Removal of cadmium and arsenic from water through biomineralization

Due to anthropogenic activities, heavy metals such as cadmium (Cd) and arsenic (As) are one of the most toxic xenobiotics contaminating water, thus affecting human health and the environment. The objective of the present investigation was to study the effect of ureolytic bacteria Bacillus paramycoides-MSR1 for the bioremediation of Cd and As from contaminated water. The B. paramycoides showed high resistance to heavy metals, Cd and As, with minimum inhibitory concentration (MIC) of 12.84 μM and 48.54 μM, respectively. The urease activity and calcium carbonate (CaCO3) precipitation were evaluated in artificial wastewater with different concentrations of Cd (0, 10, 20, 30, 40, 50, and 60 μM) and As (0, 20, 40, 60, 80, and 100 μM). The maximum urease activity in Cd-contaminated artificial wastewater was observed after 96 hours, which showed a 76.1% decline in urease activity as the metal concentration increased from 0 to 60 μM. Similarly, 14.1% decline in urease activity was observed as the concentration of As was increased from 0 to 100 μM. The calcium carbonate precipitation at the minimum inhibitory concentration of Cd and As-contaminated artificial wastewater was 189 and 183 mg/100 ml, respectively. The percentage removal of metal from artificially contaminated wastewater with varied concentrations was analyzed using atomic absorption spectroscopy (AAS). After 168 hours of incubation, 93.13% removal of Cd and 94.25% removal of As were observed. Microstructural analysis proved the presence of calcium carbonate in the form of calcite, confirming removal of cadmium and arsenic by microbially induced calcium carbonate precipitation (MICCP) to be promising technique for water decontamination.


