Effective Use of Plant-Derived Urease in the Field of Geoenvironmental/ Geotechnical Engineering

Geotechnical and geo-environmental engineering applications currently being explored through bio mineralization process include cementation of sands to improve mechanical properties and hence, to enhance bearing capacity and liquefaction resistance, sequestration of carbon, soil erosion control via surficial stabilization, groundwater flow control and remediation of soil and groundwater impacted by metals and radionuclides. Bio mediated system broadly refers to a chemical reaction series that are managed and controlled through biological activities and byproducts resulting from those reactions alter the properties of material in the system. Bio mediated methods such as microbial induced calcite precipitation (MICP), biofilm formation, biogas generation and biopolymers have been developed by injecting or stimulating microbes. MICP is the most widely explored method. Microbes having urease activity enhance the hydrolysis of urea and it helps to control the pH and to precipitate carbonate. Although it is a widely explored bio mediated method it has some disadvantages. The plant-derived urease may offer many benefits over microbial urease to induce carbonate cementation. Therefore, it is advantageous to explore the knowledge regarding such a technique as an eco-friendly method for different applications in the fields of geoenvironmental/geotechnical Engineering. The main objective of this paper is to provide an overview of the importance of plant-derived urease for different applications in the fields of geoenvironmental/ geotechnical engineering. The information presented in this paper may be important for biotechnologists/geoengineers to have wide ranging updates on the current situation. *Corresponding author: Dilrukshi RAN, Graduate School of Engineering, Hokkaido University, Kita 13, Nishi 8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan, Tel: +81-11706-6318; E-mail: nadilrukshi@yahoo.com Received November 08, 2015; Accepted December 14, 2015; Published December 24, 2015 Citation: Dilrukshi RAN, Kawasaki S (2016) Effective Use of Plant-Derived Urease in the Field of Geoenvironmental/Geotechnical Engineering. J Civil Environ Eng 6: 207. doi:10.4172/2165-784X.1000207 Copyright: © 2016 Dilrukshi RAN, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
Geotechnical engineering studies the strength and deformation of soil for successful designs of foundations and environmental engineering studies contaminants within the surrounding environment. Successful planning to improve the soil characteristics by different ground improvement techniques by assuring minimum damage to the environment, and cleaning the contaminated soil, water and air by various treatment methods are being implemented worldwide via sustainability concepts. Current ground improvement techniques in practice include grouting via cement, chemical, compaction, fracture and jet, micro piles, jacked piers, driven piers, ground anchors, shoring, soil nailing, vibro compaction, concrete columns, piers, etc. [1]. Voids of soil are filled with fluid chemical grouts during the chemical grouting. The most common chemical grouts are sodium silicate, acrylate, lignin, urethane, and resin grouts. All of these synthetic chemical grouts are hazardous and/or toxic and hence, ground water pollution may be a potential problem. On the other hand, the cement-based concrete system is not preferred due to the high costs of cement and the high amount of CO 2 that are released during the cement production [2,3]. Technology development and its productivity are necessary, in order to maintain the sustainability and to reduce the production of CO 2 emission in order to maintain eco-friendly environment. Hence, in recent years, biotechnology is practiced an increased level of interest. This requires a multidisciplinary perspective that holds the science of biology, chemistry and physics together and applies this knowledge to multifunctional geotechnical and environmental engineering applications. The idea of bio geotechnical engineering started spreading through the field of geotechnical engineering, in 2005, and bio geotechnical engineering is identified as an important research topic by the National Research Council [4]. Mitchell and Santamarina have presented the first detailed discussion of bio geotechnical engineering. The major topic of interest in these bio geotechnical studies is the improvement of soil via carbonate precipitation induced by urea hydrolysis, with most studies focusing on microbially induced calcite precipitation (MICP) [5]. The evidence of microorganism involvement in carbonate precipitation has led to the development of bio technology in the field of construction material [6,7]. In addition to that, the reduction of water hardness [8], heavy metal removal from soil and water [9,10] has been investigated using the formation mechanism of bio cement with aid of different sources of urease. The reagents for bio cement are produced at relatively low temperatures compared to ordinary cement that involves heating ingredient to temperatures up to about 1500°C. Therefore, bio cement has a potential to be used as an eco-friendly cement material with advantages of less energy to produce the cement and less release of greenhouse gases [8]. This cementation process can be conducted in-situ without disrupting the structure of original soil and has a potential to be used in both unsaturated and saturated applications [11]. There are three main groups of microorganisms that can induce the carbonate precipitation. They are photosynthetic microorganisms such as cyanobacteria and microalgae, sulphate reducing bacteria, and some species of microorganisms involved in nitrogen cycle (Bacillus pasteurii, Bacillus cereus, etc.) such as nitrogen fixing bacteria, nitrifying bacteria, denitrifying bacteria and saprobiotic bacteria through the processes of ammonification