P-CHLORO META XYLENOL (PCMX) TOLERANT BACTERIA AND …
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Proceedings of 13 th Research World International Conference, Singapore, 4 th March 2016, ISBN: 978-93-85973-55-0 17 P-CHLORO META XYLENOL (PCMX) TOLERANT BACTERIA AND THEIR BIODEGRADATION 1 BHANUPRIYA BRAHMA, 2 PRIYABRATA SARKAR 3 UJJAINI SARKAR 1,3 Jadavpur University, Chemical Engg. Dept. 2 University of Calcutta, Department of Polymer Science and Technology E-mail: 2 [email protected], 1 [email protected] Abstract- Pharmaceuticals are bioactive compounds generally resistant to biodegradation and thus create health hazard to humans and animals. One such important and universally used antiseptic is Dettol, the main ingredient of which is p- chlorometaxylenol (PCMX). We have isolated and characterized a number of PCMX resistant bacteria. They showed excellent PCMX uptake within 24 h. The use of different microbial culture in biodegradation of PCMX has shown attractive results. Microbes degraded PCMX via metabolic pathways and finally reduced them into non-toxic end products. The following four bacteria were used for this study: BT102, (accession No. JQ80423), BTUA (accession No. GU265556), BT201 (accession No. JQ595990) and BT302, (accession No. JQ782891). The preliminary results showed that out of all these, two bacteria i.e. BT102 and BT201 could withstand a very high concentration of PCMX (10 mM) concentration. The biomass of these two bacteria was collected from the broth and encapsulated in beads made of biocompatible polymer (Calcium Alginate). These bacteria loaded beads were used to remediate PCMX from ppm level to ppb from synthetic as well as real environmental samples in a batch operation. Satisfactory results were obtained in the remediation within 24 hr. Index terms- Pharmaceuticals, Dettol, Chloroxylenol, Bioremediation I. INTRODUCTION Para Chloro Meta Xylenol (PCMX) or Chloroxylenol (4-chloro-3, 5-dimethylphenol), is a halogenated phenolic and is considered one of the oldest antimicrobials in use today [1]. It is a broad spectrum antimicrobial chemical compound used to control bacteria, algae, fungi and virus. It is used in hospitals and households for disinfection and sanitation. It is a constituent of some disinfectant formulations and is also used as a topical and urinary antiseptic and as a preservative in pharmaceutical and cosmetic products [2]. PCMX disrupts microbial cell membranes, causing leakage of cell constituents [3]. Phenol or phenolic compounds are widely distributed in the environment partly as a result of natural processes and more importantly, due to human and industrial activities. These compounds originate mainly from industrial processes such as resin manufacturing, oil refineries, petrochemicals, pharmaceutical dyes, textiles and plastic industries [4]. As they are persistent in nature and due to its toxic, mutagenic and carcinogenic characteristics they are classified as highly hazardous chemicals. According to the USEPA [5] maximum concentration level of total phenolic compounds in the treated industrial effluent and water supplies should be less than 1μg/ml. The permissible limit of phenol and phenolics for the treated industrial effluents is 1mg/L as mentioned by Central Pollution Control Board (CPCB), India [6][7]. So it is an urgent need for remediation of toxic Chemicals from the wastewater. PCMX is toxic and a mild skin irritant and may trigger allergic reactions in some individuals. The main objective in the present study has been to observe the bioremediation potential of BT102, Gram negative, round shaped Pandoraea sp. (accession No. JQ80423) and BT201, Gram negative, round shaped Delftia sp. (accession No. JQ595990) and use them to uptake PCMX from the waste water. II. MATERIALS AND METHODS Chemical and Culture Medium All chemicals used in the study were of analytical grade unless otherwise specified and obtained from Merck (Darmstadt, Germany) and Sigma Aldrich Chemical (St. Louis, MO, USA). The culture media were prepared using MilliQ water (18.2 MΩ, Millipore Corporation USA) and sterilized by autoclaving at 121◦C, under 105 K Pa pressure for 15 min. Composition of Nutrient Media For regular maintenance and growth of bacteria, a media was prepared using peptone (5g/L), beef extract (3g/L) and sodium chloride (2g/L) without PCMX at pH 7 and the bacteria were subcultured for 15 days. For each 100 ml of Nutrient Broth, 2% (v/v) of bacteria from culture was poured inside a Laminar Cabinet and incubated for 24 hours at 37 ̊C under 110 rpm in orbital shaker for bacterial growth. Plate culture is also done in regular basis of 30 days for preservation of bacteria. In case of Plate culture, Agar (18 g/L) was added to Nutrient at pH=7 and same procedure was followed for bacterial growth. Standard Curve of PCMX To assess the remediation, measurement of PCMX was necessary. A standard curve (Fig.a) was prepared using standard 4 Amino Antipyrene (AAP) Assay. 