Introduction
Heavy metal pollution primarily refers to the accumulation of heavy metals, such as lead (Pb), mercury (Hg), chromium (Cr), cadmium (Cd), nickel (Ni), lanthanum (La), strontium (Sr), and other biologically toxic heavy elements in the environment in quantities higher than permissible values (Abd-Alla et al., 2012).Contact with low concentrations of most heavy metals can lead to dangerous health consequences (Azimi et al., 2017).Over the last few decades, heavy metal production and release have increased due to various anthropogenic activities that cause severe damage to the environment (Ojuederie & Babalola, 2017).In areas such as grasslands, where industrialization is Vol:.( 1234567890) relatively less of an issue, the primary anthropogenic disturbance is the grazing by farm animals that plays a considerable part in the build-up of toxic metals (Li et al., 2021;Soomro et al., 2023a).Heavy metals progressively seep into the environment and contaminate the water below (Macdonald, 1994).They can endanger human health because they build up in plants and animals, polluting the food supply (Amin et al., 2021;Baki et al., 2020;Kamzati et al., 2020;Karimi et al., 2020).Due to the potential for their accumulation in biosystems as a result of contaminated water, soil, and air sources, the bioaccumulation of the HMs in the food chain has become a significant issue in recent years (Bedassa & Desalegne, 2020;Bhavya et al., 2021;Soomro et al., 2023b).These elements are considered hazardous because of their persistent and bioaccumulative nature (Akindele et al., 2020;Amin et al., 2021).Many industries, such as electroplating, metal finishing and polishing, mining and metallurgy, electronic circuit production, iron and steel processing, pesticide and insecticide application, and fine chemical and pharmaceutical production, discharge many toxic heavy metals into water resources that have a deleterious impact on the health of living organisms (Eccles, 1999;Kubier et al., 2019).One of the main routes for the movement of heavy metals over long distances in less time is through a river ecosystem (Liang et al., 2018;Muhammad & Usman, 2022).Arsenic and cadmium disrupt the metabolic activities of the body in different ways.Cd can accumulate because of its ability to bioaccumulate in essential body organs such as the kidney, liver, heart, and brain, upsetting regular biological activities and leading to many serious ailments such as cancer, neurological illnesses, liver damage, central nervous system malfunctioning, and cardiovascular diseases (Tayang & Songachan, 2021).One of the biggest risks for heavy metal contamination in living beings is directly consuming water polluted with heavy metals (Deng et al., 2020;Muhammad & Ahmad, 2020).The International Agency for Research on Cancer (IARC) has classified arsenic, cadmium, and related compounds as highly carcinogenic to humans (Smoke & Smoking, 2004).Several cellular toxicities have been reported to be induced by continuous exposure to excess arsenic (> 0.05 ppm) (Dey et al., 2016).Cadmium is highly toxic even in meager quantities, frequently found in the environment as a divalent cadmium cation, which has severe effects on most living organisms (Fang et al., 2021;Ghorbanzadeh et al., 2020).Additionally, it causes immune system illness, which has repercussions from enzyme interference.Intense Cd toxicity in living things can result in high blood pressure, organ failure, and possibly the growth of tumors (Engwa et al., 2019;Soomro et al., 2023a).Arsenic is a metalloid with very high toxicity worldwide (Mandal & Suzuki, 2002).The trivalent form of arsenite is readily soluble and contaminates the groundwater (Anbu et al., 2016).Pesticides and insecticides incorporate various arsenic compounds (Rahman et al., 2004).Several conservative methods for detoxifying heavy metals include oxidation/reduction, reverse osmosis, membrane filtration, electrochemical methods, and ion exchange.Physical methods include magnetic separation, electrostatic techniques, material screening, flotation, and density separation (Azimi et al., 2017).Chemical methods involve reactions between heavy metal reagents and ions, reduction-oxidation processes, and electrochemical methods.(Race, 2017).However, it has many disadvantages, such as low efficiency, high demand for chemicals, increased expenses, by-product formation of poisonous sludge, and dangerous disposal of substances.Other limitations include sample pre-treatment, labor-intensive, and feasibility (Achal, Pan, & Zhang, 2012).
Removing toxic metals from water is more challenging than other pollutants that can be removed using chemical and biological wastewater treatment (Knox et al., 2002).Therefore, appropriate techniques must be developed to remove heavy metals from polluted wastewater.Microorganisms can break down and detoxify xenobiotics, degrade them into comparatively less toxic compounds, or immobilize them by utilizing their transformation or biomineralization abilities (Ayangbenro & Babalola, 2017;Cheng et al., 2016).In extremely unfriendly environments, such as media polluted with heavy metals, microorganisms are appropriate for detoxifying heavy metals (Maity et al., 2019;Qian & Zhan, 2016).Microorganisms transform heavy metals by changing their physicochemical characteristics (Liu et al., 2021).Biomineralization is a technique through which organisms synthesize inorganic substances through their metabolism (Niedermeier et al., 2018).Various researchers have demonstrated microbially induced Vol.: (0123456789) calcium carbonate precipitation to be an effective method for eliminating heavy metal pollution (e.g., Achal et al., 2011Achal et al., , 2013;;Achal, Pan, Fu, & Zhang, 2012;He et al., 2019;Kang et al., 2014;Kim et al., 2021;Kumari et al., 2014;Mitchell & Ferris, 2005) because calcium carbonate is a suitable host matrix for many heavy metals (Callagon et al., 2014).Microbially induced calcium carbonate precipitation (MICCP) is a type of induced biomineralization involving extracellular carbonate production by bacteria, most commonly through the ureolytic pathway.Bacteria play a significant role in MICCP by serving as nucleation sites for carbonate crystals (Lin et al., 2018).Bacteria release carbonate, forming bonds with cations to create minerals during MICCP (Almajed et al., 2021).Microbes, such as Bacillus licheniformis, can precipitate high concentrations of metal ions, such as calcium and magnesium, in a highly alkaline environment (Zhao et al., 2019).Urease enzyme (urea amidohydrolase; EC3.5.1.5)is quite prevalent in a huge diversity of microbes.The urease enzyme catalyzes the cleavage of urea to carbonic acid and ammonia in the presence of water.An equilibrium of these products is established in water to form bicarbonate and two moles of ammonium and hydroxyl ions.The pH increases due to the presence of hydroxyl ions, which causes a shift in the bicarbonate equilibrium; as a consequence, carbonate ions are formed.Due to this change, precipitation of heavy metals occurs, which has been summarized in Eq. 1, 2, 3, 4, 5, and 6.Mineralization of heavy metal ions is caused by the reaction of a heavy metal-cell complex, after which the ultimate conversion to metal carbonate precipitate takes place (Khadim et al., 2019).Prokaryotic microorganisms take part in oxidation-reduction reactions and alter the valency of heavy metals, thus altering their activity, which impacts their mobility or toxicity (Gavrilescu, 2004).Cadmium and arsenic are more toxic in ionic form than the metal carbonate form as the latter is relatively more insoluble and inert due to decreased bioavailability (Chen et al., 2021).The insoluble precipitate is formed when cadmium and arsenic ions in the contaminated water or soil system react with the chemical species formed during the precipitation.Heavy metal ions get surrounded by carbonate in the mineral structures through coprecipitation and are generally more stable and less toxic over Time (Achal et al., 2013).
Bioremediation of heavy metals by ureolytic microorganisms effectively eliminates metals from the environment through precipitation or coprecipitation in carbonate minerals regardless of the toxicity of metal, redox potential, or valency (Li & Gadd, 2017).Many researchers have reported studies on calcium carbonate-derived bioremediation of metals such as Pb, Ni, Cd, Cr, and La and through the use of ureolytic microbes (Dhami et al., 2017;Horiike et al., 2017;Li & Gadd, 2017;Qian et al., 2017;Zhao et al., 2017;Zhu et al., 2016) Recently, microbial-induced calcium carbonate precipitation (MICCP) has received significant attention in the bioremediation of heavy metals.Therefore, the present study focused on applying the MICCP technique to remove the heavy metal contamination from artificially prepared wastewater with different concentrations of cadmium (Cd) and arsenic (As).The metal toxicity in distilled water was artificially introduced by inoculating water with varying concentrations of metal.Ureolytic bacteria Bacillus paramycoides was used in this study.The alkaliphilic bacteria B. paramycoides was assessed for its urease enzyme activity and calcium carbonate precipitation under metal stress.The biomineralization potential of B. paramycoides for removing Cd and As from artificial wastewater was measured through atomic absorption spectroscopy (AAS).The biomineralized end product was verified through field emission scanning electron microscopy (FESEM) and electron dispersive spectroscopy (EDS). (1)

