Isolation and screening of heavy metal-resistant Aeromonas sanarellii
Depending on how their cell walls are constructed, bacteria can be divided into two main types using the fundamental microbiology technique known as gramme staining (Yao et al. 2023). By assisting in the initial identification and distinction of bacteria, this knowledge facilitates the choice of suitable diagnostic procedures and therapeutic approaches. Gramme staining offers important insights into the bacterial cell wall, which have an impact on the sensitivity to antibiotics (Liang et al. 2019). Biochemical assays and colony morphology were used to identify the bacteria (Yao et al. 2023). In this study, Gram staining of all the 10 isolates was done and visualized at 100x. Morphology and optical density (OD) of the isolates were tabulated. It may be seen from Table 1 that isolate 7 only showed the maximum growth at the minimum concentration of cadmium (OD of 0.3971 mM) and zinc (OD of 0.9756 mM). The maximum bacterial growth found at the minimum concentration of zinc and cadmium revealed that bacteria may not survive in the presence of cadmium and zinc. Hence, the isolate 7 was selected and subjected to PCR amplification for identification of 16S rRNA gene sequence. The identified partial sequence of 16S rRNA was assigned to the species A. sanarellii based on the phylogenetic tree analysis and the result reveals that the isolate 7 is the closest to A. sanarellii strain A2-67 (Fig. 2).
Table 1
Absorbance values of isolates grown in minimum concentration of Cadmium and Zinc
Isolate
|
OD at 600nm (Cd 0.1 mM)
|
OD at 600nm (Zn 1 mM)
|
Isolate 1
|
0.2981
|
0.7612
|
Isolate 2
|
0.0412
|
0.0671
|
Isolate 3
|
0.0189
|
0.0913
|
Isolate 4
|
0.1498
|
0.1298
|
Isolate 5
|
0.0812
|
0.0627
|
Isolate 6
|
0.0198
|
0.4714
|
Isolate 7
|
0.3971
|
0.9756
|
Isolate 8
|
0.1876
|
0.0891
|
Isolate 9
|
0.0917
|
0.5618
|
Isolate 10
|
0.1781
|
0.0971
|
Evaluation of heavy metal removal by Aeromonas sanarellii
Aeromonas sanarellii isolated in this study and stranded Azospirillum brasilense MTCC 4034 aquired from the Microbial Type Culture Collection and Gene Bank in India, were used to evaluate the identified heavy metal removal from the soil. The effects of different concentration on the growth of both orgainsms for Cd and Zn are shown in Fig. 3. The exposure to Cd and Zn had an impact on the pace of bacterial growth. Rapid growth reduction was seen at higher cadmium and zinc concentrations. Considering the outcomes of bacterial growth Azospirillum brasilense MTCC 4034 and the EC50 values for Cd and Zn at various concentrations of Aeromonas sanarellii were computed. In Azospirillum brasilense MTCC 4034 and Aeromonas sanarellii, the observed EC50 for Cd was 0.1 mM, but the values for Zn were 3 and 6 mM in those two species.
In Fig. 3, When compared to the control Azospirillum brasilence MTCC 4034 showed a lesser growth rate but were able to survive at the elevated level of the heavy metal concentration. In case of Aeramonas sanarellii which was isolated from the environment showed a decreased pattern of growth which may be due to the acclimatation of the bacteria to the invitro condition but still it managed to grow with a pattern of fluctuation. The bacteria demonstrated resistance to both Cd and Zn after being isolated from metal that was polluted with both metals and the ability to grow in a medium amended with 0.1 mM Cd and 3 and 6 mM Zn (Matilda et al. 2019).
According to the morphological, cultural, biochemical, and 16S rDNA partial sequence of the Aeromonas sanarellii, this study isolated. This is similar to earlier studies (Vassallo et al. 2018; Hyang et al. 2021; Uqab et al. 2022; Jebril et al. 2023). By observing how different concentrations affect the growth of Aeromonas sanarellii and Azospirillum brasilense bacteria, it is possible to infer that the bacteria species may not survive at greater concentrations of Cd and Zn than at low concentrations of both Cd and Zn. Figures 3a and 3b shows that Aeromonas sanarellii tolerated Cd and Zn concentrations up to 0.2 mM and 4 mM, respectively, for 45 minutes of incubation. Similar to Figs. 3c and 3d, Azospirillum brasilense MTCC 4034 tolerated up to 0.2 mM of Cd and 4 mM of Zn for 20 minutes of incubation..
