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Microbiology and Biotechnology Letters

Research Article(보문)

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Environmental Microbiology (EM)  |  Biodegradation and Bioremediation

Microbiol. Biotechnol. Lett. 2024; 52(4): 416-427

https://doi.org/10.48022/mbl.2407.07006

Received: July 8, 2024; Revised: October 6, 2024; Accepted: October 19, 2024

Cadmium Resistance and Bioremediation Potential of Bacteria Isolated from Hospital Wastewater Samples of Bangladesh

Taslin Jahan Mou1,2†*, Rahat Ara Mun1†, Farhana Haque1, Nadim Sharif1, Abdul Kadir Ibne Kamal3, Md. Fokhrul Islam2,4, Md. Shahedur Rahman5, Shuvra kanti Dey1, and Anowar Khasru Parvez1*

1Department of Microbiology, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh
2Institute of Quantitative Biology, Biochemistry and Biotechnology, School of Biological Sciences, The University of Edinburgh, United Kingdom-EH9 3FF
3Department of Environmental Sciences, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh
4Department of Pharmacy, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh
5Department of Genetic Engineering and Biotechnology, Jashore University of Science and Technology, Jashore-7408, Bangladesh

Correspondence to :
Taslin Jahan Mou,              moumicro@juniv.edu
Anowar Khasru Parvez,        khasru73@juniv.edu

†These authors contributed equally to this work.

Abstract

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Cadmium (Cd) pollution is considered as a pressing concern from both human and environment health perspective owing to its high toxicity and persistence nature. This study aimed to explore the Cd pollution scenario in Hospital wastewater (HWW) of Bangladesh and to screen out potential Cd-resistant bacteria for their possible application in cadmium bioremediation. Cd concentration of the Hospital wastewater samples (HWW, n = 7) was determined by Atomic Absorption Spectroscopy (AAS). The Cd content of the samples were in the range of 0.6624-5.0482 mg/l exceeding the acceptable standard limit in the environment. Preliminary, 93 Cd-resistant bacteria were isolated of which 88.1 % (n = 82) were gram negative whereas 11.82% (n = 11) were gram positive. 86% (n = 80) of the Cd-resistant isolates was observed with co-resistance against three other metals-zinc, cobalt, and nickel. Polymerase chain reaction detected the presence of cadmium resistant genetic determinant (czc gene) in 34.4% (n = 32) of the isolates. Cd-resistant isolates were distributed into 13 major genotypic groups according to Randomly Amplified Polymorphic DNA (RAPD) analysis. Genotypically diverse groups of bacterial genera belonged to Proteus, Pseudomonas, and Providencia were identified in the phylogenetic analysis using partial 16S rRNA gene sequence. 13.9% (n = 13) of the Cd-resistant isolates were noted to have high tolerance level for cadmium and the potential for Cd removal in range of (10-90%). Higher percentage of Cd removal was determined for Pseudomonas sp. (90%) and Proteus sp. (82%) in the laboratory testing. The Cd removal capacity of these native isolates could be exploited for its prospective application in the bioremediation of Cd from contaminated environmental sites in Bangladesh.

Keywords: Cadmium pollution, hospital wastewater, bioremediation, Pseudomonas, Proteus

Graphical Abstract


Introduction

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Cadmium (Cd) is a highly toxic metal pollutant which is a global public health concern because of its persistence in environment and bioaccumulation properties for living biota [1]. Sources of cadmium pollution could be associated with anthropogenic and industrial activities. Improper waste disposal could be also a potent contributing factor for cadmium pollution in the environment. Prolonged exposure to cadmium can have major consequences on human health such as kidney disease, osteoporosis, cardiovascular disease and cancer [1, 2]. In developing countries like Bangladesh Cadmium pollution has also been turned a growing concern due to rapid industrialization, urban development and lack of sufficient environmental regulatory frameworks [3].