Microorganism
The halophilic bacteria, Bacillus paramycoides-MSR1 was used in the current study.The bacterial strain was isolated from the water sediments of Sambhar Lake near Jaipur, Rajasthan, India, and was selected based on its ureolytic activity.The bacteria were identified as B. paramycoides based on its 16S rRNA sequence analysis.The 16S rRNA sequence obtained from B. paramycoides was submitted to GenBank of NCBI under the accession number OQ186756.The bacterial culture was grown in nutrient broth (Hi Media, India) under shaking conditions of 120 rpm at 37 °C.

Minimum inhibitory concentration
The influence of different concentrations of metal (Cd and As) on the growth of ureolytic bacteria B. paramycoides was tested by measuring the optical density (OD) after 24 hours of inoculation.Overnight grown B. paramycoides culture was reinoculated with 0.5 OD in nutrient broth containing different concentrations of Cd (0, 10, 20, 30, 40, 50, and 60 μM) and As (0, 20, 40, 60, 80, and 100μM).The flasks were incubated at 37° C for 24 hours under shaking (120 rpm).
The optical density at 600 nm was recorded after 24 hours using a spectrophotometer (UV-1800 Shimadzu spectrophotometer).The minimum inhibitory concentration of the artificial wastewater contaminated with Cd and As was evaluated by progressively increasing the concentration of metal salts in nutrient broth until the complete inhibition of the bacterial growth.The IC 50 value for cadmium and arsenic was calculated by plotting the OD600 values obtained for the log of varying concentrations in GraphPad Prism (version 9.3.0)software.

Urease activity
Urease activity was analyzed by measuring the breakdown of urea and the production of ammonia.The urease activity of B. paramycoides was evaluated by a protocol optimized by Natarajan, 1995 until the decline in activity was observed.The overnight grown culture with OD 0.5 was reinoculated in different concentrations of Cd and As.Then, 1 ml of bacterial culture from flasks was extracted in separate autoclaved Eppendorf tubes and centrifuged at 8000 rpm for 5 minutes.Culture filtrate (250 μl) was taken in clean and dried test tubes, and added 1 ml of 0.1 M potassium phosphate buffer (pH 8.0) and 2.5 ml of 0.1 M freshly prepared urea solution and incubated at 37 °C for 5 mins.Then, 1 ml of phenol nitroprusside solution and 1 ml alkaline hypochlorite solution was then added and incubated for 25 mins at 37 °C.The optical density was recorded at 626 nm using a UV-Vis spectrophotometer.The optical density readings were recorded at intervals of 24 h for a period of 168 h.