Vassallo et al. (2018) discovered that Escherichia coli was tolerant to a Cd concentration of 6 mg/L when using a minimal inhibitory concentration (MIC) experiment with these bacteria as their model system. Brevibacillus agri C15 and Brevibacillus agri CdR species tolerate cadmium concentrations up to 16 mM, according to research on the lowest inhibitory concentration of cadmium (Jebril et al. 2022). According to Huang et al. (2021), Ralstonia Bcul-1 was able to grow and live in vegetable soil with a Cd content of 0.42 mg/kg and remove 65.76% of the cadmium over the course of nine days. Klebsiella pneumonia sp. could tolerate a Cd concentration of 900 mg/L and Enterobactor kobei sp. could tolerate a Cd concentration of 600 mg/L, according to research by Uqab et al. (2022) on the in vitro sequestration of metallophilic cadmium-tolerant bacteria for sustainable agriculture.
Microorganisms used in bioremediation have evolved into effective, modern methods for detoxifying a variety of industrial and municipal wastewaters. Moula et al. (2021) looked into the improved bioremediation of heavy metals from phosphate processing effluent using the native bacterium Serratia rubidaea NCTC12971. The results showed that Serratia rubidaea species could endure up to a Zn concentration of 3 mg/L. Bacillus altitudinis MT422188 was identified by Khan et al. (2022), and its potential utility in zinc bioremediation was investigated. According to Khan et al. (2022), the minimum inhibitory concentration for zinc was discovered to be 20 mM and could be reached at a pH of 7 and a temperature of 37°C. According to this study, Aeromonas sanarellii and Azospirillum brasilense MTCC 4034 were able to survive against Cd and Zn concentrations of 9.2 and 184 mg/L for 45 and 20 minutes, respectively. Due to the kind of species, soils, wastewater, and habit environment, there are differences in the observations of cadmium and zinc tolerance among the various studies
Measurement of heavy metal reduction on oxidative stress markers and enzymatic antioxidants
When bacteria are exposed to heavy metals, a frequent response is the creation of non-enzymatic and enzymatic chemicals. As a defense mechanism against ROS, the body produces enzymatic antioxidants such superoxide dismutase (SOD), ascorbate peroxidase, and catalase. Lipid peroxidation, protein oxidation, and oxygen absorption are indicators of oxidative stress. When compared to the control there is a significant increase in the production of lipid peroxidation. Aeromonas sanarellii showed a significant increase in Cadmium compared to Azospirillum brasilense MTCC 4034 (Fig. 2A). A 3-fold change in the production of lipid preoxidase in Aero sp. Increase in Oxygen uptake is seen in both the organism when compared to the control (Fig. 2B). Protein Oxidation is also found to be 7-fold higher in case of both (Fig. 2C). These indicate that the cell is subjected to the loss or gain of the enzymatic activity, abnormal cellular uptake and enhancement or reduction in the proteolytic activities. The catalase and the SOD studies in Figure D and Figure E shows that the cells are able to survive and a homeostatic condition is being raised up to safeguard the cells from free radicle attack.
