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Lead uptake capacity of thermophilic bacteria Aeribacillus pallidus strains isolated from Merapi volcano, Indonesia
Korean J. Microbiol. 2021;57(2):91-98
Published online June 30, 2021
© 2021 The Microbiological Society of Korea.

Anna Rakhmawati1,2, Endang Tri Wahyuni1,3, and Triwibowo Yuwono1,4*

1Biotechnology Study Program, Graduate School of Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
2Department of Biology Education, Faculty of Mathematics and Natural Sciences, Universitas Negeri Yogyakarta, Yogyakarta 55281, Indonesia
3Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
4Department of Agricultural Microbiology, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
Correspondence to: E-mail: triwibowo_y@ugm.ac.id; Tel.: +62274564305; Fax: +62274520842
Received March 18, 2021; Revised April 4, 2021; Accepted April 7, 2021.
Abstract
Lead uptake capacity of four lead-tolerant thermophilic bacteria Aeribacillus pallidus strains MRP112, MRP148, MRP272, and MRP278, originally isolated from Merapi volcano, were examined for their potential as an absorbent to uptake lead (Pb) from an aqueous solution. The growth profile and Pb uptake capacity were evaluated in Luria Bertani (LB) medium supplemented with Pb 100 mg/L. The optimum condition was evaluated under various analytical parameters, including temperature (55°C, 60°C, 65°C), pH (5, 6, 7), and contact time (6, 15, 24 h). The interaction of bacterial cell surface with ion Pb was proven using Fourier transform infrared spectroscopy (FT-IR). The optimal growth of all strains was observed at 55°C and pH 7. Aeribacillus pallidus MRP112 showed the highest uptake capacity of 97.41 ± 0.82 mg/g at 55°C, pH 5 for 15 h. FT-IR analysis displayed involved various functional groups such as hydroxyl, carboxyl, amine, and amide group ligands of protein in binding Pb ion. The A. pallidus strain represents a good candidate for the bioremediation of Pb-contaminated environment.
Keywords : Aeribacillus pallidus, cell surface, growth curve, Pb-uptake, thermophilic bacteria
Body

Environmental lead (Pb) contamination is an enduring urban health point that prominently impacts communities. Pb persistence in the environment and the non-biodegradable nature leads to Pb accumulation to toxic levels. The toxicity of Pb has been a case of importance due to its toxic reaction on plants, animals, and humans. Due to the expansion of industrial activities, a large amount of Pb is released into the environment, disturbing its fragile balance. Being one of the most toxic heavy metals, Pb ingestion via the food chain has proven to be a potential health hazard for organisms. It is, therefore, of an urgency to find suitable techniques to reduce Pb toxicity (Frank et al., 2019; Fatemi et al., 2020; Kumar et al., 2020; Zhao et al., 2020). The new techniques are needed to provide environmentally friendly and highly selective measures for Pb removal. There is an emerging trend of employing indigenous microorganisms in the Pb bioremediation, due to several benefits including excellent metal removal efficiency and eco-friendly nature, higher specificity, suitability for in situ techniques, and potential for improvement by genetic engineering (Jin et al., 2018; Tiquia-Arashiro, 2018; Brenes-Guillén et al., 2019; Han, 2020).

Bacteria-based bioremediation is highly influenced by several factors, such as survival of bacteria in contaminated soil or water, the influence of abiotic factors on bacterial growth, mechanism of metal detoxification, the expression rate of metal detoxification genes, and influence of pollutants on bacterial activity (Govarthanan et al., 2016). Thermophilic bacteria can be used in Pb removal, thus providing a promising way in bioremediation of high-temperature contaminated sites. They can be used for the bioremediation of environments contaminated with extremely recalcitrant pollutants due to their stability and persistence under adverse environmental conditions. Extremophiles are organisms able to thrive in extreme environmental conditions and show the ability to survive high doses of heavy metals to defensive mechanisms provided by primary and secondary metabolic products, i.e., extremolytes, lipids, and extremozymes (Nicolaus et al., 2016; Giovanella et al., 2020).