Removal of cadmium and arsenic
The overnight grown B. paramycoides culture was reinoculated with OD 0.5 in artificial wastewater with different Cd and As metal concentrations.All the flasks were supplemented with 2% urea and 25 mM Vol.: (0123456789) CaCl 2 and incubated at 37 °C with 120 rpm shaking.The flasks were left undisturbed for 168 hours until the complete exhaustion of urease activity.The calcium carbonate precipitates were collected in a cellulose membrane filter (pore size 0.45 micrometer) using membrane filter apparatus by connecting it to the vacuum pump, and the filtrate was quantified for the heavy metals using atomic absorption spectroscopy.To quantify cadmium, GBC 932 AA spectrophotometer was used with a level of detection of 0.01 mg/l.For arsenic quantification after removal, Agilent Technologies 4100 NPAES Spectrophotometer was used with 0.005 mg/l detection level.The percentage removal of metal was identified based on the metal contaminant present initially and after the removal measured using AAS.

Microstructural analysis
The bio-precipitates were analyzed by field emission scanning electron microscopy (FESEM) to study the topographical information and energy dispersive spectroscopy (EDS) to study the elemental characterization.The samples were coated with a thin layer of gold to increase the conductivity of the sample.Generally, the thickness of coating lies between 20 and 30 nm.The imaging was carried out by Carl Zeiss Sigma 500 Field emission microscope and eds was carried by Bruker, QUANTAX 200.

Statistical analysis
All the experiments were performed in triplicates.The data were analyzed by analysis of variance and significant differences among the means were compared by Tukey's test at p < 0.05.All the analyses were performed using GraphPad Prism version 9.3.0software.

Minimum inhibitory concentration
The growth of B. paramycoides significantly reduced with increase in concentration of both Cd and As (Fig. 1).In Fig. 1a and b, the normalized absorbance has been plotted against the increasing cadmium concentrations as a function of logarithm.The IC 50 value for Cd was found to be 12.84 μM.Inhibitory concentration 50 (IC 50 ) was determined as the concentration of heavy metal required to inhibit the growth of 50% bacterial population.As the concentration of cadmium was increased in media, a decrease in the growth of Bacillus paramycoides was observed which is in concurrence with the observations made by Kang et al. (2014) The IC 50 value for arsenic was 48.54 μM.On comparing, it was observed that as the concentration of arsenic was increased, the bacterial growth The possible reason for the decline in bacterial growth in the presence of arsenic could be the binding of arsentite ion with the membrane which could lead to the expansion of the membrane which consequently increases the number of attachment sites thus hindering the transport of materials required for cellular growth (Liu et al., 2004).Furthermore, metal ions bind to available binding site and functional groups present in bacteria and can induce conformational metabolic activity of bacteria.It can further weaken bacterial growth and metabolism (Wei et al., 2022).It was noted by Fang et al. (2021) that Cd 2+ possesses much more toxic potential than other ions of heavy metals, and the impact of anions on bacteria is not significant.The lower IC 50 value suggests higher potency of the heavy metal to inhibit bacterial growth and population (Situmorang et al., 2020).Similarly, a higher IC 50 value signifies that the bacteria can tolerate a higher concentration of the heavy metal before showing a significant decline in growth due to the interference of heavy metal with the metabolism.

