Efficient option of industrial wastewater resources in cement mortar application with river-sand by microbial induced calcium carbonate precipitation

The industrial wastewater disposal has been growing attention for environmental protection and resource substitution, current decades. Similarly, the durability enhancement of concrete has increased attention by microbial induced CaCO3 precipitation (MICP) process (biocalcification). However, ecofriendly utilization of industrial wastewater in concrete formation is unstudied so far. The present study was carried out to evaluate the effect of industrial wastewater on the formation of cement mortar, compressive strength and water absorption. The biocement mortar strength (y) increased (y = 0.5295×2 + 1.6019×+251.05; R2 = 0.9825) with increasing percentage of organic wastewater (x) (BM0 – BM100) by MICP, where highest strength (280.75 kgf/cm2) was observed on BM100 (100% wastewater), compared to control (252.05 kgf/cm2). The water absorption (y) of biocement mortar decreases (y = −0.0251×2–0.103× + 15.965; R2 = 0.9594) with increment of wastewater (x) (%) (BM0 – BM100), where a minimum-water-absorption (14.42%) observed on BM100, compared to control (15.89%). SEM micrograph and XRD shows the formation of most-distinctive CaCO3 crystallization (aragonite/calcite) (acicular, brick shape, massive and stacked structure) inside biocement mortar (BM100), which fills the pores within cement mortar to form a denser structure, by microbial organic wastewater. Thus, present findings implied a cost-effective of MICP technology to improve the concrete properties along with the mitigation of industrial wastewater pollution, which goes some way towards solving the problem of industrial wastewater pollution.

In natural environment, small sand grains solidify to become sandstone under biogeochemical process (microorganisms, time, and pressure). At adequate calcium concentration, the calcium reacts with carbonate ions to precipitate calcium carbonate that acting as a gluing agent, where the calcium carbonate helps the transformation process of sand solidification. In this process, the microorganisms metabolize and produce urease that transform the urea into ammonium and carbonate ions 10,11 . This bio-mineralization occurs by microbial induce calcium participation, (MICP) process [12][13][14][15] .
The industrial wastewater is one of the complications to ecofriendly progress in human civilization. Carelessly discharging the wastewater into water bodies are affecting the physical, chemical, and biological changes to the environment, since it (wastewater) is not only harmful to the environment but also to human health. Therefore, industrial sectors are obligated to install comprehensive wastewater processing/treatment system. Such system often requires proper equipment, ecofriendly technology, as well as funding for wastewater treatment to permissible limit, that often increase the production cost 16,17 . However, the ecofriendly wastewater treatment is a great challenge in present-day research. Thus, it is an urgent need to utilize of wastewater for beneficial purpose of ecofriendly environment pollution management. The investigator utilized the urase enzyme of urealytic microorganism (example: Sporosarcina pasteurii and Bacillus sphaericus) for MICP process, through which the mineral can precipitate [18][19][20][21][22] . The organic wastewater of food industries, contains microbial nutrients and a mass amount of microorganism (Example: bacteria), may produces urease in wastewater 23 . Thus, the urease containing wastewater can be used for MICP process by the possible replacement of the aforementioned microorganism/ bacteria that may benefits of ecofriendly utilization of industrial wastewater with cost minimization of industrial product 23 . Furthermore, the utilization of industrial wastewater could reduce the cost for wastewater processing in industries where, the main problem of MICP has always been high-cost.
Considering the background, the present study focuses on the utilization organic wastewater for MICP process to produce biocement mortar. The wastewater of food industry was used to produce biocement mortar. In addition, the mechanical properties such as strength and water absorption, as well as the physiochemical properties of the biocement mortar by SEM and XRD, were taken under study.

