A Laboratory-Scale Study: Biodegradation of Bisphenol A (BPA) by Different Actinobacterial Consortium

Abstract

The unique diversity of microbes makes them ideal for biotechnological purposes. In this present study, 16 actinobacterial isolates were screened on media supplemented with Bisphenol A (BPA). Three out of 16 isolates exhibited high biocapacity to degrade BPA as a carbon source. Four different mixed actinobacterial consortia were developed using the above strains and the effect of each consortium on biomass growth; laccase production and BPA degradation were examined. At 100-mg/L BPA concentration, the three-member consortium grew well with maximum laccase activity as well as maximal degradation rate of Bisphenol A than the other two-member consortium. The consortium of Actinomyces naeslundii, Actinomyces bovis, and Actinomyces israelii degraded 93.1% with maximum laccase activity of 15.9 U/mL, followed by A. naeslundii and A. israelii with 87.3% and 9.5 U/mL. This was followed by A. naeslundi and A. bovis with 80.4% and 8.7 U/mL, while A. bovis and A. israelii degraded 76.0% with laccase activity of 7.0. The gas chromatography–mass spectrometry (GC–MS) analysis of biodegraded BPA showed the presence of oxalic acid and new products like 1,2,4-trimethylbenzene and 2,9-dimethyldecane.

1. Introduction

One of the most pressing health and environmental issues in today’s world is the generation of cumbersome waste with toxic organic substances like Bisphenol A (BPA) from home and industrial sector. BPA poses threats to both aquatic and terrestrial animals [1, 2]. BPA (4,4-isopropylidenediphenol) is an industrial chemical that is produced through the condensation of acetone and phenol using acid or alkaline as catalyst [3, 4].

It is one of the highest production volume chemicals [5], which is widely used as an intermediate in the synthesis of polycarbonate plastics, epoxy resins, and flame retardants [6, 7]. BPA is a monomer of polycarbonate plastics and a constituent of epoxy and polystyrene resins, which are used in the food packing industry [6, 8]. Despite these relevant usages, it has a strong estrogenic property, thus, classified as part of endocrine disruptive compounds (EDCs) [9].

The intensification of anthropogenic activities in manufacturing industries has contributed to the direct or indirect release of a wide range of toxic compounds into the environment and BPA has the potential of causing significant threats to flora, fauna, and human [1, 10, 11]. Microbial degradation is described as a major approach and a natural mechanism, by which one-can clean-up pollutants from the environment in an eco-friendly manner [12, 13, 14].

Most of the exploited biodegradation research processes rely mostly on enzymes from different strain of plankton, fungi, or bacteria [15, 16, 17, 18]. It has been reported that BPA bioremediation by fungi and bacteria is mediated mainly through lignin-degrading enzymes, such as laccase and manganese peroxidase (MnP), which are produced extracellularly [19, 20, 21]. At present, actinobacteria are relatively less explored for biodegradation processes that utilize laccase for remediating BPA.

Laccase is a multicopper oxidase and catalyzes one-electron oxidation of phenolic compounds by reducing oxygen to water [22, 23]. Laccase typically contains 15–30% carbohydrate. It usually has an acidic isoelectric point and a molecular mass of 60–90 kDa [24, 25, 26]. Laccase is encoded by a family of genes and produced in the form of multiple isozymes [27, 28]. It has been proven that genes encoding laccase isozymes were differentially regulated [29, 30].

Laccase is an important industrial enzyme. It can be applied extensively in many fields, which include textile dye transformation, waste detoxification and demineralization, and production of biofuels [31, 32, 33]. In the case of laccase, BPA metabolism is faster in the presence of mediators such as 1-hydroxybenxotriaxzole (HBT) and 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonate) than in laccase alone. Thus, the objective of this research work was to investigate the microbial growth and laccase activity from the different actinobacterial consortium during the biodegradation process. The metabolites from the BPA biodegradation were also analyzed using gas chromatography–mass spectrometry (GC–MS).

