Effects of heavy metals on bacterial growth parameters in degradation of phenol by an Antarctic bacterial consortium

Abstract

Antarctica has often been perceived as a pristine continent until the recent few decades as pollutants have been observed accruing in the Antarctic environment. Irresponsible human activities such as accidental oil spills, waste incineration and sewage disposal are among the primary anthropogenic sources of heavy metal contaminants in Antarctica. Natural sources including animal excrement, volcanism and geological weathering also contribute to the increase of heavy metals in the ecosystem. A microbial growth model is presented for the growth of a bacterial cell consortium used in the biodegradation of phenol in media containing different metal ions, namely arsenic (As), cadmium (Cd), aluminium (Al), nickel (Ni), silver (Ag), lead (Pb) and cobalt (Co). Bacterial growth was inhibited by these ions in the rank order of Al < As < Co < Pb < Ni < Cd < Ag. Greatest bacterial growth occurred in 1 ppm Al achieving an OD₆₀₀ of 0.985 and lowest in 1 ppm Ag with an OD₆₀₀ of 0.090. At a concentration of 1.0 ppm, Ag had a considerable effect on the bacterial consortium, inhibiting the degradation of phenol, whereas this concentration of the other metal ions tested had no effect on degradation. The biokinetic growth model developed supports the suitability of the bacterial consortium for use in phenol degradation.

Introduction

Antarctica is a remote region representing one of the Earth’s most extreme environments [1, 2]. The Antarctic’s ice sheets contain a unique record of climate over the past million years [3]. It has largely been unaffected by human intervention until very recently. The pristine continent is experiencing consequences of multiple local and distant sources of human impacts progressively, including tourism and mining industries [4]. The establishment of the Protocol on Environmental Protection to the Antarctic Treaty provides guidelines to the Treaty’s signatory nations for the protection of the Antarctic environment, including minimising the impacts of human activities. Despite this effort, there are clear evidence of the detrimental effects on the polar environment and ecosystems due to human activities [4–7].

In Antarctica, most human activities take place in ice-free areas, which represent only ~ 0.2–0.4% of the continent’s area [8, 9]. These regions also host the vast majority of the terrestrial flora and fauna of Antarctica. Hughes [10] highlighted that Antarctic soils are extremely vulnerable to the impacts of construction of research stations, roads, runways and other facilities. It has been estimated that the extent of contaminated soil in Antarctica is between 1 and 10 million m³ [11]. Phenol contamination was generally due to the oil spills on land and improper waste disposal by research stations, where these can be the reasons to carry out a study on the bioremediation of phenol using Antarctic bacteria from soil. The increasing number of research stations can cause fuel spill pollution on the ice-free lands of the Antarctic [50]. Plus, the phenolic compounds can be detected in daily waste discharged from coastal Antarctic research station. This waste also was dumped in the soil of Antarctic, which led to phenol pollution in the land of continent.

Anthropogenic pollutants in Antarctica which includes organic and inorganic compounds originate from both global (via long-range atmospheric transport (LRAT)) and local sources [12]. Inorganic contaminants consist predominantly of heavy metals especially cadmium (Cd), chromium (Cr), mercury (Hg), lead (Pb), arsenic (As) and copper (Cu). Heavy metals are elements that have a high atomic mass and density, are undegradable and persistently exist in nature [13]. Phenol and phenolic compound are common organic constituents in wastewaters emanating from many chemical-based industries including pharmaceutical, and petroleum and coal refineries. These compounds are toxic even at relatively low concentrations, causing great harm not only to aquatic creatures but also to humans by instigating several systemic diseases. In most cases, organic contaminants coexist with inorganic elements such as heavy metals in industrial wastes. The presence of these contaminants in the environment leads to serious concern on their potential effects on living organisms and on natural ecosystems.

