Accepted Manuscript Title: Biodegradation of 2-hydroxyquinoxaline (2-HQ) by Bacillus sp. Author: G.V. Subba Reddy B.R. Reddy M.G. Tlou PII: DOI: Reference:
S0304-3894(14)00425-7 http://dx.doi.org/doi:10.1016/j.jhazmat.2014.05.080 HAZMAT 15987
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
6-3-2014 16-5-2014 28-5-2014
Please cite this article as: G.V.S. Reddy, B.R. Reddy, M.G. Tlou, Biodegradation of 2-hydroxyquinoxaline (2-HQ) by Bacillus sp., Journal of Hazardous Materials (2014), http://dx.doi.org/10.1016/j.jhazmat.2014.05.080 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
HIGHLIGHTS 2-HQ degrading strain (HQ2) was isolated & identified as Bacillus sp.
•
The generation time of Bacillus sp. in log phase on 2-HQ is 0.79 h or 47.4 min.
•
The sp. degraded 2-HQ under conditions of 1.0 OD inoculum, pH-6-8, & 37-45°C
cr
ip t
•
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temp.
Bacillus sp. degraded 2-HQ at concentration as high as 500 ppm.
•
Formation of dimers from 2-HQ appears to be initiation of 2-HQ degradation.
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•
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Title: Biodegradation of 2-hydroxyquinoxaline (2-HQ) by Bacillus sp. Subba Reddy, G.V., aReddy, B.R. and bTlou, M.G.
a. Dept. of Microbiology, Sri Krishnadevaraya University, Anantapuram – 515 003, A.P., INDIA. b. Present address: Faculty of Science, Dept. of Biochemistry, University of Johannesburg, PO Box-524, APK Campus, Johannesburg 2006, South Africa. *Corresponding author G.V. Subba Reddy Post-Doctoral Research Fellow Dept. of Biochemistry University of Johannesburg, PO Box-524 APK Campus, Johannesburg 2006, South Africa. Email: [email protected] Mobile No. 0027-745235195
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Abstract
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An aerobic gram +ve bacterial strain capable of utilizing 2-Hydroxyquinoxaline (2-
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HQ) as sole source of carbon and energy was isolated from Chrysanthemum indicum Indian agricultural soil and named as HQ2. On the basis of morphology, physicobiochemical characteristics and 16S rRNA sequence analysis, strain HQ2 was identified as Bacillus sp. The generation time of Bacillus sp. in log phase during growth on 2-HQ is 0.79 h or 47.4 min. The optimal conditions for 2-HQ degradation by Bacillus sp. were inoculum density of 1.0 OD, pH of 6-8, temperature of 37-45°C and 2-HQ concentration of 500ppm. Among the additional carbon and nitrogen sources, carbon sources didn’t influence the degradation rate of 2-HQ, but nitrogen sources – yeast extract marginally enhanced the rate of degradation of 2-HQ. GC-MS analysis of the culture Bacillus sp. grown on 2-HQ indicated the formation of dimers from 2 molecules of 2-
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hydroxyquinoxaline. The formation of dimer for degradation of 2-HQ by the culture appears to be the first report to our scientific knowledge.
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Key words: 2-Hydroxyquinoxaline, Bacillus sp., 16S rRNA sequence analysis, GC-MS analysis.
