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Environmental Sustainability

, Volume 2, Issue 4, pp 381–389 | Cite as

Potential of Bacillus subtilis from marine environment to degrade aromatic hydrocarbons

  • Daisy VelupillaimaniEmail author
  • Arunachalam Muthaiyan
Original Article
  • 608 Downloads

Abstract

Microbial degradation of aromatic pollutants has been a promising method for bioremediation and restoring environmental damage. Protocatechuate (PCA) is a common intermediate in the microbial degradation of several aromatic compounds. The present study reports the identification of protocatechuate 3,4-dioxygenase (3,4-PCD)—a key enzyme of the β-ketoadipate pathway, in Bacillus subtilis isolated from coastal water containing waste discharge from paper mills, textile industries, and timber processing factories. The strain efficiently degraded PCA up to 20 mM concentration. Enzyme assay indicated the production of 3,4-PCD and cleavage of the benzene ring at ortho position and the formation of keto compounds of the beta- ketoadipate pathway. The utilization of PCA by plasmid cured cells indicates PCA metabolism is encoded by chromosomal genes. The metabolic potential of the B. subtilis isolate makes it a promising tool for bioremediation of aromatic pollutants present in marine environments.

Keywords

Bacillus subtilis Protocatechuate Intradiol cleavage Protocatechuate 3,4-dioxygenase Plasmid Bioremediation 

Introduction

Biodegradation by the natural population of microorganisms is cost-effective and environmentally sustainable treatment strategy (Bjerketorp et al. 2018). Although microorganisms capable of degradation of aromatic compounds have been investigated extensively, information about microbial degradation in marine environments is still very limited (Vieira et al. 2018).

Lignin, is the most abundant renewable aromatic material on earth and the largest source of aromatic building blocks (Ponnusamy et al. 2019; Sun et al. 2018). It is a complex, chemically stable, aromatic heteropolymer present in the cell walls of vascular plants (Abdelaziz et al. 2016). Despite its recalcitrance to breakdown, studies indicate the degradation of lignin by soil microorganisms (Ayeronfe et al. 2018; Xu et al. 2018).

Lignin degradation by fungi has been extensively studied when compared to bacteria (DeAngelis et al. 2013; Fang et al. 2018). In bacterial lignin degradation, most of the current knowledge is still based primarily on studies of soil bacteria (Xu et al. 2019).

Lately, studies on bacterial lignolytic enzymes in the degradation of lignin and intermediate compounds have been intensified (Abdelaziz et al. 2016; de Gonzalo et al. 2016; Fisher and Fong 2014; Hogancamp and Raushel 2018). Microbial degradation of lignin employs different pathways to yield aromatic acid intermediates such as protocatechuate (PCA) and catechol, which further get metabolized via beta-ketoadipate pathway (BKP) (Wu et al. 2017). Ring-cleaving dioxygenases catalyze intradiol cleavage of aromatic compounds such as catechol and PCA to produce intermediates that enter the carbon cycle (Tian et al. 2017).

Protocatechuate 3,4-dioxygenase (3,4-PCD), a key enzyme in the BKP, plays an essential role in the breakdown of numerous aromatic and hydroaromatic substances (Zhu et al. 2018). Recent research has shown the potential use of immobilized 3,4-PCD in degradation of the toxic 3,4-dihydroxybenzoic acid (3,4-DHBA) from industrial food processing waste water effluents (Zhang et al. 2017). 3,4-PCD from Stenotrophomonas maltophilia (KB2) reportedly shows wide substrate specificity that makes it a useful tool in applications for bioremediation or bioaugmentation purposes (Garrido-Sanz et al. 2018; Guzik et al. 2014).

The dissimilation of ferulic acid, an important constituent of lignin, has been reported in Bacillus subtilis (Gurujeyalakshmi and Mahadevan 1987; Ravi et al. 2017). Clearly B. subtilis has metabolic potential to degrade aromatic substances that are part of the lignin polymer. Moreover, B. subtilis displays exceedingly low pathogenicity to humans and was granted GRAS (generally regarded as safe) status by the U.S. Food and Drug Administration (De Boer and Diderichsen 1991; Jeżewska-Frąckowiak et al. 2018). Therefore, the wide use of B. subtilis in the fermentation industry makes it a perfect choice for use in increased production of enzymes. In this study, the breakdown of PCA—one of the precursors of lignin degradation, by B. subtilis isolated from the marine environment—was investigated and the results are reported.