of amino acids, dissimilatory reduction of NO 3 and degradation of urea or uric acid [6,12]. Currently, urease enzyme activity in most of bacteria metabolism process has been used widely as a tool to precipitate calcium carbonate. In addition to the microorganisms, urease is a widely occurring hexameric protein found in many higher order plants and some invertebrates as well as wide variety of tissues in humans. Several families of common plants are very rich in urease, including some varieties of beans, melons and squash, and the pine family [13]. Seed urease has been purified to homogeneity from several leguminous plants [14], while leaf urease has also been subjected to investigations [15]. Extraction of urease enzyme from most urease containing plant species is simple and the enzyme is readily available from laboratory suppliers also. The enzyme is approximately 12 nm in dimension [16]. The small size of a solubilized urease enzyme gives distinct advantage over ureolytic microbes during the penetration into very small pore spaces as nearly all known bacteria are greater than 300 nm in diameter, with the majority in the range of 500-5000 nm. Therefore, plant-derived urease can be applicable to much finer soils. Furthermore, exogenously added urease (i.e. Urease added as a free enzyme) has a limited lifespan and its activity and function decreases with time [17,18]. This limited lifespan is potentially advantageous in some engineering applications as the enzyme can naturally degrade and eliminate long term impacts to the ecosystem. However, microbial based urease will leave the organisms behind. Therefore, injection of ureolytic bacteria in to the ground involves many problems such as the obtaining approvals and licenses from government and the continuous monitoring of microbial ecology for safety [19]. MICP treatment may be limited to deep soil due to limitations of bacterial growth and movement in sub soil. MICP may also be limited to the soils containing limited amounts of fines due to the reduction in pore spaces in fine soils. Based on the size of microorganism, the applicability of bio modification is limited to GW, GP, SW, SP, ML, and organic soils [5]. Bacteria are not expected to enter through pore throats smaller than approximately 0.4 µm. In general, the microbial abundance is found to increase with the increase in particle size [20]. The habitable pores and traversable pore throats are found in coarse sediments and some clayey sediment at shallow depth. In clayey soil, bacteria are capable of reorienting and moving clay particles under low confining stress (at shallow depths). However, inability to make these rearrangements under high confining stresses limits bacterial activity at larger depths. Furthermore, sediment-cell interaction may cause puncture or tensile failure of the cell membrane. Similarly, at larger depths, silt and sand particles may crush and cause a reduction in pore spaces, reducing the biological activity [20]. Moreover, the production of urease active bacteria and isolation of pure ureolytic bacteria is one of the main costs for applying to bio cementation technology [21] without enrichment of urease positive bacteria from local environment [22]. The urease enzyme that is extracted from plant species is little expensive when purchased from lab supply, but lab grade enzyme is very effective [23]. However, plant urease can be applied as a crude extract instead of commercially purified urease. Crude extracts of jack bean (Canavalia ensiformis) have the potential to be used as a replacement for commercially available purified urease [24]. These are the factors that give some conceptions to the geotechnologists or biotechnologists to think and investigate about bio mineralization of calcite from plant-derived urease as a promising alternative for microbial urease for making bio cement.

Urease Activity
Urease (EC 3.5.1.5) is a nickel dependent metalloenzyme which catalyzes the hydrolysis of urea. One mole of urea is hydrolyzed intracellularly to one mole of ammonia and one mole of carbamate (Equation 1), which is spontaneously hydrolyzed to one mole of ammonia and one mole of carbonic acid (Equation 2). Ammonia and carbamate subsequently equilibrate in water to form bicarbonate and 2 moles of ammonium and hydroxide ions as described in Equation 3 and 4 [7]. CO (NH 2 ) 2 +H 2 O→ H 2 COOH + NH 3 (1) Total reaction: The presence of calcium ion in the system will lead to the precipitation of calcium carbonate once a certain level of super saturation is reached. Hausinger has shown that urease accelerates the hydrolysis of urea by a factor of 10 14 in comparison with the spontaneous reaction [25]. This fifth reaction raises the pH of the solution. This raise in pH creates an optimum condition for carbonate precipitation. The NH 4 + and CO 3 2produced from this reaction actually represent the final products of series of reactions. The NH 4 + ions actually start out as NH 3 . When the ammonia reacts with water, it creates OH-ions, which raise the pH of the system. This raise in pH causes the carbonate precipitation. Functionally, ureases belong to the superfamily of amidohydrolases and phosphotriestreases [26]. The members of the amidohydrolases superfamily catalyze a variety of hydrolysis reactions utilizing amides, urease, phosphotriestreases and substituted triazines. The primary common feature of the urease enzymes is the presence of metal centers in their active sites, whose task is to activate the substrate and water for the reaction. Among other dinuclear metallohydrolases in the superfamily, ureases are unique in that they possess Ni (II) ions in the active site. Plant and fungal ureases are homo-oligomeric proteins of 90-kDa identical subunits. Bacterial ureases are multimers of two-or three-subunit complexes. The bacterial and plant ureases have high sequence similarity, suggesting that they have similar threedimensional structures and a conserved catalytic mechanism [27,28].