There is limited information on bioremediation agents by thermophilic bacteria. Previously, we have isolated thermophilic A. pallidus strains which demonstrated lead-tolerance from solfatara of Merapi volcano, Indonesia. Four strains of A. pallidus MRP112, MRP148, MRP272, and MRP278 showed the activity to tolerate 100 mg/L of Pb both in solid and liquid Luria Bertani (LB) media for 24 h at 55°C. It is interesting to further explore the use of A. pallidus strains for the removal of Pb from an aqueous solution as an approach for the bioremediation of Pb-contaminated sites. The present study aims at investigating the effect of temperature, pH, and contact time, on the uptake capacity and growth profile of the lead-tolerant thermophilic bacterium A. pallidus MRP112, MRP148, MRP272, and MRP278.

Materials and Methods

Bacterial strains and cultural condition

Aeribacillus pallidus MRP112, MRP148, MRP272, and MRP278 used in this study, was isolated from solfatara of Merapi volcano, Central Java, Indonesia, and deposited in the Laboratory of Microbiology, Department of Biology Education, Faculty of Mathematics and Natural Sciences, Universitas Negeri Yogyakarta, Indonesia with National Center for Biotechnology Information (NCBI) accession numbers of MT422124, MT422122, MT422121, and MT422120, respectively. The bacteria were routinely cultured at 55°C in Luria Bertani (LB, Oxoid) containing (g/L): tryptone 10, yeast extract 5, and NaCl 10 at pH 7 on LB agar supplemented with Pb 10 mg/L to maintain Pb tolerance.

Pb solutions and media preparation

Pb(II) 1 g/L stock solution was prepared by dissolving 1.59 g Pb(NO3)2 (Merck) in 1 L aquabidest, and 1 ml/L of 0.1 mol/L HNO3 (Merck) was added to the solutions to avoid precipitation of Pb(II). The media used for this experiment was a solution containing NaCl at 5 g/L concentration to prevent PbCl2 precipitation with modification by Peens et al. (2018). The LB media and Pb(NO3)2 stock solution were autoclaved at 121°C for 15 min separately and the pH of the culture medium was adjusted with NaOH and HNO3 before the autoclaving.

Growth profile of A. pallidus strains

The culture growth was determined by measuring samples turbidity using a spectrophotometer (GENESYS 10S, Thermo Fisher Scientific) at 600 nm every 3 h for 48 h at 55°C, 60°C, and 65°C under different Pb concentrations (0, 50, 100 mg/L) and pH (5, 6, 7). All experiments were carried out using LB media and bacterial inoculum size of OD600 0.8 in a reciprocating shaking waterbath (GFL 1083) at 100 rpm.

The Pb uptake capacity assay

The Pb uptake capacity assay on A. pallidus MRP112, MRP148, MRP272, and MRP278 strains were firstly cultivated at least for 24 h before being used as inoculum. Three 100 ml Erlenmeyer flasks containing 30 ml of autoclaved LB medium were inoculated with 3 ml (OD600 0.8) of inoculum and supplemented with 100 mg/L of Pb. Cultures were then incubated at 55°C pH 5, 6, and 7 in a reciprocating shaking waterbath (GFL 1083) at 100 rpm for 6, 15, and 24 h. LB media supplemented with corresponding concentrations of Pb in each treatment was used as the blank control. Following Pb uptake experiment, bacterial cells were harvested by centrifugation at 2,400 × g for 10 min, the supernatant was collected and Pb concentration was measured using atomic absorption spectrophotometer (AAS 7000, Shimadzu). All of the experiments were conducted in triplicates. In addition, lead uptake capacity was calculated by using the following formula (Konig-Péter et al., 2014):

Lead uptake capacity (mg/g)=V(CoCt)/m

where Co and Ct are initial Pb concentration in the solution (mg/L) and the Pb concentration after time t in the solution (mg/L), respectively; V is the solution volume (ml) and m is the bacterial cell mass (mg).

Determination of surface characteristics

Fourier Transform Infrared spectroscopy (FT-IR) was carried out on A. pallidus MRP112 before and after contact with 50 mg/L and 100 mg/L Pb to determine surface molecules of the bacteria. The samples were pulverized in an agate mortar before being conditioned in KBr pallets and tested using 8201PC (Shimadzu) in the 4,000 to 400 cm-1 wave region.

Statistical analysis

Statistical analysis for Pb uptake capacity for each strain was carried out using SPSS 22.00 (IBM Corporation). All experiments were carried out in triplicates and values are shown as a mean ± SD. Comparisons among multiple groups were done via analysis of variance (ANOVA), p < 0.05 was considered significant.

