Study on Bioremediation of Heavy metals Present in Cement

Cement, crucial for construction, poses significant environmental risks due to heavy metal contamination, including lead, cadmium, chromium, nickel, etc. These metals, released during cement production, endanger both human health and ecosystems. An innovative approach involves utilizing microorganisms for bioremediation, converting pollutants into less harmful forms. Microbes develop resistance mechanisms against heavy metals, reducing their concentration and mobility within cement. In this project, various brands and types of cement were collected, and different bacteria were cultured. A comparison was made regarding the mechanical properties, concentration of heavy metals, elemental composition, surface morphology, and particle size of the cement before and after bioremediation using Atomic Absorption Spectroscopy (AAS), Energy Dispersive X-ray Analysis (EDAX), and Scanning Electron Microscopy (SEM), respectively. The results obtained from the tests conducted were compared between the conventional and bacteria-induced cement samples. Implementation of such biotechnological approaches not only addresses environmental concerns but also promotes the development of innovative and sustainable solutions in construction.


INTRODUCTION 1.1 CEMENT
Cement stands as a pivotal binding agent in construction, renowned for its capacity to set, harden, and effectively adhere to other materials.Comprised mainly of calcium, silicon, aluminium, iron, and various other components, cement is available in diverse forms, ranging from the widely utilized Portland cement to specialized blends tailored to specific construction needs.However, cement production significantly contributes to global CO₂ emissions.The manufacturing process involves heating raw materials in a cement kiln, resulting in the release of CO₂ stored in calcium carbonate.Nonetheless, hydrated cement products, such as concrete, gradually reabsorb atmospheric CO₂ through the carbonation process, thereby mitigating initial emissions.Cement's essential characteristics profoundly influence the load-bearing capacity of structures, the setting time upon mixing with water, and the ease of handling and placement in construction field.Yet, the environmental impact of cement production has garnered considerable attention due to concerns surrounding carbon dioxide emissions and resource-intensive manufacturing practices.Furthermore, the presence of heavy metals in cement poses significant environmental challenges, emphasizing the urgent need for sustainable practices and innovative solutions.

HEAVY METALS
Heavy metals, characterized by their elevated atomic weights and densities, pose significant environmental and health concerns.These metals, including lead, mercury, cadmium, arsenic, and chromium, exhibit toxic properties with adverse effects on both human health and ecosystems.Their ability to accumulate in the food chain, coupled with their persistence in the environment, necessitates strict regulatory measures mitigate potential exposure.Remediation techniques, such as bioremediation, offer avenues for mitigating contamination, contribute to addressing the challenges associated with heavy metal pollution.In the realm of construction, heavy metals find their way into cement, either as inherent components or introduced during the production process, with metals like lead, cadmium, and chromium being notable examples.This presence raises environmental concerns, regarding potential leaching into surrounding soil and water during the lifecycle of cement-based structures.Including the use of microorganisms for bioremediation to reduce heavy metal content in cement.The below mentioned table 1.1 illustrates the adverse health impacts of each heavy metal that require reduction.

BIOREMDIATION
Bioremediation is an eco-friendly approach that utilizes living organisms, such as bacteria, fungi, and plants, to mitigate environmental pollution.This technique leverages the metabolic capabilities of these organisms to either degrade or immobilize pollutants, transforming them into less harmful forms.It offers a sustainable alternative to traditional remediation methods, reducing the environmental impact of pollution while promoting ecosystem restoration.
In the context of heavy metal contamination, bioremediation involves harnessing the unique abilities of microorganisms to reduce the concentration of metals in the environment.The application of bioremediation techniques to heavy metals, such as lead, cadmium, and chromium, has shown promise in mitigating the environmental and health risks associated with these pollutants.This environmentally conscious approach is particularly relevant in industries like construction, where heavy metals may be present in materials like cement.Research in this area focuses on optimizing bioremediation strategies for heavy metals, contributing to the development of sustainable and effective solutions for environmental cleanup.