Plant-derived Urease
Several families of common plants are very rich in urease, including some varieties of beans, melons and squash, and the pine family [13]. It includes jack beans (Canvalia ensiformis), soybean (Glycine max) leaf and seeds, pigweeds (Chenopodium album) and mulberry leaf (Morusalba), etc. [29] and they help to catalyze the reaction of urea hydrolysis to form ammonium and carbonate ions. Many researches are investigating new plant species such as leaves rather than the seeds having urease activity. Some plant species having urease activity gathered from the literature are summarized in Table 1. The beststudied plant urease is that from jack bean [30] which is identified as the first nickel metalloenzyme [31] and the urease from jack bean is the first enzyme crystallized [32]. In 1926, Sumner shows that urease is a protein. Molecular weight of jack bean urease is 480,000 Da (480 kDa) [33]. There are forty seven -SH groups in the urease species of molecular weight 480,000 Da (480 kDa) and it has been estimated that 4 to 8 of these groups are essential for the activity [34]. Maximum catalytic activity of jack bean urease occurs at 65°C [35] and it is inactive at temperatures above 70°C [36]. The optimum pH for jack bean urease usually lies between 6.0-7.0 [37,38]. The urease isolated from chickpea seeds (Cicer arietinum L.) yields maximum activity at pH 7.2 [39]. This result is similar to those reported for urease from jack bean [40] and pigeon pea [13] but different from that isolated from mulberry leaves [15]. Maximum urease activity of chickpea seeds obtains at temperature of 55°C and beyond that the enzyme denatures rapidly and thus loose its activity. This result is closely related to those reported by Das et al. [13] and Srivastava et al. [41] but differs from that stated by El-Shora [42]. Urease activity increases with substrate concentration. After reaching an optimum value, the urease activity decreases with rising urea concentration [39]. The rate of hydrolysis of urea is increased with an increase in urea concentrations until a maximum is reached, beyond which hydrolysis is decreased. The results can be explained by substrate inhibition at higher urea concentrations [43,44]. Pervin et al. has also studied the effect of various metal ions and chemicals on the activity of chickpea urease. The urease activity has increased in the presence of calcium ion at low concentrations like 3 mM or less and decreased at higher calcium ion concentrations. Divalent cations like Ba 2+ and Mg 2+ slightly stimulate the enzyme at a concentration of 1-3 mM and Na + and K + produce little or no effect on the activity [42]. Heavy metals, such as Cu 2+ , Zn 2+ , Pb 2+ and Hg 2+ almost completely inhibit the enzyme activity. This has also been proved for pigeon pea urease [45] and jack bean urease [46,47]. The urease enzyme activity is completely inactivated when the concentration of Hg 2+ is high enough [48]. Muskmelon (Cucumis melo) is another plant source of urease enzyme [49]. Kharbooza and Banggi are common names for muskmelon. Recently, urease from mulberry leaves (Morus alba) has also purified and characterized [15].

Microbial urease
Microbial (bacteria, fungi and algae) urease is an enzyme produced by ureolytic microorganisms and hydrolysis of urea is catalyzed in to ammonium and carbonate by this enzyme. The metabolic activities of these microbial sources promote the precipitation of calcium carbonate in the form of calcite crystals using produced carbonate, and calcium ions in the system. These crystals form bonds between

Soil urease
Soil ureases are partly extracellular being liberated during microbial and plant root metabolism and death. They are also intracellular as part of the soil biomass [54]. Soil urease activity is related to the vegetation of the soil [55]. Higher urease activity in soils under vegetation compared to vegetation free soils has been observed by Reddy et al. [56] and they attributed it to the higher microbial proliferation and microbial activity at the rhizosphere. The major factor that influence for the level and distribution of urease activity through the soil profile is the content of soil organic carbon [57,58]. However, this relationship is obviously modified within particular horizons by other soil properties such as reaction, gleying and soil texture [57]. According to the Myers and McGarity, urease activity in calcareous soils is lower than in non-calcareous soil. They have reported that in saline soils, urease activity is low [57]. Skujins has reported that in alkaline soils, the activity is much less, and it is also much less in carbonate rich soils, apparently because of detrimental effect of Ca 2+ on the urease producing organisms [59]. Numerous studies show that the urease activity in soils increases with the increase in temperature from 10°C to 40°C. In some soils, urease activity increases very markedly with the increase in temperature from 40°C to 70°C [37,60,61] but decreases rapidly above this temperature range [61,62]. The optimum pH of soil urease activity reported in past is 6.5 to 7.0 [63] and it is similar to that of jack bean urease [37,64,65].