Results and Discussion

Growth profile

Growth profile under different conditions of Pb concentration, temperature, and pH, was compared to obtain a clear view on the effect of such parameters on the growth of four A. pallidus strains. It was observed that there were no differences between the four strains in the extent of each phase. The increase in Pb concentration results in a reduction of bacterial growth as shown in Fig. 1B and C; Fig. 2B and C; Fig. 3B and C; Fig. 4B and C. The addition of Pb at 50 mg/L and 100 mg/L resulted in the reduction of growth of all bacterial strains as evidenced by a lower OD value and longer lag phase of around 6–9 h. The response to the increase of Pb concentration was marked by two modes, first a steep decrease in optical density in all cultures, followed by a subsequent slow decrease of growth which reflects the growth inhibition. Growth response to Pb is also demonstrated by a slight increase in or stable optical density of all cultures, followed by a steep decrease (Poli et al., 2009). A. pallidus strains exhibited different growth patterns in the presence of Pb over an experimental time of 3 to 24 h until the growth of bacteria reached stationary phase. The increase in lag phase due to the toxic levels of Pb led the cells to undergo acclimatization to adapt to the environment (Singh et al., 2019). However, the growth curve pattern suggested that these bacteria exhibited tolerance towards Pb stress. A lower value of optical density indicated that the biomass growth of these bacterial isolates was highly affected due to the presence of Pb in the growth medium. Similarly, a previous study also suggested that bacterial growth is Pb concentration-dependent. The decrease of optical density followed the increase of Pb concentration (Marzan et al., 2017).

Fig. 1. Aeribacillus pallidus MRP112 growth profile (A) without Pb at 55°C and various pH; (B) 50 mg/L Pb at 55°C and various pH; (C) 100 mg/L Pb at 55°C and various pH; (D) at 60°C, pH 7, and various Pb concentration; (E) at 65°C, pH 7, and various Pb concentration.

Fig. 2. Aeribacillus pallidus MRP148 growth profile (A) without Pb at 55°C and various pH; (B) 50 mg/L Pb at 55°C and various pH; (C) 100 mg/L Pb at 55°C and various pH; (D) at 60°C, pH 7, and various Pb concentration; (E) at 65°C, pH 7, and various Pb concentration.

Fig. 3. Aeribacillus pallidus MRP272 growth profile (A) without Pb at 55°C and various pH; (B) 50 mg/L Pb at 55°C and various pH; (C) 100 mg/L Pb at 55°C and various pH; (D) at 60°C, pH 7, and various Pb concentration; (E) at 65°C, pH 7, and various Pb concentration.

Fig. 4. Aeribacillus pallidus MRP278 growth profile (A) without Pb at 55°C and various pH; (B) 50 mg/L Pb at 55°C and various pH; (C) 100 mg/L Pb at 55°C and various pH; (D) at 60°C, pH 7, and various Pb concentration; (E) at 65°C, pH 7, and various Pb concentration.

Bacterial growth decreased as the temperature of the medium increased from 60°C to 65°C as shown in Fig. 1D and E; Fig. 2D and E; Fig. 3D and E; Fig. 4D and E. The pH value, however, gave a contrast effect on growth compared to the effect of temperature, whereby bacterial growth increased as the pH value increased (Fig. 1A, B, and C; Fig. 2A, B, and C; Fig. 3A, B, and C; Fig. 4A, B, and C). It was observed that the optimal growth of A. pallidus was obtained at 55°C and pH 7, while at pH 5 there was no growth recorded. This result was in line with previous studies which suggested that A. pallidus optimum growth occurred 55–60°C and at pH 7–8 (Yasawong et al., 2011; Mnif et al., 2014; Mnif et al., 2014, 2015; Bose and Satyanarayana, 2016; Ma et al., 2019).

