Bioremediation of Cr(VI) using indigenous bacterial strains isolated from a common industrial effluent treatment plant in Vishakhapatnam

AARD average absolute relative deviation

DBC dry biomass concentration

EDS energy-dispersive spectroscopy

FTIR Fourier-transform infrared

IC initial concentration

LB Luria broth

MIC minimum inhibitory concentration

OFAT one-factor-at-a-time

RMSE root mean square error

Heavy metal pollution from wastewater poses a severe environmental and health concern, as the discharge of industrial and domestic sewage can introduce toxic heavy metals like chromium (Cr), lead (Pb), zinc (Zn), cadmium (Cd), nickel (Ni), copper (Cu), mercury (Hg), and arsenic (As) into water bodies, endangering ecosystems and human well-being (Mitra et al. 2022). According to the Agency for Toxic Substances and Disease Registry (ATSDR), hexavalent chromium (Cr(VI)) holds the 17th position among the most hazardous chemicals posing a severe threat to human health (ATSDR 2022). Electroplating, textile dyeing and printing, leather tanning, metal finishing, and paint manufacturing industries are some that are responsible for releasing Cr(VI) into wastewater (Kerur et al. 2021). India, contributing to 13% of global leather production, is becoming one of the largest leather goods exporting countries, and it is noteworthy to mention that leather industry wastewater is one of the significant contributors of Cr(VI) to the environment (Princy et al. 2020; Venkatesan et al. 2021). Due to its specific chemical properties, including solubility and reactivity, Cr(VI) has carcinogenic effects, causing oxidative stress and targeting multiple organs through bioaccumulation (Wani et al. 2019). The World Health Organization (WHO) has set a maximum permissible limit of 0.05 mg/L for Cr(VI) in treated wastewater (Prabhakaran et al. 2019). Conventional methods such as membrane filtration, photocatalysis, adsorption, precipitation, and ion exchange have been employed for Cr(VI) removal (Tang et al. 2021). However, these methods are associated with several disadvantages, such as high operational costs, energy consumption, the generation of secondary pollutants, and reduced efficiency, when targeting low concentrations of Cr(VI) (Diep et al. 2018). Green technologies employing microorganisms for removing or converting toxic heavy metals into nontoxic substances from the contaminated environment are considered more advantageous and sustainable than conventional methods.

Microorganisms, including bacteria, algae, fungi, and yeast, can efficiently bioaccumulate heavy metals in their living cells. Bioremediation is a process that utilizes different biological mechanisms to convert pollutants into nontoxic forms using live microorganisms (Sharma & Kumar 2021). In recent years, bioremediation has been perceived to be a promising technology for removing Cr(VI) in an efficient, eco-friendly, and cost-effective manner owing to several advantages of microorganisms, such as ease of handling, culture, and implementation (Kholisa et al. 2022, 2023). The performance of living biomass in binding metal ions depends on the nutrient and environmental status and the cell age. Over the last few years, bacteria have been widely researched and employed due to their ability to adsorb (biosorption), solubilize (bioaccumulation), and precipitate (bioprecipitation) heavy metals (Fernández et al. 2018). Some reported works on the bioremediation of Cr(VI) using bacteria are as follows: Escherichia sp., Acinetobacter sp., etc., could remediate Cr(VI) by biosorption; Bacillus sp. could remove Cr(VI) by bioreduction; and Bacillus sp. and Streptomyces sp. could abate Cr(VI) by biotransformation (Marzan et al. 2017; Kumaresan Sarankumar et al. 2020; Sanjay et al. 2020). Since tannery waste, chromium mines, etc., are rich in Cr(VI), the organisms isolated from such effluents exhibited high resistance toward Cr(VI). Several studies have primarily concentrated on isolating and identifying chromium-removing bacteria from tannery effluents, chromium mines, etc. (Yaashikaa et al. 2019; Sanjay et al. 2020; Elahi et al. 2022).