Hospitals generate a significant quantity of wastewater that may have detrimental effects on the environment when discharged without any treatment. The discharged wastewater is a major source of diverse pollutants in the environment, which are generated from different practices in the hospital like drug treatment, diagnostic laboratory and research activities, radiology and trough the patients from Operation Theater [4, 5]. These substances include metabolized pharmaceuticals, disinfectants, solvents, heavy metals, toxic chemicals, radioactive substances and pathogenic microorganisms [5, 6]. Heavy metals enter hospital wastewater through the use of medicines, photographic materials, and medical tools with the heavy metals as an ingredient and their improper disposal without treatment [7]. Indiscriminate discharge of these pollutants into the environment imposes significant threat to the living organisms and for the environment due to their toxic, non-biodegradable, and persistent characteristics [8]. Heavy metals such as cadmium, lead, chromium, etc. are highly soluble in aqueous solutions and readily absorbed by the living organisms, which could be carcinogenic and mutagenic as well [9]. Among the heavy metals, cadmium should be considered with special attention as it can enter the food chain very easily and deleterious for living organisms at minute quantity (0.001−0.1 mg/l) [10, 11].

In Bangladesh, medical waste disposal is not well established and regulated; therefore, this could further disseminate the Cd contamination in the surrounding soil as well as the aquatic environment. Numerous traditional methods have been explored to remove cadmium from wastewater, primarily relying on chemical precipitation, ion exchange, membrane technology, and adsorption. However, these approaches are not cost-effective, require high-energy consumption, and result in negative consequences for the environment with the production of more toxic residual byproduct [12, 13]. From these perspectives, they are not convenient to be applied in developing countries like Bangladesh. More straightforward and relatively inexpensive approaches are based on biological methods such as microbial bioremediation and phytoremediation, which concurrently are more environmentally friendly and cost-effective [14].

Many investigators have reported the potential application and utilization of cadmium resistant bacteria for Cadmium bioremediation [15]. Microbes employ various survival strategies to counteract cadmium stress, such as sequestering cadmium ions and actively expelling them, while others utilize enzymatic detoxification and accumulate cadmium [16, 17]. Detoxification mechanisms can target a single metal or a cluster of chemically similar metals and drugs. Additionally, these mechanisms may differ depending on the microorganism types. In gram-negative bacteria, a multi-protein chemiosmotic antiport system contributes to the development of Cd resistance, which is the Czc system [17, 18]. The system is comprised of three subunits named czcC, czcB, and czcA [19]. The structural genes czcCBA are regulated by six proteins which are- CzcD, CzcR, CzcS, CzcN, CzcI, and ‘‘RpoX’’ (an unknown sigma factor). Within the trio of structural genes, it is suggested that czcA serves as the functional efflux-transport protein. Nevertheless, its selectivity for ion exportation appears to be controlled by the existence of two additional structural proteins [20].

In a prior review, cadmium contamination levels were evaluated in the diet and river water of Bangladesh, finding that they fell within the guidelines set by the Bangladesh Standard Testing Institute and World Health Organization [21]. However, higher level of cadmium was detected in the agricultural soils of Bangladesh [22]. As far our knowledge concern, there is no study reported on cadmium pollution scenario of hospital wastewater of Bangladesh. Therefore, we designed the present study to determine the cadmium level in the hospital wastewater samples of Bangladesh and to explore the bioremediation potential of potential cadmium resistant bacteria isolated from the samples. Herein the study, the sampling sites were mainly based on Savar, which comprises 34 different medical facilities, which provide health care support for approximately 1,387,426 population from the area, as well as patients from other parts of the country.

Materials and Methods

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Sampling and study area

Savar is a rapidly rising hubs of urbanization and industrial activities in Bangladesh which is located between 23°44´N and 23°12´N latitude and between 90°11´E and 90°22´E longitude. There are a number of medical facilities and hospitals in around Savar, where in majority cases the waste disposal system is not well regulated. Most of the wastes from the hospitals in this area are disposed into the surrounding water habitats without any proper treatment. Therefore, seven hospitals (Fig. 1) were selected in this study, from where the wastes are being directly discharged into nearby water system. Water samples were collected in sterile sampling bottles which were washed, dried and then treated with 20% HNO3 to avoid contamination and the absorption of trace metals. The sampling points, collection date, and coding numbers of the sample were marked on the container appropriately. The collected samples were labeled as E, D, G, S, A, P, and R according to the name of the source hospital. The temperature and pH of the samples were measured on the spot. The collected samples were kept in an icebox for transportation to the laboratory.

