents a number of advantages when compared to traditional soil stabilization techniques. In particular it should be relatively low-cost and simple to administer since its only components are SDS and calcium chloride. Additionally, these constituents are relatively more sustainable than chemicals associated with more-traditional loose soil stabilization techniques.

The study of Microbial-induced carbonate precipitation (MICP) on organic soil has remained limited. This PhD study intends to fill the knowledge gap by studying MICP of tropical peat. Based on the finding, it was possible (i) to isolate bacteria strains from acidic tropical peat with high urea hydrolysis activities and capable of bio-cementation; (ii) to induce bio-cementation in acidic peat, which leads to strength gain and reduction of permeability; (iii) to improve consolidation behaviour.

The possibility of using microbiological processes to improve the mechanical properties of soil by undisturbed in-situ application has gained attention over recent years. This study has contributed to the technology of biocement, based on microbially induced carbonate precipitation (MICP), for the purpose of soil reinforcement application. MICP involves both the hydrolysis of urea by bacterial urease enzyme and calcium carbonate precipitation in the presence of dissolved calcium ions. Other previously published approaches were based on saturated flow (submersed flow), which is accomplished by pumping solutions from an injection point to a recovery point which is limited exclusively to water saturated soil. This work describes a new variation of in-situ soil reinforcement technology by using surface percolation via – for example – spray irrigation onto dry, free draining ground, such as dunes or dykes. In order to accomplish bacterial immobilization by surface percolation, it was necessary to alternately percolate bacterial suspension and cementation solution (CaCl2 and urea) to form sequential solution layers within the sand columns. By allowing Ca2+ ions diffusion between each layer bacterial immobilization could be enhanced from 30% to 80%. For a limited number of about 3 to 4 treatments this novel application method of cementation allowed homogeneous strength over the depth of the entire 1 m sand column. Although the strength was homogenous, CaCO3 analysis showed that about 3 times less crystals were precipitated in the top layer compared to the bottom layers suggesting differences in efficiency of the calcite crystal to provide strength. This work demonstrated that this efficiency of calcite crystals was related to the pore water content of the continuously drained column with less water content enabling more efficient strength formation. The geotechnical properties of bio-cemented sand samples under different degrees of saturation confirmed that higher strength could be obtained at lower degrees of saturation. To our knowledge, this study was the first study to demonstrate that the calcite crystals formed under a lower degree of saturation had more crystals formed in the contact points, contributing to the strength of the cemented samples. These preferred crystal formation was caused by the retained cementation solution situated in the form of menisci between sand particles at low degree of saturation. Scanning electron microscopy supported the idea that lower water contents lead to selective positioning of crystals at the bridging points between sand grains. After biocementation treatment, fine sand samples exhibited significant increase in cohesion from 1.1 to 280 kPa and friction angle from 23o to 41o. Similar improvements were also obtained for coarse sand samples. Overall, fine sand sample indicated higher cohesion but lower friction angle than coarse sand samples having similar CaCO3 content. The performance of cementation in large (2 m) laboratory scale trials indicated that subsequent treatments of more 4 times in fine sand caused clogging close to the injection end, resulting in limited cementation depth less than 1 m. This clogging problem was not observed in the 2 m treated coarse sand column, which had strength varying between 850 to 2067 kPa. This showed that the surface percolation technology was more applicable for coarse sand soil. The laboratory large scale application (80 L) of fine sand cementation indicated that relatively homogenous cementation in the horizontal direction could be achieved with 80% of cemented sand having strength between 2 to 2.5 MPa. This suggested that although the liquid infiltration flow paths could not be controlled in the surface percolation method, self-adjusting flow paths were triggered by the changed internal flow resistance caused by the precipitated crystals, favoring the homogeneous cementation. A simple mathematical model demonstrated that the cementation depth is dependent on the infiltration rate of cementation solution and the immobilized urease activity. Higher infiltration rate and lower urease activity will enable in deeper cementation. The model also predicted that repeated treatments will enhance sand clogging close to the injection point. The traditional production of ureolytic bacteria used for biocementation is very expensive, because of strictly sterile processing. This study described the sustainable, non-sterile production of urease enzyme using activated sludge as inoculum. By using selective conditions (high pH and high ammonia concentration) for the target ureolytic bacteria plus the presence of urea as the enzyme substrate, highly active ureolytic bacteria, physiologically resembling Bacillus pasteurii were enriched and continuously produced from chemostat operation of the bioreactor. When using a pH of 10, and about 0.17 M urea in a yeast extract based medium ureolytic bacteria developed under aerobic chemostat operation at hydraulic retention times of about 10 h with urease levels of about 60 U/ml culture. This activity is six times higher than required for successful biocementation. The protein rich yeast extract medium could be replaced by commercial milk powder or by lysed activated sludge, which could make the industrial production less costly. A method of in-situ production of urease activity was developed. This method involved providing selective growth medium to allow ureolytic bacteria to proliferate and produce urease activity in-situ of sand column. The aerobic ureolytic bacteria inoculum could only be enriched in unsaturated coarse sand column, where sufficient oxygen was available. However, high urease activities of 20 and 10 U/mL were obtained by growing soil bacteria under aerobic and anaerobic conditions respectively. The successful enrichment of highly urease active bacteria under anaerobic conditions could allow the in-situ production of urease activity at water logged soils. The in-situ produced urease activities by the enriched soil ureolytic bacteria were sufficient to allow successful cementation of fine (>500 kPa) and coarse (>1000 kPa) sand columns. The strength and CaCO3 analysis indicated that the common obstacle of surface clogging in deeper fine sand column was avoided, explained by avoiding bacterial accumulation at the top of the column. In combination, all findings of the present study imply that the cost of MICP technology can be reduced by optimizing the conditions for effective crystals precipitation by providing low saturation conditions when the cementation is operated. The cost reduction can also be achieved by producing urease activity more economically by omitting the requirement of sterilization (non-sterile cultivation) and bioreactor (in-situ growth). These are expected to make this technology more readily acceptable for field applications.