2. Material and methods

2.1 Chemicals and reagents

BPA, hydrochloric acid, sodium acetate, sodium hydroxide, calcium chloride, iron sulphate, potassium phosphate, hydrogen peroxide, ammonium nitrate, and 2,2′azino-di-(3 ~ ethyl benzothiazoline-6-sulphonic acid) (ABTS) were products of Sigma-Aldrich, Germany. Ethanol and yeast extract were ordered from BDH Chemicals Limited, England, Scharlaun Chemie S.A. Barcelona, Spain, and Biomark Laboratories Pune, India. All the other chemicals and reagents used were of analytical grade.

2.2 Microorganism

Sixteen actinobacterial isolates that were obtained from the culture collection of the Enzyme and Microbial Technology laboratory, Department of Biochemistry, Federal University of Technology, Akure, Nigeria, were screening for this study. Three (A. naeslundii, A. bovis, and A. israelii) of the isolates with best-growing ability on media supplemented with 100 mg/L of BPA were selected from the culture bank for further studies.

2.2.1 Seed culture preparation

Seed culture for each actinobacterial strain was prepared by growing a loopful portion from the slant in sterile media containing (5 g/L), yeast extract (1.5 g/L), beef extract (1.5 g/L), and NaCl (5 g/L) with pH adjusted to 7.4. The media were incubated in an orbital incubator at 30°C for 24 h at 160 r⋅min⁻¹ in a shaking incubator (Stuart, UK). After 24 h, the seed culture was used as inoculum for the production media. Each seed inoculum (constituting 5% v/v) was transferred into 500-mL Erlenmeyer flask containing 100 mL of production media. Three different actinobacterial consortia were developed as follows: A. naeslundii, A. israelii, and A. bovis used as inoculant for the degradation processes.

2.2.2 Preparation of mineral salt medium

The mineral salt medium used in this study was composed of KH₂PO₄ (0.2 g/L), K₂HPO₄ (0.2 g/L), CaCl₂.H₂O (0.1 g/L), NaCl (0.8 g/L), MgSO₄.7H₂O (0.2 g/L), MnSO₄.7H₂O (0.01 g/L), FeSO₄.7H₂O (0.02 g/L), and yeast (2.0 g/L) with pH adjusted to 7.0. The basal mineral media was supplemented with 100 mg/L of BPA. These were sterilized in an autoclave at 15 psi and (121°C) for 20 minutes.

2.2.3 Actinobacterial growth during the biodegradation process

Biomass concentration was estimated at every 24 h from the absorbance of appropriately diluted culture medium at 620 nm according to the predetermined correlation between optical density and dry weight of biomass [34, 35]. The media were incubated at room temperature for 312 h at 160 r⋅min⁻¹.

2.3 Effect of actinobacterial consortium on laccase production and BPA degradation

The effect of actinobacterial consortium on laccase production and BPA degradation was investigated for a period of 312 h in sterilized mineral salt media supplemented with 100 mg/L of BPA at pH 7.0. The consortia were developed using equal volume of individual seed culture. BPA degradation efficiency and laccase production were monitored, as described below, at 24-h interval throughout the incubation period.

2.3.1 Enzyme assays

The activity of laccase was measured using the common substrate, ABTS (E₄₂₀ = 36,000 M⁻¹ cm⁻¹) a modified method of Bourbonnais and Paice [36]. This was done by monitoring spectrophotometrically change in absorbance at 420 nm (A₄₂₀) correlated with the rate of oxidation of 1-mM ABTS in 1-mM Tris–HCl buffer pH 7.0. The experiment was performed in 1-mL cuvettes at 30°C. Reaction mixture contained 750-μL ABTS and 250 μL of enzyme solution.