Being one among the developing industrial cities, Vishakhapatnam is facing severe pollutant discharge problems. Common industrial effluent treatment plants are used to treat the effluents from different industries in Vishakhapatnam. The pollutants being treated in such plants vary in wide ranges both in concentration and nature. Therefore, microbes isolated from such effluent water tend to have high resistance to various contaminants ranging from heavy metals to organic pollutants. Considering the toxicity of Cr(VI) and the role of bacteria in its removal, the current work has been designed based on the following three main objectives, such as the isolation and identification of suitable bacterial strain(s), a parametric study on the removal of Cr(VI), and modeling of bioremediation. High Cr(VI)-resistant bacterial strains were initially isolated and identified. The parametric study was done using a one-factor-at-a-time (OFAT) analysis to achieve the maximum removal of Cr(VI). Finally, the logistic model of population growth was used to predict the variation of biomass growth with time. The novelty of the current study lies in utilizing high Cr(VI)-resistant indigenous bacterial strains isolated from a common industrial effluent treatment plant in Vishakhapatnam for the bioremediation of Cr(VI). This approach aims to contribute toward a greener world.

All experiments were replicated thrice, and the results were statistically analyzed using mean and standard deviation.

The Luria broth (LB) (Himedia, India) medium comprising of tryptone (10 g/L), yeast extract (5 g/L), and sodium chloride (10 g/L) was used as a liquid medium for bacterial cultivation. Solid agar plates, essential for bacterial growth, were prepared by supplementing the LB medium with 12 g/L agar. In order to prepare a stock solution of Cr(VI) with a concentration of 1,000 mg/L, 2.82 g of K₂Cr₂O₇ (Merck, India) was dissolved in 1,000 mL of sterile distilled water. The indigenous bacterial strains were cultured in the LB medium. When assessing bacterial growth in the presence of Cr(VI), the required dilution of the Cr(VI) solution was achieved by adding an appropriate volume of the sterile LB medium to the Cr(VI) solution.

An effluent sample was obtained from an industrial effluent treatment plant situated at a contaminated site in Vishakhapatnam (N 17.6707°, E 83.0930°). This treatment plant manages wastewater from multiple small pharmaceutical industrial units. Initially, the wastewater is gathered and stored in a tank. An API (American Petroleum Institute) separator then separates oil and grease molecules from the wastewater. The resulting treated effluent is then directed into an equalization tank. The sample was collected from this equalization tank. The temperature of the effluent sample was kept at 4 °C to preserve its chemical and biological characteristics (Nokman et al. 2019). The sample was serially diluted (10⁻¹⁰ times), and the diluted sample was spread on the surface of the agar plates and incubated at 35 ± 2 °C for 24 h. Colonies exhibiting distinct morphological characteristics were identified and chosen for further experiments. These selected colonies were subcultured using the same media (Seragadam et al. 2021). To prepare the stock solution of Cr(VI) of 1,000 mg/L concentration, 2.82 g of K₂Cr₂O₇ (Merck, India) was dissolved in 1,000 mL of sterile distilled water. The required dilution of the Cr(VI) solution was achieved by adding an appropriate volume of sterile distilled water. To isolate Cr(VI)-resistant bacterial strains, a nutrient agar medium was supplemented with the Cr(VI) solution having a 50 mg/L concentration. The nutrient agar was sterilized at 121 °C for 15 min and cooled to room temperature. Three bacterial strains with high resistance to Cr(VI) concentration were isolated and termed S1, S2, and S3, and such strains were selected for further studies.

The isolated metal-resistant bacterial strains were analyzed based on colony characterization, biochemical characteristics, and carbohydrate fermentation as described in Bergey's Manual for identifying the species of the bacteria (Bergey & Holt 2000). As per the standard protocol to determine the genus of the isolated bacterial strains, biochemical characterization tests like Gram stain, oxidase, catalase, methyl red, indole production, and citrate tests were done. The analysis also involved studying the utilization of carbohydrates such as glucose, sucrose, maltose, xylose, and lactose by the isolates (Marzan et al. 2017). Selected strains were sent to Cytogene (Lucknow, India) for molecular identification through 16S rRNA gene sequencing. The resulting nucleotide sequence was subjected to BLAST analysis using the online tool provided by the NCBI. Subsequently, the obtained sequences were submitted to the NCBI to acquire accession numbers for the isolated strains (Sahoo & Chaudhuri 2021).