Microbial-Induced Calcium Carbonate (CaCO3) Precipitation (MICP) is a biological process in which microbial activities alter the surrounding aqueous environment and induce CaCO3 precipitation. Because the formed CaCO3 crystals can bond soil particles and improve the mechanical properties of soils such as strength, MICP has been explored for potential engineering applications such as soil stabilisation. However, it has been difficult to control and predict the properties of CaCO3 precipitates, thus making it very challenging to achieve homogeneous MICP-treated soils with the desired mechanical properties. This PhD study investigates MICP at both micro and macro scales to improve the micro-scale understandings of MICP which can be applied at the macro-scale for improving the homogeneity and mechanical properties of MICP-treated sand. A microfluidic chip which models a sandy soil matrix was designed and fabricated to investigate the micro-scale fundamentals of MICP. The first important finding was that, during MICP processes, phase transformation of CaCO3 can occur, which results in smaller and less stable CaCO3 crystals dissolving at the expense of growth of larger and more stable CaCO3 crystals. In addition, it was found that bacteria can aggregate after being mixed with cementation solution, and both bacterial density and the concentration of cementation solution affect the size of aggregates, which may consequently affect the transport and distribution of bacteria in a soil matrix. Furthermore, bacterial density was found to have a profound effect on both the growth kinetics and characteristics of CaCO3. A higher bacterial density resulted in a quicker formation of a larger amount of smaller crystals, whereas a lower bacterial density resulted in a slower formation of fewer but larger crystals. Based on the findings from micro-scale experiments, upscaling experiments were conducted on sandy soils to investigate the effect of injection interval on the strength of MICP treated soils and the effects of bacterial density and concentration of cementation solution on the uniformity of MICP treated soils. Increasing the interval between injections of cementation solution (from 4 h to 24 h) increased the average size of CaCO3 crystals and the resulting strength of MICP-treated sand. An optimised combination of bacterial density and cementation solution concentration resulted in a relative homogeneous distribution of CaCO3 content and suitable strength and stiffness of MICP-treated sand. This thesis study revealed that a microfluidic chip is a very useful tool to investigate the micro-scale fundamentals of MICP including the behaviour of bacteria and the process of CaCO3 precipitation. The optimised MICP protocols will be useful for improving the engineering performance of MICP-treated sandy soils such as uniformity and strength.