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1. Introduction
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Quinalphos [O,O-diethyl-O-(2-quinoxalinyl)-phosphorothioate;Organophosphorous insecticide group, with commercial trade name as Ekalux] is a broad-band based
an
pesticide with insecticidal and acaricidal properties. For this reason, quinalphos is frequently used in many countries over many crops in agriculture for control of insects
M
and represents a source of toxicity to humans and vertebrate animals. A 3-year evaluation (2000-2002) [1] of forensic pesticide and herbicide intoxications in Portugal indicated
d
about 29% of total cases due to quinalphos. Primary toxicity due to acetyl cholinesterase
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inhibition, is terminated by hydrolysis of quinalphos and its metabolic products
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containing phosphorous detected in human serum and urine [2]. Hydrolysis of the ester bond connecting the aromatic moiety to diethyl phosphorothioate in quinalphos leads to 2-hydroxyquinoxaline (2-HQ), which has also been identified as the main metabolite in soil, in water and on crops [3-6]. In vitro, 2-HQ is capable of photocatalytically destroying antioxidant vitamin C and E and oxidizing various biogenic amines such as serotonin, melatonin, N1-acetyl-5-methoxykynuramine, dopamine, epinephrine and norepinephrine and also anthranilic acid derivatives [7]. 2-HQ is also mutagenic, carcinogenic and genotoxic to organisms [7-9]. 2-Hydroxyquinoxaline appears to be as a secondary source of toxicity, as 2-HQ even in small concentrations leads to growth inhibition, induction of oxidative stress and genotoxicity in test organisms. 2-
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hydroxyquinaxoline is an aromatic and bicyclic compound with fusion of one benzene ring and one pyrazene ring and is getting accumulated in the environment- soil due to its
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slow degradation upon repeated applications of the parent compound – quinalphos [3]. Quinoline, a heterocyclic compound, distinct from 2-HQ, was also detected in the
cr
environment due to production and application of quinoline-based drugs, quinoline-based
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dyes and petroleum products [10]. It is desirable to have microbial/bacterial cultures in our stockpile for mitigation of N- heterocyclic compounds associated toxicity by
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enhancing degradation of N-heterocyclic compounds in the environment. Number of bacterial cultures such as Bacillus sp. [10], Pseudomonas sp. [11, 12], Burkholderia
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pickettii [13] and Rhodococcus sp. [14] with capacity to degrade quinoline were reported. Microbial metabolism of 2-hydroxyquinoxaline, a metabolite formed during degradation
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of quinalphos received less attention than p-nitrophenol, a metabolite in biodegradation
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of other organophosphates - Methylparathion. In this direction, efforts have been put to
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isolate 2-hydroxyquinoxaline - degrading bacteria from soil by selective enrichment technique method. Focussing on biodegradation of 2-HQ, provides a base of scientific knowledge. This is the first case we are reporting in this article isolation, identification and characterization of bacterium able to degrade 2-HQ as the sole source of carbon and energy source.
2. Materials & Methods 2.1. Soil sample
Soil sample [organic matter (%) - 0.42; nitrogen (%) - 1.54; pH - 8.1] was collected from Chrysanthemum indicum agriculture field of Pendlimarry village with prehistory use of quinalphos in YSR Kadapa district, Andhra Pradesh, India.
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2.2. Chemicals 2-Hydroxyquinoxaline was purchased from Sigma-Aldrich Fluka (99%). This 2-HQ
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was used for bacterial growth as sole source of carbon and energy. All other chemicals and solvents used in the present study are of analytical reagent grade/HPLC grade.
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2.3. Culture Medium
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The mineral salts medium(MSM) contained (gL-1) 1.5 NH4NO3, 1.5 K2HPO4. 3H2O, 0.2 MgSO4. 7H2O, 1.0 NaCl and 1 mL of trace elements stock solution. The trace element
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stock solution contained (gL-1): 2.0 CaCl2.2H2O, 0.2 MnSO4.4H2O, 0.1 CuSO4.2H2O, 0.2
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ZnSO4.H2O, 0.02 FeSO4.7H2O, 0.09 CoCl2.6H2O, 0.12 Na2MoO4.2H2O and 0.006 H3BO3.
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2.4. Isolation of bacterial strain
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The enrichment culture technique was set up by inoculating 100 mL of sterile MSM
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containing 0.005% 2-HQ with 5 gram of agricultural soil in 250ml Erlenmeyer flask. The flask was incubated in an orbital shaker (Orbitek LE-IL Model) at 37°C and 175 rpm. After 5 days of incubation period, 5 mL portion of the culture was transferred to fresh medium with high 2-HQ concentration up to 0.05% in Erlenmeyer flasks and the flasks were incubated for 5-days. After 5 more transfers, the culture was purified by serial dilution transfer method and streak plating onto solidified MSM containing 0.005% of 2HQ. Finally, a pure bacterial strain was obtained and designated as HQ2. 2.5. Identification and characterization of bacterial strain 2.5.1. Morphological and biochemical characterization
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Morphological observations of bacterial isolate were made with optical compound microscope. Physiological and biochemical properties of the strain was determined by the
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2.5.2. Sequence analysis and construction of a phylogenetic tree
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procedures as described by Bergey’s Manual of Determinative Bacteriology [15].