Various disease states and toxicities in living things have been connected to oxidative stress, according to Alkharashi et al. (2017). The most common environmental heavy metal pollutants are cadmium and zinc, and exposure to them in people and animals comes from both anthropogenic and natural sources, including mining, smelting, and other industrial activities (Patra et al. 2011). Increased oxidative stress following Cd exposure is associated with two diseases: hypertension and insulin resistance (Johns et al. 2023). Cd poisoning impairs lysosome function by preventing their fusion with autophagosomes (Yuan et al. 2021). The neurotoxic effects of Cd in a mouse neuro-crest cell line were found to be caused by these changes. Another method by which Cd causes injury to lung cells is by up regulating apoptosis (Pi et al. 2017). The oxidative stress identified in this study can be used to select from a variety of ameliorative tactics that could stop the oxidative harm to the body systems that results from or happens during exposure to these toxicants (Patra et al. 2011). Reactive oxygen species are thought to be produced more effectively by cadmium and zinc, which could cause serious harm to cellular components. In polluted environments, the linked heavy metals Cd and Zn nearly often coexist (Chandran et al. 2005). Given that Cd and Zn are contaminating the environment more and more, it is crucial to understand the mechanisms underlying toxicity in order to better understand how these metals operate. The results of this study imply that Cd and Zn may change the physiology of the lysosomes in the digestive glands by promoting lipid peroxidation, as seen by the increased amount of lipofuscin granules.
Mechanism of heavy metal uptake by Aeromonas sanarellii
Using GSH-functionalized gold nanoparticles, the amount of cadmium was determined using the UV-visible spectrophotometric technique, and absorbance was measured at 660 nm. Fraction 1 contains the metal concentration in the medium. Fraction 2 corresponds to the amount of metal present on the surface of the organism. The amount absorbed by the organism is fraction 3. Fraction 4 is the amount of metal internalised, and fraction 5 is the metal adsorbed onto the surface of the culture vessel. In the Fig. 5a depicts that the in Azospirillum brasilense MTCC 4034 the cadmium is adsorbed to the surface, in case of aero the cadmium is found distributed in F2, F3 and F4 which shows that cadmium is in the medium, adsorbed and absorbed. The internalization of Cadmium (F4) is high in Aeromonas sanarellii when compared to Azospirillum brasilense MTCC 4034 which shows that cadium had entered the cell. This coincides with the results of fluctuation in ROS. From the results obtained, it is seen that F2 had the highest amount of cadmium compared to all others. Fraction 2 represents the amount of cadmium present on the extracellular surface. It is seen that the accumulation of cadmium is higher on the outer surface.
Internalization of zinc was quantified using 8-hydroxyquinoline, its absorbance was measured at 384 nm, and a graph was plotted. Zinc was quantified by measuring absorbance at 384nm. In the case of zinc, fraction 4 had the highest amount of zinc. Fraction 4 was obtained after treatment with concentrated nitric acid. Hence, this fraction would contain the internalized metal. Zinc accumulation is higher on the intracellular surface (Fig. 5b). Zinc being one of the essential metal for the growth of algae is found to be internalized more when compared to cadmium. Internalization is 2 fold higher in Azospirillum brasilense MTCC 4034 when compared to Aeromonas sanarellii.
A catalyst activity is induced by the internalisation of Cd and Zn, and this catalyst activity ultimately results in DNA damage. This showed that even at sublethal concentrations, the internalisation of Cd and Zn in Aeromonas sanarellii and Azospirillum brasilense has the capacity to generate oxidative stress, which may be one of the underlying causes of the observed DNA damage. M. macrocopa may withstand cadmium concentrations up to 10 g/L before DNA damage occurs due to cadmium-induced increases in glutathione S-transferase (GST) levels (Wang et al. 2023). Contrarily, isolated Aeromonas sanarellii and Azospirillum brasilense bacteria can withstand cadmium concentrations of 9.2 mg/L and 184 mg/L before DNA damage is caused by glutathione S-transferase (GST) levels being elevated. The findings of this study advise taking corrective action if the concentrations of Cd and Zn are above 9 mg/L and 180 mg/L, respectively. In addition, this study recommends choosing biochemical biomarkers to evaluate the toxicity of Cd and Zn and to evaluate quick reactions even during brief exposure to low concentrations (Samarakoon et al. 2023). When the cell membrane interacts with the heavy meals, endocytosis is primarily triggered, causing the heavy meals to be internalised and transported to the extracellular environment (Felix et al. 2016). The internalisation of Zn can affect the cellular uptake pathway. When Cd and Zn concentrations were high, dark, packed cellular lysosomes could be seen in comparison to unaffected Cd and Zn cells (Felix et al. 2016).