Results and Discussion
Microbiologically induced calcite precipitation by industrial wastewater. Microbiologically induced calcite precipitation and characterization of industrial wastewater. The basic characteristics of industrial water is shown in Table 1. The industrial wastewater was noticed slightly higher pH at 8.40 ± 0.5. COD of wastewater was found significantly high as 1200.10 ± 2.5 mg/l compare to BOD (10.50 ± 0.12 mg/l). This is indicated that the higher amount of oxygen required to chemically oxidize organic compounds compare to the amount of oxygen required to biologically oxidize the organics in the industrial wastewater. The temperature of wastewater was observed 30.90 ± 0.89 °C. The CFU of wastewater was noticed as 10 3 to 10 5 (cfu/ml), where as the urease activity was observed as 0.894 ± 0.01 (mol/l). The results of the varied parameters of urea and Ca(NO 3 ) 2 with 40 ml of wastewater in different experimental conditions (C 1 -C 4 ) ( Table 2) shows that the precipitation was increased with the increasing urea and Ca(NO 3 ) 2 concentration, significantly (y = 0.3568ln(x) + 0.0241; R² = 0.9747) (Fig. 1). The precipitation was not occurred in control condition due to absence of urea and Ca(NO 3 ) 2 . Thus, the urea and Ca(NO 3 ) 2 are essential for microbiologically induced calcite precipitation in wastewater, which are very active in precipitation process. Hammes et al. 24 , reported that the strain-specific calcification occurred during ureolytic microbial carbonate precipitation by Bacillus sphaericus. In another study, the Ca 2+ remove from industrial wastewater by MICP process through ureolytic microorganisms 25   Formation and characterization of biocement mortar. Formation of biocement mortar and compressive strength. The organic wastewater, which contain microbial consortium that influences on water adsorption rate and compressive strength of biocement mortar ( Fig. 3a-d). The study highlights the effect of organic wastewater on the compressive strength of cement mortar. The effect of organic waste water on compressive strength of cement mortar are shown in Fig. 3a Table 2). Bar diagram represents mean ± SD, where n = 3. www.nature.com/scientificreports www.nature.com/scientificreports/ BM 60 , BM 80 and BM 100 ). The regression analysis in between independent variable as wastewater percentage (x) and dependent variable compressive strength (y) reflects a positive polynomial relation (R 2 = 0.9825) (i.e. compressive strength increases with the increasing of wastewater percentage). The cement mortar treated with 100% wastewater was observed the highest strength (280.75 kgf/cm 2 ) compared to control after 28 days. Therefore, the strength of biocement mortar and CaCO 3 precipitation were increased with the increasing amount of organic wastewater (Fig. 3a,b). The current findings of compressive strength are comparable with the finding of Chahal et al. 18,28 , where researchers were indicated the compressive strength was increased in presence of microorganism (S. pasteurii). The optimum compressive strength was reported 10 5 cells/ml, whereas the matrix integrity disrupts due to excessive bacterial activity at 10 7 cells/ml 18,28 . In another research 29 , the compressive strength of bacterial concrete was reported to be increased in 10 3 -10 5 (cfu/ml), whereas the strength was found to be decreased in/ after 10 7 (cfu/ml), compared to the concrete sample without bacteria. In the present study, 10-10 3 (cfu/ml) of bacteria are survived after mixing with cement, which produced the urease enzyme. It is confirming that the reason may be that urease enzyme (produced by bacteria in organic wastewater in Table 1) reacts with urea and www.nature.com/scientificreports www.nature.com/scientificreports/ calcium nitrate which can produce calcium carbonate precipitation (see XRD of material in previous section) 22 . The cement mortar provides additional pores (during hydration reaction), where the calcium carbonate is predicated and fully filled the porosity of biocement mortar. Although the chemical substances contained in organic wastewater which decreases the binding rate of calcium and citrate in hydration reaction as well as produces a retarding effect. However, in addition of organic wastewater totally (100%) that can effectively improve the strength of cement mortar compare to control.