Materials and methods

Bacterial strain, growth conditions, and media

B. subtilis (MDB1), isolated from the coastal waters (Chennai, Tamilnadu, India) containing waste discharge from paper mills, textile industries, and timber processing factories, was obtained from Prof. A. Mahadevan, CAS in Botany, University of Madras, Chennai 600,025 and used in this study.

The culture was maintained in minimal medium (MM) containing (NH4)2HPO4—3.0 g, K2HPO4—1.2 g, NaCl—0.6 g in 1 l of water (pH 7.5) with glycerol (2.6%) as the sole carbon source. At 48 h, the cells were routinely subcultured to get uninduced cells. For short-term storage, the culture was grown in Luria–Bertani (LB) medium (Sambrook and Russell 2001) amended with PCA (5 mM). The media were adjusted to pH 7.5 and solidified with 2% agar when needed. For testing its ability to grow on glycerol, glucose, and PCA as sole carbon sources, 48-h culture of B. subtilis was used to inoculate MM broth amended with either PCA (5 mM), or glycerol (10%), or glucose (10 mM). The carbon sources were filter-sterilized through 0.45 µ Millipore filter and added to the autoclaved media before inoculation. The culture was prepared by aseptically transferring 0.1 ml of stock culture into 20 ml of the medium and incubated at 37 °C on a rotary incubator at 150 rpm. Growth was monitored at regular intervals by measuring the OD at 540 nm by spectrophotometer (Phillips, PU 8400).

Preparation of cell-free enzyme

PCD-grown B. subtilis cells were harvested by centrifugation at 12,000×g at 4 °C for 10 min. The cells were washed and suspended in 10 ml of phosphate buffer (0.1 M, pH 7.0) containing 17% sucrose (w/v), 0.1% ascorbic acid (w/v), and 0.1% cysteine–HCl. The cells were disrupted in an ultrasonic disintegrator (Braun, Model 2000) for 1 min and centrifuged at 15,000×g for 20 min at 4 °C to separate the cell debris and unbroken cells. The supernatant was further saturated with equal volume of ice-cold acetone and incubated at 0 °C for 24 h to maximize precipitation. The sample was dialyzed for another 48 h in Tris.HCl buffer (pH 7.2) to remove salt contamination, lyophilized, dissolved in 0.5 M Tris.HCl (pH 7.2), and assayed immediately for enzyme activity.

3,4-PCD enzyme assay

To screen for the presence of intradiol ring cleavage dioxygenase activity in crude cell extracts, Rothera’s test was performed. 3,4-PCD was assayed by the method of Stanier and Ingraham (1954). The reaction mixture contained PCA 0.1 mL (10 μg), 1 mL enzyme, and 1.9 mL 0.5 M phosphate buffer (pH 7.2). Cleavage of PCA by 3,4-PCD was assayed spectrophotometrically by following the disappearance of substrate at 290 nm and appearance of the product, beta- carboxy cis,cis–muconic acid at 260 nm. In the controls, the enzyme was replaced with glass-distilled water. The standard curve for PCA was determined by plotting the gradient concentration of PCA against their absorbance at 290 nm.

Antibiotic susceptibility test

The antibiogram of B. subtilis was determined by using antibiotic sensitivity discs (Hi Media; Mumbai). The antibiotics and concentration used are listed in Table 1. The antibiotic susceptibility spectrum was determined by Kirby-Bauer disc-diffusion method. LB-grown B. subtilis culture (100 μL) with OD 1.0 at 540 nm was used to inoculate Mueller–Hinton agar plates. The antibiotic discs were placed on the inoculated agar surface and the plates were incubated at 37 °C for 24 h. The zone of inhibition for different antibiotics was measured to obtain the susceptibility profile.
Table 1

Antibiotic sensitivity pattern of B. subtilis

Antibiotic

Concentration (µg)