The production of building materials is a significant contributor to anthropogenic greenhouse gas emissions with conventional kiln brick production being one of the most energy intensive processes. In addition, phosphorus is a resource that is required by all living organisms and is a key ingredient in many fertilisers. The demand for building materials and global natural phosphate rock (phosphorous) are increasing and decreasing respectively as urbanization increases. Naturally occurring phosphorous is expected to experience a peak in the near future after which it will be completely depleted. Urine has been identified as a potential source of phosphorous for fertiliser production as well as urea for microbial induced calcium carbonate precipitation (MICP) applications. MICP is a natural process that has the ability to produce bio-building material. Urine accounts for a small percentage of the total volume of domestic wastewater but contains a large percentage of the nutrients wastewater treatment plants (WWTP) seek to remove before they adversely affect receiving water bodies. The unprecedented rate of climate change and the associated pressures, coupled with the increased awareness around the depletion of natural resources, presents a significant challenge for which innovative and sustainable solutions are required. The reason for engaging in this project was to investigate if the urea present in human urine could be used in the natural MICP for the production of bio-bricks while at the same time recovering phosphorus from urine. Firstly, a thorough review of literature was conducted to assess current innovations pertaining to the dissertation topic. The process of bio-brick production by MICP requires a urea rich solution which could be recovered from urine. However, the urea present in urine naturally degrades and this process needs to be delayed if urine is to be used as a urea source for MICP. This was achieved by “stabilising” the urine with calcium hydroxide. Sporosarcina pasteurii (S. pasteurii) was the bacteria strain used to help drive the MICP process. The bacteria degraded the urea present in the urine to form carbonate ions which then combined with the calcium ions present in the urine solution to produce calcium carbonate. This calcium carbonate was then used as a bio-cement to glue loose sand particles together in the shape of a brick. The cementation media was made by adding calcium chloride and nutrient broth to the stabilised urine, and lowering its pH to 11.2. The purpose of adding calcium chloride was to improve the efficiency of the process since the stabilised urine did not have enough calcium ions. Ordinary sand mixed with Greywacke aggregate and inoculated with S. pasteurii bacteria was used as the media for the MICP process. Bio-brick moulds were filled with the sand mixture and sealed. The cementation media was pumped through the bio-brick mould to fill its’ pore volume. The media was retained in the moulds for a defined retention time ranging from 1-8 hours. At the end of every retention time, new cementation media was pumped through the bio-brick to fill it’s pore volume again. iv To establish an optimal starting influent calcium concentration the influent calcium concentration changed between experiments. Additionally, in subsequent experiments, the calcium concentration was raised in a stepwise manner during an experiment to establish the maximum amount the influent calcium concentration could be raised to before the microbial community experienced adverse effects. Additionally, experiments explored the effects a range of retention times had on the bio-brick system in order to establish an optimal retention time. Another experiment was set up to investigate the relationship between the number of treatments and the resultant compressive strength. The findings from the above-mentioned experiments further guided subsequent experiments which singled out and tested certain factors thought to be affecting the bio-brick system. The factors tested include after treatment washing, ionic strength, pH and calcium concentration of the influent cementation media. Possible alternative nutrient medias (ANMs) were also investigated for a cheaper alternative to the laboratory grade growth media used to grow the bacteria. Lastly, an integrated system that produced both fertilisers and bio-bricks was developed. Its basic economics of raw material inputs and outputs were used to assess the financial implications of the proposed system, and the social and policy barriers likely to affect the implementation of an integrated urine treatment system were examined. Urine treated with calcium hydroxide offers a urea-rich solution that can be used for MICP processes. This resulted in the worlds’ first bio-brick “grown” from human urine. The starting influent calcium concentration reached a maximum of 0.09 M before adverse effects to the microbial community were experienced. Furthermore, in terms of a stepwise increase during the treatment cycle, the influent calcium concentration could be raised to 0.12 M without any adverse bacteria effects. The minimum retention time the bio-brick system could withstand was 2 hours which allowed the treatment cycle to be completed in a shorter time. The highest compressive strength obtained was equal to 2.7 MPa. To produce this strength about 31.2 L of stabilised urine was used. The relationship between the number of treatments and the compressive strength showed that an increase in the number of treatments increased the compressive strength. Both the pH and ionic strength of the urine were identified to have an inhibiting effect on the ureolytic activity and MICP process. Additionally, using an influent cementation media with an optimal pH for urea hydrolysis, improved the bacteria’s ability to operate at higher ionic strengths. However, when the stabilised urine was stored, urea hydrolysis occurred earlier likely because of external contamination by naturally occurring bacteria in the lab. LML (Lactose mother liquor) was identified as alternative growth media for S. pasteurii growth which could reduce raw material costs considerably. The bio-brick production process was found to be more cost-effective if it was incorporated into the integrated urine treatment process system. The integrated system included fertiliser production by recovering calcium phosphate fertilisers and ammonium sulphate fertilisers before and after the bio-brick production respectively. Producing 1000 bio-bricks a day would require 23% of Cape Towns’ population daily urine production and would incur a profit of ZAR 7330 per day between the raw material cost and the revenue from sales. For implementation in a South African context, certain policy barriers need to be overcome. Potential paths for implementation are reclassifying the urine for its use in an industrial process and obtaining an operating permit or seeking an exemption for a permit through the ECA (Environment Conservation Act). Research suggests that products from the integrated system are likely to be socially v accepted and that a combined appeal to people's environmental sensitivities and targeted marketing messages would enhance people’s acceptance. Finally, recommendations for further paths to take to build on the research established in this dissertation were made. It is recommended that additional characteristics of the bio-bricks should be tested, recycled material should be used as media for bio-bricks, the bacteria strain should be modified and methods for reducing the ionic strength of urine should be investigated. Additionally, it is recommended that consumers’ willingness to use urine-based products should be further studied, the legislative options for implementing bio-brick and fertiliser production should be investigated and a more detailed and expansive economic analysis should be performed.