Formation of biocement mortar and water absorption. Similar to the compressive strength, the organic wastewater influences on the water absorption capacity of biocement mortar formation. The effects of organic wastewater on water absorption in cement mortar are shown in Fig. 3. The water absorption of biocement mortar was noticed as 15.89% at 28 days without organic wastewater, whereas 15.51%, 15.58%, 15.06% and 14.87% of water absorption in biocement mortar were observed in the treated group of 20%, 40%, 60% and 80% organic waste water, respectively at 28days. The biocement mortar treated with 100% wastewater was observed a water absorption of 14.42% after 28 days. A polynomial relationship (y = −0.0251× 2 -0.103×+ 15.965) was observed between water absorption and increment of wastewater percentage (%) (BM 0 , BM 20 , BM 40 , BM 60 , BM 80 and BM 100 ). The regression analysis in between independent variable as wastewater percentage (x) and dependent variable water absorption (y) reflects a negative polynomial relation (R 2 = 0.9594) (i.e. water absorption decreases with the increasing of wastewater percentage). Thus, the water absorption decreases as the proportion of wastewater increases in the treatment process of biocement mortar formation. Chahal et al. 28 observed a four-times reduction of water absorption in fly ash concrete with 10 5 cells/ml of S. pasteurii. In another study, Chahal et al. 18 reported a maximum reduction of water absorption with 10 5 cells/ml for 10% silica fume concrete at 91 days; however, concrete with 5% silica fume gave 0.1% water absorption (minimum) at 91 days, which was 0.3% at 28 days. The waterproofing effect was reported to increase with increasing calcium dosages in the presence of Bacillus sphaericus LMG 225 57, whereas for a while the calcium dosage of 17 g Ca 2+ m −2 the water absorption was reported similar to that of untreated cases. in a 50% decrease of the rate of water absorption was reported at a concentrations of 67 g Ca 2+ m −2 7 . In another report, the surface deposition of calcium carbonate crystals decreased the water absorption from 65% to 90% depending on the porosity of the material by B. sphaericus 30 . The ureolytic bacteria such as Bacillus sphaericus are able to precipitate CaCO 3 in their micro-environment by conversion of urea into ammonium and carbonate. Thermogravimetric analysis showed that bacteria were able to precipitate CaCO 3 crystals inside the cracks, as a result the permeability of the biocement mortar decreased 31 . In present study shows that the urease activity plays an important role of the CaCO 3 formation. The urease activity (mol/l) was observed in wastewater, which was increased significantly upto 60 min; however, the activity was decreased a bit with the decreasing concentration of wastewater (Fig. 3d). On the other hand, the urease activity was noticed higher in the mixer of cement with wastewater, compare to only waste water (Fig. 3e). Therefore, urease activity helps to precipitate the calcium carbonate to the mixture of biocement mortar. The water absorption decreased with the increasing of wastewater concentration or CaCO 3 formation (Fig. 3c). Thus, these results reflect the formation and precipitation of calcium carbonate from urea and calcium nitrate in presence of urease from bacteria in organic wastewater. The Fig. 3f shows that the porosity of the biocement mortar decreases with the CaCO 3 precipitation and it is confirmed that the precipitated calcium carbonate effectively fills pores on and within (inside) the surface of the biocement mortar. Therefore, the investigation documents the calcium carbonate precipitation as a result reduction of water absorption on and within biocement mortar, which provides a hopeful solution for durability of cement. Furthermore, the precipitation of calcium carbonate could also fill the pores inside the cement mortar which increases the density and structural strength of the cement mortar.
X-ray diffraction (XRD) analysis of biocement mortar. Figure 4 shows the of XRD result of the cement mortar. The quartz phase was observed the peak intensity at 2θ for the value around 26.63°, and 68.3° representing www.nature.com/scientificreports www.nature.com/scientificreports/ the Miller index of (101) and (301), respectively. The peak intensity at 2θ for the value around 29.399°, 39.42°, 43.17°, 60.68°, and 81.5° representing the Miller index of (104), (113), (202), (214), and (2110), respectively for the formation of calcite phase. The observed aragonite and calcite are the products of calcium carbonate, which are formed in biocement mortar, influences by microbial organic wastewater; and further confirmed of a white powder which is calcium carbonate. The calcite peak intensities of biocement mortar (Fig. 4) treated from BM 20 to BM 100 are noticed evidently higher compare to BM O , which indicates the addition of organic wastewater is relevant to changes the amount and crystallization form of calcium carbonate. In comparison of compression strength and water absorption, the present result confirms the calcium carbonate precipitation through a biochemical process in presence of urea, calcium nitrate and urease (which generated from microorganisms in wastewater). Furthermore, the precipitated calcium carbonate can fill the pores of cement mortar that formed during cement-hydration reactions.