5 ml of filtrate (PCMX) was taken in a volumetric flask and volume was made up to 100 ml with ultra pure water and transferred into a beaker. 2ml Ammonium Chloride solution was added to this and pH was
Transcript of P-CHLORO META XYLENOL (PCMX) TOLERANT BACTERIA AND …
Proceedings of 13th Research World International Conference, Singapore, 4th March 2016, ISBN: 978-93-85973-55-0
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P-CHLORO META XYLENOL (PCMX) TOLERANT BACTERIA AND THEIR BIODEGRADATION
1BHANUPRIYA BRAHMA, 2PRIYABRATA SARKAR 3UJJAINI SARKAR
1,3Jadavpur University, Chemical Engg. Dept.
2University of Calcutta, Department of Polymer Science and Technology E-mail: [email protected], [email protected]
Abstract- Pharmaceuticals are bioactive compounds generally resistant to biodegradation and thus create health hazard to humans and animals. One such important and universally used antiseptic is Dettol, the main ingredient of which is p-chlorometaxylenol (PCMX). We have isolated and characterized a number of PCMX resistant bacteria. They showed excellent PCMX uptake within 24 h. The use of different microbial culture in biodegradation of PCMX has shown attractive results. Microbes degraded PCMX via metabolic pathways and finally reduced them into non-toxic end products. The following four bacteria were used for this study: BT102, (accession No. JQ80423), BTUA (accession No. GU265556), BT201 (accession No. JQ595990) and BT302, (accession No. JQ782891). The preliminary results showed that out of all these, two bacteria i.e. BT102 and BT201 could withstand a very high concentration of PCMX (10 mM) concentration. The biomass of these two bacteria was collected from the broth and encapsulated in beads made of biocompatible polymer (Calcium Alginate). These bacteria loaded beads were used to remediate PCMX from ppm level to ppb from synthetic as well as real environmental samples in a batch operation. Satisfactory results were obtained in the remediation within 24 hr. Index terms- Pharmaceuticals, Dettol, Chloroxylenol, Bioremediation I. INTRODUCTION Para Chloro Meta Xylenol (PCMX) or Chloroxylenol (4-chloro-3, 5-dimethylphenol), is a halogenated phenolic and is considered one of the oldest antimicrobials in use today [1]. It is a broad spectrum antimicrobial chemical compound used to control bacteria, algae, fungi and virus. It is used in hospitals and households for disinfection and sanitation. It is a constituent of some disinfectant formulations and is also used as a topical and urinary antiseptic and as a preservative in pharmaceutical and cosmetic products [2]. PCMX disrupts microbial cell membranes, causing leakage of cell constituents [3]. Phenol or phenolic compounds are widely distributed in the environment partly as a result of natural processes and more importantly, due to human and industrial activities. These compounds originate mainly from industrial processes such as resin manufacturing, oil refineries, petrochemicals, pharmaceutical dyes, textiles and plastic industries [4]. As they are persistent in nature and due to its toxic, mutagenic and carcinogenic characteristics they are classified as highly hazardous chemicals. According to the USEPA [5] maximum concentration level of total phenolic compounds in the treated industrial effluent and water supplies should be less than 1µg/ml. The permissible limit of phenol and phenolics for the treated industrial effluents is 1mg/L as mentioned by Central Pollution Control Board (CPCB), India [6][7]. So it is an urgent need for remediation of toxic Chemicals from the wastewater. PCMX is toxic and a mild skin irritant and may trigger allergic reactions in some individuals. The main objective in the present study has been to observe the bioremediation potential of BT102, Gram negative, round shaped Pandoraea sp. (accession No.