Figure 1.Study area and sample collection sites.

Determination of physicochemical parameters

Physicochemical parameters analysis of the samples was conducted according to the standard methods recommended by APHA (2005). All the analysis were performed immediately after collection of the samples.

Cadmium concentration of the samples were measured using Atomic Absorption Spectrometry (AAS). For the AAS analysis, the samples were digested with a combination of HNO3 (conc.) & HClO4 (conc.) Then the samples were heated at 200℃ until about dry in a hot plate. Finally, the samples were cooled, passed through a nitrate cellulose filter, and analyzed using a Shimadzu Atomic Absorption Spectrophotometer (AAS) with a Furnace system controlled by AAWizard software. Temperature of the samples were determined using a mercury thermometer. EC values of the samples were recorded by the EC meter (HANNA, EC 241 Conductivity meter). TDS meter (Model HANNA HI 8734) was employed to measure the TDS value, whereas the determination of TSS was conducted through gravimetric analysis, employing evaporating dishes in conjunction with Whatman filter paper with a pore size of 11 μm. Modified Winkler's method was followed to measure the BOD and COD value. Microprocessor turbidity meter, DO meter and glass electrode pH meter were used to determine the DO and pH value respectively. The coefficient of correlation between the physicochemical parameters of the samples was determined the Pearson correlations test using GraphPad prism software (version 8.0.1).

Isolation and preservation of the bacterial isolates

Cadmium-resistant bacteria were isolated by serially diluting the samples in normal saline and inoculated on nutrient agar (NA) medium (HIMEDIA) supplemented with a 0.5 mM of cadmium sulfate (3 CdSO4·8H2O). Plates were incubated at room temperature (25℃) for 3 days. The colonies observed were quantified as colony-forming units per milliliter (CFU/ml), and visually distinct colonies were sub-cultured for purification. Pure colony of the isolates were preserved at -20℃ with nutrient broth in 50% glycerol.

Biochemical characterization

Biochemical characterization & identification of bacterial isolates were done by Gram's staining & biochemical tests such as IMViC tests (indole, methyl red, and Voges-Proskauer and citrate utilization tests), carbohydrate fermentation assay using Kligler Iron Agar (KIA), Oxidase test and Catalase test. All the procedures were followed as described in Bergey's Manual of Determinative Bacteriology [23].

Analysis of cadmium tolerance and Co-resistance to Zinc, Cobalt, Nickel

For the cadmium tolerance test, all the bacterial isolates were streaked on the Luria Bertani (LB) (HIMEDIA) agar media supplemented with cadmium sulfate hydrate (3CdSO4·8H2O). The concentrations of Cd used for the tolerance assay were from 1−8 mM. ANOVA was performed to evaluate if there were significant differences in cadmium resistance between the bacterial isolates. To analyze co-resistance to other heavy metals, the Cd-resistant isolates were subcultured into LB agar media incorporated with zinc, cobalt & nickel metal salt solution (0.5 mM) separately. Each plate was incubated at room temperature (25℃). Growth of the isolates on the media was observed for three days [24].

Bacterial DNA extraction by boiling method and RAPD genotyping of the Isolates

For molecular characterization, the isolates were subjected to genomic DNA extraction by the modified boiling method described by Sultana et al. 2017 [25]. Briefly, single colony was grown overnight in 5 ml nutrient broth at the shaker incubator for (25℃). Culture was then taken in an eppendorf tube (1 ml) and centrifuged for 10 min at 12,000 rpm. Pellets were collected and suspended in distilled water and re-centrifuged for washing. After centrifugation, the pellets were dissolved in 200 μl PCR water and kept at 100℃ boiling temperature for 10 min. Then the samples were immediately kept on ice for 10 min. Then the eppendorf’s were again centrifuged at 10,000 rpm for 10 min. Fresh eppendorf tube was used to collect the supernatant.