Urease activity
The urease activity significantly decreased with increasing concentration of Cd and As at different time intervals (Fig. 2).In Cd, the highest urease enzyme activity was reported at 96 hours, while in As, the most increased urease enzyme activity was observed at 120 hours.The maximum urease enzyme (722 U/ml) was observed in control without any metal.The urease enzyme activity at cadmium (Cd) concentrations of 10 μM, 20 μM, 30 μM, and 40 μM was noted to be 616 U/ml, 602 U/ml, 562 U/ml, and 525 U/ml after 96 hours of incubation (Fig. 2).A sharp decrease in the urease enzyme activity, i.e., 278 U/ml was noted upon reaching the cadmium concentration of 50 μM followed by 60 μM, i.e., 172 U/ ml.The urease activity declined tremendously by 14.6% at 10 μM concentration to 76.17% at 60 μM.This might be due to the presence of metal, hindering the metabolic activity of bacteria (Chandrangsu et al., 2017).The variation of urease enzyme activity was observed with increasing concentrations of arsenic for 168 h (Fig. 2b).After 24 h of inoculation, the urease activity is almost negligible and increases fivefold within the next 24 h.The urease enzyme activity at arsenic concentrations 0 μM, 20 μM, 40 μM, 60 μM, 80 μM, and 100 μM was 855 U/ml, 847 U/ml, 836 U/ml, 824 U/ml, 797 U/ml, and 734 U/ml, respectively.In the presence of As toxicity, the urease activity declined by 14.1% at 100-μM concentration after 120 h of incubation.The statistical analysis inferred that time intervals and different concentrations of Cd and As significantly affected the urease activity by the bacterial strain (Table 1).Urease-producing bacteria break down urea, forming carbonate ions, which react with heavy metal ions to form precipitates of heavy metal carbonate (Jalilvand et al., 2020).Many studies show a proportional relationship between increased heavy metal concentration and urease enzyme activity and bacterial growth (Cheng et al., 2017;Fan et al., 2020).Similar results were corroborated by Wei et al. (2022) and Qiao et al. (2021).The effectiveness of urease-facilitated calcium-cadmium carbonate precipitation in the bioremediation of cadmium by Salmonella mucilaginosa and Enterobacter cloacae was observed by Bhattacharya et al. (2018).

Calcium carbonate precipitation
Calcium carbonate precipitation was observed for a period of 168 hours from the time of inoculation in nutrient broth in presence of urea and calcium chloride.The quantification of calcium carbonate precipitation was done after an interval of 24 h using the EDTA titration method.As seen in Fig. 3a, after 24 hours, significantly less calcium carbonate was precipitated, increasing successively with every 24-hour interval.The increase in calcium carbonate precipitation was observed until the 168th hour, which was recorded to be the highest for every cadmium concentration.Upon completion of 168 hours of incubation, the maximum amount of calcium carbonate precipitated was measured for 0 μM to be 204 mg/100 ml, followed by 189 mg/100 ml (10 μM), 174 mg/100 ml (20 μM), 159 mg/ 100 ml (30 μM), and 134 mg/100 ml (40 μM).Almost a threefold drop was observed in the precipitation of calcium carbonate at cadmium concentration above 50 μM (50 mg/100 ml) followed by 45 mg/100 ml in 60-μM cadmium concentration.As the concentration of heavy metals in the solution was increased, the precipitation of calcium carbonate decreased because heavy metal ions act as an inhibitor to the normal cellular functions of bacteria.Similar observations were reported by Li et al. (2014).At 96 hours and 120 hours, a significant increase in the precipitation of calcium carbonate can be seen, which can also be attributed to the fact that the urease enzyme activity was found to be at its peak at the same number of hours post-inoculation.
In case of As, it was observed that calcium carbonate precipitation showed an increasing trend during the 168 h of incubation.The maximum calcium carbonate precipitation was observed in As concentration of 0 μM (208 mg/100 ml) followed by 20 μM (198 mg/ 100 ml), 40 μM (183 mg/100 ml), 60 μM ( 178Table 1 The factorial analysis of variation (ANOVA) results indicating the effects of urease activity and calcium carbonate at different time periods (24-168 h) in artificial waste water with different concentrations of cadmium and arsenic.The P values < 0.001 indicates the values are highly significant.Two-way ANOVA revealed significant variation between two factors, i.e., metal concentration and time.The interactions are highly significant mg/100 ml), 80 μM (163 mg/100 ml), and lastly, 100 μM (141 mg/100 ml).The statistical analysis inferred that time intervals and different concentrations of Cd and As significantly affected the calcium carbonate precipitation by the bacterial strain (Table 1).There was an increase in calcium carbonate precipitation with a rise in time intervals of incubation.This might be because bacteria inoculated in the medium could utilize the urease enzyme to precipitate the calcium carbonate even in the presence of different concentrations of the As.During the urease enzyme activity, the medium showed higher urease activity until 120 h of incubation.

