Extra-cellular Polysaccharides and Microbially Induced Carbonate Cementation Using Indigenous Microbes in Cattle Manure

Abstract

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).