RAPD typing is based on the amplification of random segments of bacteria genomic DNA employing short single-stranded primers. It can rapidly detect genetic diversity and strain differentiation among bacterial isolates even among closely related species. For RAPD genotyping, random primer (1283) was used and PCR reactions mixtures were prepared in 25-μl volumes as follows-12.5 μl G2 green mix, 2 μl primer (10 pmol), and 4 μl template DNA and nuclease-Free Water up to the volume. Individual reaction components were obtained commercially (Promega, USA) and amplification was performed using a PCR thermocycler (Biometra, Germany). PCR conditions included the steps - 94℃ for 5 min (initial denaturation), followed by 30 cycles of 1 min at 94℃, 1 min at 39℃, and 2 min at 72℃ with a final extension of 5 min at 72℃ [26]. Amplified PCR product was subjected to agarose gel electrophoresis for 30 min at 100 V with 100 bp DNA ladder (Promega). Gels were stained with 0.4 μg/ml ethidium bromide (Sigma, USA). Gel was visualized under UV light and digitalized by the Alpha Imager HP System Versatile Gel Imaging (USA).

Molecular identification and PCR Detection of Cadmium resistance gene

16s identification of the bacterial isolates was carried out using the primer sets-8F (5-AGT TTG ATC CTG GCT CAG-3) and 1492R (5-ACC TTG TTA CGA CTT-3). PCR amplification conditions were maintained according to the procedure described in elsewhere [27]. Cadmium resistance gene czcC was amplified from the isolates using the primer sets czcF (AAC CAG ATC TCG CGC GAG AAC) and czcR (CGG CAA CAC CAG TAG GGT CAG) [18]. PCR mixture (25 μl) comprised 0.5 μM of each primer, 200 μM dNTPS, 1.0 U Taq DNA polymerase, PCR buffer supplied with the enzyme and 1 μl of template DNA. Nuclease free water was used to balance the volume of reaction mixture. PCR reactions involved the following conditions: 95℃ for 5 min for denaturation, 36-cycle programmed denaturation at 94℃ for 1 min, 61.6℃ for 30 to anneal, 72℃ for 1 min in extension and a final extension at 72 ℃ for 10 min. PCR amplicons were visualized in 1.5% agarose gel electrophoresis.

Nucleotide sequence and phylogenetic analysis

Big-Dye terminator cycle sequencing kit and an ABI Prism 310 Genetic Analyzer (Applied Biosystems Inc., USA) were used to determine the nucleotide sequences of PCR amplicons (DNA). The chromatogram of sequences was analyzed using Chromas 2.6.5 (Technelysium, Australia). The sequences were blasted for the identification of the isolates. ClustalW Multiple Alignment algorithm in the BioEdit 7.2.6 softwarce was employed to conduct the Multiple sequence alignment and the sequences were submitted to the NCBI (National Center for Biotechnology Information) for the accession numbers. The accession numbers are as follows- OP860655, OP860656, OP860657, OP860658, OP860659, OP860660, OP860661, OP860662, OP860663, OP860664, OP860676, and OP860676. The MEGA 11 was used to conduct the phylogenetic relationship analyses of 16S rRNA genes using a partial sequence of these amplicons and the reference sequences. Phylogenetic trees were constructed using neighbor-joining method with1000 bootstrap replicates.

Determination of cadmium accumulation capacity of the isolates

Study isolates were grown in 5 ml Luria Bertani broth and kept in shaker incubator with 200 rpm. After 24 h, the OD600 of the bacterial cultures were measured, adjusted to OD600 = 1 and then 5 ml was transferred to 50 ml of LB broth containing 100 μg/ml of cadmium. These were kept again at shaker incubator for 24 h with 200 rpm. LB media without the bacterial culture was kept as a control for the assay. Throughout the growth phase, a 4 ml aliquot of bacterial culture was collected at six-hour intervals up to 24 h, then centrifuged at 6000 rpm for 30 min. Supernatant was collected and digested with HNO3 & HClO4, heated at 200℃ until dry in a hot plate, and finally were cooled and filtrated through a nitrate cellulose filter. The samples were then analyzed using standard AAS to quantify the Cd accumulation level of the isolates [28].