The total genomic DNA was extracted from the potential bacterial isolate (HQ2)
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following a standard phenolic extraction procedure [16]. Phylogenetic analysis based on
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16S rRNA gene sequences were made as described by Qin [17]. The 16S rRNA gene sequences of strain HQ2 was amplified on their genomic DNA with universal conserved as
primers
–
AGACTCAGGTTTGATCCTGG-3′
16
forward
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sequence
and
16
primer
reverse
sequence primer sequence
-
5′- 5′-
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ACGGCTACCTTGTTACGACTT-3′. Both forwarded and reverse sequences were
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generated and combined to get assembled partial sequence of 16S rRNA gene. The
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determined sequence was compared with those in the GenBank/EMBL data base using the online tool BLAST program [18] accessible through the web (NCBI). FASTA sequences of the closely related bacterial sp. to the present bacterial isolate was collected and aligned and constructed phylogenetic tree using the Robust Phylogenetic [19, 20] online tool.
2.6. Growth of bacteria on 2-HQ For growth of the bacterial isolate on 2-hydroxyquinoxaline, 50 mL of sterile MSM in sterile 250 mL Erlenmeyer flasks was spiked with 2-hydroxyquinoxaline at concentration of 50 µg mL-1. Meanwhile, potential bacterial isolate inoculum was prepared by growing the bacterial strain in 50ml of MSM with yeast extract (0.1%) and
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supplemented with 50 ppm of 2-HQ per ml of MSM and incubated in an orbital shaker at 175 rpm at 37°C for overnight. The overnight culture was harvested aseptically (8000g,
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15min, 4°C) and thoroughly washed with MSM and suspended in sterile MSM to get suspension with the desired OD. Flasks with test chemical - 2-hydroxyquinoxaline in
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MSM and inoculated with the potential bacterial culture at the final OD of 1.0 per ml of MSM. Uninoculated flasks with fortified medium served as control. All flasks were
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incubated in an orbital shaker at 175 rpm at 37°C for 24 h. Five milliliter aliquots from
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growing culture broth were withdrawn after every 6 h for determining OD value at wavelength 600nm in UV-Visible spectrophotometer (Chemito-UV-2600) and total
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number of viable bacterial colony forming units by serial dilution technique method on nutrient agar medium plates. The specific growth rate of Bacillus sp. was calculated in
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the logarithmic phase. 2.7. Biodegradation of 2-HQ
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Experiments for biodegradation of 2-hydroxyquinoxaline by the bacterial isolate
were carried out in 250-mL Erlenmeyer flasks in the same manner as done for growth experiments (2.6). At regular intervals of 24 hrs, 10mL of culture filtrate was aseptically withdrawn from flasks for growth measurement and residue analysis. Culture/medium from both uninoculated and inoculated flasks was centrifuged at 8000g for 15 minutes in a refrigerated centrifuge. Supernatants collected in this fashion were thrice extracted with dichloromethane with equal volume of supernatant. The extracts were pooled together dried over anhydrous (NH4)2SO4, filtered, and evaporated to dryness at room temperature. The residue was dissolved in methanol for HPLC and GC-MS analysis. 2.7.1. Factors influencing on biodegradation of 2-HQ
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Appropriate modifications in growth conditions of the bacterial culture on 2-HQ were made in order to assess the effect of various factors on degradation of 2-HQ by the
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potential bacterial isolate. For this purpose, MSM spiked with 2-HQ at 50 mg L-1 was distributed in 250-mL flasks at the rate of 100 ml per flask. Additional carbon source
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(glucose/sodium acetate) or additional nitrogen source NH4Cl, (NH4)2SO4, urea or yeast
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extract was included in certain flasks at 0.01% concentration. Flasks were inoculated with bacterial suspension to get OD of 1.0 in the inoculated media. Flasks with/without
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supplementation, devoid of inoculums were maintained as appropriate controls. After incubation of all flasks at 37°C and 175 rpm in a shaker, flasks were withdrawn at regular