MICRO ORGANISMS AND THEIR ROLE
Microorganisms, play fundamental roles in various ecological processes, from nutrient cycling to decomposition, highlighting their significance in maintaining environmental balance.In addition to their natural functions, microorganisms have gained attention for their applications in biotechnology, medicine, and environmental management, showcasing their adaptability and versatility across disciplines.Various microorganisms play integral roles in the reduction of heavy metals, contributing to the process of bioremediation.Pseudomonas aeruginosa, renowned for its versatility, actively participates in reducing and detoxifying heavy metals such as nickel.Bacillus aluvi, another notable microorganism, is effective in the reduction of lead contamination by absorbing, accumulating, and precipitating lead ions, thus contributing to the remediation of lead-polluted environments.Actinobacteria, a diverse bacterial group, engages in reducing heavy metals like chromium through various metabolic pathways, rendering them less harmful.Shewanella oneidensis is recognized for its ability to reduce and precipitate metals like uranium, using electron transfer mechanisms to convert soluble uranium into insoluble forms.

CALCIUM CARBONATE PRECIPITATION
• When calcium ions (Ca 2+ ) and carbonate ions (CO3 2-) come together in a solution, they can react to form solid calcium carbonate.

• The chemical equation for this reaction is:
• This process is known as precipitation because the calcium carbonate comes out of the solution and forms a solid, often seen as a white, chalky substance.

HEAVY METAL PRECIPITATION
• When heavy metal ions (M 2+ ) are present in a solution along with carbonate ions (CO3 2-), they can also und • The general chemical equation for this reaction is: • The specific metal carbonate formed depends on the type of heavy metal present.For example, if the heavy metal is lead (Pb), the resulting compound would be lead carbonate (PbCO 3 ).
In both cases (Calcium precipitation and metal precipitation), the carbonate ions act as a "glue" that binds withs the calcium ions or heavy metal ions, causing them to come out of solution and form solid carbonate compounds.This precipitation process is essential for immobilizing heavy metals, reducing their mobility and potential harm to the environment.AAS enables the identification and quantification of heavy metals, such as lead, cadmium, mercury, and chromium, in diverse matrices, including water, soil, and biological samples.The precise measurement capabilities of AAS contribute significantly to understanding and managing the presence of heavy metals, facilitating informed decision-making in environmental and public health contexts.

SCANNING ELECTRON MICROSCOPY (SEM)
Scanning Electron Microscopy (SEM) provides a valuable glimpse into the intricate world of the microscopic domain.It allows for detailed examination of the surface morphology, texture, and particle size of cement specimens with exceptional resolution.Through SEM analysis, researchers can delve into the microstructural features of cement materials, unraveling critical insights into their physical properties and composition.This comprehensive analysis not only facilitates a deeper understanding of the inherent characteristics of cement but also sheds light on the efficacy of bioremediation processes.By scrutinizing the interactions between microorganisms, cement, and heavy metal pollutants at the microscopic level, SEM offers invaluable insights into the complex dynamics at play during remediation efforts.• To analyze the reduction in concentration of heavy metals in cement using AAS after the bioremediation process.• To prepare bacteria induced cement mortar cubes.
• To compare the compressive strength before and after bioremediation.
• To examine the elemental composition and interactions between the bacteria and cement before and after the bioremediation using Energy Dispersive X-ray Analysis (EDAX) and Scanning Electron Microscopy (SEM) respectively.Fatini Mat Arisah, Amirah Farhana Amir (2021) demonstrated that Pseudomonas aeruginosa RW 9 effective removal of up to 85% of 10 mg/L chromium (VI).Extracellular sequestration was the primary mechanism for chromium (VI) removal, accounting for over 50% of total removal of chromium (VI) induced the synthesis of biosurfactants, identified as rhamnolipids.Xiaoxia Yu, Jintong Zhao (2021) discussed about soil ecosystems rely on microbes for various processes, especially in heavy metal-contaminated environments.Cadmium-tolerant phyla like Proteobacteria and Gemmatimonas play vital roles, promoting ecosystem resilience in the presence of heavy metals.Mahendra Aryal (2020) investigated Bacterial bio sorption is effective for removing heavy metals (HMs) from contaminated sites.Operating conditions such as pH, biomass concentration, contact time, temperature, and initial metal concentrations influence bio sorption performance.Bacterial biomass shows superior performance compared to other biomaterials.Prakash Mallappa Munnoli, Sudisha Jogayya (2020) experimented that A 40% suspension exhibited higher compressive strength compared to 20% and 60% in all cases B. Subtilis with optimized 40% suspension (CFU 10x10^8/ml) showed increases in CS of 4.32%, 5.56%, and 3.81% for 3 days, 7 days, and 28 days respectively.Overall, B. Subtilis resulted in a 5.92% increase in CS compared to the control cube at 3 days.Ya-Nan Xu, Yinguang Chen (2020) demonstrated the enhanced sulfate reduction focus on improving sulfate reduction for efficient heavy metal removal by sulfate-reducing bacteria (SRB).The heavy metal is removed by immobilizing SRB, creating a protective barrier between them and the harmful metals.
When different types of bacteria work together, they can be more efficient.Some bacteria can even soak up heavy metals.Yogesh Jayant, Saresh K Jain (2020) investigated that Bacteria aid healing by producing calcite, improving compressive strength, durability, and reducing water permeability.Maintain low bacterial concentration and use 0.50% calcium lactate for effective results.2007) studied that natural adsorbent materials, without activation, Or nickel (II) and aluminium (III) ions, the ideal pH is 7.5 and 6.5, respectively.Clay (bleaching earth) is particularly effective for removing these ions.The pseudo-first-order chemical reaction model provides the best fit for the data.• Desulfovibrio alaskensis strain 6SR showed a remarkable ability to remove 98% of lead in solution.