Results

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Physicochemical parameters and bacteriological quality of the hospital effluent samples

The temperature of the collected samples ranged from 27.9℃ to 29.4℃. The highest EC (1014 μs/cm) and TDS (647 mg/l) was detected in sample R, whereas TSS (830 (mg/l)) value was highest in Sample D. The highest BOD value (189 mg/l) was detected in sample G. The highest COD value (413 mg/l) was detected in sample R. Highest level of Cadmium content (5.0482 mg/l) was found in the in sample G (Table 1). Total viable bacterial count (TVBC) of the samples was in the range of 1.3 × 106 to 5.6 × 107 CFU/ml (Table 1). The Pearson correlation coefficients matrix of the water parameters are presented Table 2. The correlation coefficients, which range from -1 to 1, illustrate the relationships between these parameters, highlighting significant trends and interactions.

Table 1 . Physicochemical parameters and Cadmium concentration of the Hospital waste water samples.

Sample IDTemp. (℃)EC (μs/cm)TDS (mg/l)TSS (mg/l)Turbidity (FTU)pHDO (mg/l)BOD (mg/l)COD (mg/l)Cd (mg/l)Total viable bacterial count (TVBC) cfu/ml
E29.361739366551.207.515.0916410.91403.5 × 106
D28.372846789063.607.515.3217360.66241.3 × 106
G28.529218945093.207.765.181891645.04824 × 106
S27.962821317055.807.895.51341774.90921.8 × 107
A28.456836536087.707.495.53482345.01403.3 × 107
P27.9559357165102.807.255.49262704.09105.6 × 107
R29.4101464783066.107.765.251824135.03563.2 × 106


Table 2 . Correlation coefficients among the physicochemical parameters and Cadmium concentration of the samples.

TemperaturepHECTDSTSSTurbidityDOBODCODCdTVBC
Temperatur1.000
pH0.1731.000
EC0.4640.1091.000
TDS0.619-0.1760.8871.000
TSS0.7040.0820.5510.7191.000
Turbidity-0.426-0.460-0.504-0.258-0.4741.000
DO-0.764-0.174-0.009-0.204-0.6510.3511.000
BOD0.4170.5320.0290.1290.2240.243-0.3861.000
COD0.1400.1280.4250.398-0.1630.3700.3070.5541.000
Cd-0.2070.391-0.122-0.233-0.5560.4910.4450.5730.7701.000
TVBC-0.278-0.4520.1910.199-0.5100.5800.632-0.0530.7380.5181.000


Screening of cadmium resistant bacteria, cadmium tolerance and Co resistance to Zinc, Cobalt, Nickel

Ninety-three Cd-resistant bacteria were isolated from the hospital wastewater samples. The heterotrophic Cd-resistant strains in the biochemical tests showed different characteristics. Among the isolates, 87% (81 of 93) were Gram-negative, and 13% (12 of 93) were Gram-positive. In the cadmium tolerance assay, all the isolates showed tolerance to 1 mM Cd, but only six isolates showed tolerance up to 3 mM Cd. According to the ANOVA test, for the cadmium tolerance label of the isolates F-value was 1.72 with a corresponding p-value of 0.069 which indicate the difference are not statistically significant. Co-resistance to 0.5 mM zinc, cobalt, and nickel were detected in 86% (80 of 93) of the isolates, among them a very small proportion of isolates showed mono-resistance at 3.78% (3 of 80) and dual-resistance at 13.75% (11 of 80) while resistance to all of the three heavy metals was observed in 82.5% (66 of 80) metal resistant isolates.