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intervals after extraction with dichloromethane for residue analysis. Influence of concentration of 2-HQ on its degradation was assessed by growing the bacterial isolate
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on 2-HQ in MSM at different concentrations (50-500 ppm). In another experiment, flasks
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containing 2-HQ at 50 mg L-1 in MSM at pH 7.0 and bacterial cell suspension at initial
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OD of 1.0 were incubated in a shaker at 175 rpm and at different temperatures - 30-45°C in order to study influence of temperature on degradation of 2-HQ. Flasks with MSM after adjustment to different pH and spiked with 2-HQ at 50 mg L-1 was incubated with cell suspension of bacterial isolate at initial OD of 1.0 in a shaker at speed of 175 rpm and 37°C in order to find out influence of pH on degradation of 2-HQ. 2.8. Analytical methods
2.8.1. 2-HQ residue analysis by High Performance Liquid Chromatography (HPLC) Residue of 2-HQ extracted and finally dissolved in methanol in different experiments was analyzed by HPLC (Shimadzu, Japan) equipped with a ternary gradient pump, programmable variable-wave length UV detector, column oven, and electric
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sample valve and ODS-2, C18, reverse phase column (4.6mm×250mm×5μm). The 2-HQ residue analysis was conducted by using an isocratic mobile phase of methanol. Sample
1
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injection volume was 20 µL, the mobile phase was programmed at flow rate of 1 mL minand 2-hydroxyquinoxaline was detected at 345 nm wave length with retention time of
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0.833 minutes.
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2.8.2. Identification of 2-hydroxyquinoxaline metabolites by GC-MS analysis MSM was spiked with 2-hydroxyquinoxaline at 50 µg mL-1 in the same manner as
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mentioned earlier in section 2.6. The flasks were incubated under optimal conditions at 37°C and 175 rpm for 5-days. At regular intervals 10 mL aliquots of culture was
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withdrawn aseptically and extraction with dichloromethane for metabolites identification
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as in the same manner as specified in section 2.7. Dried residues were subjected to Gas
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Chromatography-Mass Spectrometry (GC-MS) analysis. Residues finally recovered in methanol from control and bacterial cultures were analyzed in GC-MS-QP-5050
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chromatograph (M/s. Shimadzu Instruments, Japan). Initially the residues extracted from the controls were analyzed. The data obtained on GC-MS from control was taken as reference for the identification of any new peaks found in the sample recovered from bacterial culture grown on 2-hydroxyquinoxaline. 3. Results & Discussion
3.1. Isolation and identification of a 2-HQ degrading bacterium A soil bacterial strain capable of utilizing 2-HQ as its sole carbon and energy source was isolated (HQ2) by an enrichment technique. Cell morphology of HQ2 is gram-
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positive and its colonies are opaque, glistering and powdery undulate. Biochemical characteristics of (HQ2) such as IMVIC tests, glucose and lactose fermentations, catalase
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activity, casein hydrolysis and gelatin liquefaction whereas urease, nitrate reducatse and starch hydrolysis are positive. Morphological and biochemical characteristics suggest that
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the strain HQ2 falls in to Bacillaceae group.
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3.2. Phylogenetic analysis
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Analysis of 16S rRNA sequence, using Robust Phylogenetic tree [19, 20] online tool, the neighbor-joining dendrogram was constructed and shown in the (Fig. 1). Based
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on the dendrogram, along with morphological and biochemical characteristics potential bacterial strain (HQ2) was 69% identical with Bacillus firmus and is tentatively identified
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as Bacillus sp. The nucleotide sequence coding for 16S rRNA of potential strain HQ2
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(1476 bases) were deposited in the GenBank database with accession number
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Bankit1538194 Seq1JX239694.