At an interval of 1 minute for 5 minutes, the absorbance of the mixture was measured. One unit of laccase activity was defined as the amount of enzyme that oxidized 1 mM of ABTS per minute under standard assay conditions. Laccase activity was expressed as U/mL. The enzyme activity was calculated using the Eq. (1).

$$\mathit{Enzyme}\mathit{activity}=\frac{\frac{\mathit{Absorbance}}{\mathit{minute}}\times \mathit{Total}\mathit{volume}\mathit{of}\mathit{mixture}}{\mathit{Total}\mathit{time}\times \mathit{Extinction}\mathit{coefficient}\times \mathit{volume}\mathit{of}\mathit{enzyme}}$$E1

2.3.2 Determination of BPA degradation

The percentage removal of BPA was determined using Folin–Ciocalteu reagent according to the method of Yordanova et al. [37]. The residual of BPA supplemented in the mineral salt media was determined at 24-h intervals throughout the degradation period. Aliquots of the culture media were withdrawn at intervals and centrifuged for 10 min at 3500 r⋅min⁻¹. One milliliter of the supernatant was added to 10 mL of distilled H₂O and 1 mL of Folin–Ciocalteu reagents. The mixture was left for 5 min, and 2 mL of 20% Na₂CO₃ (w/v) was added to the mixture. The solution was kept in the dark for 60 min, and therefore, absorbance at 750 nm was measured [37]. The degradation rate was expressed as the difference between the initial and final absorbance. This was estimated in percentage as follows (Eq. (2)):

$$BPA\mathit{Degradation}\left(\%\right)=\frac{\mathit{Initial}\left(BPA\right)\mathit{concentration}-\mathit{Final}\left(BPA\right)}{\mathit{Initial}\left(BPA\right)\mathit{concentration}}\times 100$$E2

2.4 Gas chromatography: mass spectrometry (GC: MS) analysis of BPA degradation metabolites

From the quantitative confirmation analysis employing a Shimadzu gas chromatograph GC-2010 series connected to a Shimadzu spectrometer GCMS-QP2010 PLUS (Japan), the separation of the compounds was achieved by employing a DB5MS capillary column (60 m) (Supelco), the carrier gas was helium and maintained at constant flow of (0.9 mL⋅min⁻¹). A sample volume of 1 μL was injected in the splitless mode at an inlet temperature of 280°C. The MS transfer line temperature was maintained at 280°C, whereas the ion source temperature was 180°C.

3. Results and discussion

3.1 Actinobacterial growth

Of all the six actinobaterial isolates examined, only three display the potential to grow on BPA. All the three actinobacterial isolates (A. naeslundii, A. bovis, and A. israelii) grew in the presence of BPA indicating their ability to metabolize BPA as a carbon and energy source. Likewise, the actinobacterial growth suggests that the necessary enzymes are required for the degradation of BPA; thus, the production of laccase in basal salt mineral medium confirmed the BPA degrading activity.

3.2 Influence of consortium on enzyme production and BPA degradation

3.2.1 Effect of consortium on laccase production

The influence of actinobacterial consortium on the production of laccase was studied over the degradation period of 312 h, as shown in Figure 1. The consortium of A. naeslundii, A. bovis, and A. israelii supported a maximum laccase yield of 15.9 U/mL followed by the consortium of A. naeslundii and A. israelii (9.5 U/mL), A. naeslundii and A. bovis (8.7 U/mL), while A. bovis and A. israelii (7.0 U/mL). Initially, at the incubation time of 24 h, the laccase activity observed for the three-member consortium had the lowest enzyme activity; this may be because the three individual strains are adjusting for one another to coexist in the culture media. Interestingly, at this period, the laccase activity increases steadily up to 168 h where maximal laccase activity was recorded. This result suggested that laccase production increases proportionately with the growth of the actinobacterial consortium. This result is similar to the findings of Bogan and Lamar [38], who observed that extracellular enzymes of organisms are produced in response to their growth phases. Tsioulpas et al. reported that maximum laccase activity was measured in the growth medium, while 69–76% of phenolic compounds were removed by Pleurotus spp. [39]. This present work recognized that enzyme secretion also depends on the physical factor, nutritional, physiological, and biochemical nature of the microorganism.