Geological repositories are being considered as the best feasible solution for the storage of hazardous materials such as high level nuclear waste throughout the world, including the UK. However; when crystalline rock is the chosen storage medium, the construction of the underground tunnels and caverns can enhance discontinuities within the rock. These discontinuities can be pathways by which radio-nuclides can reach the biosphere, due to their higher permeability, connectivity and density (Blyth and Freitas, 1992). Thus, depending on aperture, density and predicted travel times, it may be necessary to grout all fractures, even small aperture ones, which over thousands of years can contribute significantly to subsurface flow. Conventional cementitious and chemical grouts are unsuitable within some regions of a geological disposal facility due to concerns regarding longevity, toxicity, reactions with other barriers and/or workability issues. The four main requirements of a grout are; to be of low viscosity as the lower the viscosity the easier it is to achieve good penetration, to have a controllable gel/setting time, to be chemically inert to prevent reactions within the subsurface or have any toxic consequences during preparation, and to be durable thus able to withstand exposure to varying physic-chemical condition. MICP (Microbially Induced Calcite Precipitation) and Colloidal Silica are novel grouts which may be suitable for the sealing of fine aperture fractures in rock. MICP research has been predominantly focussed on its application in sediments, whilst colloidal silica has shown its potential for reducing the liquefaction potential of non-cohesive soils and for sealing fractures. This research examines the influence of hydraulic controls (velocity, flow rate, aperture) on the spatial distribution of microbially induced calcite precipitation (MICP) within simulated fractures using flocculated Sporosarcina pasteurii. The experimental results show that under flowing conditions, the spatial distribution of microbially induced calcite precipitate on fracture surfaces is controlled by fluid velocity. Even for a uniform initial fracture aperture with a steady flow rate, a feedback mechanism existed between velocity and precipitation that resulted in a precipitate distribution that focussed flow into a small number of self-organizing channels which remained stable. Ultimately, this feedback mechanism controlled the final aperture profile which governed flow within the fracture. To use MICP for field scale sealing operations (e.g., in aquifers and host rock surrounding nuclear waste storage sites), it is important to develop an injection strategy that ensures microbially precipitated calcite is distributed homogenously throughout the rock body to avoid preferential flow through high porosity pathways. Sporosarcina pasteurii was found to be able to hydrolyse urea for several days before the bacteria became encased within calcite preventing access to the cementing fluid. The higher rates of urea hydrolysis occurred within the first 9 hours, though significant rates of urea hydrolysis still occurred after this period. By reducing the size of bacterial flocs it is possible to reduce the impact of sedimentation and straining, promoting a more even distribution of bacteria thus calcite precipitate throughout the plate. By increasing the length of time that the bacteria flow through the fracture, more bacteria can become entrained upon the fracture surface giving a better distribution. The introduction of a filler (colloidal silica) that can also act as a nucleation site for calcite precipitation was examined as a way of reducing the time it takes for the sealing of a fracture. Both Sporosarcina pasteurii and colloidal silica have negative surface charges thus colloidal silica could be used as a nucleation surface, this plus its nanometre size which could allow for a better distribution of and could enhance calcite precipitation. A clear difference in the mass of grout retained within the fracture was seen, with MICP alone showing the greatest weight increase. During the 8 grouting cycles with MICP + colloidal silica there appeared to be pieces of calcite travelling through the open channels. This would indicate that the calcite is unable to attach to the fracture surface. Thus, adding a small amount of colloidal silica to the cementing solution as a filler was not an efficient way to produce calcite fill. However, Sporosarcina pasteurii produces ammonium ions from the hydrolysis of the non-ionic urea, which as a cation can destabilise the silica sol resulting in gelation. Batch tests were used to determine what differences in gel point, gel rate and shear strength were created by different cations, including the chemical addition of ammonium ions and the biological production of ammonium ions by the bacterium Sporosarcina pasteurii. The sensitivity of colloidal silica to calcium chloride can result in dramatic differences in gel time with small changes in molarity having great impact on whether the colloidal silica gels or not. The direct addition of ammonium salts requires ten times the concentration, compared to CaCl2, to achieve similar shear strength values. However; this concentration produces very short gel times, potentially reducing the radius of penetration. The bacterial in-situ production of ammonium ions gives the greatest gel times yet still produces the same shear strength as that of a sodium chloride accelerator. This increasing of gel times, without adversely impacting grout properties, could be beneficial for penetrating greater distances into fractured rock reducing the number of injection points required. This would be particularly useful for subsurface engineering applications where large volumes of rock are required to be grouted.