OBSERVATIONS FROM THE LITERATURE STUDY
• Paracoccus denitrificans AC-3 was able to remove 46.19% of nickel.
• The bio sorption capacity of Pseudomonas aeruginosa is 98%, and Pseudomonas aeruginosa RW 9 demonstrated effective removal of up to 85% Chromium.• Based on various literature, it has been observed that bioremediation is a cost-effective and eco-friendly alternative to physicochemical methods.• Many studies have implemented bioremediation using bacteria such as Pseudomonas species, Bacillus species, Rhizobacterium, etc., which have demonstrated effective results in reducing metals like chromium, lead, and cadmium present in soil and water bodies.Therefore, the application of these bacteria in cement could potentially yield positive outcomes.• The bacteria have been observed to not only reduce the concentration of heavy metals but also alter the properties of soil or water bodies.Consequently, the introduction of these bacteria into concrete could potentially modify its properties as well.• Biosorption, a cost-effective technology, involves transport across cell membranes, complexation, ion exchange, and precipitation, playing a vital role in chromium removal from water.• Factors such as pH levels, biomass density, and duration of contact, temperature, and initial metal levels directly impact the effectiveness of biosorption.• B. Subtilis resulted in a 5.92% increase in Compressive strength compared to the control cube at 3 days.

FINE AGGREGATE
Fine aggregate confirming to IS: 383-1970, a fundamental component in concrete mixtures, consists of small, granular particles such as sand.Its primary role is to fill the voids between coarse aggregates and bind together the cement paste, contributing to the overall strength and durability of the concrete.The quality and grading of fine aggregate significantly influence the workability, cohesion, and finish of the concrete mix.The appropriate selection and proportioning of fine aggregate play a crucial role in achieving the desired properties of the concrete, making it a key consideration in construction practices.Pseudomonas aeruginosa plays a vital role in reducing nickel contamination, showcasing its potential in bioremediation efforts.Bacillus aluvi is recognized for its contribution to the reduction of lead, demonstrating its efficacy in mitigating the impact of this heavy metal.Actinobacteria, with its distinctive characteristics, actively contributes to the reduction of chromium, further highlighting the diverse and beneficial roles that bacteria play in addressing environmental challenges associated with heavy metal pollutants.

CULTIVATION OF BACTERIA
• The cultivation of bacteria involves a series of precise steps to create a conducive environment for their growth.• Initially, in a conical flask, 2.9 grams of nutrient broth are meticulously mixed with 100 ml of distilled water, forming a nutrient-rich medium essential for bacterial proliferation.• The conical flask is then carefully sealed with cotton and subjected to sterilization in an autoclave at a temperature of 121⁰ C, ensuring the elimination of any potential contaminants.• After the sterilization process, the prepared sample is inoculated with 1 ml of bacteria under the controlled conditions of a laminar airflow chamber.• This step is crucial to introduce a controlled amount of bacterial culture into the nutrient broth, initiating the growth process.• The inoculated culture is then transferred to an incubator set at a specific temperature, in this case, 35 degrees Celsius, for a defined period, typically 24 hours.• The incubation period allows for the optimal conditions needed for bacterial replication and the formation of a robust culture.• Test for Initial Setting Time: 1. Place the test block under the needle-bearing rod and quickly release the needle at 2-minute intervals.
2. Record the time (T2) when the needle fails to pierce the block by about 5 mm.
• Test for Final Setting Time: 1. Replace the needle with an annular attachment on the Vicat apparatus.2. Cement is considered finally set when the final setting needle makes an impression on the test block, while the attachment fails to do so.3. Record the time (T3) as the final setting time.The standard consistency test assesses the optimal water content for normal consistency in cement.It determines the right balance between fluidity and cohesion in cement paste, crucial for quality control in construction.Achieving standard consistency ensures optimal performance in various applications, guiding water-to-cement ratios for effective concrete mixes.

