Biodegradation of aromatic hydrocarbons using microbial adsorbed bioreactor

Abstract

Polyurethane (PU) tubular coil-based bioreactor was constructed and evaluated for the effective biodegradation of benzene, toluene, xylene and phenol (BTXP). Herein, the removal of BTXP was done with a formulated bacterial consortium adsorbed on the inner surface of the PU coil. The formulated consortium consisted of four bacterial strains namely, Alcaligenes sp. d₂, Enterobacter aerogenes, Raoultella sp. and Bacillus megaterium. The adsorption ability of the bacterial cells onto the coil surface was assessed by spectrophotometric and Scanning Electron Microscopic (SEM) analysis. BTXP degradation performance was evaluated by Ultra-Violet spectroscopy and the degradation was confirmed by Fourier Transform Infrared Spectroscopy (FT/IR). The bioreactor constructed using polyurethane (PU) tubular coil with adsorbed bacterial cells exhibited 70% degradation capacity of 250 µL of 5% benzene, toluene, xylene and phenol (BTXP) at a pH of 6 within 8 h of treatment. FT/IR spectra of the treated sample indicated the production of ketonic, carboxylic acid/esters during biodegradation. The innovative technology proposed in the current study with the formulated bacterial consortium and the novel bioreactor opens up new possibilities for the better removal of BTXP mixture from contaminated sites and industrial effluents.

Introduction

The dominant pollutants of the ecosystem are the chemicals used in day to day life activities and these pollutants are produced as by-products through various industrial activities. Recalcitrant xenobiotics compounds are non-degradable and resistant to being broken down through chemical processes. The aromatic hydrocarbons have been generally used as a primary energy source by different industries. Because of their widespread use, aromatic hydrocarbon became one of the most common environmental pollutant present in the terrestrial and aqueous environment. Simple aromatic compounds consist of mono-aromatic hydrocarbons such as Benzene, Toluene, Xylene and Phenol commonly known as BTXP compounds, which are commonly found as a mixture in crude petroleum and different petroleum products. In the modernized society, 67% of pollution occurs due to contamination with these aromatic compounds and the main source of such pollution is petrochemical industries (Singh et al. 2016, 2017). According to the United States, Environmental Protection Agency (USEPA) these organics are classified as priority environmental pollutants (Farhadian et al. 2008; Bernal-Martinez et al. 2009; Zhang et al. 2018).

Normally aromatic compounds like Benzene, Toluene Xylene and Phenol frequently co-occur as mixture along with many other toxic chemicals and this mixture is well known toxic, carcinogenic and mutagenic agent. The widespread use of BTXP in various industrial operations and the discharge of mixture without effective treatment had led to widespread environmental pollution (Rajamanickam et al. 2017). Techniques like physicochemical and biological methods have been in employment for the treatment or removal of varieties of xenobiotics. The physical and chemical methods are expensive and often produce undesirable by-products which require further treatment steps to reduce toxicity (Sridevi et al. 2011; Tian et al. 2016).

One of the most significant processes for the removal of pollutants is biodegradation. Biodegradation is the application of microbial cells for the degradation of hazardous pollutants from soil, sediments, water, or other contaminated materials. Different types of microorganisms have been isolated from different habitats for the degradation of toxic pollutants (Shekhar et al. 2015; Ismaeil et al. 2018; Roy et al. 2018). Various xenobiotics in industrial effluents occur as a mixture and withstand biodegradation because single microorganism has not evolved the apt catabolic pathway to degrade them. Due to this serious dilemma, it is more appropriate to develop an effective method for the removal of a mixture of organic compounds rather than a single toxic compound. Hence a consortium designed properly can degrade various mixtures of organic compounds. Formulation of a syntropic bacterial consortium with broad metabolic capabilities (Mukherjee and Bordoloi 2011; M’rassi et al. 2015; Li et al. 2016; Gurav et al. 2017) is an effective approach for the biodegradation of complex organic mixture.

A wide variety of treatment techniques have been used for the removal of BTXP from contaminated water. A significant number of researches is being directed towards the designing of bioreactors for the treatment of wastewater. Bioreactors with immobilized microbial cells have received rising interest in the field of wastewater treatment. Such techniques offer advantages like high microbial growth, high metabolic activities, and strong resistance to different chemicals. Several synthetic and natural polymers are used as supporting materials for cell immobilization. Supporting materials like PVC, polystyrene, polyurethane, etc. increases the treatment performance with low energy consumption and gives stable degradation performance. Polyurethane based bioreactors were used successfully for the treatment of pollutant effluents (Chu and Wang 2011; Shalini and Pydi 2019).