The current manufacturing of clay-fired and cement bricks has contributed greatly to anthropogenic global emissions and environmental damages. A possible solution that could be used to alleviate such environmental pressures is through the adoption of carbon neutral, microbial induced calcium carbonate precipitation (MICP) bio-bricks as a replacement for traditional bricks. MICP produced bio-bricks are formed by exploiting the ability of the microorganism, Sporosarcina pasteurii, to produce a biocement capable of binding sand particles (or any aggregate) together into a solid. Furthermore, such bio-bricks can be grown from otherwise ‘waste' resources such as human urine. This significantly reduces energy inputs whilst creating value by ‘upcycling' waste streams, resulting in a product which is sustainable whilst promoting the modern ethos of implementing environmentally friendly circular economies. However, the environmental benefits of MICP bio-bricks are hindered by the use of sand in their production. Sand, after water, is by volume the worlds most exploited and traded raw material and as such the supply of sand is being rapidly depleted globally. Added to this, sand extraction processes are known to cause extensive environmental damages. A possible solution to this issue is to replace the sand aggregate used to grow bio-bricks with mine tailings. The increasing global demand for metal products has resulted in the concurrent production of vast volumes of waste mine tailings which, if left untreated, pose a potential risk of leaching toxins into surrounding populations and biota. As such it was postulated that this risk to surrounding populations and the environment could be mitigated by repurposing mine tailings, as a replacement for sand, into MICP bio-bricks. Both a technical and economic study was conducted to determine the feasibility of repurposing copper mine tailings into bio-bricks. As bio-bricks were resource intensive to produce (reagents, chemicals etc.), bio-columns were used as a proxy in studying the technical feasibility of such a process. The technical aspect of this study involved characterising copper mine tailings received from Columbia in terms of physiochemical make-up, particle size distribution and the development of a MICP submergent technique used in growing the bio-columns. This was necessitated by the fact that it was noted during the characterisation of the mine tailings that the cementation media could not be pumped through the columns filled with mine tailings aggregate, resulting in the traditional pumping method used to grow MICP bio-solids being impractical. The submergent technique was used to compare the MICP efficiency of growing biocolumns from either beach sand or copper mine tailings. In addition, the toxicity of copper to S. pasteurii was investigated and an attempt was made to acclimate a culture of S. pasteurii to the copper concentration found within copper mine tailings. Furthermore, the copper mine tailings were screened to determine if there were any indigenous, anaerobic and copper tolerant ureolytic extremophiles contained within, which had the potential to grow more robust bio-columns.

Soft soils are composed of large portions of silts and clay and are characterized by low strength and high compressibility owing to their high-water content. Biocementation techniques are emerging as a strong alternative to conventional methods owing to their eco- friendliness and sustainability. Biocementation techniques bonds soil particles through biologically precipitated cementing products, such as extracellular polymeric saccharides (EPS) and microbially induced calcite precipitation (MICP). Extracellular polymeric saccharides are constituents of protective biofilms that shield microorganisms from environmental stress and are responsible for cohesion of microorganisms and adhesion of biofilms to surfaces. They are bound to surfaces by growth of fibrous bridges, filling of voids and formation of van der Waal’s, hydrogen, and ionic bonds. Existing studies have relied on extraneous polymer addition to overcome the need for microbial and nutrient injection, time for cultivation and excrement secretion and compatibility of the microbes with host mineral. A major drawback of extraneous addition is the inability of polysaccharides to penetrate the deeper layers of a porous solid owing to formation of surficial crust and/or resistance by the micro-porosity of the system. The problem of binding particles located in deeper layers can be addressed by in-situ secretion of polysaccharides in the connected voids network of a solid ensuring cementation through entire depth. Microbially induced calcite precipitation (MICP) is another preferred biocementation technique in which bacteria possessing urease enzyme hydrolyses solution of externally injected urea to produce bicarbonate ions. The anions react with calcium ions in soil to form calcium carbonate that bond soil particles and produce stabilized soil. Flushing of microbes during repeated injection resulted in uneven distribution of precipitated calcite. Further, competition with native bacteria for nutrients led to starvation and diminished population of injected microbes. These factors motivated researchers to examine indigenous microbes for calcite precipitation. An alternative to urease pathway is microbially induced denitrification that relies on anaerobes/facultatively anaerobes to oxidise organic matter using nitrate ions. The CO2 produced from anaerobic decomposition of organic matter transform to carbonates upon hydrolysis in alkaline pH environment. The focus of this thesis is to examine biocementation process such as in-situ EPS secretion and in-situ microbially induced