PROCEDURE
1. Weigh 400 grams of cement and mix it gradually with a calculated amount of water until a uniform paste is achieved.2. Transfer the cement paste into the Vicat mould, ensuring thorough filling without voids.3. Lower the Vicat plunger gently onto the paste surface and release it quickly, repeating until no impression is left.4. The consistency is considered standard when the Vicat plunger penetrates the paste to a depth of 10 to 12 mm under a 50 N load.5. Record the water amount used and the achieved consistency, providing crucial information for quality control and further assessments.

DRY SEIVING METHOD (As per IS 4031 part 1: 1996)
The fineness of cement refers to the particle size distribution and surface area of cement particles.It is a crucial property that influences the hydration and setting characteristics of cement, as well as the strength and durability of concrete produced with it.Finer cement particles provide a larger surface area for hydration reactions to occur, leading to faster setting times and increased early strength development.The fineness of cement is typically measured by specific surface area, which is determined through methods such as the Blaine air permeability test or the laser diffraction technique.A higher specific surface area indicates finer cement particles, while a lower specific surface area indicates coarser particles.The fineness of cement is an important quality control parameter in cement production to ensure consistent and optimal performance in concrete applications.4.1.3.1 PROCEDURE 1.Take 1000 grams (1 Kg) of cement for the test sample and name it as (w1).2. Rub the cement particle well with your hands so that no lumps are left.3. Pour the 1 Kg cement content in the sieve and close it perfectly with the sieve lid. 4. Put the sieve in the shaking machine and start the machine for 15 minutes.5. Brush the sieve base gently with the bristle brush so that nothing is left on the sieve surface.6. Weigh the retained amount of cement on the sieve and note it as (w2).7. Find the percentage of the weight of cement-retained on the 90 µm sieve.

SPECIFIC GRAVITY OF CEMENT (As per IS 4031 PART-11 1988)
Specific gravity of cement refers to the ratio of the mass of a given volume of cement to the mass of an equal volume of water at a specified temperature.It is a fundamental property that helps assess the density or compactness of cement particles.Specific gravity is an important parameter in determining the quality and consistency of cement, as it can influence various properties of concrete, including its strength, durability, and workability.A higher specific gravity indicates denser cement particles, while a lower specific gravity suggests lighter particles.

PROCEDURE
1.The flask is allowed to dry completely and made free from liquid and moisture and the weight of the empty flask is taken as W1. 2. The bottle is filled with cement to its half (Around 50gm of cement) and closed with a stopper and it is weighed with stopper and taken as W2. 3. To this kerosene is added to the top of the bottle.The mixture is mixed thoroughly and air bubbles are removed.The flask with kerosene, cement with stopper is weighed and taken as W3. 4. Next, the flask is emptied and filled with kerosene to the top.The arrangement is weighed and taken as W4. 5. Specific Gravity of Cement is given by the formula,  The compressive strength test on cement mortar cubes is a pivotal assessment, gauging the material's ability to withstand axial loads and compression.This test provides essential information on the mortar's structural performance, influencing decisions regarding its suitability for construction applications.The measured compressive strength signifies the maximum stress the mortar can endure under uniaxial compressive loads, serving as a critical parameter for ensuring the durability and structural integrity of constructed elements.4.1.5.1 PROCEDURE 1. Use a cement-to-sand ratio of 1:3 for the test.2. Take 200 gm of cement, and mix it with 600 gm of sand for 1 minute.3. Calculate the water needed for standard consistency using the formula, with P set to 30 cement, 4. Add water to the mixture and mix for three minutes.5. Assemble the cube mould on a vibrating machine, applying mould oil before pouring in the mortar.6. Vibrate at a rate of 12000 ± 400 per minute for 2 minutes.Remove the mould, level the top surface with a trowel, and repeat for other cubes.7.After 24 hours, demould the cubes, mark with date and number, and submerge in a freshwater tank for curing.8. Test three cubes each on the third and 28th days for compressive strength.9. Measure cube weight and record data.Place cubes in a compression testing machine and apply a load at a rate of 35 N/mm²/mi.Note the load at which the cube is crushed.