Equilibrium between ammonium ion and ammonia gas occurs in aqueous solutions of ammonium salts [66]. When, enzyme activity is high in soil, the rate of hydrolysis of urea is increased and high soil pH and greater losses of gaseous ammonia can be observed. Although high ammonium ion concentration in soil is non-toxic, ammonia gas is extremely toxic to the plant [67]. Therefore, the loss of volatile ammonia and ammonia toxicity to germinate seedlings are major problems in urea fertilization.

Methods for Evaluating Urease Activity
There are several methods to determine urease activity such as manometric, titrimetric, colorimetric, potentiometric method as well as spectrophotometric method. Some of those methods are summarized in Table 2. In these methods, urease can be examined by measuring substrate/products of the ureolytic reaction or byproducts of this reaction, such as ammonia, an increase in pH or conductivity as well as generated heat. A number of excellent assays are available for quantifying urease activity and analyzing its kinetic behavior [28]. More popularly, ammonia release during the ureolytic reaction can be detected with phenol-hypochlorite [68], Nessler's reagent [32], diacetyl monoxime method or Ehrlich's reagent method to allow colorimetric determination of activity and can be detected by ion-selective electrodes. It is possible to include pH indicators/pH sensitive dyes in the assays or simply observe changes with pH electrodes since release of ammonia results in an increase in pH. The amount of ammonia can also be monitored spectrophotometrically using a couple system with NADH-dependent glutamate dehydrogenase (ammonia is a substrate for this enzyme). The other product of the reaction, carbon dioxide, can be trapped and monitored by radiological methods with 14 C-labeled urea as a substrate [69]. All of the methods described above are distinct in their sensitivities, ease of use, and susceptibility to interference. Two commonly employed assays mentioned above paragraph namely phenol-hypochlorite and Nessler's method are time consuming, incompatible with common buffers, require high temperature, non-homogenous true color solution and have the disadvantage of using harmful chemicals. Croston et al. explain some advantages, disadvantages and the cares that should be followed at titrimetric, pH change and conductimetric methods. The end point of the titrimetric method is not sharp and good results require extreme care in titration. The titration method requires appreciably more time than the others but gives direct measure of urease activity. The pH change method is simple in operation, however, the recorded differences between samples at room temperature may be very small and frequent checking of the meter is required to minimize errors. The sensitivity of the titrimetric and pH-change methods can be improved by using 50°C or 60°C reaction temperatures. However, from laboratory experiences of Croston et al. and his team, their opinion is that maximum precision is obtained by the conductimetric method [70]. It is easy to identify urease activity. However, quantifying the urease activity is time consuming. Although there are some analytical techniques to determine urease activity, the use of spectro-photometric method is still well popular. Specific urease activity can also be obtained quantitatively from conductivity method proposed by Whiffin [71]. This method of measuring conductivity is simple when compare with other methods found in literature. Wiffin's above method is suitable for measuring urease activity quantitatively, because this method is simple and not using expensive and harmful chemicals. Initially, this method has been used to measure urease activity of microorganisms [71]. We have used this conductivity method for measuring urease activity of watermelon (Citrullus vulgaris) seeds extract [72].

Immobilization of Urease Enzyme
The use of enzymes is limited due to their limited availability, high cost, instability and the limited possibility of economic recovery of these soluble biocatalysts from a reaction mixture. These problems can be solved by immobilizing of enzymes [73]. Enzyme immobilization can be defined as confining the enzyme molecules to a distinct phase Soil Absorbance (optical density) is measured at 690 nm wavelength, using the spectrophotometer *Urease activity is expressed as µg N hydrolyzed/g dry soil per 2 hour at 37°C. [133] Soil Absorbance (optical density) is measured at 690 nm wavelength, using the spectrophotometer *Urease activity is calculated as mole of ammonium released per hour per gram of soil. [134] Pararhodobacter sp.