Figure 1.

Laccase activity of the different consortia. Key: An+Ai = Actinomyces naeslundii and Actinomyces israelii; An+Ab = A. naeslundii and A. bovis; Ab+Ai = A. bovis and A. israelii; and An+Ab+Ai = A. naeslundii, A. bovis, and A. israelii.

3.2.2 Effect of consortium on BPA degradation

The influence of the actinobacterial consortium on BPA degradation was studied over the degradation period of 312 h, as shown in Figure 2. The consortium of Actinomyces naeslundii, A. bovis, and Actinomyces israelii supported a maximum BPA degradation of 93.1% followed by that of A. naeslundii and A. israelii (87.3%), A. naeslundi and A. bovis (80.4%), and A. bovis and A. israelii (76.0%). Microbial consortia have the capability of degrading a wide range of hydrocarbons. This research was able to link actinobacterial growth and laccase activity to the rate of BPA degradation because it can be deduced that at 24 h, all the two-member consortium growth gradually increases until 120 h when a steady decrease set in. However, a different growth pattern was observed for the three-member consortium where the growth steadily decreased at 24 h and then sharply increases at 48 h, and the growth increases until 168 h when there was a gradual decline to the incubation period of 312 h. The decrease in growth and laccase activity might be due to the depletion of nutrients or the production of waste or toxic substances into the culture media during this period. BPA degradation rate hardly increases after the 168-h incubation period. However, the enhanced BPA degradation performance displayed by the three-member actinobacterial consortium is due to synergic in the secretion of laccase compared to the two-member consortium. Although all the actinobacterial strains under study exhibited promising potentials to degrade BPA, their interactive or compatibility test was not investigated.

Figure 2.

Degradation of BPA by different consortia (a) An+Ai; (b) An+Ab; (c) Ab+Ai; and (d) An+Ab+Ai.

3.3 Degradation by-product of BPA

The mineral-salt-medium-supplemented BPA as a sole carbon source at pH 7.0 inoculated in each actinobacterial consortium was analyzed after an incubation period of 312 h. Cultures were extracted for GC–MS analysis to determine the biodegradation products of BPA. The by-products of BPA degradation were investigated, and each metabolite produced during the biodegradation process is shown in Figure 3. The GC–MS analysis showed that the metabolites could be identified as concerned compounds by comparisons with known authentic compounds using the NIST Chemistry library. The mass peak is found at 38 for 1,2,4-trimethylbenzene, and its relative molecular mass is 120 at the retention time of 4.0 min, while the base peak value was observed at 105. In addition, 2,9-dimethyldecane was identified at mass peak of 21 at the retention time of 4.9 min. The relative molecular mass of the compound 2,9-dimethyldecane was observed as 170 and base peak was noticed at 43. Oxalic acid was also identified as one of the intermediate products, the relative molecular mass of the compound was 216 with a retention time of 7.0 min, and the mass and base peak values were recorded at 12 and 57, respectively. According to Kusvuran and Yildrim [40], oxalic acid was identified as organic intermediate from the degradation of BPA, which is similar to our observation in this study. However, intermediates such as p-hydroxyacetophone, hydroquinone, p-hydroxybenzaldehyde, and p-hydroxbenzoic identified by previous studies [41] were not detected.

Figure 3.

Chromatogram generated by GC–MS.

4. Conclusion

This study focused on the actinobacterial isolates identified as Actinomyces naeslundii, A. bovis, and Actinomyces israelii, which showed adaptive and biocapacity mechanisms to survive on culture media supplemented with BPA. A direct relationship was found between the microbial growth, laccase activity of the actinobacterial consortium, and BPA degradation. From the evidence presented in this research work, it can be concluded that the investigated actinobacterial strains could be considered as good prospects for their application in the bioremediation of BPA-contaminated environments as they revealed promising potential.