QUANTITY CALCULATION
Six cubes were casted from each collected cement sample for the compressive strength test, which was conducted on the 7th and 28th days.4.5 represents the results of compressive strength for cement mortar cubes from eleven distinct cement samples, along with their average outcomes.

TEST CONDUCTED ON BACTERIA 4.2.1 COLONY FORMING UNITS
Colony-forming units (CFUs) of bacteria are viable bacterial cells capable of reproducing and forming visible colonies on a solid growth medium under specific conditions, used to estimate bacterial numbers in samples.

PROCEDURE
1. Prepare the agar medium by sterilizing by autoclave.2. Pour the prepared agar into Petri dishes.3. Prepare a series of dilutions by transferring specific volumes of the sample into sterile dilution blanks or test tubes containing sterile saline or distilled water.
4. Mix well after each dilution.5. Spread the inoculum evenly over the surface of the agar using a sterile spreader or by rotating the plate gently.6. Incubate the plates for 24 hours, depending on the growth characteristics of the bacteria at the temperature of 37°C.7. Count the colonies on each plate and record the number of colonies for each dilution.

COMMUNITY COMPOSITION ANALYSIS
A compatibility test of bacteria, often referred to as bacterial compatibility testing or bacterial susceptibility testing, is a method used to determine the sensitivity of bacteria to specific antimicrobial agents.This test involves exposing bacterial isolates to different antibiotics or antimicrobial compounds to assess their effectiveness in inhibiting bacterial growth or killing the bacteria.

PROCEDURE
1. Prepare the agar medium of three bacteria's namely pseudomonas aeurginosa, bacillus alluvi and action bacteria.2. Pour the melted agar into sterile Petri dishes and allow it to solidify.3. Spread the inoculum evenly over the surface of the agar using a sterile spreader or by rotating the plate gently.
4. Incubate the plates for 24 hours, depending on the growth characteristics of the bacteria at the temperature of 37°C. 5. Assess the growth of all bacterial strains.If there are no issues with their growth, we can utilize the three bacteria together.Otherwise, they cannot be used together.

TEST CONDUCTED ON BACTERIA INDUCED CEMENT MORTAR CUBES
The examination of the cement involved conducting tests to evaluate its mechanical and physical properties such as, Compressive strength test.Additionally, Atomic Absorption Spectroscopy (AAS) analyses were carried out to further characterize the presence of heavy metals in cement.

COMPRESSIVE STRENGTH TEST (As per IS 4031 Part 6: 1988)
The compressive strength test on cement mortar cubes is a pivotal assessment, gauging the material's ability to withstand axial loads and compression.This test provides essential information on the mortar's structural performance, influencing decisions regarding its suitability for construction applications.The measured compressive strength signifies the maximum stress the mortar can endure under uniaxial compressive loads, serving as a critical parameter for ensuring the durability and structural integrity of constructed elements.

PROCEDURE
1. Use a cement-to-sand ratio of 1:3 for the test.
2. Take 200 gm of cement, and mix it with 600 gm of sand for 1 minute.
3. Calculate the water needed for standard consistency using the formula, with P set to 30 cement.4. All the bacteria of 10ml, is also combined with the calculated amount of water. 5. Add water to the mixture and mix for three minutes.6. Assemble the cube mould on a vibrating machine, applying mould oil before pouring in the mortar.
7. Vibrate at a rate of 12000 ± 400 per minute for 2 minutes.Remove the mould, level the top surface with a trowel, and repeat for other cubes.8.After 24 hours, demould the cubes, mark with date and number, and submerge in a freshwater tank for curing.9. Test three cubes each on the third and 28th days for compressive strength.10.Measure cube weight and record data.Place cubes in a compression testing machine and apply a load at a rate of 35 N/mm²/mi.Note the load at which the cube is crushed.4.6 displays the compressive strength results of bacteria-induced cement mortar cubes, showcasing an increase in strength compared to conventional cement cubes.This improvement is attributed to the addition of Actino bacteria, Bacillus alluvi and Pseudomonas aeruginosa contributing to enhanced strength.