One of the approaches to remediate pollution is through the utilisation of highly resistant microbes. This approach has been proven efficacious and environmentally sustainable [14]. In cold climatic regions, cold-adapted microorganisms are capable in adapting metabolically to counterweigh the undesirable effects of low temperatures [15–17]. Natural marine microbial community, including the ones from Antarctic, have demonstrated the ability to degrade major fractions of spilled petroleum, thus consequently reducing the impacts of oil spills [18, 19]. The spotlight now falls onto heavy metal–resistant bacteria to obtain greater bioremediation efficacy. In a phenol degradation study, Arthrobacter bambusae has been reported to degrade phenol and tolerate most 1 ppm of heavy metals except for Ag and Cd [20]. A similar study has revealed that Rhodococcus baikonurensis was not only able to tolerate heavy metals present at concentration of 1 ppm but was also effectively degrading phenol [21].

This current work proposes the kinetic studies of Antarctic psychrotolerant microbial consortium that can degrade phenol in the presence of heavy metals. In biodegradation kinetics, substrate (phenol) concentration is customarily affected by the microbial enzymatic reactions. Hence, substrate degradation is directly proportional to the number of microorganisms [22]. Hence, in the presence of heavy metals, the extent of the microbial growth inhibition in correlation to the degradation of phenol will be assessed in the present work.

Materials and methods

Microorganisms and media

The bacterial consortium used in this study was obtained from soil samples collected near Chilean Bernardo O’Higgins Riquelme Base Station on Trinity Peninsula, northwest of Antarctic Peninsula. The soil sample (63°19′16″S 57°53′ 57″W) was collected with the depth of the soil around 2 to 5 cm. Then it was placed in sterile 50 mL of Falcon tube and stored at 4℃ for 2 months after collection and during transport to Malaysia. A sub-sample (approximately 0.5 g) from the soil sample was inoculated and incubated in the nutrient broth (NB) (Friendmann Schmidt, Germany) on a 150 rpm orbital shaker at 10℃ for 3–4 days. This step was repeated for three times and used for the bacterial culture stock. The consortium was screened for phenol-degrading activity in a minimal salt medium (MSM) containing the following (g/L): KH₂PO₄ (0.4), K₂HPO₄ (0.2), NaCl (2.0), MgSO₄ (0.1), (NH₄)₂SO₄ (1.0), MnSO₄·H₂O (0.001), Fe₂(SO₄)·H₂O (0.001) and NaMoO₄·H₂O (0.001), adjusted to pH 7.0. The MSM was supplemented with 0.5 g/L phenol as the sole carbon source. The effects of seven heavy metals, Ni, Co, Al, Pb, Ag, Cd and As, were investigated. Each of the media was spiked with one type of heavy metal at 1 ppm. The media was aseptically inoculated with 2% (v/v) standardised inoculum at ~ 1.0 optical density (OD_600nm). Samples were incubated on a shaking incubator at 150 rpm for 120 h at 10°C. A set of media without the presence of heavy metal ions incubated under the same conditions acted as a control. All treatments were conducted in triplicates.

Determination of phenol degradation in the presence of heavy metals

Determination of the remaining phenol concentration at the end of the incubation period was carried out using 4-aminoantipyrine colorimetric assay following the American Public Health Association protocol [23]. Samples were centrifuged using a tabletop centrifuge (CR1512, CAPPRondo, Germany) at 150 rpm for 10 min. The supernatant was carefully transferred into a test tube. pH of the supernatant was adjusted to 10 by the addition of ammonium buffer solution into each test tube using a dropper. One hundred microlitres of aminoantipyrine solution was pipetted into each test tube followed by 100 µL of potassium ferricyanide (C₆N₆FeK₃) solution. The test tubes were incubated for 15 min in a dark room before measuring the absorbance at 510 nm. Phenol degradation was assessed relative to the phenol concentration in the positive control and the remaining phenol concentration was expressed as a percentage.

Kinetic study of bacterial growth

The modified Gompertz equation consists of three phases, i.e. lag, exponential and stationary phases, and was applied to analyse the effects of different heavy metal ions on cell growth parameters of bacterial consortium. The kinetics of the bacterial growth was described by the modified Gompertz model equation [24, 25];

$$y=A\times exp\left\{-,e,x,p,\left[\frac{{\mu }_{\mathit{max}}\times e}{A},\times ,\left(\lambda ,-,t\right),+,1\right]\right\}$$

where y is the bacterial growth (λ600), A is the initial bacteria growth, µ_max is the maximum growth rate, λ is the lag phase time and t is the time (h). The equation was fitted to triplicate sets of growth data using non-linear regression modules in GraphPad Prism software (version 8.3.1, USA). To assess the significant differences in bacterial growth in the presence of different metal ions, an analysis of variance (ANOVA) was performed using GraphPad InStat software (version 3.05, USA).