Morphology of biocement mortar. The morphological signature (SEM micrograph) of the biocement mortar containing 0% (BM O ), 20% (BM 20 ), 40% (BM 40 ), 60% (BM 60 ), 80% (BM 80 ), and 100% (BM 100 ) of wastewater are shown in Figs. 5 and 6. SEM-EDX micrograph shows the acicular, massive and stacked calcite structure in cement mortar; in particular, the needle shape, brick shape, and stacks of calcite crystals were observed inside cement mortar. Results shows at a higher proportion of waste water; the crystallization of calcite is more evident/pronounced. The most distinctive calcite crystallization is formed treated with 100% (BM 100 ) waste water, where calcite crystals can fill the pores within the cement mortar to form the denser structure. This result can be mutually confirmed with the results of the strength and water absorption of cement mortar (see previous section). It is clear that the biologically produced calcite, precipitates within the concrete void and block pores/voids, thereby increasing the strength. Ghosh et al. 32 , reported that a thermophilic anaerobic microorganism increases the compressive strength of 25% in cement mortar in 28 days with the addition of about 10 5 cell/ml of water. The strength improvement was reported www.nature.com/scientificreports www.nature.com/scientificreports/ due to growth of filler material within the pores of the cement-sand matrix by microbial growth and the process of microbiologically induced mineral precipitation 32 . In another research report, the B. sphaericus improves strength of cement concrete, where concrete-immobilized bacterial spores and able to seal the cracks by biomineral formation after being revived by water and growth nutrients 33 . The potential crack healing ureolytic bacteria (example Bacillus sphaericus,) are able to precipitate CaCO 3 in their micro-environment by conversion of urea into ammonium and carbonate; as a results the cracks were filled completely 31 . Sujatha et al. 34 reported a indigenous soil bacteria which enhance the compressive strength of cement mortar by precipitating the calcium carbonate mineral; as 18% of compressive strength was increased with 28 days, where the bacteria transformed soluble organic nutrients into insoluble inorganic calcite crystals (applicable for repair for concrete cracks). Hence, the present investigation reflects a positive direction of the application of microbial consortium of wastewater, which can be applicable and improve the strength, durability and repair of concrete cracks of cement concrete (Fig. 7).

Conclusion
The industrial wastewater (10 3 -10 5 cfu/ml) was applied to enhance the durability of biocement mortar such as compressive strength, water absorption by microbial-induced calcium carbonate precipitation (MICP) (biocalcification). The 'strength' of biocement mortar increased (R 2 = 0.9825) and 'water absorption' of biocement mortar decreases (R 2 = 0.9594) with the increasing percentage (%) of organic wastewater by MICP process. The highest 'strength' (280.75 kgf/cm 2 ) and lower 'water absorption' (14.42%) was noticed in addition of 100% wastewater after 28 days. Morphological study reveals the acicular, massive and stacked calcite structure in cement mortar samples; in particular, the needle shape, brick shape, and stacks of calcite crystals were observed inside cement mortar. XRD analysis indicated the formation of calcium carbonate (aragonite and calcite) in biocement mortar which influences by hydrolysis of urea, catalyzes by microbial enzyme of urease in MICP process using microbial organic wastewater. The crystallization of calcite is more evident/pronounced in higher proportion of wastewater. The most distinctive calcite crystallization is formed in the samples of 100% (BM 100 ) waste water, where calcite crystals fills the pores within the cement mortar to form the denser structure. Thus, the findings implied a cost-effective of MICP technology to improve the permeability of concrete and thereby enhancing the life of concrete structures along with the mitigation of industrial wastewater pollution, which also goes some way towards solving the problem of industrial wastewater pollution.