The goal of the present study was to design a lab-scale bioreactor adsorbed with bacterial cells for the effective biodegradation of BTXP.

Materials and methods

Microbial consortia

The bacterial consortium for the study was formulated by mixing four different bacterial strains namely, Alcaligenes sp. d₂, Enterobacter aerogenes SBS1 (Genbank Accession no.: KC758848), Raoultella sp. SBS2 (Genbank Accession no.: KC758849) and Bacillus megaterium SBS3 (Genbank Accession no.: KC758849) in equal proportions. The four strains used in the study were isolated from detergent contaminated soil and reported in the previous study (Vijayan et al. 2014).

Sample

BTXP containing mineral salt (MS) medium with 1 g KH₂PO₄, 1 g (NH₄)₂SO₄, 0.5 g Mg SO₄·7H₂O and 0.001 g CaCl₂ in 1 L of distilled water were used as the basal medium. 100 mL MS medium with 250 µL of 5% of benzene, toluene, xylene and phenol (BTXP) at a pH of 6 was used as the sample for the present study. The bacterial consortium showed better growth in BTXP at pH 6. The chemicals were purchased from SRL and Hi-media Laboratories, India. The medium was sterilized at 121 °C for 20 min and the organic mixture was added after sterilization.

Characterization of control MS-BTXP medium

The uninoculated control MS-BTXP medium for the biodegradation studies was mainly characterized by wavelength scanning and FT/IR analysis. The control MS-BTXP medium was scanned between 200 and 300 nm with a UV–visible spectrophotometer (Hitachi U-2900/U-2910) just after the preparation (0 h) to determine the particular wavelength at which the sample exhibit maximum absorption. The absorption maximum, i.e. the λ_max and the FT/IR spectrum of the control medium were used to analyze the biodegradation efficiency of the bioreactor.

Designing of bioreactor

Biodegradation of BTXP was carried out using a polyurethane (PU) tubular coil-based bioreactor. The PU tubular coil was selected because of its better mechanical strength and for its possible industrial applications. The PU coil has an internal diameter of 8 mm with 5 m total length. The total working volume of the reactor was 120–125 mL (Fig. 1a, b). Steel wire with 6 m length and 4 mm thickness was inserted into the PU coil to make scratches on the inner surface and one end of the wire was sharpened and crooked to rub the smooth inner surface of the coil. The other end of this steel wire was connected to a power tool and rotated the wire through the inner surface of the coil. The same procedure was repeated twice to deepen the scratches.

Fig. 1.

Images of polyurethane tubular coil used for the construction of bioreactor. a Sectional view of polyurethane coil with an internal diameter of 8 mm. b Polyurethane tubular coil used for the construction of bioreactor with a total length of 5 m and a total working volume of 120–125 mL

The laboratory-scale bioreactor was designed and fabricated with a polyurethane (PU) tubular coil unit and a peristaltic pump, as shown schematically in Fig. 2. The saline suspension of bacterial consortium and the MS-BTXP medium was fed to the reactor and the flow rates through the reactor were controlled by a peristaltic pump (Pharmacia Biotech Pump P-1). The conical flask with bacterial-saline suspension was connected to the inlet of the peristaltic pump with a sterile tube and the outlet of the peristaltic pump connected to the inlet of the PU coil. The suspension was fed through the PU coil unit at a selected flow rate of 1 mL/min for the adsorption of bacterial cells onto the rough inner surface of the PU coil. The MS-BTXP media was then fed through the consortium adsorbed PU coil at specific flow rates for the bacterial degradation of BTXP present in the medium. Further, the outlet of the PU coil unit was connected to a conical flask to collect the bacterial treated sample.

Fig. 2.

Image of polyurethane tubular coil bioreactor used for the degradation of BTXP. PU coil was with 8 mm internal diameter, 5 m length and 120–125 mL working volume. The sample was passed through the PU coil at different flow rates of 1 mL/min, 2 mL/min, 3 mL/min, 4 mL/min and 5 mL/min with a peristaltic pump and the sample after treatment was collected to a conical flask for further analysis

Scanning Electron Microscopic (SEM) examination of adsorbed cells

Electron micrographic analysis of bacteria-treated (control) and untreated (test) PU coil was performed to examine the effective adsorption of bacterial strains during biodegradation studies. The PU coil was cut into small pieces with a sharp blade. A small portion of the PU coil was placed in 100 mL nutrient broth with 250 µL of BTXP. The flask was inoculated with the formulated consortium and incubated at room temperature for 48 h. The bacteria adsorbed coil piece was considered as the test sample. The control and test samples were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer with pH 7.3 for 2–3 h. Further, the samples were dehydrated with ethanol solutions. After drying, the samples were coated with gold using a JEOL-JFC-1600 auto fine coater and examined under JEOL-JSM-6390 scanning electron microscope.