SCANNING ELECTRON MICROSCOPY ANALYSIS (SEM)
Scanning Electron Microscopy (SEM) offers a window into the microscopic realm.Through SEM analysis, we can scrutinize the surface morphology and particle size of the cement specimens.This detailed analysis helps us better understand how well the bioremediation process works.It helps us see the complex ways microorganisms, cement, and heavy metal pollutants interact with each other.The above Table 4.7 displays the SEM images of the cement mortar cubes of Maha cement (OPC) that represents the presence of rod-shaped bacteria involved in calcite production, which contributes to an increase in the compressive strength of cement mortar cubes, where as in the conventional cement mortar cubes there is no calcite precipitation.
The above Table 4.8 displays the SEM images of the cement mortar cubes of ultratech OPC cement that represents the presence of rod-shaped bacteria involved in calcite production, which contributes to an increase in the compressive strength of cement mortar cubes, where as in the conventional cement mortar cubes there is no calcite precipitation.

ENERGY DISPERSIVE X-RAY ANALYSIS (EDAX)
Energy Dispersive X-Ray Analysis (EDAX), is an X-ray technique used to identify the elemental composition of materials.It provides valuable insights into the chemical characterization of a sample by analyzing the X-rays emitted during interactions between X-ray excitation and the sample.

ACID DIGESTION PROCESS OF CEMENT SAMPLES
The acid digestion process is vital for the chemical analysis of cement samples.Through finely grinding the solid cement and using a mixture of strong acids like hydrochloric acid and nitric acid, this process dissolves the cement matrix.The resulting solution, obtained after cooling and filtration, contains the dissolved components ready for further analysis.Here is the procedure for the acid digestion process: • Weigh 1 gram of cement from each of the 8 samples and place them under a fume hood.
• Combine 12 ml of hydrochloric acid with 4 ml of nitric acid in a 3:1 ratio and add the mixture to the cement samples.• Boil the samples to eliminate fumes, then let them cool for one hour.
• After cooling, introduce 50 ml of distilled water to each sample.
• And proceed to filter each sample using filter paper.

GRAPH
The Atomic Absorption Spectroscopy (AAS) results graph visually represents the concentration of elements in a sample.Peaks on the graph indicate absorption intensity, directly correlating with element concentration.The calibration curve establishes a relationship between absorbance and known concentrations, facilitating quantification of unknown samples.Reproducibility, noise reduction, and adherence to calibration standards ensure result accuracy.The graph's interpretation involves comparing unknown sample peaks to the calibration curve, with higher peaks signifying higher concentrations.Monitoring instrument performance and determining the limit of detection (LOD) contribute to quality control in AAS analysis.Above in Table 5.1, the outcomes of the AAS analysis reveal the concentrations of nickel, chromium, and lead in the eleven cement samples.

BACTERIA INDUCED CEMENT
Atomic Absorption Spectroscopy (AAS) analyses were carried out to further characterize the presence of heavy metals in cement.
Each bacteria of 1ml is added to every cement sample, with additional glucose provided as nutrient to sustain bacterial viability.The cement samples are then covered with Parafilm and incubated for four weeks to facilitate interaction.After this period, the samples undergo acid digestion before being analyzed using AAS.

ACID DIGESTION PROCESS
The acid digestion process is vital for the chemical analysis of cement samples.Through finely grinding the solid cement and using a mixture of strong acids like hydrochloric acid and nitric acid, this process dissolves the cement matrix.The resulting solution, obtained after cooling and filtration, contains the dissolved components ready for further analysis.Here is the procedure for the acid digestion process: • Weigh 1 gram of cement from each of the 8 samples and place them under a fume hood.
• Combine 12 ml of hydrochloric acid with 4 ml of nitric acid in a 3:1 ratio and add the mixture to the cement samples.• Boil the samples to eliminate fumes, then let them cool for one hour.
• After cooling, introduce 50 ml of distilled water to each sample.
• And proceed to filter each sample using filter paper.• It demonstrates that the bacteria-induced cement mortar cubes exhibit greater strength compared to conventional cement, indicating that the addition of bacteria has enhanced the mechanical properties of the cement mortar cubes.

SCANNING ELECTRON MICROSCOPY ANALYSIS (SEM)
• The observed rod-shaped bacteria in the SEM images corroborate previous studies indicating their role in bio mineralization processes within cementitious materials.• The formation of calcite by these bacteria within the cement mortar matrix demonstrates their potential to enhance the mechanical properties of construction materials, particularly in terms of compressive strength.