Molecular characterization of cadmium resistant isolates

About 34% (32 of 93) of isolates had cadmium resistant genetic determinant (czc gene) with an amplicon size approximately 650 bp (Fig. 2). RAPD analysis discriminated 93 isolates into 13 different major patterns with the combination of nine different fragments varied in size from 250 bp to >1.1 kbp (Fig. 3). Individual bands were scored and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) was employed to analyze the Cluster (Fig. 4). From 13 genotypic groups, one representative isolate was selected for detailed 16S rRNA gene sequence identification (approximately 1484 bp) to correlate the phylogenetic relationship to the nearest species-level. However, the similarity threshold for one sequence was not good for the phylogenetic analysis. Therefore, twelve sequences from the representative genotypes were included in the phylogenetic analysis. According to the analysis, the majority of the isolates in this study are closely related to Proteus mirabilis, followed by Pseudomonas aeruginosa, Providencia retgerri, and Bacillus sp. (Fig. 5).

Figure 2.PCR amplification of cadmium resistance gene (czc) (Reaction mixture without any template DNA was used as negative control and marker used was 100 bp DNA ladder).

Figure 3.RAPD profiling pattern of the isolates in 1.5% agarose gel (Reaction mixture without any template DNA was used as negative control and marker used was 100 bp DNA ladder).

Figure 4.Cluster analysis of the RAPD genotypes of the study isolates.

Figure 5.Phylogenetic tree of 16S rRNA gene sequences of cadmium resistant isolates.

Determination of cadmium accumulation capacity

In this study, thirteen isolates (from 13 RAPD group-P2, P3, P22, A60, P39, A55, A35, P5, A50, S1b, S1i, P21, and G1c) with high cadmium tolerance were tested to determine the capacity to accumulate cadmium from media. Among these isolates, P2, A50, P3 and A55 had better Cd removal capacity than other isolates, and all of these four isolates were positive for the presence of the czc gene. They removed 70−90% of Cd from the media. G1c, P21, P22, A35, removed 30−70% Cd, and P5, A60, S1b, S1i removed 10-20% Cd (Figs. 6 and 7). All the isolates removed the maximum amount of Cd from 6 hours to 18 h.

Figure 6.(A) Cadmium removal by the bacterial isolates (P3, A55, P39, S1b, S1i) with initially exposed to 100 mg of cad-mium in the aqueous media (B) Cadmium removal by the bacterial isolates (P2, P22, A35, A60, P5, A50) with initially exposed to 100 mg of cadmium in the aqueous media.

Figure 7.Cadmium Removal (%) efficiency of the bacterial isolates (P3, A55, P39, S1b, S1i, P2, P22, A35, A60, P5, A50).

Discussion

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Environmental pollution with heavy metals is a critical concern in Bangladesh due to rapid urbanization, industrialization, and the improper waste disposal system. Bioremediation is a sustainable and environmentally friendly solution for heavy metal pollution like cadmium, arsenic, chromium, etc. [14] Bioremediation using indigenous microbial community could be potential alternative for the conventional physical and chemical treatment methods. Therefore, the study focused to screen out cadmium resistant bacteria from hospital wastewater samples of Bangladesh for their possible application and utilization in the bioremediation of cadmium.

Firstly, we determined the physicochemical parameters of the hospital wastewater sample. The findings reveal that, Cadmium was present in seven hospital wastewater samples in significant concentrations ranging 0.6624−5.0482 mg/l. The measured concentrations of Cd in samples were higher than the permissible limit 0.005 mg/l in water standards of Bangladesh [29]. The report of cadmium pollution in hospital wastewater is quite rare in Bangladesh and the source of this pollution is elusive. In a review article, it was reported that cadmium content in river water and sediments of Bangladesh were within the Bangladesh Standard Testing Institute limit [30]. However, our findings divulge cadmium pollution in hospital wastewater in Bangladesh for the first time and thus unveil a new environmental threat. Therefore, the cadmium pollution scenario in Bangladesh should be monitored properly for protecting biodiversity as well as public health [21].