Treatment of MS-BTXP medium with the consortium adsorbed bioreactor

The % degradation of BTXP was calculated at different flow rates and residence time. 150 mL of the bacterial consortium was prepared for the adsorption process. The optical density (OD) of the bacterial-saline suspension was measured at 650 nm. The bacterial-saline suspension passed through the coil at a speed of 1 mL/min. After complete filling, the coil was incubated for about 2 h for the adsorption of bacterial strains onto the inner surface of the coil. After incubation, the bacterial consortium was completely drained out from the coil and the OD of the collected sample was measured at 650 nm. Then the coil was washed with saline at a speed of 1 mL/min and the OD of the collected sample was again measured at 650 nm and the percentage of adsorption efficiency of the bacterial consortium was calculated.

The MS-BTXP medium was passed through the PU coil at different flow rates of 1 mL/min, 2 mL/min, 3 mL/min, 4 mL/min and 5 mL/min with the peristaltic pump. Three ml of treated samples were collected from the outlet of the PU coil for four times. Each sample was denoted as 1st, 2nd, 3rd and 4th collections and used for calculating the % removal of BTXP during treatment. Each of these samples was extracted with diethyl ether and used for further analysis. The effluent was completely removed from the coil and the treatment was continued with the next flow rate. Aromatic hydrocarbons have strong absorbance in the UV range and BTXP medium used in the present study showed maximum absorption at 251 nm and the absorbance reading was selected as the initial OD to calculate the degradation % of BTXP during treatment. The % removal of BTXP at different flow rates were calculated as,

$$\%\text{removal}\text{of}\text{BTXP}=\left[\frac{\text{Initial}\text{OD}\text{at}251\text{nm}-\text{Final}\text{OD}\text{at}251\text{nm}}{\text{Initial}\text{OD}\text{at}251\text{nm}}\right]\times 100,$$

where the λ_max of control MS-BTXP medium was used as the initial OD.

Results

Characterization of control MS-BTXP medium

The wavelength scanning of the control MS-BTXP medium between 200 and 300 nm instantly after preparation (0 h) showed maximum absorption at 251 nm, therefore 251 nm was selected as the λ_max for calculating the % degradation of BTXP at different flow rates and residence times. At 251 nm the control medium showed the maximum absorption value as 3.98, which indicated the concentration of BTXP in the sample.

Scanning Electron Microscopic (SEM) examination of adsorbed bacterial cells

Small pieces of PU coil with scratches were placed in nutrient broth with 250 µL of 5% BTXP and incubated for about 48 h at room temperature to allow the attachment of bacterial cells. The adsorption of the bacterial cell onto the PU coil was confirmed by Scanning Electron Microscopic (SEM). SEM examination of control and the test sample indicated significant variations in the appearance. The micrograph of the control sample showed the presence of scratches on the inner surface of the coil. The micrograph of test sample indicated the microbial colonization on the scratches which made on the inner surface of the PU coil (Fig. 3a, b). The adsorbed bacterial cells utilize the nutrients and BTXP mixture present in the medium as carbon and energy sources and were metabolically active viable cells.

Fig. 3.

Scanning Electron Micrographic (SEM) images of small polyurethane coil pieces. a SEM showing scratches on the inner surface of polyurethane coil piece. b SEM showing the attachment of bacterial cells as monolayer on the scratches made in the inner surface of polyurethane coil piece. The PU piece was inoculated with the formulated consortium and incubated at room temperature for 48 h for the adsorption of bacterial cells

Treatment of MS-BTXP medium

Operation of bioreactor

The evaluation of the efficiency of bacterial cell adsorption onto the PU coil surface was done spectrophotometrically and the percentage of biomass attachment were calculated. The Adsorption efficiency was calculated with the absorbance of bacterial-saline suspension at 650 nm before and after treatment. The suspension showed absorbance of 0.6 (initial) and 0.26 (final) and the % attachment of bacterial cells onto the coil was calculated as 56.6 after 2 h of incubation. An absorbance of 0.158 at 650 nm was observed after washing the PU coil with saline at a flow rate of 1 mL/min. This low absorbance indicated that a few numbers of adsorbed cells were washed out during the continuous process.