Methods
Characterization of industrial wastewater. The food industrial wastewater was collected from "Grape King Bio" company (wastewater release 8462 tons per month) and used to produce biocement mortar by MICP process. The basic wastewater parameter such as pH (HI 9828 Multiparameter, HANNA, Taiwan), COD (Chemical Oxygen Demand) (mg/l) (NOVA-60, MERCK), BOD (mg/l) (Biological Oxygen Demand) (NOVA-60, with shaking at 120 rpm for precipitation. The precipitates were collected by centrifuging at 5000 rpm and dry at 50 °C for 3 days. The dry powder was weighted by gravimetric method and store for further study. The chemical character synthesized powder particle was measured by XRD analysis. The urease activity in wastewater was measured following the procedure of Chen et al. 14 . Preparation of biocement mortar. A standard Portland cement (produced by "Taiwan Cement"; Type-Ι, specific weight: 3.15) ( Table 3) and natural river sand (Table 4; Fig. 8) was used for biocement mortar experiment. Both of natural water (as control) and industrial wastewater was used for the formation of biocement mortar considering the ratio or proportion as 0.6 [water to cement (W/C)] (Table 5). Since the formation of pores in cement mortar by cement-hydration reactions are small to survive 8,31,35 the microorganisms, it is necessary a significant larger pore size within the cement mortar for MICP process in building materials 5,8,31,35 . Therefore, in the present study, the river sand (<0.075 mm) (grains size distribution is shown in the Fig. 8) was used into the mortar to form larger pores that could improve the survival of microorganisms for MICP. The biocement mortar was prepared using fixed concentrations of urea and Ca(NO 3 ) 2 at 1.1 M (consider as per standardized results of the highest precipitation in MICP process from section "Experimental procedure of MICP process"), while 40% of river sand was used in mortar. The industrial wastewater was used in the range of 20-100% (with 20% interval), where a control experiment (BM 0 ) was design with 100% natural water. The different composition of cubic shapes of biocement mortar (BM 0 , BM 20 , BM 40 , BM 60 , BM 80 and BM 100 ) were prepared to optimized the MICP within the biocement mortar cube. In each composition (as per Table 3) of biocement mortar, the Portland cement and natural sand was mixed with low speed (140 ± 5 rpm) for 1 min, and then the natural water and organic wastewater was mixed as well as stirred for 1.5 minutes before switched to medium speed (285 ± 10 rpm) for 1 min. The mixture was cast in a 125 cm 3 (5 cm×5 cm×5 cm) cube mold for 24 hours with the water-cement ratio (W/C) of 0.6. After demolding, the cubic sample preserved for 28 days at 70 ± 2% RH and 20 °C ± 2 °C for further study. The urease activity of different percentage (%) of wastewater in different set of samples (BM 0 , BM 20 , BM 40 , BM 60 , BM 80 and BM 100 ) were measured following the procedure of Chen et al. 14 . Also, the urease activity of the mixer of cement and waste water was estimated. The porosity of the bicement mortar was measured following the procedure of Emamian and Eskandari-Naddaf 36 .
Assessment of biocement mortar properties. Estimation of compressive strength of biocement mortar. The measurement of compressive strength of cubic biocement mortar (BM 0 , BM 20 , BM 40 , BM 60 , BM 80 and BM 100 ) are conducted according to CNS1010 R3032 37 . The measurement of cubic biocement mortar was conducted with 3 replicates; repeated for 3 times (YS/5001-25 T, YENSTRON, Taiwan). Center of the samples is placed in the compression testing machine for testing and the compression load is increased at a speed of 0.5 mm/min until the sample can no longer sustain the compression, and the structural integrity is damaged. To calculate the compressive strength of the sample, the maximum load was recorded, and divided by the cross-section area of the sample. www.nature.com/scientificreports www.nature.com/scientificreports/ Estimation of water absorption of biocement mortar. The change of water absorption by CaCO 3 precipitation that may occurs by the MICP process within the biocement mortar cube and fills the pores of the cement mortar samples. The water absorption test was conducted on cement mortar samples (BM 0 , BM 20 , BM 40 , BM 60 , BM 80 and BM 100 ) following the procedure of ASTM C642 38 . The measurement of water absorption of the biocement mortar cube was carried out by drying the biocement mortar cube to a constant temperature at 110 °C in an oven, and the gravimetric weights were measured at 24 h intervals until the mass balance between initial and final weight less than 0.5%. The dry biocement mortar cubewas then immersed in water at 21 °C for 48 h, and after taking out, the surface was wiped dry, and the mass of the saturated substance after the immersion was calculated.

Test results Specification
Type-Cement