To characterize the dry biomass of both bacterial biomasses of each strain, one grown with Cr(VI) and the other without Cr(VI), Fourier-transform infrared spectroscopy (FTIR) studies were conducted. The analysis was performed using a BRUKER Alpha II FTIR Spectrometer. For the FTIR studies, the dry biomass sample was finely ground and thoroughly mixed with potassium bromide (Merck, India) to prepare transparent pellets. These pellets were utilized to determine functional groups (Rai et al. 2021). An energy-dispersive spectroscopy (EDS) study was conducted to examine the elemental mapping on the surface of both bacterial biomasses of each strain, one with Cr(VI) and one without Cr(VI), using a TESCAN-VEGA 3 LMU instrument, which was operated at an accelerating voltage of 25 kV. The Cr(VI)-loaded bacterial biomass was prepared as follows: LB medium containing 50 mg/L Cr(VI) was prepared by diluting the stock solution of Cr(VI) with the necessary amount of LB medium. The bacterial isolates were then inoculated into such a solution and incubated in a BOD incubator (Modern Instruments, Kolkata, India) at 35 ± 2 °C for 24 h. Finally, the biomass was harvested by centrifugation (Eltek TC 8100F centrifuge) at 5,000 rpm for 15 min. The harvested biomass was subsequently washed three times with distilled water. The resulting pellet was dried in a Universal Hot Air Oven (Kolkata, India) at 50 ± 1 °C for 12 h and was used for characterization later. To get the native bacterial biomass without Cr(VI), each bacterial strain was incubated separately in the LB medium (Himedia, India) at 35 ± 2 °C for 24 h.

Isolated bacterial strains were grown in Cr(VI)-laden LB agar plates to check the minimum inhibitory concentration (MIC) of the metal. To prepare LB agar plates, initially agar (Himedia, India) was added to simulated the LB medium containing a definite amount of Cr(VI) solution. The mixture was then poured into a number of Petri dishes, and the standard protocol was followed to prepare agar plates (Marzan et al. 2017). Cr(VI) concentrations in the LB agar plates were increased gradually from 50 to 200 mg/L, and bacterial isolates were streaked on the plates. For each transfer, the culture from the lower concentration was streaked onto an agar plate containing a higher concentration of the metal. In cases where the isolates did not exhibit growth on the Petri plate, the MIC was determined following the standard protocol (Al-Ansari et al. 2021). The experiments were conducted individually with each of the three isolated Cr(VI)-resistant bacterial strains.

Three isolated bacterial strains were grown individually in a 25-mL Cr(VI)-laden LB medium having an initial concentration (IC) of 50 mg/L to test whether the bacteria could remove the metal from the medium. A loop full of inoculum of each strain was added separately at a pH value of 7 and kept in a BOD incubator at 35 ± 2 °C for 24 h. After the incubation period, the sample was collected and subjected to centrifugation at 5,000 rpm for 15 min. The resulting supernatant was then collected and analyzed for Cr(VI) concentration. The concentration of Cr(VI) was determined spectrophotometrically using 1,5-diphenyl carbazide at 540 nm using a UV–VIS spectrophotometer (Thermo Fisher Scientific, Genesys 20, India) (Sen et al. 2018).