Color change due to variation in pH *pH indicator: Cresol red solution (yellow to purple has been observed from pH 7.2 to pH 8.8) *Medium used to isolate microorganism in ZoBell2216E medium [135] Bacteria -*Christensen's urea medium was used [136] Bacteria Absorbance (optical density) is measured at 430 nm and 560 nm wavelengths, using the spectrophotometer *Urease activity as Units/ml enzyme (one unit of urease activity corresponds to the amount of enzyme that hydrolyzes 1µM of urea per minute) [137] from the one in which the substrates and the products are present. This may be achieved by fixing the enzyme molecules to or within some suitable material. It is critical that the substrates and the products move freely in and out of the phase to which the enzyme molecules are confined. Immobilization of enzymes can be done by various methods such as entrapment, physical adsorption, membrane confinement and covalent binding. Enzyme molecules do not necessarily render them immobile in some methods of immobilization. They move freely within their phase in entrapment and membrane confinement, and they are in fact, immobile in adsorption and covalent binding [74]. The materials used for immobilization of enzymes are called carrier matrices and they are usually inert polymers or inorganic materials. The ideal carrier matrix has low cost, inertness, physical strength, stability, and regenerability after the useful lifetime of the immobilized enzyme, enhancement of enzyme specificity, reduction in product inhibition, a shift in the pH optimum for enzyme action to the desired value for the process, and reduction in microbial contamination and non-specific adsorption. However, most matrices possess only some of the above features. Therefore, carrier matrix for the immobilization of an enzyme must be chosen with care keeping in view the properties and limitations of various matrices. In the process of adsorption, the enzyme molecules adhere to the surface of carrier matrix due to a combination of hydrophobic effects and the formation of several salt links per enzyme molecule. The binding of enzyme molecules to the carrier matrix is usually very strong, but it may be weakened during use by many factors such as addition of substrate, pH or ionic strength.
In covalent binding, the enzyme molecules are attached to the carrier matrix by covalent bonds. As a result the strength formation occurs with the side chains of amino acids of the enzyme, their degree of reactivity being dependent on their charged status. Roughly, the following relation is observed in reactivity. -S > -SH > -0 > -NH 2 >-COO > OH >> -NH 3 + . In the case of entrapment, enzyme molecules are held or entrapped within suitable gels or fibers and there may or may not be covalent bond formation between the enzyme molecules and the matrix. A non-covalent entrapment may be viewed as putting the enzyme molecule in a molecular cage just as a caged bird/animal. When covalent binding is also to be generated, the enzyme molecules are usually treated with a suitable reagent. In membrane confinement, enzyme molecules in an aqueous solution may be confined within a semipermeable membrane which, ideally, allows a free movement in either direction to the substrates and products but does not permit the enzyme molecules to escape [74]. Among the techniques used for immobilization, entrapment is favored due to various reasons. Non toxicity of the matrix, variation in the bead size of gel and high percentage of immobilization are some reasons. Considering these things, calcium alginate mediated entrapment has attracted much attention [75]. Immobilization changes original enzyme properties like storage stability, kinetic parameter and customizes them for specific applications [76,77]. Furthermore, immobilization of enzymes enhances their thermal stabilities [54]. For example, glucose isomerase denatures at 45°C in solution, however, it is stable for about 1 year even at 65°C when suitably immobilized. Recovery of enzyme may also reduce effluent handling problems [74]. Enzymes can be used repeatedly only if they can be recovered from the reaction mixtures. Immobilization permits their repeated use since such enzyme preparations can be easily separated from the reaction system. Immobilized enzymes can be used in non-aqueous systems as well. Continuous production systems can be used, which is not possible with free enzymes and enzymes can be used at much higher concentrations than free enzymes.

Use of Plant-derived Urease in Geoenvironmental/ Geotechnical Engineering
Nowadays, the urease enzyme is widely used in different fields of industries such as medicinal, construction, agricultural, food, etc. Different kinds of ureolitic bacteria and micro algae, soil urease and plant urease have applied for the above fields. Currently, the potential for using plant-derived urease enzyme to precipitate carbonate (mainly calcite) through urea hydrolysis in several environmental and geotechnical engineering applications has been demonstrated. The aim of this section is to summarize the existing and potential applications in CaCO 3 precipitation using plant-derived urease.