ENERGY DISPERSIVE X-RAY ANALYSIS (EDAX)
• The EDAX analysis provides valuable insights into the elemental composition of the cement mortar cubes, revealing a notable increase of 20.9% and 4.7% in calcium content in the bacterial-induced samples compared to their conventional counterparts.• The higher calcium content in the bacterial-induced cement mortar cubes underscores the active role played by bacteria in facilitating calcium precipitation, a crucial process for enhancing the mechanical properties of the material.

CONCLUSION
• Bioremediation employing microorganisms to reduce heavy metal content in cement shows significant promise.
• The physical properties of cement were evaluated as per IS codal provisions.
• Table 6.1 highlights a substantial decrease in heavy metal concentrations in bacteria-treated cement compared to conventional cement.• Atomic absorption analysis in Table 6.2 indicates heavy metal concentrations generally within WHO limits.Addition of bacteria proves effective in reducing heavy metal concentrations, demonstrating bioremediation's potential to address environmental concerns related to heavy metal leaching in cement.• Enhanced mechanical properties in bacteria-treated cement mortar cubes, as shown in Table 6.3, suggest potential for more durable and sustainable construction materials, aligning with industry sustainability goals and prompting further research into microbial interventions for broader applications.This study lays groundwork for future investigations, highlighting bioremediation's dual impact on environmental and structural aspects of cement materials.• SEM images confirm the presence of rod-shaped bacteria, indicating their role in bio mineralization within cementitious materials.Calcite formation by these bacteria enhances mechanical properties, especially compressive strength.• Additionally, EDAX analysis reveals a notable increase of 20.9% and 4.7% in calcium content in bacterial-treated samples, underscoring the bacteria's crucial role in strengthening materials through calcium precipitation.This evidence highlights the potential of bacterial interventions in enhancing the mechanical integrity of cementitious materials for more sustainable construction practices.

Fig 1 . 1
Fig 1.1 Different brands of cement bags

Fig. 1 . 9
Fig.1.9Energy dispersive x-ray analysis (EDAX)This meticulous analysis offers invaluable insights into the alterations taking place within the cement matrix due to microbial activity.Moreover, EDAX enables us to identify any potential changes in the chemical structure of the cement, shedding light on the mechanisms underlying microbial interactions with heavy metals.

Fig. 1 .
Fig.1.10Scanning electron microscopy (SEM) N.K. Srivastava, C.B. Majumdernove (2019) focused on the advances in microbial cloning techniques can enhance removal efficiency, reducing treatment costs.Biofilters can remove heavy metals down to ppb levels, making them cost-effective for industries like chemicals, fertilizers, textiles, and more.Hitendra Shivhare, Prof. Vijay Kumar Shrivastava (2018) examined that the Concrete containing 100% bacteria solution demonstrated a compressive strength of 23.98 N/mm2 after 7 days, while concrete with 70% bacteria solution exhibited a strength of 33.95 N/mm2 after 28 days, both surpassing conventional concrete.Inclusion of bacteria increased concrete compressive strength, mainly due to microbiologically induced calcium carbonate precipitation (MICCP), filling pores within concrete cubes.Joseph Thatheyus, D. Ramya (2016) studied that wastewater treatment, involving methods like chemical precipitation.The traditional high-pH approach generates waste and is less effective at low chromium (VI) concentrations.Bacteria, such as Pseudomonas and Bacillus, detoxify chromium by reducing chromium (VI) to chromium (III) with diverse resistance mechanisms.They show promise for environmental cleanup.Wasiatus Sa'diyah, Endang Suarsini (2016) concluded that bacterial consortium (B.alvei and B. pumilus) achieves 93.58% lead reduction at a 7% culture concentration, meeting quality standards.The consortium's success arises from the synergistic action of B. alvei and B. pumilus, enabling higher lead reduction.Aparna K Sathyan (2015) concluded the Mortar cube strength increases up to 10^7 cells/ml bacteria concentration but decreases thereafter.Optimal bacteria doses boost compressive strength by 58% (7 days) and 23% (28 days) over controls.SEM analysis confirms bacterial involvement in calcite production.A. Singh, S. M. Prasad (2015) carried out the growing awareness of pollution motivates the development of clean-up technologies, particularly for heavy metal contamination.Promoting eco-friendly solutions is essential in addressing heavy metal contamination.To combat heavy metal pollution, various techniques are used, including low-cost absorbents, chelating agents, phytoremediation, and even molecular and nanotechnology approaches Mireille Bruschi, Larry L.Barton (2015) reported the mechanisms behind how sulfate-reducing bacteria detoxify toxic metals like mercury, chromate, and arsenate are not entirely clear.Sulfate-reducing bacteria provide an eco-friendly option for metal detoxification.They can be used for soil and groundwater treatment, and their enzymes can create biosensors to measure metal bioavailability.Paul B. Tchounwou, Clement G. Yedjou (2012) investigated heavy metals like arsenic, cadmium, chromium, lead, and mercury exist naturally, but human activities significantly contaminate the environment with them.It's vital to understand how these metals interact on a molecular level and health management when dealing with mixtures of toxic elements.Pratik M. Choksia, Vishal Y. Joshib (