Result

Effects of metals on phenol degradation

The effects of different metal ions on phenol degradation by Antarctic soil bacterial consortium were assessed after 120 h of incubation. Figure 1 exhibits that phenol degradation was significantly affected in the presence of 1 ppm Co and Ag with 22% and 100% inhibition, respectively. Meanwhile, in the presence of 1 ppm Al, bacterial growth was the highest, followed by As, Co, Pb, Ni, Cd and Ag. All heavy metals, except Ag, has shown able to promote bacterial growth as compared to control. This proved that these heavy metals can be utilised in certain metabolic processes required for cell growth.

Fig. 1.

The effects of 1 ppm concentration of different heavy metal ions on Antarctic soil bacterial consortium growth and phenol degradation. Triplicate samples were incubated at 10°C for 5 days. Data represent mean ± SEM

Growth kinetics

The modelled results of the modified Gompertz equation were plotted against the bacterial growth measured upon exposure to seven heavy metals (Fig. 2). The parameters determined from the non-linear regression analysis are presented in Table 1, including the coefficient determination (R²), and degree of freedom (DF), as well as the sum of square (SS) and standard deviation of the residuals (Sy.x). The R² values allow the model comparison in terms of experimental fit against the experimental values. A high R² value indicates that the predicted regression curve was close to the experimental values obtained, showing the best model fit, mathematically. The SS value indicates the regression curve minuses the SS of the distance of the points from the curve, while Sy.x value is the standard deviation of the vertical distances of the experimental point from the regressed line. Ideally, the SS and Sy.x values should be lower in any regression model, which indicates a better model fit to the data. The result exhibited the highest R² value for Co at 0.9770, followed by Pb, Ni, Cd and Al at 0.9733, 0.9593, 0.9326 and 0.9264, respectively. Meanwhile, the values of SS and Sy.x for Pb, Ni, Cd and Al were higher compared to Co (SS, 0.04652; Sy.x, 0.05569).

Fig. 2.

Non-linear regression models obtained using the Gompertz growth equation for a 1 ppm Al with maximum growth rate, μ_max = 18.85 h⁻¹; b 1 ppm As, μ_max = 19.31 h⁻¹; c 1 ppm Cd, μ_max = 16.89 h⁻¹; d 1 ppm Co, μ_max = 22.06 h⁻¹; e 1 ppm Pb, μ_max = 17.03 h⁻¹; f 1 ppm Ni, μ_max = 15.95 h⁻¹; g 1 ppm Ag, μ_max = 6.825 h⁻¹; and h biotic control, μ_max = 14.81 h⁻¹. All incubations carried out in triplicate. All data points represent mean ± SEM

Table 1.

ANOVA table (goodness of fit) for non-linear regression curve fit of the bacterial consortium in different types of heavy metals

Heavy metal	Goodness of Fit
	R2	DF	SS	Sy.x
Control	0.8732	15	0.0965	0.0802
Ni	0.9593	15	0.0637	0.0652
Co	0.9770	15	0.0465	0.0557
Al	0.9264	15	0.1608	0.1035
Cd	0.9326	15	0.1193	0.0892
Pb	0.9733	15	0.0528	0.0594
As	0.6221	15	0.9374	0.2500
Ag	0.8395	15	0.0025	0.0127

Table 2 exhibits the best-fit values for non-linear regression curve fit of the bacterial consortium in different types of heavy metals. In general, λ value indicates the capacity of the strain to adapt to new environmental conditions. In relation to this, the bacterial growth presented the shortest lag phase when incubated with 1 ppm of Ni (15.95 h) (not considering Ag). This revealed the capability of the bacteria to reach exponential phase faster as compared to other heavy metals. The µ_max indicates the better capacity of the strain to absorb nutrient in the medium. The bacterial growth in 1 ppm of Al presented the highest maximum growth rate at 0.9792 h⁻¹. Indicating the adaptive capabilities of the bacterial consortium in the presence of 1 ppm of Al.