3.3. Growth of bacteria on 2-hydroxyquinoxaline Growth experiment was performed in MSM fortified with concentration of 50 µg
mL-1 of 2-HQ (2.6). Viable cell count in growing culture of bacteria was measured at regular intervals (supplementary table 1). At beginning (0 time) immediately after inoculation the viable cell count of 28×109 CFU/mL was recorded in Bacillus sp. After incubation for 6 h, initial total viable cell count increased to 50×109 CFU/ml in the culture of Bacillus sp. During this log phase of bacteria growth rate and generation time calculated as per the equation - K = log Nt - log No/log 2×t. Doubling time of Bacillus sp. at log phase is 0.79 h or 47.4min/generation.
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Growth of Arthrobacter sp. HY2 was observed after incubation in MSM containing 50 mg L-1 PNP in MSM after 6 h with concomitant decrease in PNP and was further
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increased by two folds within 12 h [21]. Similarly, commencement of growth of Arthrobacter protophormiae RKJ100 on PNP was noticeable after 2 h and reached
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maximum on 12-16 h [22, 23]. Optimum growth of Serratia sp. DS001 was found at a
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concentration of 0.3mM PNP with doubling time of 13.8 h [24]. However, growth of potential culture in the present study was different on 2-hydroxyquinoxaline and had
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longer generation time.
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3.4. Biodegradation of 2-hydroxyquinoxaline (2-HQ) by Bacillus sp. Capacity of the biodegradation of 2-HQ by bacterial isolate - Bacillus sp. was
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assessed under different conditions.
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3.4.1. Influence of additional carbon source on biodegradation of 2-HQ Biodegradation of 2-HQ by the potential bacterial isolate - Bacillus sp. in the
presence or absence of glucose or sodium acetate in MSM was compared. The extent of 2-hydroxyquinoxaline disappeared in the culture - Bacillus sp. was determined and is presented in (Fig. 2a).
Disappearance of 2-hydroxyquinoxaline in uninoculated medium with/without
additional carbon was not appreciable and didn’t exceed 1% of added 2hydroxyquinoxaline even at the end of 1-day incubation. But about 90% of added 2hydroxyquinoxaline disappeared in culture of Bacillus sp. (inoculated) in the presence of glucose or sodium acetate at end of 1-day incubation. There was no significant difference
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in figures of % of degradation of 2-hydroxyquinoxaline caused by Bacillus sp. culture in the presence or absence of additional carbon. It is clear from the results of the present
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study that the degradation of 2-HQ by the potential bacterial strain was not influenced by the additional carbon.
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In contrast, Feng [25] reported that Pseudomonas sp. strain ATCC 700113
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stimulated mineralization of 3,5,6-trichloro-2-pyridinol (TCP) in MSM at the concentration of 100 mg L-1 by the addition of carbon sources such as glucose, maleic
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acid and succinic acid. Similarly, addition of glucose (100 mg L-1) as co-substrate for EBN-12 mutant strain resulted in complete degradation of 100 mg L-1 of p-nitrophenol
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(PNP), a metabolite in degradation of parathion/methyl parathion in 20 h rather than in 24
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h in its absence [26]. Addition of glucose at low concentration (0.1-0.5%) enhanced the
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degradation of PNP by Pseudomonas sp. [27] and Arthrobacter sp. HY2 [21]. In the present study inclusion of glucose at low concentration in MSM had no influence on
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degradation of 2-hydroxyquinoxaline by Bacillus sp. 3.4.2. Influence of additional nitrogen source on biodegradation of 2-HQ In order to find out a suitable nitrogen source from - inorganic forms - ammonium
chloride and ammonium sulphate, organic forms - urea and yeast extract
for
biodegradation of 2-HQ by the potential bacterial isolate - Bacillus sp. was grown on 2HQ in MSM amended with the additional nitrogen source. Appropriate controls - MSM devoid of both the additional nitrogen source and inoculum, MSM devoid of the additional nitrogen source with receipt of inoculum and inoculated MSM amended with additional nitrogen source were used.