SEM analysis was done using Thermosceintific Apreo S. SEM analysis revealed the changes that occur in the structure and size of bacteria due to metal exposure. For Aeromonas sanarellii, control samples, no accumulation was seen. In the case of cadmium, metal occurrence was observed at the outer surface of bacteria (Fig. 6). This is in accordance with the results obtained from internalization studies. Zinc treated cells, a reduction in size was observed in comparison to the control. The change in size has occurred due to the uptake of metal. Intracellular metal uptake is higher in zinc treated cells in correlation with the internalization studies.
Using Bradford's reagent, the amount of proteins in the isolated samples was determined. The BSA served as a point of reference. Using an appropriate extraction process, protein was recovered from the grown cells. Bradford's reagent was then used to estimate the extracted protein. In the instance of Azospirillum, 624 g of protein were obtained in the control. 571 g of protein in cadmium and 596 g of protein in zinc were obtained. In Aeromonas, 608 g of protein were collected from untreated cells, while 511 g were obtained from cells that had been exposed to cadmium. Using cells treated with zinc, 499 g of protein were produced (Fig. 7).
The various functional groups, including phosphates, sulphates, amides, hydroxyl, or negatively charged proteins, that are found in isolated Aeromonas sanarellii and Azospirillum brasilense bacteria and that exchange ions with the metal ion to produce strong ionic contact. Metal ions may interact with cellular membranes in the periplasm by entering the region through porins that are present on the surface during the adsorption process (Kanamarlapudi et al. 2018). The protein complex in Clostridium sp. increased the species' tolerance threshold for different heavy metal concentrations. Reductive precipitation was used to complete the affinity of Clostridium sp. for heavy metals (Long et al. 2021). There may be an elevated cadmium content in the extracellular media, as evidenced by the greater expression levels of the natural resistance-associated macrophage protein Auxeochlorella protothecoides (Tripathi and Poluri 2021). According to Wang et al. (2021), the siderophore binding protein, also known as EC20, increases the affinity of E. coli towards the metal ions copper, nickel, and lead. According to this study's findings (Jeyakumar et al. 2023), the rice protein in Aeromonas sanarellii and Azospirillum brasilense may be the primary factor influencing their affinity for Cd and Zn. The findings of this experiment using Aeromonas sanarellii and Azospirillum brasiliense bacteria showed that monodentate and polydentate complexes are the two types of complexes that are most frequently discovered in the biosorption between Cd and Zn.
The gel used in this study was 10%. Utilizing Coomassie Brilliant Blue, staining was carried out. According to the data, metal-treated cells saw a decrease in the intensity of various protein bands. While some proteins were also seen to express more in the treated cells. This demonstrated that some proteins are upregulated in response to metal treatment whereas other proteins are downregulated. The diversity in protein expression is what gives bacteria their tolerance to heavy metals (Fig. 8).
SDS-Page analysis is one of the commonly used techniques for calculating molecular mass (Arul et al. 2022). The process described by Debelian et al. (1996) was followed for doing the protein electrophoresis. In comparison to approaches based on charge parameters, the SDS-PAGE technique appears to detect broader taxonomic associations, notably at the species and subspecies levels (Dao et al. 2023). This is because it separates proteins by the more stable criterion of molecular weight SDS-PAGE analysis of Leptotrichia isolates revealed the genetic and phenotypic diversity. This was in line with earlier research we conducted (Eribe et al. 2002) based on cellular fatty acid levels and biochemical/enzymatic reactions. The reproducibility of SDS-PAGE analysis of Leptotrichia whole-cell proteins was optimized for L. buccalis, S. termitidis, and F. perfoetens (Eribe and Olsen 2002). The Bacillus subtilis cell samples and SDS-PAGE generation were prepared by Dao et al. (2023). In the cytoplasm of B. subtilis, a powerful promoter was used to assess the expression of a protein that is anti-S. aureus, according to Dao et al. (2023). The findings of this study may aid researchers in collecting samples and creating SDS-PAGE experiments, as well as support the consolidation of research on protein expression in a variety of bacteria species other than Aeromonas sanarellii and Azospirillum brasilense. MALDI TOF analysis was done to identify variations in proteins that occur upon the uptake of metal by bacteria. For Azospirillum (Fig. 9b-c), control samples had seven spectral peaks between three thousand and nine thousand m/z ion range. Cadmium treated cells had eleven spectral peaks between two thousand five hundred and fifteen thousand m/z ion range. Zinc-treated cells had nine spectral peaks in the ion range of two thousand to thirteen thousand m/z. The increase in the number of peaks in metal treated cells indicates the expression of new proteins that give the cells the ability to tolerate heavy metal.