JQ80423) and BT201, Gram negative, round shaped Delftia sp. (accession No. JQ595990) and use them to uptake PCMX from the waste water. II. MATERIALS AND METHODS Chemical and Culture Medium All chemicals used in the study were of analytical grade unless otherwise specified and obtained from Merck (Darmstadt, Germany) and Sigma Aldrich Chemical (St. Louis, MO, USA). The culture media were prepared using MilliQ water (18.2 MΩ, Millipore Corporation USA) and sterilized by autoclaving at 121◦C, under 105 K Pa pressure for 15 min. Composition of Nutrient Media For regular maintenance and growth of bacteria, a media was prepared using peptone (5g/L), beef extract (3g/L) and sodium chloride (2g/L) without PCMX at pH 7 and the bacteria were subcultured for 15 days. For each 100 ml of Nutrient Broth, 2% (v/v) of bacteria from culture was poured inside a Laminar Cabinet and incubated for 24 hours at 37 ̊C under 110 rpm in orbital shaker for bacterial growth. Plate culture is also done in regular basis of 30 days for preservation of bacteria. In case of Plate culture, Agar (18 g/L) was added to Nutrient at pH=7 and same procedure was followed for bacterial growth. Standard Curve of PCMX To assess the remediation, measurement of PCMX was necessary. A standard curve (Fig.a) was prepared using standard 4 Amino Antipyrene (AAP) Assay. 5 ml of filtrate (PCMX) was taken in a volumetric flask and volume was made up to 100 ml with ultra pure water and transferred into a beaker. 2ml Ammonium Chloride solution was added to this and pH was
P-Chloro Meta Xylenol (PCMX) Tolerant Bacteria And Their Biodegradation
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adjusted to 10±0.2 followed by addition of 2ml each of 4 amino antipyrene and potassium ferricyanide solution. The intensity of the colour was measured at 510nm. Bacterial Growth Pattern in presence of Dettol (PCMX) and Determination of MIC for Isolates For bacterial growth in presence of PCMX, Nutrient Broth was taken in 15ml test tube and PCMX was added. A concentration from 1mM PCMX to 10mM PCMX in nutrient broth was used and 100µl of bacteria culture (BT102, BT201, BT302 and BTUA) was added in each in this PCMX solution in the laminar flow chamber. All these samples were incubated for 24 hours at 37 ̊ C under 110 rpm. The growth of the bacteria was monitored by measurement of optical density after regular interval at 600 nm. Fig. b, below shows the growth of bacteria in PCMX after 24 hours. BT102 and BT201 bacteria shows maximum growth in presence of PCMX in our study. Further, to detect the level of PCMX tolerance, minimum inhibitory concentrations (MIC) of BT102 and BT201 isolates were determined. The MIC is defined as the lowest concentration of PCMX which suppresses visible bacterial growth. For MIC, spreading of bacterial strain was done in Agar plates and 1mM to 25mM concentration of PCMX was checked. The solution of PCMX was made in sterilised tap water (STW).
Figure a: Standard curve for PCMX at 510nm using 4 AAP
assay In Fig. c, it is seen that BT102 strain could withstand less than 10 mM PCMX i.e for 1mM PCMX and 5mM PCMX, there is no inhibition of bacterial growth but for 10 mM PCMX, inhibition hole could be seen. Fig. d (i) and d (ii) on the other hand,
showed that BT201 strain could withstand upto 15mM of PCMX and no inhibition hole were seen upto this concentration.