Table 2.

Best-fit values for non-linear regression curve fit of the bacterial consortium in different types of heavy metals

Heavy metal	Best-fit values
	Growth (λ600)	µmax (h−1)
Control	14.81	0.5582
Ni	15.95	0.8424
Co	22.06	0.9776
Al	18.85	0.9792
Cd	16.89	0.8684
Pb	17.03	0.9408
As	19.31	0.8740
Ag	6.825	0.1019

Discussion

Growth responses and degradation abilities of different microorganisms may vary in response to different heavy metals [26]. It has been reported that high concentration of heavy metals can be toxic as they can reduce the respiration rate of bacteria [27]. Heavy metals have also been reported affecting the cell in various ways including disturbing the electron transport chain, thus impacting the structure of cell membrane [28]. This might lead to structural and functional characteristic changes of microbial community as species sensitive to heavy metals are eradicated [29]. On the other hand, some metals when present at low concentrations have been observed to increase the respiration rate of metal-tolerant bacteria and promote cell growth [27]. Hence, the presence of heavy metals in the Antarctica could disturb the activity of the bacteria used to degrade phenol. Numerous studies reported that high amounts with various type of heavy metals have been found in Antarctica, whether it is from natural sources or due to the anthropogenic activities. A study informed that there were heavy metals present in the soil at the Artigas base research station and in the topsoil collected in 2015 to 2016, where this includes As, Zn, Cu, Mn, Ni, Cr and Pb. The concentration of these metals varied according to their type within a range of 1.96 to 740.93 ppm [49]. On the other hand, the most affected anthropogenic activity area, which was near to the station of General Bernardo O’Higgins Riqulme, Chile, has high concentrations of copper (201 ppm), zinc (163 to 771 ppm) and strontium (733 to 1279 ppm) in fine-earth and skeleton, yet there is no data reported on the exact concentration of heavy metal pollution from soil [51].

The data obtained in this study confirms the capability of the bacterial consortium to completely degrade phenol in the presence of As, Pb, Cd, Ni and Al at 1 ppm concentration. Ahmad et al. [30] similarly reported that Acinetobacter sp. strain AQ5NOL 1 could degrade phenol in the presence of various heavy metal ions.

In the case of phenol degradation, heavy metals were described capable in hampering bacterial growth and/or inhibiting enzymes that play major roles in degradation pathways [31]. According to Percival et al. [32], Ag exhibits inhibition activity across broad ranges of microbial species. Ag ion reacts with amino acid in proteins by attaching to sulphydryl, amino, imidazole, phosphate and carboxyl groups of membrane or enzyme proteins, leading to protein inhibition or denaturation. Ag is also known to be able to puncture bacterial cell walls by reacting with the peptidoglycan component which in turn can cause a disruption to the DNA and its replication cycle [32–34]. Dibrov et al. [35] stated that Ag can induce massive proton leakage through the bacterial membrane, resulting in complete de-energisation and, ultimately, cell death. This result is consistent with the findings of Ibrahim et al. [36] and Zakaria et al. [21]; both authors reported significant growth inhibition of Rhodococcus sp. AQ5-07 and R. baikonurensis strain AQ5-001 in 1 ppm of Ag.

Generally, bacterial growth is significantly related to the degradation of pollutants. This is due to the bacterial ability to utilise pollutant as a source of nutrient to support their growth [37]. Certain types of heavy metals have also been acknowledged essential to living organisms in carrying basic metabolic functions. In this study, Co exhibits slight inhibition on phenol degradation, but promotes bacterial growth. This is because Co is an essential metal indispensable for cell functioning such as amino acid formation and coenzyme for cell mitosis [38]. However, excess of this metal can affect organisms unfavourably. Cobalt is widely known to be toxic at high concentration and can cause groundwater contamination, leading to serious negative impacts to the ecosystem [39, 40]. Cobalt is known to disturb the course of redox reaction, inhibit biopolymer hydrolysis processes and reduce the rate of enzymatic hydrolysis of compounds [41]. Therefore, this result demonstrated the supressing aptitude of Co towards the bacterial efficiency in degrading pollutants, in parallel with previous studies [21, 42].