Susceptible (+)/Resistance (−)

Novobiocin

5

+

Erythromycin

10

+

Carbenicillin

100

+

Tetracycline

30

+

Streptomycin

25

Chloramphenicol

30

+

Kanamycin

10

+

Neomycin

10

+

Bacitracin

10

+

Ampicillin

25

+

Nalidixic acid

30

+

Isolation and curing of plasmid DNA

Indigenous plasmid DNA from B. subtilis was isolated from cells grown for 16 h at 37 °C in LB medium amended with ampicillin (75 μg/ml). Extracted plasmid DNA was purified using the Qiagen-Qia prep mini column (Qiagen, Germany). Agarose gels (0.8% or 1% w/v) were used to resolve the DNA samples and stained with ethidium bromide (0.5% v/v).

Plasmid curing was carried out by growing B. subtilis cells in LB medium supplemented with mitomycin C (20 μg/mL). The treated culture was incubated at 37 °C in a rotary shaker at 150 rpm. To select for plasmid cured B. subtilis, log phase cells were serially diluted, spread on LB agar plates and incubated at 37 °C. Colonies were randomly picked and grown in 10 mL LB and screened for plasmids. The cured colonies were sub-cultured in growth medium containing streptomycin (100 µg/mL) and PCA to check for their degradative ability.

Results

Growth of B. subtilis on single carbon-sources

The growth pattern of B. subtilis on glycerol, glucose, and PCA as sole carbon sources was determined (Fig. 1). On MM amended with glycerol (10%), the cells required a prolonged period of multiplication after a lag phase that extended to 8 h. The log phase was attained after 34 h of incubation. The stationary phase was reached at 36 h. When glucose (10 mM) was substituted for glycerol in the medium, the log phase was reached even before 6 h and continued until 32 h of incubation. Thus, glucose favored good growth of B. subtilis. The ability of B. subtilis to utilize PCA as the sole source of carbon and energy was tested by replacing the glycerol with 5 mM PCA. B. subtilis exhibited significant growth on media containing PCA as the sole carbon and energy source. PCA was readily utilized by B. subtilis cells. The cells had a lag phase of 8 h; the growth curve reached its maximum at 32 h of incubation and thereafter the cells entered the stationary phase. Growth increased with an increase in the concentration of PCA and reached maximum at 20 mM. However, increasing the concentration to 40 mM was toxic to the cells. PCA at 40 mM was inhibitory to the growth of B. subtilis (Fig. 2).
Fig. 1

Growth of B. subtilis on different carbon-sources. This replicated growth pattern (n = 3) of B. subtilis on MM amended with glycerol (10%), glucose (10 mM) and PCA (5 mM) shows good growth on all three carbon sources. The cells recorded an extended lag phase in glycerol and PCA while glucose favored good growth. Error bars represent the mean of data from triplicate assays

Fig. 2

Growth of B. subtilis on different concentrations of PCA. This replicated growth pattern (n = 3) of B. subtilis cells cultured on different concentrations of PCA shows an increase in growth corresponding to an increase in concentration. Maximum growth was recorded at 20 mM PCA. Error bars represent the mean of data from triplicate assays

Growth of B. subtilis on dual carbon-sources

Cells cultured on glycerol when transferred to MM medium containing both glycerol (10%) and PCA (5 mM) exhibited a prolonged lag phase. However, the growth rate of PCA-grown B. subtilis cells on glycerol + PCA was greatly enhanced. This growth pattern indicates the role of glycerol as a co-metabolite for the utilization of PCA as the sole source of carbon and energy in B. subtilis (Fig. 3). Additionally, the influence of glucose (10 mM) on the utilization of PCA by B. subtilis was investigated. Cells grown on PCA (5 mM) multiplied rapidly when transferred to MM containing PCA alone. However, glucose-grown cells when transferred to medium containing PCA alone, exhibited a lag phase of 8 h (Fig. 4). The growth patterns for PCA and glucose-grown cells on glucose were almost similar (Fig. 5). Cells exposed to PCA and transferred to medium containing both glucose and PCA continued to grow up to 32 h without any lag phase. The growth pattern of glucose-grown cells on glucose + PCA was the same up to 8 h, thereafter the cells entered the decline phase (Fig. 6). The initial growth pattern on dual carbon sources was almost identical to the growth on glucose alone. This indicated the preferential utilization of glucose during the log phase. Regardless of their prior induction on PCA, B. subtilis utilized PCA only after a lag phase of 4 h (Fig. 3). The presence of glucose (10 mM) along with PCA (5 mM) resulted in diauxic growth of B. subtilis.
Fig. 3