Variation in pH
Due to the ureolytic action of the enzyme urease, urea is hydrolyzed, and as a consequence, ammonium and hydroxyl ions are generated, leading to a rise in the pH of the medium.This phenomenon leads to binding Ca 2+ ions to the heavy metal in the solution.The trend observed in the change of pH over time showed a slight reduction in the pH upon adding urea and calcium chloride for the first 5 h, considering the addition of urea and calcium carbonate to be done at 0 h.This pH reduction can be explained by considering the dissociation of the ions in water upon addition.The pH showed an increasing trend till 96 hours, after which a plateau was observed.Figure 4 shows variation in pH for different Cd and As concentrations for the introductory period (0-10 hours) and the secondary period (24-168 hours).The pH variation in control sample was more abrupt than the cadmium concentrations from 10 to 60 μM.The pH of the artificially contaminated wastewater with Cd before incubation was 6.98 ± 0.6 which increased to 8.75 ± 0.14 after 168 h of incubation.Similar results were observed in the medium inoculated with different As concentrations (0-100 μM).Initially, the pH of the artificially contaminated waste water was 7.07 ± 0.7 which was observed to increase to 9.04 ± 0.21 after 168 h.The pH falls upon adding the calcium chloride, which might be due to the absorption of hydroxide ions, leaving free protons in the medium (Sidhu et al., 2022).However, in the experiment's second phase, the medium's pH starts increasing because of bacterial metabolic activity.Statistically, the results were observed to be significant at a 5% level of significance.Achal, Pan, and Zhang (2012) reported an increase in the pH due to the presence of ammonium ions and the evolution of carbon dioxide during ureolysis.Due to alkaline conditions, the rate of metal oxidation was amplified while the solubilization of metal prevented (Ayangbenro et al., 2018).

Removal of cadmium and arsenic
The quantification of heavy metals was done by atomic absorption spectroscopy.In this technique, the sample is made to atomize, and the free atoms thus produced absorb UV or visible light and make transitions to higher electronic energy levels.The concentration of the heavy metal is determined from the extent of absorption.It was observed that B. paramycoides was able to remove more than 80% of Cd and As after 168 h (Fig. 5).The Cd removal was 80.4, 89.8, 91, 93.1, 78.14, and 67.7% from the water with respect to 10, 20, 30, 40, 50, and 60 μM concentration of the metal, respectively (Fig. 5a).Similarly, in the case of the arsenic 88.1, 93.2, 94.2, 93, and 90.1% of arsenic was removed from the water with 20, 40, 60, 80, and 100 μM of As concentration, respectively (Fig. 5b).
Removal of Cd and As increased with increased contact time, and after equilibrium is attained, it essentially remains unchanged.This might be due to unavailability of nutrients.After, the equilibrium is reached, the urease activity by live and dead biomass in the artificial water supplemented with nutrient broth, urea, and CaCl 2 was observed till 168 h of incubation.The biosorption and bioremediation efficiency of ureolytic bacteria in the presence of metal toxicity to effectively hydrolyze urea depends upon an increase in the pH to an alkaline range (8.0-9.1) and form carbonate ions to eliminate all heavy metal ions from the water (Danjo & Kawasaki, 2016;Mwandira et al., 2017).In the current study, the percentage removal of metal varied at different metal concentrations from artificial wastewater.This might be because metal ions can compete with the functional groups present in bacterial cells for binding sites.Bacterial cells have various functional groups, such as sulfhydryl (-SH), amino (-NH 2 ), carboxyl (-COOH), and hydroxyl (-OH) groups, which can potentially bind to metal ions (Chillé et al., 2022;Javanbakht et al., 2014).When metal ions are in high concentrations, they can occupy and saturate all the available binding sites on the bacterial cell surface or within the cell (Zhao et al., 2019).This saturation of binding sites prevents the functional groups from interacting with other essential molecules or performing their normal functions within the cell (Ayele & Godeto, 2021).Bhattacharya et al. (2018) reported 98% Cd 2+ mineralization using nutrient broth spiked with 5 mg/l of Cd 2+ .Up to 92% arsenic elimination by Enterobacteriaceae was reported by Nagvenkar and Ramaiah (2010) after a period of 5 days.Removal efficiency of 86% for both Pb 2+ and Cr 6+ was achieved in a NB medium spiked with 25 mg/l initial concentrations of toxic metals by He et al. (2019).Heavy metals are more toxic in ionic form than metal carbonate as the latter is relatively more insoluble and inert.Through precipitation, microorganisms aid in metal immobilization, leading to chemical changes in metal compounds (Kumari et al., 2016).