CaCO 3 precipitation
CaCO 3 (calcite) precipitation may be achieved by many different processes and enzymatic hydrolysis of urea is the most energy efficient process [78] and it has been proposed as an engineering technique in the early 1990 [79][80][81]. Calcite precipitation is a relatively straightforward chemical process controlled mainly by four key elements. These are the concentration of calcium ions, concentration of dissolved inorganic carbon (DIC), pH and availability of nucleation sites [71,82,83]. In addition, several environmental parameters such as salinity, temperature may also affect for the performance of calcite precipitation [81,[84][85][86]. The rate of calcite precipitation is also depending on the injecting rate of chemicals to the soil and the rate of pH rise to prompt precipitation [87]. The rate of precipitation helps to achieve uniform cementation in situ. Higher rates of calcite precipitation lead to plugging at the injection source and slower rates of precipitation allow uniform delivery of the chemicals. Cementation between soil particles can be improved by adding impurities which can change crystal form as found in nature [88]. As an example, the presence of Mg can create Mg-calcite having different crystals from those of calcite [89]. Larger amount of Mg contribute to form dolomite (CaMg (CO 3 ) 2 ) or magenesite (MgCO 3 ). Both of them can resist against acid more than calcite [90]. The resistance against acetic acid is stronger for the cementation with Mg/Ca ratio of 0.5 than calcite without magnesium, and UCS value estimate as 3.2 MPa for Mg/Ca ratio of 0.5 and 1 MPa for calcite without magnesium [91]. Ismail et al. explain that the shape and the angularity of the soil particles have a great influence on the strength increase. The rounder and smoother particles have larger contact point result in a lower consumption of calcite crystals in order to achieve the same strength as for sharp and pointy particles [92]. Soil particle size is also a factor that effects for the precipitation of calcium carbonate [87]. Well graded and coarser sands have a higher rate of precipitation than finer and poorly graded sands. Very coarse and very fine soils like gravel and silts respectively also take a much longer time to increase shear wave velocity due to the limited rate of permeability in fine soils and the limited number of particle contacts in the very coarse soils. Calcium carbonate forms three anhydrous polymorphs named as calcite, aragonite and vaterite, three other hydrated crystalline phases named as mono hydrocalcite (CaCO 3 •H 2 O), ikaite (CaCO 3 •6H 2 O), and amorphous calcium carbonate (ACC) with differences in short range order and degree of hydration [93][94][95][96][97]. Rodrigues-Navarro et al. explains that the efficiency of bio deposition treatment depends on the type and structure of the precipitated CaCO 3 polymorphs (Vaterite or calcite). More pronounced consolidating effect can be seen in welldeveloped calcite crystals rather than tiny acicular vaterite crystal [98].
have made during past years to diminish the hydraulic conductivity of soils, to reinforce the loose soils and to prevent soil erosion. However, all of these applications are limited to laboratory scale and any kind of field application has not reported so far. Calcite precipitation using jack bean urease has been investigated to improve the mechanical properties of sand [99]. As a result of that, unconfined compressive strength (UCS) of 317 kPa has been achieved, compared to that of sand without jack bean urease. The highest strength of the specimen has been obtained using calcium chloride stock solution rather than the other stock solutions of calcium sources (calcium hydroxide and calcium nitrate). Nemati and Voordouw have investigated plant urease induced calcite formation, plugging studies in an unconsolidated porous media system and plugging studies of Berea sandstone in a core-flooding system [100]. Jack bean urease having urease activity of 26,100 Units/g solid has been used for the study. According to the results of the study on plugging of unconsolidated porous media by enzymatically formed CaCO 3 , a significant decrease in permeability of porous media has been observed. At low concentration of enzyme (0.03 g/l), the extent of plugging enhance with the increase in temperature from 22°C to 30°C. Although the temperature is a factor that influence for carbonate precipitation, its influence is less with high concentrations of enzyme used in this study. However, Nemati and Voordouw, explain that proportional increase of reactants and enzyme concentrations above a certain level inhibit the urease activity and decrease the quantity of produced CaCO 3 and plugging of porous media. Another attempt to find a method to create building materials using precipitated CaCO 3 has been done by Bull with the aid of jack bean (Canavalia ensiformis) urease from Sigma-Aldrich Co. LLC. Specific activity of the jack bean urease used in the study is 15,000-50,000 Units/g solid. They have obtained uniform cemented specimens by dissolving urea and CaCl 2 in one solution and urease in another solution. After curing, and air drying for 2 weeks, maximum UCS of 319 kPa and an elastic modulus of approximately 10 MPa have been obtained. Bull has also observed that CaCO 3 cementation is not successful when solutions with high concentrations of CaCl 2 and urea [101]. Hence, it is beneficial to study tolerable limits of concentrations of each solution for a better output. Neupane et al. have considered plant urease (jack bean) induced CaCO 3 precipitation technique for both small scale laboratory samples and samples in drum cans with a diameter of 56 cm and a height of 85 cm to evaluate the applicability of this technique for larger-scale applications. They have reported that the amount of precipitated calcite helps to modify the mechanical properties of sandy soil. However, mechanical tests using the same grouting condition have not conducted [102]. Hamdan et al. have conducted laboratory column