The other physicochemical parameters were also divergent in our study of hospital water samples, which significantly affect the water quality. The average temperature of the hospital effluent was 28.5℃. The measured EC value of this study (559 to 1014 μs/cm) was found lower than the accepted standard value for wastewater, which is 1200 μs/cm in Bangladesh. Low EC shows that a small number of ionic substances are present in hospital effluent. TDS value of the samples was measured in the range of 189−647 mg/l which was also within the permissible limit for Bangladesh. TSS value of the samples ranged from 165 to 890 mg/l, and all were above the standard value 150 mg/l for inland surface water [15]. In some previous studies, the TSS in the hospital wastewater was in a range of 36 to 269 mg/l [32]. The turbidity of hospital wastewater varied from 51.20 FTU to 102.80 FTU. The turbidity values obtained from the effluent were much higher than the acceptable standard in Bangladesh which is <10 FTU [32]. The pH levels of the wastewater samples varied between 7.25 and 7.89, indicating a slightly alkaline nature. This falls within the acceptable range for a variety of domestic and recreational purposes, as defined by the Department of Environment (DoE), which sets the standard pH range for such uses between 6 and 9 [29]. Low pH affects the solubility of cadmium in the water environment, here in our study the higher cadmium found in the samples does not correlate with the pH. According to the Bangladesh standard, the wastewater should have a DO in the range of 4.8−8 mg/l [27], whereas in this study the DO values were higher as ranged from 5.09 to 5.53 mg/l. The BOD value ranged from 16 to 189 mg/l. As per the Environment Conservation Rules, 1997 the permissible limit for BOD in wastewater discharge is 50 mg/l [31]. Except for two samples R and G, all the samples had lower BOD values than the standards, which might indicate the effluents' good quality. COD value of the samples were above the standard (50 mg/l) except for samples D and E [30].

In the correlation analysis, temperature exhibited a strong positive correlation with TSS (0.704) and a strong negative correlation with DO (-0.764), suggesting that higher temperatures are associated with increased suspended solids and decreased oxygen levels. pH shows moderate positive correlations with Cd (0.391) whereas moderate negative correlations with Turbidity (-0.460) and TVBC (-0.452), indicating potential influences on heavy metal presence and biological activity. EC is strongly positively correlated with TDS (0.887) and moderately with COD (0.425), while TDS also has strong positive correlations with TSS (0.719), reflecting the interdependence of dissolved ion concentration measures. Turbidity displays strong negative correlations with Temperature (-0.426), pH (-0.460), EC (-0.504), and TSS (-0.474), implying that clearer water tends to have lower levels of these parameters. DO's strong negative correlations with Temperature (-0.764) and TSS (-0.651) underscore the impact of warmer, more turbid waters on oxygen levels. BOD and COD show positive correlations with Cd and TVBC, indicating higher organic and chemical pollution levels associated with heavy metals and bacterial counts. Finally, Cd's strong positive correlation with COD (0.770) and TVBC's correlations with multiple parameters suggest that heavy metal pollution correlates with increased bacterial counts, reflecting reduced water quality. Significant increase in COD levels compared with BOD also indicates the possible presence of toxicant levels, e.g., heavy metals may be possibly present in the wastewater [32, 33].

In the cadmium tolerance assay, six isolates belonging two group identified by 16S rRNA sequencing, Proteus mirabilis (P3, A55, and P39) and Pseudomonas aeruginosa (P2, A50 and P21) were found to be tolerant against 3 mM level of cadmium in the media which is very high compared to the standard acceptable limit in the environment. High level of cadmium tolerance of the isolates might be correlated with their survival habitat heavily contaminated with cadmium as observed in the physicochemical parameter analysis. In 2014, similarly, a highly Cd-resistant Pseudomonas sp. was reported from wastewater discharge in Malaysia [34]. The higher level of cadmium resistance in these bacterial isolates could upgrade their possible application field level cadmium bioremediation in the contaminated sites.

In our study, RAPD revealed thirteen different patterns among 93 bacterial isolates. Cluster analysis through Unweighted Pair Group Method with Arithmetic Mean (UPGMA) revealed that the thirteen group patterns are divided into main two clusters, where the second cluster contains different subgroups (Fig. 4). 16S rRNA sequencing confirmed the presence of bacterial genera of Pseudomonas, Proteus, and Providencia among the study isolates [34]. From the Phylogenetic tree, every isolate forms a different clade indicating that they differ in strain level, and each has individual clustering. Identifying the rest of the isolates according to their genotype remains to be carried out.