Heavy metal such as aluminium (Al), cadmium (Cd), lead (Pb) and arsenic (As) do not have any biological role and are toxic to living organisms. They pose adverse effect on microbes by jeopardising the DNA integrity. This transpires from the displacement of other essential metals from their native binding sites or ligand interactions due to the similar charges of the metal ions [43]. Functional disturbances such as inhibition of enzyme activity and oxidative phosphorylation lead to major changes in microbial morphology and metabolism, which eventually impede bacterial growth [44, 45]. However, according to Dixit et al. [46], microorganisms can adopt different mechanisms to interact and survive in the presence of inorganic metals. Various mechanisms used by microbes to survive metal toxicity are biotransformation, production of exopolysaccharide, extrusion and synthesis of metallothioneins [47]. This might explain the reason behind the unaffect growth and phenol-degrading activity of the Antarctic microbial consortium used in this study in the presence of certain metals. Meanwhile, nickel (Ni) is an essential nutrient for selected microorganisms where it participates in a variety of cellular processes. Many microbes are capable of sensing cellular Ni ion concentration and taking up this trace element via Ni-specific permeases or ATP-binding cassette-type transport systems [48].

Bacteria groups such as Rhodococcus, Arthrobacter, Acinetobacter, Pseudomonas and Bacillus [52–55] were identified as phenol-degrading bacteria in past research. In addition, Pham et al. [56] also summarised that all these bacteria also have their ability to tolerate the presence of heavy metals for them to grow. These bacteria group might be the bacterial consortium responsible in degrading phenol. Besides, there were several cold-adapted Antarctic bacteria identified as phenol degrader, such as Arthrobacter spp. strain AQ5-05 and AQ5-06 and Rhodococcus sp. strain AQ5-07, where these bacteria were isolated from Antarctic soil contaminated with phenol (Lee et al., 2017). Meanwhile, bacteria such as Arthrobacter sp. strain AQ5-15 (isolated from Antarctic base soil), Arthrobacter bambusae strain AQ5-003 (contaminated Antarctic soil) and Rodococcus boikonurensis strain AQ5-001 (contaminated Antarctic soil) were discovered able to degrade phenol effectively [20, 21, 57]. Cold-adapted bacteria that can degrade phenol also have been reported identified. These include Pseudomonas sp. strain ATR208 (isolated from mountain in the northeast of Iran), Pseudomonas putida LY1 (Northern-east Poland) and Arthrobacter psychrophenolicus (Alpine ice cave) [58–60].

The results of this study suggested that each of the heavy metals analysed affected the growth of the bacteria and were able to stimulate or inhibit phenol degradation. Each growth curve can perform different regression fit with different best-fit values for the result as presence of heavy metals changes the behaviour of bacterial cell growth. The results obtained for different growth kinetic regression curves indicated that Gompertz model was able to facilitate understanding on the growth kinetics of Antarctic bacterial consortium with faster rate of bacterial growth, high value of R² and low error, which could be used for modelling the bacterial growth kinetics under heavy metal stress.

Conclusion

In this study, we provided the first report on the effects of various heavy metal ions on the growth kinetics of Antarctic soil bacterial consortium. This consortium tolerated and grew rapidly, under exposure to five heavy metals, namely Ni, Co, Al, Pb, Cd and As, which are common co-pollutants in phenol pollution events. We found that the phenol degradation ability of our bacterial consortium was not significantly reduced upon exposure to these metals. In contrast, exposure to Ag resulted in almost complete inhibition of growth and phenol degradation. These data support the potential of bacterial consortium in bioremediating polluted soils and wastewater in Antarctica as well as other cold environments.

Funding

This study was supported by Universiti Putra Malaysia, PUTRA Berimpak (9660000), and Centro de Investigacion y Monitoreo Ambiental Antàrctico (CIMAA). Peter Convey is supported by NERC core funding to the BAS ‘Biodiversity, Evolution and Adaptation’ Team.

Declarations

Conflict of interest

The authors declare no competing interests.