tests (Acrylic tube tests and Triaxial tests) for cemented specimens made using silica sand, urea, calcium chloride and plant-derived urease, and obtained significant strength increase over non-cemented specimens at the same relative density. X-Ray Diffraction (XRD) analysis and Scanning Electron Microscopy (SEM) observations confirm that calcium carbonate (specifically calcite) is the cementing agent, and acid digestion perform to determine the amount of CaCO 3 [103]. In addition to the urease from jack bean, some investigations have been done by using other types of seeds urease. Sword beans (Canavalia gladiate) urease is an example. The permeability as well as strength characteristics have been examined using sword beans urease [104]. The permeability of the improved samples has been reduced by more than one order of magnitude. Maximum unconfined compressive strength value obtained is 1620 kPa. An increase in compressive strength up to 1.6 MPa has also been obtained for oven dried specimens made using commercially available urease, with the activity of 2970 Units/g [105]. The strength enhancement and permeability reduction of subsurface soils are essential features for geotechnical engineering applications. It is necessary to achieve UCS value of 100 kPa to avoid ground liquefaction during earthquakes [106]. This target value is successfully achieved by the past research studies based on plant-derived urease induced calcite precipitation. Hence, this technique can be applied for ground reinforcement. However, it is necessary to investigate the initial ground condition for soil type, soil pH, contaminants in the soil, permeability characteristics, etc. before applying this technique in-situ in large scale. The homogenous, well-controlled distribution of CaCO 3 should also be achieved prior to the real field applications. Therefore, future investigations should focus on these areas to get maximum output from this technique. We studied about plant-derived urease (seeds of watermelon) induced Calcium Phosphate Compound (CPC)  precipitation instead of CaCO 3 precipitation [72]. CPC precipitation mechanism is highly dependent on its pH ( Figure 1) [107] which can be increased by catalyzing the hydrolysis of urea using plant-derived urease. There are 11 CPCs with various calcium-to-phosphate (Ca/P) molar ratios in the ternary system of Ca(OH) 2 -H 3 PO 4 -H 2 O (Table 3). All CPCs are easy to dissolve in acidic solution and tend to precipitate in pH around 8-10. As an example, highest precipitation (least Ca 2+ ion concentration) of DCPA can be obtained at pH around 8 as indicated by arrows in Figure 1. It is required alkaline pH more than around 8 for precipitating CPCs other than DCPA and DCPD. Gel-like or amorphous CPCs change into HA over time ( Figure 2) [107,108]. The change rate is dependent on both pH and the concentrations of calcium and phosphate ions. HA forms directly in solutions with low super saturation levels (indicated by arrow 1 in Figure 2), as the concentration of phosphate ions increase, less stable phases of calcium phosphate compounds (e.g. ACP and OCP) form initially and transform into HA over time (indicated by arrows 2-4 in Figure 2). Therefore, CPCs hardens after injection into soil and rock because of the self-setting mechanism [109] and hardening rate can be controlled by pH as well as phosphate ion concentrations. Out of all CPCs, the highest strength can be obtained from HA. CPCs exist as phosphate rocks (mainly fluoroapatite) in the natural environment and as an important inorganic substance (mainly hydroxyapatite, HA) in living organisms [110]. Phosphate and calcium stock solutions can be made from fertilizers, and calcium and phosphate can also be extracted from the bones of livestock and the shells of marine animals, [111]. CPCs that precipitate after grout injection are non-toxic. Unlike concrete, reexcavated muck that consists of soil, rock, and CPC grout is recyclable as an agricultural fertilizer. These advantages make it suitable for geotechnical applications [111]. In our study, UCS of more than 100 kPa was obtained by selecting superlative proportions of urea and urease enzyme with calcium acetate and dipotassium phosphate as calcium and phosphate stock solutions, respectively. In CPC precipitation method, it is very important to select superlative proportions of urea and urease enzyme for obtaining a pH for highest precipitation of CPC. This novel application may be important for the biocementation using plant-derived urease in future. The present study was carried out as a fundamental study on improving the strength of sand by using CPCs. We are planning to conduct further laboratory studies on CPCs for different types of soils having different permeability characteristics to use as a practical-scale experiment in actual ground in future. Most of the studies done in literature were focused on urea as ammonia source to complete the carbonate precipitation process. However, urea is a key raw material to produce fertilizer. Hence, the price of urea may become high with the demand for fertilizer. Under economical point of view, it is problematic to use urea at the carbonate precipitation process [112]. Therefore, it is economical to investigate carbonate precipitation using novel ammonia sources. Soil erosion due to wind and surface water runoff is a major environmental issue as it contaminates rivers, streams and ground water, and deposition of eroded soil in streams and rivers cause to unexpected flooding. Plantderived urease induced carbonate cementation has been proposed as a promising application for the stabilization of soil against wind and surface water erosion [23]. Knorr has obtained significant strength and relatively high resistance to water erosion due to carbonate cementation. This is a significant evidence to study this method deeply as an alternative method for controlling dust through carbonate precipitation. It is clear that the urease induced calcite precipitation can be expected at the presence of calcium ion in the system