Growth of B. subtilis on glycerol + PCA. This replicated growth pattern (n = 3) of B. subtilis cells cultured on glycerol and transferred to MM amended with Glycerol (10%), and PCA (5 mM) shows prolonged lag phase. However, growth rate of PCA- grown cells was greatly enhanced on MM containing glycerol + PCA. Error bars represent the mean of data from triplicate assays

Fig. 4

Growth of B. subtilis on PCA + Glucose. This replicated growth pattern (n = 3) of B. subtilis cells cultured on PCA (5 mM) and transferred to MM with PCA alone multiplied rapidly. However, glucose-grown cells when transferred to medium containing PCA alone, exhibited a lag phase of 8 h. Error bars represent the mean of data from triplicate assays

Fig. 5

Growth of B. subtilis on PCA (alone) and glucose (alone). This replicated growth pattern (n = 3) of B. subtilis for PCA- and glucose-grown cells on glucose alone were almost similar without a lag phase. Error bars represent the mean of data from triplicate assays

Fig. 6

Growth of B. subtilis on glucose + PCA. This replicated growth pattern (n = 3) of glucose-grown B. subtilis cells on glucose + PCA showed similar growth rate up to 8 h but the growth rate decreased thereafter. Error bars represent the mean of data from triplicate assays

3,4-PCD enzyme assay

PCA up to 20 mM favored growth of B. subtilis cells. Clearly, PCA induced the enzymes associated with its oxidation. Hence, 3,4-PCD enzyme activity was assayed. The crude enzyme from B. subtilis cells grown on PCA did not develop deep yellow color, indicating the absence of meta cleavage of the aromatic ring. The contents turned deep purple in color indicating ortho cleavage of PCA and appearance of keto compounds of β-ketoadipate pathway.

The presence of beta- carboxy cis, cis–muconic acid was indicated by the increase in absorbance at 260 nm and confirmed the ortho cleavage of PCA by 3,4-PCD (Fig. 7).
Fig. 7

Intradiol cleaving enzyme assay. Enzyme was assayed in a spectrophotometer based on the disappearance of the substrate PCA at 290 nm (λ max for PCA) and appearance of the product, β-carboxy cis, cis–muconate at 260 nm (λ max for β-carboxy cis, cis–muconate). Increase in absorbance at 260 nm indicated appearance of β-carboxy cis, cis–muconate and confirms ortho cleavage of PCA by 3, 4-PCD. Error bars represent the mean of data from triplicate assays

Antibiotic susceptibility test

The antibiotic susceptibility spectrum of B. subtilis to antibiotics was evaluated. Depending upon the zone of inhibition and using the standard chart for antibiotic discs, it was evident that B. subtilis was resistant to streptomycin (25 μg/disc). The cells were sensitive to novobiocin, erythromycin, carbenicillin, tetracycline, chloramphenicol, kanamycin, neomycin, bacitracin, ampicillin, and nalidixic acid (Table 1).

Isolation and curing of plasmid DNA

B. subtilis MDB1 was screened for the presence of plasmids. The yield of plasmid DNA varied from 5 to 10 μg/mL culture. A single plasmid with low mobility was observed on the gel (Fig. 8). To check whether the PCA degradation trait of B. subtilis is encoded in plasmid DNA or genomic DNA, plasmid curing was performed (Fig. 9). Plasmid-cured cells were sub-cultured in medium containing streptomycin (100 µg/mL) and PCA to check for degradative ability. Growth of colonies indicates the Streptomycin resistant and PCA degrading genes are not carried on the plasmid but encoded in chromosomal DNA.
Fig. 8