Pb uptake capacity

Among four strains, A. pallidus MRP112 showed the highest uptake capacity at 55°C, pH 5 for 15 h (Table 1). Pb uptake capacities were performed best at pH 5. The pH of the solution has a significant impact on the uptake of Pb since it determines the surface charge of the adsorbent, the degree of ionization, and the speciation of the adsorbate (Sudha and Srinivasan, 2016). The adsorption capabilities and bioavailabilities of Pb are more easily achieved at lower pH values (Gupta and Joia, 2016). The concentration of hydrogen ion (pH) has a significant influence on Pb uptake capacity. Under pH conditions above 6, the accumulation of hydroxyl group (OH-) results in the chemical precipitation of Pb as Pb(OH)2 and affecting the removal capacity. In contrast, high acidic conditions (pH < 2) decreased the removal capacity as a result of competition for negatively charged binding sites between heavy metal cation and protons (H+). However, the optimal pH condition also depends on the metal-accepting groups on the bacterial surface. For example, phosphoryl groups are mainly in an unprotonated form already at pH 2, therefore bacteria with these uptaking groups can remove Pb at lower pH (Cephidian et al., 2016). García et al. (2016) reported that the fast initial Pb biosorption rate was attributed to the surface binding and the following slower sorption was attributed to the interior penetration. At pH 5 the increase in uptake capacity increases with time, but at pH 6 and 7 tends to decrease, suggesting the relation with the increase of the number of cells at pH 6 and 7. Despite the fact that the number of Pb removal is the same, yet the ability to take up Pb is less due to the higher cell number. The growth phase is an important parameter that can affect ion-binding by bacteria, therefore growth phase should be considered when developing or employing chemical models for bacteria-bearing systems (Heinrich et al., 2008).

Pb uptake capacity (mg/g) by Aeribacillus pallidus MRP112, MRP148, MRP272, and MRP278 at various contact time and pH, 100 mg/L Pb, 55°C

Time (h) pH MRP112 MRP148 MRP272 MRP278
6 5 67.58 ± 1.29b 45.62 ± 0.98b 34.78 ± 1.34b 47.05 ± 1.62b
6 47.78 ± 0.74c 26.49 ± 0.32c 27.95 ± 0.19c 39.41 ± 1.19c
7 25.75 ± 0.42e 23.28 ± 1.44e 16.49 ± 1.08e 21.68 ± 0.54f
15 5 97.41 ± 0.82a 45.72 ± 0.58b 34.98 ± 0.63b 47.89 ± 0.85b
6 48.03 ± 0.58c 26.69 ± 0.66c 27.39 ± 1.19c 37.15 ± 0.65d
7 15.54 ± 0.45f 14.72 ± 0.50f 10.19 ± 0.35f 12.02 ± 0.32g
24 5 97.41 ± 0.75a 51.77 ± 1.11a 39.79 ± 1.48a 49.55 ± 1.34a
6 30.31 ± 0.46d 24.75 ± 0.64c 24.97 ± 0.59d 28.58 ± 0.57e
7 9.72 ± 0.50g 10.03 ± 0.12g 9.64 ± 0.64f 10.77 ± 0.67g

* Means sharing the same superscripted letters at each bacterial isolates are statistically non-significant at p < 0.05



Surface characteristics

FT-IR analysis was performed on A. pallidus MRP112 to give a qualitative and preliminary analysis of the main functional groups present on the cell wall which may be responsible for Pb uptake. Comparison of A. pallidus MRP112 cells spectra loaded with Pb metal ions is shown in Fig. 5. In general, the results did not show systematic shifts of the bands after Pb uptake, but punctual and discrete shifts of certain bands allowing to infer the possible groups involved in Pb adsorption. The transmittance of the peaks in the loaded biomass is substantially lower than those in the raw sample of the bacterial biomass (Joo et al., 2010). Results of FT-IR revealed the region between 3,440 and 3,485 cm-1 corresponds to hydroxyl stretches in several compounds, and amines and amides in amino acids. This band is broad because of the high number and large density of hydrogen bonds, mainly hydroxyl radicals in the polysaccharide pyranose rings. Hydrocarbon stretches also occur between 2,360 and 2,380 cm-1. Furthermore, stretches of carboxylate groups between 1,620 and 1,660 cm-1 (Oliveira et al., 2014; Vimalnath and Subramanian, 2018). Thermophilic bacteria A. pallidus strain MRP112 used in this study showed the highest activity in Pb uptake suggesting its potential as a good candidate for the bioremediation of Pb-contaminated environment.

Fig. 5. FT-IR Spectra of Aeribacillus pallidus MRP112 without, loaded with 50 mg/L, and 100 mg/L Pb.
Acknowledgments

This study was funded by the Indonesian Endowment Fund for Education (LPDP), Ministry of Finance, Republic of Indonesia. The authors greatly aknowledged the support of Universitas Gadjah Mada, Yogyakarta, and Universitas Negeri Yogyakarta for the completion of this study.

Conflict of Interest

We have no conflicts of interest to report.

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June 2021, 57 (2)