The microenvironmental factors, such as the concentration of inoculum, ICs of Cr(VI), and pH, affect the removal efficiency of Cr(VI) and the growth of microorganisms (Rai et al. 2020). Therefore, the effects of such factors for the removal of Cr(VI) using the test strains (S1, S2, and S3) were analyzed using the OFAT method to maximize the removal of Cr(VI). For the present study, pH (5–9), the IC of Cr(VI) (20–100 mg/L) and the concentration of inoculum (1–4%) of S1, S2, and S3 were varied separately. The IC of Cr(VI) of simulated wastewater was varied first, keeping both pH of the medium and the concentration of inoculum constant at 7 and 5% (Bailey & Ollis 1986), respectively, for each of the three strains separately. The concentration of inoculum was varied next, keeping the initial pH at 7 and an IC of 60 mg/L for S1 and S3 and 80 mg/L for S2 where maximum removal was obtained. Finally, the pH of the solution was varied, with an IC of 60 mg/L Cr(VI) and the concentration of inoculum 3% for S1 and S3 and 80 mg/L and 2% for S2. The temperature of incubation was kept constant at 35 ± 2 °C, and isolates were incubated for 24 h. Following incubation, the samples were centrifuged at 5,000 rpm for 15 min. The resulting supernatant was then analyzed to determine the residual Cr(VI) concentration. The cell mass at the bottom of the centrifuge tube was rinsed three times using distilled water. Subsequently, the rinsed cell mass was dried in a hot air oven at 50 °C for 12 h, and its dry weight was measured. The most preferred operating conditions were chosen based on the highest Cr(VI) removal and biomass growth after the OFAT studies (Seragadam et al. 2021).

The isolated bacterial strains were inoculated individually into a 25-mL Cr(VI)-laden LB medium (Cr(VI) solution diluted with a definite amount of LB medium) taken in several 100 mL Erlenmeyer flasks. The flasks were then incubated for 24 h at 35 ± 2 °C in an incubator. Samples were withdrawn after every 2 h to analyze the residual concentration of Cr(VI) and the dry cell biomass. The kinetics of Cr(VI) removal were investigated at different ICs ranging from 20 to 60 mg/L. As analyzed during the parametric study, the pH and inoculum concentration were maintained at their optimal values.

The morphological and biochemical characteristics of the three potential metal-resistant isolates (S1, S2, and S3) are shown in Table 1.

Table 2 presents the elemental composition of the biomass of three bacterial isolates, such as Bacillus subtilis NITSP1, Rhizobium pusense NITSP2, and Pseudomonas aeruginosa NITSP3, grown in the LB medium (with and without Cr(VI)). The weight percent of chromium after treatment with Cr(VI) laden LB medium was 0.27% for Bacillus subtilis NITSP1, 0.17% for Rhizobium pusense NITSP2, and 0.01% for Pseudomonas aeruginosa NITSP3, whereas for treatment with LB medium only, the concentration of Cr(VI) was found as 'zero'. Comparing the elemental composition data, it is evident that Bacillus subtilis NITSP1 displayed the highest uptake capacity of chromium. Similar results were obtained by Elahi et al. (2022) in their studies involving the Bacillus cereus strain b-525k. Gram-positive bacteria, such as Bacillus subtilis and Bacillus cereus, lack an outer membrane, and cell walls are characterized by multiple layers of peptidoglycan. This peptidoglycan-rich cell wall is the primary interaction site with metal ions. It plays a crucial role in determining the ability of bacteria to bind metal ions.

Table 3 shows that Bacillus subtilis NITSP1 is most efficient in removing Cr(VI) from the synthetic wastewater at specific conditions. It is well known that Gram-positive bacteria are more efficient in removing hexavalent chromium (Banerjee et al. 2019). Hence, Bacillus subtilis NITSP1 showed the same. A key factor contributing to this is the composition of the peptidoglycan layer in Gram-positive bacteria. Such bacteria possess multiple layers of peptidoglycan enriched with unique teichoic acids, amino acids like alanine and glutamate, and meso-diaminopimelic acid. This layer consists of enzymes, glycoproteins, lipopolysaccharides, and phospholipids. These molecules act as ligands and offer active sites for metal binding. Teichoic acid and other acidic groups in the cell wall are sources of carboxyl groups that play a crucial role in metal uptake (Nanda et al. 2019). The EDS study also proved the highest capability in removing Cr(VI) using Bacillus subtilis NITSP1 (Table 1).