DNA profile of B. subtilis. Lanes 1 & 2—arrow indicates the mega plasmid DNA above the chromosomal DNA; Lane 3—marker λ Hind III digest

Fig. 9

Curing of plasmid DNA in B. subtilis cells. Lane 1 arrow indicates plasmid DNA cured; Lane 2- marker λ Hind III digest

Discussion

Lignin, present in agricultural and industrial waste is a complex, recalcitrant compound and poses a significant challenge to the environment (Ayeronfe et al. 2018). Marine sediments serve as a suitable sink for aromatic compounds derived from industrial wastes and sewage effluents in aquatic environments (Zhuang et al. 2019). Hence, there is a great need for increasing the knowledge about the microbial degradation of recalcitrant compounds present in the marine environment, especially mechanisms, enzymology and genetic basis of degradation. In present study, we have described the aromatic degradation ability of the marine isolate, B. subtilis MDB1.

Since this B. subtilis strain was isolated from polluted coastal water, it might have survived along with other easily metabolizable carbon compounds that serve as an initial energy source required for breakdown of complex recalcitrant aromatic compounds. PCA has been reported as an intermediate in the biodegradation of many aromatic substances (Arunachalam et al. 2003; Guevara et al. 2019; Wu et al. 2017). Therefore, we studied the effect of simple carbon sources on the utilization of PCA by B. subtilis. According to our results, glucose supported good growth of B. subtilis, while an extended lag phase was observed in glycerol and PCA. This is similar to reports of preferential utilization of glucose as a carbon source by Serratia marcescens, Klebsiella pneumonia and Citrobacter sp. (Chandra et al. 2011).

Microorganisms readily metabolize preferred carbon sources which results in accumulation of non-preferred, complex aromatic compounds that are not easily degraded (Buffing et al. 2018). Moreover, some bacteria utilized another carbon compound to derive energy required for lignin degradation (Zhu et al. 2017). However, in our study B. subtilis was able to utilize PCA (20 mM) as a sole carbon source without the support of easily metabolizable glucose. Our results are similar to reports of Stenotrophomonas sp. and B. subtilis isolated from a palm oil plantation that reduced alkali lignin without the requirement of additional carbon sources (Azman et al. 2019).

In our growth study on single carbon sources, PCA (20 mM) favored the growth of B. subtilis whereas a higher concentration (40 mM) had an inhibitory effect. This result is similar to reports of inhibitory effect of high concentration of PCA due to interference with substrate transport across the cell membrane in Pseudomonas putida and disruption of cytoplasmic membranes in Escherichia coli (Bernal-Mercado et al. 2018; Harwood et al. 1994).

Easily metabolizable sugars such as glucose when added to the culture media serve as cometabolites and enhance utilization of aromatic compounds by lignin degrading microbial population (Chandra et al. 2011; Kuang et al. 2018; Xu et al. 2018). Therefore, in this study we tested the utilization of PCA by glucose-grown cells of B. subtilis. In dual carbon source growth study, B. subtilis cells preferentially utilized glucose and upon its completion, a second phase of growth occurred with the utilization of PCA. This diauxic pattern of growth occurs in cells when induction to the less preferred carbon source was prevented in the presence of the preferred substrate (Wang et al. 2019). The preferential utilization of substrates causes carbon catabolite repression of complex compounds, increasing their recalcitrance in the environment (Choudhary et al. 2017; Martínez-Valenzuela et al. 2018). Hence, the utilization of glucose and PCA by B. subtilis in the present study suggests its potential application in degradation of aromatic pollutants present with other nutrients in the marine environment.

Dioxygenases are natural enzymes for lignin degradation (Wang et al. 2017). In our study, the enzyme assay results indicated the ortho-cleavage of PCA by 3,4-PCD. The present study  reports the presence of 3,4-PCD, a key enzyme of the PCA degradation in B. subtilis. The intradiol or ortho- ring cleavage pathway commonly known as the BKP has a significant role in the degradation of many aromatic substances in nature (Tian et al. 2017; Żur et al. 2018). 3,4-PCD, has great potential for environmental bioremediation (Zhang et al. 2017). The reported efficiency of immobilized 3,4-PCD paves a future application for water purification (Das et al. 2016).