with suitable conditions such as pH, temperature, etc. It has a potential to be used to reduce water hardness. The amount of water hardness depends upon the amount of dissolved ions of calcium and magnesium in the water. The presence of a high concentration of calcium ions (500-1500 mgl -1 ) in the water causes severe problems [113]. The hard water can cause to precipitate minerals as carbonate, phosphate and/or gypsum in pipelines causing many problems [113] and reduce the effectiveness of use of soap for bathing and laundry. There are many commercial ways of treating hard water including water filters, water softeners, electromagnetic water conditioners and reverse osmosis [114]. These methods are complex and expensive to use in large scale. Plant-derived urease induced calcite precipitation may be a good solution. It is nontoxic and no need to remove bacteria as use during microbial urease. However, it is necessary to investigate the functionality of plant urease under different temperatures, and tolerable concentrations of calcium ions in the system. Hammes et al. (2003) have considered the potential of removing Ca 2+ ions from industrial waste waters by MICP. The calcium ions removal up to 90% from the waste water can be achieved by adding low concentrations of urea (0-16 gl −1 ) [115]. This kind of approach can also be considered using plant urease. Future investigations should be focused on novel sources of urease to deal with many environmental issues with sustainable and eco-friendly manner. Jroundi et al. explain that a lot of the world heritage listed buildings and statuaries are built out of carbonate stones especially marble and limestone. These are typically susceptible to physical erosion, chemical weathering and bio-deterioration which lead to a threat for the cultural and historical heritage. It has a possibility to apply the plant-derived urease induced carbonate precipitation technique to create a consolidated surface layer by spraying the biogrout solution straight onto the surface of the buildings or statuaries in order to prevent further erosion. This kind of preservation would take place through the strengthening of the existing bonds of the sediments in the existing stone [116]. With rapid urbanization, soil and water pollution are threatening not only to human health but also to the entire living organisms in environment. In order to maintain a good quality of soil and water and keep them free from contamination, continuous efforts have made to develop technologies to remove contaminants. Physicochemical methods are widely used for remedying polluted soil and water, especially at a small scale. However, high costs and side effects may cause to limit those methods for a large scale of remediation. Ureolytically-driven calcium carbonate precipitation is a basis for a promising in-situ remediation of contaminants. Co-precipitation in calcium carbonate is one attractive in-situ remediation strategy for divalent radionuclide and trace metal ions, such as 60 Co, 137 Cs, and 90 Sr. The reactions involved in this process are as follows [117].
(1) Enzymatically catalyzed urea hydrolysis produces NH 4 + and   During calcite precipitation, heavy metal ions with ion radious close to Ca 2+ , such as Sr 2+ , Pb 2+ , Cd 2+ and Cu 2+ may be incorporated in to the CaCO 3 crystal by substituting the Ca 2+ in the lattice or entering the interstice or defect of crystal. Therefore, plant-derived urease induced CaCO 3 precipitation can be used as a remediation technique in heavy metal removal.

Conclusions
This paper reviews intensively about the great potential of plantderived urease induced calcium carbonate precipitation as providing the way forward for the problems encountered in the field of geoenvironmental/geotechnical engineering as sustainable and ecofriendly manner. There are several potential applications of carbonate precipitation via plant-derived urease. Some of these applications are ground improvement by strength development of weak unconsolidated soil, removal of contaminants (e.g. radioactive pollutants) and calcium ions from groundwater and wastewaters, protection and restoration of limestone monuments and statuaries, plugging the pores of the oil-recover reservoir rock, stone formation (like sandstone), etc. The methodological summary and results of laboratory scale practices using plant-derived urease presented in this paper will have important implications for the design of technologies using plant-derived urease and to embark on the current research work as a possible alternative to the well-known bacterial approaches. In literature, there are limited amount of laboratory studies on plant-derived urease induced carbonate precipitation. More exploratory works at laboratory scale should be carried out to determine the effectiveness of the carbonate precipitation via plant-derived urease for industrial/ field applications. The major challenges in this novel area for field applications include assessment of subsurface soil condition including soil type, pH, mineralogy and their interaction with the available fluids and minerals, ground water flow and available minerals. The optimal balance of substrates use for various applications should be studied to increase the economic feasibility and to reduce the production of unwanted byproducts (ex. ammonia production during hydrolysis of urea). Therefore, additional researches are also necessary to overcome these problems. It is necessary to investigate the durability, longevity and reversibility of the carbonate precipitation process under economical point of view. Furthermore, comparative studies should be done to check the feasibility of this method with that of the chemical methods which include environmental impacts as well as high cost. The potential of these bio minerals has brought a new revolution in various engineering applications but still there are some things to explore in order to bring this method environmentally safe, cost effective and to develop as a convenient technology from lab to field scales.