Cadmium resistant genetic determinant (czcC gene) is reported to be linked with high tolerance of cadmium in bacteria [24]. It is also responsible for resistance to other heavy metals [35, 36]. It was reported that czcC gene was found in Alcaligenes sp. and Pseudomonas sp. in some previous studies [37]. In the present study, we found 32 isolates were positive for the presence of czc gene. Interestingly, all of these isolated were from the hospital wastewater samples with higher cadmium concentration in range of 4−5 mg/l. Considering this finding, it could be inferred that the higher level of cadmium in the environment might be associated with metal resistance in the microbiota through different mechanisms including the presence of genetic determinants like czc gene.

In our study, four isolates identified as Pseudomonas sp. (P2 and A50) and Proteus mirabilis (P3 and A55) were revealed with convincing Cd removing potential (70−90%) in the bioaccumulation assay (Fig. 7). All the isolates removed maximum amount of cadmium during 6 h to 18 h. When a microorganism is introduced into a growth media with toxic substances, the eventual outcome that happen, the microbial cell will eventually be stressed due to pollutant toxicity [21]. After that, in the log phase (6−18 h), isolates will remove the maximum amount of the cadmium as the bacteria rapidly multiply with time, so the accumulation of the cadmium into the bacteria could also be higher. In a previous investigation, Pseudomonas sp. was reported to bio-accumulate 75% cadmium [19]. A minor lessening in cadmium removal was observed in the medium after 16 h in our study. It is described somewhere else that once metal bioaccumulation peaks, there is a subsequent decrease in growth, leading to a reduction in the number of viable cells within the culture [19]. Further, determination of Cd removal capacity and molecular characterization remains to be carried out to fully appreciate the cadmium remediation potential of the isolates.

Hospital wastewater samples of this study contained significant amount of cadmium exceeding the standard limits, which is hazardous to the environment and public health. Proper monitoring of the waste disposal is crucial to prevent the heavy metal pollution in aquatic environment. Ninety-three Cd-resistant bacteria screened from the samples revealed a wide variety of microbial species distributed in thirteen genera. The Cd accumulation rate of Pseudomonas (P2, A50) and Proteus (A55, P3) were high enough to be considered for bioremediation potential. Further molecular profiling and characterization of these potential isolates such as their resistance and removal mechanism would pave the way for their maximum possible application in the field experiments.

Limitation: In this study, a limited number of samples were collected from a particular area in Bangladesh, which might not comprehensively represent the overall situation of cadmium pollution in country. This obviously necessitates a more robust sampling strategy. Further investigations focusing on large scale sampling and advanced molecular analyses are crucial for developing effective bioremediation strategies and addressing cadmium pollution in Bangladesh.

Acknowledgments

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This study has been funded by University Grants Commission (UGC), Bangladesh and Jahangirnagar University. Furthermore, the authors express their gratitude to Mr. Nikhil Chandra Bhoumik from the Wazed Mia Center of Excellence, Jahangirnagar University, for his assistance in detecting the cadmium content of the samples.

Author Contributions

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Conceptualization: Taslin Jahan Mou

Data curation: Rahat ara Mun, Nadim Sharif, Abdul Kadir Ibne Kamal, Md. Fokhrul Islam

Formal analysis: Taslin Jahan Mou, Rahat Ara Mun, Farhana Haque,

Funding acquisition: Taslin Jahan Mou, Md. Fokhrul Islam

Investigation: Rahat Ara Mun, Taslin Jahan Mou

Methodology: Taslin Jahan Mou, Rahat ara Mun

Resources: Md. Shahedur Rahman

Software: Md. Shahedur Rahman

Supervision: Taslin Jahan Mou, Anowar Khasru Parvez,

Validation: Shuvra kanti Dey, Anowar Khasru Parvez

References

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