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Figure b: Bacterial growth after 24 hours in PCMX at 600nm
Figure c: Growth of BT102 strain in 1mM, 5mM and 10mM
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Figure d(i): Growth of BT201 strain in 1mM, and
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Figure d(ii): Growth of BT201 strain in 15mM, 20mM and
25mM of PCMX concentration Preparation of Calcium Alginate Beads with Whole Cell Bacteria BT102 and BT201 bacteria were previously isolated from crude source and were characterised and identified [9]. In the present study, whole cells of BT102 and BT201 strains were immobilized in sodium alginate beads and used for the bioremediation studies in a batch mode. The biomass of the strains was collected after 24 hours incubation of the broth and preserved at -20 ºC in .89% NaCl for use in bioremediation process. Before using the bacterial mass, it was washed with Milli-Q water twice to remove NaCl and centrifuged at 6000 rpm and 27ºC. The collected mass was then poured in sodium alginate solution for preparation of beads. The adsorbent should have a distinct pore structure which enables fast transport. Calcium Alginate beads were prepared by dropwise dispensing 3% (w/v) sodium alginate aqueous solution having 4% (w/v) extracted mass of BT102 and BT201 strains (separately) into a continuously stirred (250 rpm) 2% (w/v) CaCl2 solution (Fiol et al. 2006). Beads thus produced were kept in CaCl2 solution overnight and were washed three times with reverse osmosis (RO) water and excess water was soaked by filter paper. These modified alginate beads of both strains were used to remediate PCMX in Dettol. Fig. e(i) shows arrangement for making calcium alginate beads. Fig. e(ii) and e(iii) display the calcium alginate beads with and without bacterial strain respectively.
Figure e: Assembly for formation of Calcium Alginate Beads
Figure e(ii): Bacterial strain encapsulated Calcium Alginate
Beads
Figure e(iii): Calcium Alginate Beads
III. BIOREMEDIATION STUDIES OF PCMX USING BACTERIA BT102 AND BT20 The data obtained from bioremediation of PCMX shows that both the strains ie. BT102 and BT201 could remediate PCMX approximately very efficiently to ppb level from very high concentrations of 5ppm, 25ppm, 50ppm and 100 ppm PCMX within 16 hrs. The bioremediation capacity could be seen from Fig. f and Fig. g for both the strains (BT102 and BT201) at 510 nm with respect to time. CONCLUSION Powerful PCMX degrading bacteria have been isolated and identified. The two strains BT102 and
Figure f: Bioremediation of PCMX in Dettol with BT102 strain
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Figure g: Bioremediation of PCMX in Dettol with BT201
Strain BT201 could tolerate PCMX upto 10mM concentration. The bioremediation of PCMX using these two bacteria (BT102 and BT201) encapsulated in calcium alginate beads has shown fantastic efficiency in batch operation mode by reducing PCMX from 10ppm to 100 ppb (i.e 99%). This study could show that a cost effective method could be viable by application of native bacterial strains for
PCMX degradation from municipal waste water for a possible reuse of water. REFERENCES [1]. [1] Bruch MK. 1996. Chloroxylenol: an old-new
antimicrobial. In: Ascenzi JM (Ed), Handbook of Disinfectants and Antiseptics. Dekker Inc., New York, pp. 265-294.
[2]. [2] Wallhauser KH. 1984. Antimicrobial preservatives used in the cosmetic industry. In: Kabara JJ (Ed), Cosmetic and Drug Preservation, Principles and Practice, Marcel Dekker Inc., New Zealand, pp. 684-686.
[3]. [3] Reckitt & Colman. 1996. Dettol. Product Monograph. [4]. [4] Kumaran P and Paruchuri YL. 1997, Kinetics of phenol
biotransformation, Water Research, 31(1):11-22 (Elsevier) [5]. [5]http://www.epa.gov/waterscience/standards/rules/ntr.html#
sectionG3 [6]. [6] S Suvra, SinghaSomdtta and Sarkar Ujjaini. 2009,
Adsorption of PCMX in composite adsorbent beds: Parameter estimation using nonlinear least square technique, Chemical Engineering Journal, pp. 361-366
[7]. [7] CPCB, 1995.General Guidelines for Discharge of Effluents (Inland Surface Water)
[8]. [8] G Tanushree and Sarkar Priyabrata, 2014, Isolation of a novel uric-acid-degrading microbe Comamonas sp. BTUA and rapid biosensing of uric acid from extracted uricase enzyme, J. Biosci. 39, pp. 805-819
[9]. [9] Lejin Xu1 and Jianlong Wang1, 2013, Degradation of 4-Chloro-3, 5-Dimethylphenol by a Heterogeneous Fenton-Like Reaction Using Nanoscale Zero-Valent Iron Catalysts, Environmental Engineering Science Volume 30, pp. 294-301
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