carbonate cementation (MICC) to improve the unconfined compressive strength of synthetic soft soil using native microbes of cattle manure. The thesis has three objectives. In the first objective, the thesis explores in-situ EPS secretion to improve the unconfined compressive strength of a synthetic soft soil specimen. Small amount of cattle manure is mixed with the synthetic soil to facilitate supply of organic C and native EPS producing bacteria under anaerobic condition. The soft soil is prepared in the laboratory by mixing equal proportions of kaolinite (50%), sand (50%) and small amount of cattle manure (10% of kaolinite-sand mass). The mix is remoulded into cylindrical specimens at adequately high-water content using ultra-pure water. Sand inclusion facilitates sites for bacterial adhesion during curing of the soil under anoxic/anaerobic conditions. The environmental stress caused by restricted availability of electron acceptors (dissolved oxygen, nitrate ions) induces EPS secretion by the native microbes of cattle manure in the porous network of the synthetic soil specimen. Evidence for the growth of EPS producing bacteria and the bonding mechanisms of EPS with soil particles is obtained by performing bio-chemical analysis with slurry samples and micro-structural studies with slices obtained at mid-depth of cylindrical specimens. The unconfined compressive strength of the synthetic soil specimen increased from 19 kPa to 132 kPa after 28-days of curing. Besides van der Waals and hydrogen bonds, interfacial frictional resistance between mineral units mobilized by bridging of sand particles and embedment of cattle manure fibres in kaolinite aggregates cause immediate increase in unconfined compressive strength of the treated specimen. Frictional bonds between mineral grains/aggregates and cattle manure fibres, EPS bonds between soil aggregates and hydrogen and van der Waals bonds contributed 46, 39 and 14% to the unconfined compressive strength (132 kPa) of the treated soil specimen. The stress-strain characteristics of the specimens exhibit progressive failure which is attributed to the ductility of the bridge-forming fibres and the viscoelastic nature of EPS deposited in soil pores and on particle surfaces. In the second objective, the thesis explores microbially induced calcium carbonate precipitation (MICC) in the synthetic soft soil specimen using the anaerobic denitrification pathway by native microbes. Cattle manure is again used as organic C and native denitrifying bacteria source in the soft soil specimen. Required amount of calcium nitrate salt is extraneously added to the soil to provide nitrate source (electron acceptors) for metabolism of denitrifying bacteria, while Ca2+ ions participate in calcium carbonate precipitation. Small amount of magnesium oxide (MgO) is added to counter the reduction in pH caused by volatile fatty acids (VFAs) produced during microbial degradation of cattle manure. The presence of cattle manure also facilitates EPS cementation in the soft soil specimen. The unconfined compressive strength of the soil specimen increased from 19 kPa to 169 kPa after 28-days of curing. Hydrogen and van der Waals bonds, frictional bonds between sand grains/kaolinite aggregates and cattle manure fibres, and EPS + MICC bonds contribute 13, 40 and 47% to the unconfined compressive strength (169 kPa) of the treated soil. Presence of microbially induced carbonate cementation between aggregate contacts, tend to impart brittle behaviour and greater rigidity to the stress-strain characteristics of the treated soil specimens. In the third objective, the thesis explores combining pozzolanic reactions with microbially induced carbonate cementation (MICC) to improve the unconfined compressive strength of the synthetic soft soil. Cattle manure is used as source of native microbes and organic matter reservoir for growth and sustenance of microbes contained in cattle manure. It also provides calcium (Ca2+) and magnesium (Mg2+) ions necessary for carbonate precipitation and pozzolanic reactions. Carbon dioxide (gas) is formed as by-product of degradation of cattle manure particles by facultative anaerobes. Dissolution of evolved carbon dioxide (gas) provides the alkalinity for mineral precipitate formation. Volatile fatty acids (VFAs) produced during microbial degradation of cattle manure are neutralized by addition of varying (0.5 to 10%) amounts of magnesium oxide (MgO). Pozzolanic reactions are facilitated by strong alkaline pH from MgO presence. Bridging of soil aggregates by CM fibers and short-term reactions contribute to immediate gain in strength of treated specimens. At MgO contents ≥ 3.5%, deposition of carbonate precipitates and pozzolanic reaction products at aggregate contacts over-ride the ductile nature of CM fibers and impart brittle stress-strain behavior. The gain in strength from interfacial resistance mobilised by CM fibres bridges, short-term modification reactions, pozzolanic reactions and MICC cementation transform the very soft soil (UCS < 25 kPa) to hard (UCS > 383 kPa) soils. At lower MgO contents (0.5 and 3.5%) pozzolanic reactions and MICC modes near equally (48 and 52%) contribute to compression strength of the specimens. At higher MgO contents (5 and 10%), pozzolanic reactions are dominant (60 and 71%) contributors as the slightly more alkaline pH of these specimens may have suppressed microbial activity. Based on the classification of compression strengths, EPS bonding transforms very soft kaolinite to stiff kaolinite (UCS range 96-192 kPa). EPS plus MICC bonding also transforms the very soft specimen to stiff specimen. Finally, MICC plus pozzolanic reactions transform the very soft soil to hard specimens (UCS range > 383 kPa).