The MS medium with 250 µL of BTXP was prepared and OD readings at 251 nm of each trial were used to determine the efficiency of designed bioreactor. The subsequent decrease in the OD reading of 1st, 2nd, 3rd and 4th collections of each flow rates after treatment indicated the increase in the % removal of BTXP with an increase in residence time. The maximum BTXP degradation of 70% was attained in the 4th collection with a flow rate of 1 mL/min and residence time of 8 h (Table 1). The MS-BTXP medium showed an absorbance of 0.782 at 251 nm. An absorbance of 0.233 was observed in the 4th collection and the maximum % removal of BTXP was calculated as 70.204. This is the outcome of the study and indicated in bold. The data indicated that with an increase in flow rates, there was a subsequent decrease in the efficiency of the reactor and the % removal of BTXP.

Table 1.

% Removal of BTXP using polyurethane based bioreactor adsorbed with bacterial consortium

Speed (mL/min)	Residence time (h)	% Removal of BTXP
		1st	2nd	3rd	4th
Treatment of MS-BTXP medium
1	8	48.337	60.74	65.217	70.204a
2	6.50	43.989	49.104	51.278	55.754
3	6.35	42.583	48.721	50.00	54.731
4	6.05	33.887	45.268	54.731	56.138
5	5.55	26.982	41.176	54.731	56.777

^aThe maximum BTXP degradation of 70% was attained in the 4th collection of treated sample with flow rate of 1 mL/min at residence time of 8 h

FT/IR analysis of BTXP treatment with consortium adsorbed bioreactor

FT/IR analysis of control MS-BTXP medium showed specific bands representing Benzene, Toluene, Xylene and Phenol. The H bonded O–H stretch (3329.28 cm⁻¹), C–H stretch (3043.96 cm⁻¹), C=C stretch (1594.09, 1496.00, 1472.90 cm⁻¹), C–O stretch (1217.80, 1167.12, 1069.85, 1023.83 cm⁻¹) and C–H bends (999.33, 885.60, 809.20, 749.10, 688.39 cm⁻¹) in the FT/IR spectrum indicated the characteristic representation of BTXP in the medium. The ether extracted supernatant of treated BTXP medium with maximum degradation was also analysed by FT/IR. When compared with the uninoculated BTXP control medium, the FT/IR spectrum of BTXP medium after treatment with the bioreactor showed the absence of many of the specific bands of BTXP (Fig. 4a, b). The structural changes in the FT/IR spectra of 4th collection of treated sample with the flow rate 1 mL/min indicated the effective biodegradation of BTXP present in the medium. The library search report indicated that the vibration at 1634 cm⁻¹ represented the symmetric C=O stretch of ketones. The FT/IR spectrum of the treated sample also showed the presence of vibration in the range 1000–1300 cm⁻¹ indicating the presence of C–O stretch of esters and carboxylic acids. This indicated the production of ketonic, carboxylic acid/esters in the biodegraded sample.

Fig. 4.

Fourier Transform Infrared Spectrum of BTXP sample before and after biodegradation. a Fourier Transform Infrared Spectrum of BTXP mixture (control) before biodegradation. b Fourier Transform Infrared Spectrum of BTXP mixture after biodegradation. The MS medium with 250 µL of BTXP was used as the medium. b Represents the FT/IR spectrum of biodegraded BTXP mixture using PU based bioreactor adsorbed with bacterial consortium formulated with Alcaligenes sp. d₂, Enterobacter aerogenes, Raoultella sp. and Bacillus megaterium and the sample was fed through the coil with flow rate 1 mL/min and residence time of 8 h. The peaks between 3600–3100 cm⁻¹, 3100–3000 cm⁻¹, 1600–1450 cm⁻¹, 1300–1000 cm⁻¹ and 1000–650 cm⁻¹ indicated the presence of BTXP in the control sample and absence of these peaks in the treated sample indicated the removal of BTXP after treatment

Discussion

In the present study, the formulation of the bacterial consortium was based on the degradation efficiency of individual isolates and the FT/IR analysis of treated sample indicated the effective participation of individual strains in degradation process (Vijayan et al. 2014).

There are several reports on the potential use of immobilized bacterial cells in different matrices for the degradation of numerous toxic aromatic chemicals. Many synthetic and natural polymers are used as the immobilization matrix (de-Bashan and Bashan 2010). The superior mechanical properties, high porosity, large adsorption surface, resistance to organic solvents and microbial attack, easy handling, and cost-effectiveness make Polyurethane (PU) foam as excellent support for the immobilization of cells (Patil et al. 2006). High rates of sorption of positive charges and the hydrophobic nature of PU increases the interaction with microbial cell surfaces (Wang et al. 2009). The immobilization and performance of Thiobacillus ferrooxidans (Armentia and Webb 1992) and a hydrocarbon-degrading strain Rhodococcus sp. F92 (Quek et al. 2006) on polyurethane foam in the biodegradation of petroleum hydrocarbons was studied effectively.