Fig. 4 .S
Fig.4.4 90 microns sieve and a pan 4 represents the results of specific gravity of cement.Hence the specific gravity of cement of grade 53 & OPC and PPC should be between 3.1 and 3.15 as per IS 4031 part 11: 1998.4.1.5COMPRESSIVE STRENGTH TEST (As per IS 4031 Part 6: 1988)

1.8 METHODOLOGY LITERATURE REVIEW 2.1 LITERATURE REVIEW Roohallah Saberi Riseh ,Mozhgan Gholizadeh Vazvani (2023) explored the heavy metal accumulation in soils affects plant performance, morphology, and physiology. Heavy metals have irreversible effects on the environment. Plant Growth-Promoting Rhizobacteria (PGPRs) can mediate interactions between plants and heavy elements. Isolating metal-resistant bacteria from polluted sites offers promising
Eswar Sairam Ravipati, Nikhil Nitin Mahajan (2021) examined that heavy metals like lead pose significant toxicity risks to humans, animals, and plants.Electrochemical approaches are sensitive but lack selectivity for specific ions like lead.This review provides insights into lead's regulatory, toxicity, and analytical aspects.

Table . 2.1 Bacteria that contributes in the reduction of heavy metals S.no Name of The Heavy Metal Name of The Bacteria
The table 2.1 illustrates the observed contributions to the reduction of heavy metals found in the literature.•Bioremediation processes, which include the utilization of bacteria to decrease heavy metal levels, have the potential to alter certain properties of soil and water bodies.These changes can affect soil pH, texture and structure, nutrient content, and overall water quality.• Bacillus megaterium and Bacillus licheniformis from the Bacillus family reduced toxicity by 42% and 20% respectively.

3.1 Details of obtained cement samples S.no Name of the brand Grade of the cement Type of the cement
• Bacterial consortium (B.alvei and B. pumilus) achieves 93.58% Pb reduction at 7% culture concentration, meeting standards.• Inclusion of bacteria increased concrete compressive strength, mainly due to microbiologically induced calcium carbonate precipitation (MICCP), filling pores within concrete cubes.• Concrete containing 100% bacteria solution demonstrated a compressive strength of 23.98 N/mm2 after 7 days, surpassing conventional concrete.MATERIALS 3.1.CEMENT SAMPLES Cement samples from four distinct brands, encompassing both 53-grade Ordinary Portland Cement (OPC) Confirming to IS: 2269-1987 and Portland Pozzolana Cement (PPC) Confirming to IS: 1489-1(1991), were systematically gathered for this project.In total, eight cement samples were collected, and the table3.1 below shows the detailed list is provided Table.

Table . 4.1 Test results of initial and final testing time S.no Cement brand Grade and type of cement
The above table4.1 shows the results of initial and final setting time of cement.Hence the minimum Initial and maximum Final setting time of a cement should be 30 minutes and 600 minutes respectively as per IS

table 4 .
3 represents the results of fineness of cement.Hence the percentage of of fineness of cement of grade 53 & OPC and PPC should be less than 10 % as per IS

table 5 .
2 above shows the heavy metal concentration results in bacteria-induced cement samples.It indicates that the addition of bacteria leads to a more significant reduction in heavy metal concentrations compared to the previous results.

INTIAL AND FINAL SETTING TIME The
initial and final setting time of all the collected cement brands are within the limits as per IS 269:2015 and IS 1489: Part 1:2015.6.2STANDARDCONSISTENCY TESTThe standard consistency tests for all collected cement brands confirm that the water content for Grade 53

6.6 COMPRESSIVE STRENGTH Table.6.3 Comparison of test results -Compressive strength test •
The table 6.3 above shows a comparison of compressive strength test results between conventional cement mortar cubes and bacteria-induced cement mortar cubes.