碩士
國立臺灣科技大學
營建工程系
107
Microbial Induced Calcite Precipitation (MICP) is a new technique of improving the engineering properties of soil, a multidisciplinary technique that involves biology, chemistry and soil mechanics. This technique involves the hydrolysis of urea by urease bacteria enzyme into carbonate ions and ammonium ions that precipitate in form of calcite in presence of calcium source. The calcite precipitate in the pore space of soil sample, where they can move and find oxygen for their activity, at particle-to-particle contact. However, the pore space of soil varied with the type of soil. The pore space in coarse-grained soil are greater than pore space in fine-grained-soil. Therefore, the application of MICP in fine-grained soil is limited. The limitation of MICP application in fine-grained soil was studied in this research with Taipei silty soil. MICP method was successively applied on Taipei silty using two method, namely mixing method and injection method. The unconfined compressive strength test and the electronic cone penetrometer test was used to indicate the improvement of soil shear strength for mixing method and injection method, respectively. An increase of two fold of the shear strength of Taipei silty clay from both method was achieved. Prior to the mixing method and the injection method, the ability of Sporosarcina pasteurii, used in this study, was investigated. High concentration of S. pasteurii inside growing medium promotes high urease activity. Soil improvement by natural (coir) fiber was also studied in this research. It shows the strength of the soil increases with the addition of fiber up to 1%. However, MICP gives better result on soil improvement compare to natural fiber. A combination of the two method was attempted. However, MICP application give better response to soil improvement than the combination of fiber and MICP.

abstract: The potential of using bio-geo-chemical processes for applications in geotechnical engineering has been widely explored in order to overcome the limitation of traditional ground improvement techniques. Biomineralization via urea hydrolysis, referred to as Microbial or Enzymatic Induced Carbonate Precipitation (MICP/EICP), has been shown to increase soil strength by stimulating precipitation of calcium carbonate minerals, bonding soil particles and filling the pores. Microbial Induced Desaturation and Precipitation (MIDP) via denitrification has also been studied for its potential to stabilize soils through mineral precipitation, but also through production of biogas, which can mitigate earthquake induced liquefaction by desaturation of the soil. Empirical relationships have been established, which relate the amount of products of these biochemical processes to the engineering properties of treated soils. However, these engineering properties may vary significantly depending on the biomineral and biogas formation mechanism and distribution patterns at pore-scale. This research focused on the pore-scale characterization of biomineral and biogas formations in porous media. The pore-scale characteristics of calcium carbonate precipitation via EICP and biogenic gas formation via MIDP were explored by visual observation in a transparent porous media using a microfluidic chip. For this purpose, an imaging system was designed and image processing algorithms were developed to analyze the experimental images and detect the nucleation and growth of precipitated minerals and formation and migration mechanisms of gas bubbles within the microfluidic chip. Statistical analysis was performed based on the processed images to assess the evolution of biomineral size distribution, the number of precipitated minerals and the porosity reduction in time. The resulting images from the biomineralization study were used in a numerical simulation to investigate the relation between the mineral distribution, porosity-permeability relationships and process efficiency. By comparing biogenic gas production with abiotic gas production experiments, it was found that the gas formation significantly affects the gas distribution and resulting degree of saturation. The experimental results and image analysis provide insight in the kinetics of the precipitation and gas formation processes and their resulting distribution and related engineering properties.
Dissertation/Thesis
Doctoral Dissertation Civil, Environmental and Sustainable Engineering 2019

博士
國立中興大學
土木工程學系所
105
The research focus on using Microbial induce carbonate precipitation (Microbial Induced Calcium Carbonate Precipitation, MICP) to repair the microcrack in concrete. In this research we tried to use Bacillus pasteurii for the repairmen of the microcrack in concrete. The results of serial tests prove that the higher the concentration of the bacterial broth, the greater the amount of calcium carbonate precipitate was induced, while using Bacillus pasteurii broth for concrete crack rehabilitation. The flexural strengths of the repaired concrete test samples were the greatest at 100% bacterial concentration. Compared to the control group (bacterial concentration of 0%), the flexural strength had increased by 32.58% for 1-mm crack samples and 51.01% for 2-mm crack samples, and the compression strength had increased by 28.58% and 23.85%, respectively. The tests all confirm that the using bacteria in concrete crack rehabilitation can increase the flexural and compression strength of the repaired concrete due to Microbial induce carbonate precipitation (MICP).