Figure 8 illustrates the time-dependent variation of DBC during the removal of Cr(VI) using three indigenous bacterial strains at various Cr(VI) concentrations, ranging from 20 to 60 mg/L. The maximum specific growth rates (⁠⁠) for Bacillus subtilis NITSP1, Rhizobium pusense NITSP2, and Pseudomonas aeruginosa NITSP3 were found to be 0.8439, 0.406, and 0.499 h⁻¹, respectively. The highest value of for Bacillus subtilis NITSP1 reconfirmed its superiority in removing Cr(VI) from simulated wastewater. For calculating the model parameters (⁠⁠), the least absolute residuals curve-fitting method available in MATLAB version R2023a was used, and the average absolute relative deviation (AARD) between experimental (⁠ is the number of data points) and predicted data was minimized (Gwala et al. 2021). AARD is mathematically represented as follows:

The predicted DBC calculated using the logistic model was superimposed on the corresponding plots of the variation of experimental data with time. The values of kinetic parameters ⁠, and and root mean square error (RMSE) are shown in Table 4. With an increase in initial Cr(VI) concentration from 20 to 60 mg/L, the carrying capacity, ⁠, decreased from 2.27 to 2.104, 6.87 to 3.887, and 2.524 to 2.184 g/L for Bacillus subtilis NITSP1, Rhizobium pusense NITSP2, and Pseudomonas aeruginosa NITSP3, respectively. For each initial Cr(VI) concentration, the values between experimental and simulated data of DBC were in the range of 0.94–0.987, whereas the RMSE values were in the range of 0.099–0.483. The high values of and low values of RMSE proved that the proposed logistic model holds good in predicting the variation of biomass growth with time. Using the logistic model, Sen et al. (2018) found the maximum specific growth rate (⁠⁠) and maximum carrying capacity of 0.31 day⁻¹ and 0.857 g/L, respectively, for the removal of Cr(VI) at an IC of 20 mg/L using the cyanobacterial strain Limnococcus sp.

Three bacterial strains (Bacillus subtilis NITSP1, Rhizobium pusense NITSP2, and Pseudomonas aeruginosa NITSP3) having high resistance to Cr(VI) were isolated from a common industrial effluent treatment plant, a contaminated site in Vishakhapatnam. Bacillus subtilis NITSP1 and Rhizobium pusense NITSP2 exhibited growth at 200 mg/L Cr(VI) within 24 h at 35 ± 2 °C and neutral pH. The growth of Pseudomonas aeruginosa NITSP3 was observed at 100 mg/L Cr(VI) for 24 h at 35 ± 2 °C at neutral pH. Among the three isolates, Bacillus subtilis NITSP1 was found to be the most efficient in removing Cr(VI) (80.194 ± 4.0%) with an initial Cr(VI) concentration of 60 mg/L, a pH value of 7.0, an inoculum size of 2% [(v/v)], and an incubation period of 24 h. The present study suggests that the logistic model of population growth could adequately describe the variation of DBC over time, indicating the potential of the current strains to remove Cr(VI) from synthetic wastewater. However, further research is needed to explore the applicability of these findings for the treatment of real industrial wastewater, taking into account all relevant parameters in a continuous reactor setup.

The authors are thankful to the National Institute of Technology Durgapur and Gayathri Vidya Parishad College of Engineering, Vishakhapatnam, for providing all the required facilities to do research work.

S.D. and K.C.G. conceptualized the study. S.D. and B.S. carried out methodology. B.S. did formal analysis. P.S. investigated the work. P.S. wrote the original draft preparation. K.C.G., S.D., and B.S. wrote the original draft and reviewed and edited the manuscript. B.S. and S.D. found resources for the study. S.D., K.C.G., and B.S. supervised the work.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.