The number of peaks observed in cadmium treated cells is higher than in zinc-treated cells because the toxicity of cadmium is higher in comparison with zinc. In the case of aeromonas sanarellii (Fig. 10a-c), seven peaks were observed in the ion range of two thousand eight hundred to sixteen thousand m/z in control. Whereas in cadmium, only four peaks were observed between three thousand and nine thousand m/z. Zinc treatment had eight peaks between three thousand and sixteen thousand m/z. A reduction in the number of peaks in cadmium indicates downregulation of proteins to tolerate the toxicity of the metal. Despite the fact that MALDI-TOF MS, or matrix-assisted laser desorption ionization-time of flight mass spectrometry, has been used, it has emerged as a possible method for microbial identification and diagnosis (Signhal et al. 2015). This work used 16S rRNA for determining the microorganism classification. Modern approaches like as MALDI-TOF MS have taken the place of older ones in the clinical laboratory for identifying bacteria. According to Rychert (2019), this might be primarily utilized to distinguish between bacteria and fungus. The findings of this study, along with those of earlier studies, pointed to the need to develop various scenarios for novel biological replicates, novel strains, and fresh species that are not found in the culture media.
Metallothionein was estimated using dithiobis (nitrobenzoic acid). The absorbance was measured at 412 nm and a graph was plotted. Metallothionein estimation showed that there was no expression of metallothionein in untreated cells. Azospirillum that had been exposed to cadmium contained 56 µmol and Aeromonas 64 µmol of metallothionein. Following zinc treatment, 32 µmol of Aeromonas and 38 µmol of Azospirillum were produced (Fig. 11). Due to the heavy nature of cadmium, metallothionein would be produced even in small amounts. Considering that zinc is an important metal, significant amounts would be required to trigger the creation of metallothionein. As a result, cadmium expressing cells create considerably more metallothionein than zinc treated cells. Metallothionein help in the transportation of essential metals into the cell and the non-essential toxic metal present in the cytoplasm to be vacuolized for the removal. The increased concentration of metallothionin in cadmium treated Azospirillum brasilense MTCC 4034 and Aeromonas sanarellii support it since cadmium is one of the toxic heavy metal even at a low concentration.
Some bacteria have defence mechanisms, such as efflux transport, precipitation, and intracellular sequestration by metallothionein (MT), to combat the harmful consequences of intracellular Cd and Zn buildup. When zinc is distributed over the microbe, Pomorski et al. (2023) observed zinc buffering in an extensive picomolar to nanomolar range of free Zn(II) concentrations within the cell. This may be determined by MT methods, similar to Cd measurement in the microbial cell wall. This MT method made it simple to determine the levels of both Cd and Zn in the tissue as well as cellular free Cd and Zn from the affinity sites. Heavy metals and other environmental stresses can raise the expression level of these MTs (Zhou et al. 2017). According to Ma et al. (2019), engineered bacteria expressing MTs have promising bioremediation applications. Tetrahymena thermophila's expression of MTT5 may be exploited for Cd bioremediation (Zhou et al. 2017). While the recombinant E. coli improved Cd's ability to bioaccumulate through the production of MT in the cytosol (Deng et al. 2007). The results of this study showed that the single most important reason in the shift in function of these molecules over time from tight binding and storage to one that is extremely dynamic is different metal affinities.