abstract: ABSTRACT Enzyme-Induced Carbonate Precipitation (EICP) using a plant-derived form of the urease enzyme to induce the precipitation of calcium carbonate (CaCO3) shows promise as a method of stabilizing soil for the mitigation of fugitive dust. Fugitive dust is a significant problem in Arizona, particularly in Maricopa County. Maricopa County is an EPA air quality non-attainment zone, due primarily to fugitive dust, which presents a significant health risk to local residents. Conventional methods for fugitive dust control, including the application of water, are either ineffective in arid climates, very expensive, or limited to short term stabilization. Due to these limitations, engineers are searching for new and more effective ways to stabilize the soil and reduce wind erosion. EICP employs urea hydrolysis, a process in which carbonate precipitation is catalyzed by the urease enzyme, a widely occurring protein found in many plants and microorganisms. Wind tunnel experiments were conducted in the ASU/NASA Planetary Wind Tunnel to evaluate the use of EICP as a means to stabilize soil against fugitive dust emission. Three different soils were tested, including a native Arizona silty-sand, a uniform fine to medium grained silica sand, and mine tailings from a mine in southern Arizona. The test soil was loosely placed in specimen container and the surface was sprayed with an aqueous solution containing urea, calcium chloride, and urease enzyme. After a short period of time to allow for CaCO3 precipitation, the specimens were tested in the wind tunnel. The completed tests show that EICP can increase the detachment velocity compared to bare or wetted soil and thus holds promise as a means of mitigating fugitive dust emissions.
Dissertation/Thesis
M.S. Civil and Environmental Engineering 2014

abstract: In enzyme induced carbonate precipitation (EICP), calcium carbonate (CaCO3) precipitation is catalyzed by plant-derived urease enzyme. In EICP, urea hydrolyzes into ammonia and inorganic carbon, altering geochemical conditions in a manner that promotes carbonate mineral precipitation. The calcium source in this process comes from calcium chloride (CaCl2) in aqueous solution. Research work conducted for this dissertation has demonstrated that EICP can be employed for a variety of geotechnical purposes, including mass soil stabilization, columnar soil stabilization, and stabilization of erodible surficial soils. The research presented herein also shows that the optimal ratio of urea to CaCl2 at ionic strengths of less than 1 molar is approximately 1.75:1. EICP solutions of very high initial ionic strength (i.e. 6 M) as well as high urea concentrations (> 2 M) resulted in enzyme precipitation (salting-out) which hindered carbonate precipitation. In addition, the production of NH4+ may also result in enzyme precipitation. However, enzyme precipitation appeared to be reversible to some extent. Mass soil stabilization was demonstrated via percolation and mix-and-compact methods using coarse silica sand (Ottawa 20-30) and medium-fine silica sand (F-60) to produce cemented soil specimens whose strength improvement correlated with CaCO3 content, independent of the method employed to prepare the specimen. Columnar stabilization, i.e. creating columns of soil cemented by carbonate precipitation, using Ottawa 20-30, F-60, and native AZ soil was demonstrated at several scales beginning with small columns (102-mm diameter) and culminating in a 1-m3 soil-filled box. Wind tunnel tests demonstrated that surficial soil stabilization equivalent to that provided by thoroughly wetting the soil can be achieved through a topically-applied solution of CaCl2, urea, and the urease enzyme. The topically applied solution was shown to form an erosion-resistant CaCO3 crust on fine sand and silty soils. Cementation of erodible surficial soils was also achieved via EICP by including a biodegradable hydrogel in the stabilization solution. A dilute hydrogel solution extended the time frame over which the precipitation reaction could occur and provided improved spatial control of the EICP solution.
Dissertation/Thesis
Doctoral Dissertation Civil and Environmental Engineering 2015

abstract: The dissimilatory reduction of nitrate, or denitrification, offers the potential of a sustainable, cost effective method for the non-disruptive mitigation of earthquake-induced soil liquefaction. Worldwide, trillions of dollars of infrastructure are at risk for liquefaction damage in earthquake prone regions. However, most techniques for remediating liquefiable soils are either not applicable to sites near existing infrastructure, or are prohibitively expensive. Recently, laboratory studies have shown the potential for biogeotechnical soil improvement techniques such as microbially induced carbonate precipitation (MICP) to mitigate liquefaction potential in a non-disruptive manner. Multiple microbial processes have been identified for MICP, but only two have been extensively studied. Ureolysis, the most commonly studied process for MICP, has been shown to quickly and efficiently induce carbonate precipitation on particle surfaces and at particle contacts to improve the stiffness, strength, and dilatant behavior of liquefiable soils. However, ureolysis also produces copious amounts of ammonium, a potentially toxic byproduct. The second process studied for MICP, denitrification, has been shown to precipitate carbonate, and hence improve soil properties, much more slowly than ureolysis. However, the byproducts of denitrification, nitrogen and carbon dioxide gas, are non-toxic, and present the added benefit of rapidly desaturating the treated soil. Small amounts of desaturation have been shown to increase the cyclic resistance, and hence the liquefaction resistance, of liquefiable soils. So, denitrification offers the potential to mitigate liquefaction as a two-stage process, with desaturation providing short term mitigation, and MICP providing long term liquefaction resistance. This study presents the results of soil testing, stoichiometric modeling, and microbial ecology characterization to better characterize the potential use of denitrification as a two-stage process for liquefaction mitigation.
Dissertation/Thesis
Doctoral Dissertation Civil and Environmental Engineering 2016