An appropriate option of immobilized culture and careful deliberation of various design parameters are necessary during treatment with immobilized cells. In the present study, a laboratory-scale bioreactor was constructed using polyurethane (PU) tubular coil. The adsorption of bacterial consortium onto the inner surface of the PU coil was analysed by spectroscopic analysis and the adsorbed cells were observed using scanning electron microscopy. The scanning electron microscopic image (Fig. 3b) indicated the attachment of consortium onto the surface of the PU coil. The inner surface of the PU coil was smooth and this surface was made rough for the attachment of bacterial cells. A research study used SEM images to provide evidence for the immobilization of bacterial cells on polyurethane foam during the biodegradation process (Sonwani et al. 2019). The efficient adsorption of bacterial consortium onto the rough inner surface of the PU coil could be due to the presence of physical interactions between the microorganisms and the carrier polymer. The scratches on the PU surface helped the bacterial cells to form a stable monolayer of metabolically active cells. The actual mechanism of adsorption was complex and needs further investigation to explore reality. The BTXP degradation potential by the attached bacterial consortium was higher than that of free cells (Table 1). This study reports the novelty of a polyurethane (PU) tubular coil as a reactor and the adsorption of the formulated consortium on the PU coil to degrade BTXP mixture effectively. Application of bacterial cell immobilized polyurethane foam for the degradation of 97% of polycyclic aromatic hydrocarbon was reported (Alassandrello et al. 2017). The successful removal of benzene with an immobilized PUF based PBBR was reported in research work (Kureel et al. 2017). A moving bed bioreactor (MBBR) with Zeolite powder-based polyurethane sponges as biocarrier showed better performance, nearly 10% higher than the conventional moving bed bioreactor (Song et al. 2019). The amount of biofilm attachment was more in PU based bioreactor. A PU foam based MBBR showed better COD removal than polyethylene (PE)-based bioreactor (Sandip and Kalyanraman 2019).

Designing of a bioreactor with microbial adsorbed polyurethane improved the biodegradation of BTXP significantly. The better degradation of BTXP mixture was due to the fact that the adsorbed PU surface can retain viable biomass throughout the process with the least percentage of cell removal during the continuous process. The main advantage of the designed bioreactor was its tubular coil nature. The coil-shaped PU with high length column enables the cumulative biomass adsorption onto the coil surface. The increased residence time was possible with the tubular coil for the treatment of BTXP mixture and this helped in the better removal of organic load.

In the current study, FT/IR analysis was used as the analytical method to determine the changes in the functional groups in biomolecules which gave information about the biodegradation efficiency of constructed bioreactor. Many research works reported the effectiveness of microbial consortium (Vaidya et al. 2017, 2018) and application of FT/IR to establish the biodegradation process (Pathak et al. 2015; Shen et al. 2015; Patowry et al. 2016).

Comparison of FT/IR with other commonly used tools such as molecular studies, Gas Chromatography–Mass spectroscopic (GC–MS) and nuclear magnetic resonance (NMR) spectroscopic techniques are needed to throw light into the exact pathway followed for the degradation of these aromatic compounds. Analysis with these versatile analytical methods minimize the limitations of the present study and open up a way for future research work.

Conclusion

The present investigation reported that the formulated bacterial consortium with Alcaligenes sp. d₂, Enterobacter aerogenes, Raoultella sp. and Bacillus megaterium was efficient for the degradation of BTXP. The bioreactor constructed using polyurethane (PU) tubular coil with adsorbed bacterial cells showed 70% of degradation efficiency with a flow rate of 1 mL/min and residence time of 8 h. The result indicates the excellent efficacy of the lab-scale bioreactor in treating BTXP mixture. The lab-scale bioreactor designed is a novel concept in the field of wastewater treatment and the present investigation can be further exploited for large scale industrial applications.

Author contributions

The author of the manuscript has made substantial contributions and the work is an accurate representation of the trial results.

Funding

I do not have any financial relationship with any type of organization, funding agencies and project sponsors.

Compliance with ethical standards

Conflict of interest

The author declared that she has no conflict of interest in the publication.

Ethical approval: research involving human participants and/or animals

This article does not contain any studies with human participants and/or animals performed by the author.

Informed consent

Informed consent was obtained from all individual participants included in the study.