Microstructural analysis
Scanning electron microscopy was performed to study the morphology of the precipitated calcium carbonate crystal.The crystal structure was studied using FESEM and EDS to determine the percentage of calcium carbonate and heavy metal content in the precipitate to ensure that the heavy metal ions have been trapped within the calcium carbonate crystal lattice.The FESEM images obtained for the heavy metal-calcium carbonate bio-precipitates are shown in Fig. 6.The structure of calcium carbonate crystals with the morphology resembling the calcite polymorph of calcium carbonate was observed and labelled as CC in Fig. 6.Calcium carbonates behave as an effective adsorbent and have also demonstrated its ability to engulf divalent heavy metals cations (Achal et al., 2011;Callagon et al., 2014;Kim et al., 2021).Calcium carbonates have been used as heavy metal scavengers since long (e.g., Dang et al., 2017;He et al., 2019;Liu & Lian, 2019).The EDS graphs along with the elemental composition of heavy metal calcium carbonate bio-precipitates is shown in Fig. 7.
The crystal formation was in abundance and observed to be stacked over one another forming a layer of calcium carbonate.The calcium carbonate precipitation was confirmed using EDS.EDS analysis specified that the precipitated compound was Ca 2+ , oxygen, and carbon of CaCO 3 .The bacteria represent the nucleating sites for calcite-precipitation which is the main crystalline product involved in the bioremediation process.Calcite in the form of CaCO 3 as verified by EDS analysis.

Conclusions
In the present investigation, growth of ureolytic bacteria B. paramycoides strain was determined for the minimum inhibitory concentration 50 (IC 50 ) in artificial wastewater contaminated with different concentrations of metal (cadmium and arsenic).The bacteria were observed to exhibit urease activity and calcium carbonate precipitation in the presence of different metal stress.The IC 50 value for cadmium was 12.84 μM while it was 48.54 μM for arsenic.The highest urease activity was reported at 96 h in cadmium while in arsenic the highest urease activity was observed till the 120 h incubation at 37 °C.The calcium carbonate precipitation in As contaminated artificial wastewater was in the range of 208-141 mg/100 ml showing declining trend with increase in the concentration 0, 20, 40, 60, 80 μM, and 100 μM.
Similarly, in Cd-contaminated artificial wastewater calcium carbonate precipitation was in the range of 204-134 mg/100 ml with the increase in concentration from 0 to 40 μM.The removal efficiency of Cd and As from the artificial wastewater due to MICCP was evaluated using the atomic absorption spectroscopy.The maximum heavy metal removal percentage achieved for cadmium was 93.13% while it was 94.25% for arsenic.No heavy metal removal was observed in water without bacterial inoculum.The FESEM and EDX results also showed the formation of heavy metal-calcium carbonate complex which means the immobilization of the heavy metals was achieved successfully through microbially induced calcium carbonate precipitation.

Fig. 1
Fig. 1 Effect of different concentrations of (a) cadmium and (c) arsenic on the growth (OD 600 ) of B. paramycoides, and (b) normalized absorbance vs increasing cadmium concentrations, and (d) normalized absorbance vs. increasing arsenic concentrations

Fig. 2
Fig. 2 Urease activity of B. paramycoides in artificially contaminated wastewater with different concentrations of (a) cadmium and (b) arsenic.Error bars represent standard deviation (n = 3).The factorial ANOVA results indicate that heavy metal concentration and time are highly significant (P Values < 0.001)

Fig
Fig. 3 Calcium carbonate precipitation by B. paramycoides in artificial wastewater with concentrations of (a) Cd and (b) As.Error bars represent standard deviation (n = 3).The factorial ANOVA results indicate that heavy metal concentration and Time are highly significant (P < 0.001)

Fig. 7
Fig. 7 EDS analysis of (a) cadmium-calcium carbonate bioprecipitates and (b) arsenic calcium carbonate bioprecipitates.Ca was abundantly present as rhombohedral calcium carbonate crystals precipitated by B. paramycoides . concentration, df degree of freedom