Plasmids encode beneficial traits that include the ability to degrade different aromatic compounds. Genes for aromatic metabolic pathways are often plasmid-encoded (Suvorova and Gelfand 2019). Association between drug resistance and aromatic degradation pathways encoded by plasmids has been reported in bacteria (Jutkina et al. 2011; Yan and Wu 2017). Therefore, resistance of B. subtilis MDB1 to antibiotics was evaluated by antimicrobial sensitivity discs. B. subtilis MDB1 was resistant to streptomycin. However, the absence of plasmids did not affect streptomycin resistance and PCA oxidation. Plasmid cured B. subtilis cells utilized PCA as the sole carbon source. Clearly, the plasmid in B. subtilis is not associated with PCA degradation. These findings demonstrate that the PCA metabolic machinery of B. subtilis is encoded by chromosomal genes. This is similar to other reports of chromosomal catabolic pathways for the assimilation of aromatic compounds (Guevara et al. 2019; Morales et al. 2004). Chromosomal genes encoding metabolic pathways are more stable when compared to plasmid encoded metabolic machinery (Xin et al. 2019). This renders B. subtilis MDB1 a good candidate as an inexpensive and sustainable alternative for facilitating bioremediation of environmental pollutants.

In summary, besides the bioremediation aspect of lignin degradation, interest in lignin as an underexploited carbon source has significantly increased in the last two decades, as evidenced by published reports on lignin depolymerization and valorization (Brink et al. 2019; Chio et al. 2019; Henson et al. 2018; Hirose et al. 2018; Mei et al. 2019; Ponnusamy et al. 2019; Shanmugam et al. 2019; Xu et al. 2018; Zhang et al. 2019). Lignocellulose can serve as a sustainable bioenergy source to meet the growing demand for fuels and chemicals (Sun et al. 2018). Moreover, marine microbial enzymes are naturally halotolerant and better retain their conformation even under high ionic strength conditions (Woo and Hazen 2018). Findings of the present study contribute valuable information to this emerging new facet of biodegradation research that involves discovery of bacterial strains in the marine environment for production of useful products (fuels, chemicals and materials) from recalcitrant biomass.

Conclusion

This study has explored the aromatic degradation potential of a marine isolate B. subtilis MDB1. Information about microbial degradation in marine environments is still very limited. Considering the increasing interest in discovery of efficient lignin depolymerizing microbes, these findings provide new direction towards the underexplored marine environment as a potential reservoir for aromatic degrading bacteria. Although 3,4-PCD plays a pivotal role in the degradation of many aromatic substances in nature, the complete sequencing of the B. subtilis (168) genome makes no mention of PCD genes. Therefore, the preliminary findings from this study form the framework for further advanced work involving the characterization and sequencing of PCD genes in B. subtilis. This will be a valuable contribution to supplement the lack of information about protocatechuate-degrading gene sequence in B. subtilis. Microbial degradation of aromatic environmental pollutants involves complex processes in microbial communities. The ability to form spores, secrete halotolerant enzymes and its low pathogenicity to humans may confer a competitive advantage to B. subtilis in bioremediation of contaminated coastal waters. Although, lignin waste disposal can be achieved by various chemical and physical methods, microbial biodegradation is eco-friendly and the natural competency of B. subtilis will facilitate an enduring sustenance of the marine environment.

Notes

Acknowledgements

The bacterial culture and resources provided by Prof. A. Mahadevan, Center for Advanced Studies in Botany, University of Madras, Chennai, India to conduct this study is thankfully acknowledged. The authors thank Amanda Newsum, Editor, Grand Canyon Education, Phoenix, Arizona, USA, for helping with the editing needs of this manuscript.

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Copyright information

© Society for Environmental Sustainability 2019

Authors and Affiliations

  1. 1.Centre for Advanced Studies in BotanyUniversity of MadrasChennaiIndia
  2. 2.Division of Arts and SciencesUniversity of New MexicoGallupUSA
  3. 3.College of Science, Engineering and TechnologyGrand Canyon UniversityArizonaUSA

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