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Disappearance of 2-hydroxyquinoxaline in uninoculated medium irrespective of the nitrogen source was limited to only 1% even at the end of 1-day incubation. But
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degradation of 2-hydroxyquinoxaline occurred to the extent of 90-96% in the culture of Bacillus sp. at the end of 1-day incubation (Fig. 2b). Growth of Bacillus sp. on MSM
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without additional nitrogen caused 90% degradation of 2-HQ as against 90-96% by the
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same culture on MSM with different additional nitrogen source. Among the additional nitrogen sources used in the present study, yeast extract marginally enhanced the
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degradation of 2-HQ. On contrary, addition of ammonium ion at 3.8mM did not affect TCP degradation by the resting cells of Pseudomonas sp. strain ATCC 700113 but a
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concentration of 15mM had slightly inhibitory effect on TCP degradation [25]. Qiu [28] reported that addition of ammonium chloride and ammonium sulphate (1g L-1) did not
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favor the growth of Ochrobactrum sp. B2 or degradation of parathion/methyl parathion
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metabolite - PNP. Addition of sodium nitrate, ammonium sulphate (0.04%) did not exert
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significant effect on PNP degradation by P. putida [29]. Srilatha [30], reported that additional nitrogen source did not enhance the degradation rate of PNP by the bacterial cultures Arthrobacter sp. and Nocardioides sp. Quinoline is a bicyclic aromatic compound and is structurally similar to 2-HQ due to presence of one benzene ring and one heterocyclic ring but differs from 2-HQ with one nitrogen in heterocyclic ring. Zhu [14] reported that additional nitrogen source in particular (NH4)2SO4 enhanced the growth and degradation of quinoline by Rhodococcus sp. QL2. In the present study, supplementation of additional nitrogen in the form of yeast extract marginally improved degradation of 2-hydroxyquinoxaline by Bacillus sp. 3.4.3. Influence of inoculum density on biodegradation of 2-hydroxyquinoxaline
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In order to find out the optimal size of inoculum density for degradation of 2-HQ by the potential bacterial isolate - Bacillus sp. was cultivated on MSM after inoculation with
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the three different sizes of inoculum densities. The cell suspension of culture was adjusted to desired cell densities and added to MSM fortified with 2-HQ to provide initial
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cell densities at 0.5, 0.75 and 1.0 OD at A600 in inoculated medium.
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The corresponding figures of the 2-HQ degradation by Bacillus sp. upon growth with inoculum of 0.5, 0.75 and 1.0 OD sizes were 80.38, 86.5 and 90.18%, respectively at
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the end of 24 h incubation (Fig. 3). Thus, the grown bacterial culture with highest initial cell density, i.e. 1.0 OD supported the maximum degradation of 2-HQ indicating
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inoculum of 1.0 OD optimal.
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Labana [31] examined influence of inoculum size 2×105, 2×106, 2×107, 2×108 and 2×109 cells/g soil on degradation of methyl parathion metabolite - PNP by Arthrobacter
Ac ce p
protophormiae RKJ100 in soils. Among these inoculum densities, 2×105, 2×106 cells/g soil depleted PNP after 10 days of incubation whereas 2×109 cells were able to degrade PNP after 5 days of incubation. The optimum inoculum size was found to be 2×108 which achieved 98% depletion in just 2 days. Ramadan [32] reported that Pseudomonas cepacia did not mineralize p-nitrophenol at a density less than 104 cell/ml of lake water. In general, longer lag phase before onset of rapid degradation of xenobiotic by microbial cultures with a smaller inoculum density is noticeable. It has been suggested that the longer lag phase seems for multiplication of small active population of xenobiotic/pesticide-degrading bacteria to certain level so as to display degradation of the
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xenobiotic/metabolite [33]. This type of correlation between the length of acclimation period and size of inoculum density in liquid cultures may also be applicable to microbial
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degradation of metabolites of pesticides [34].
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3.4.4. Influence of concentration of 2-hydroxyquinoxaline on its biodegradation
The potential bacterial isolate - Bacillus sp. was grown on 2-HQ at different
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concentrations of 50, 100, 200 and 500 mg L-1 in MSM. Degradation of 2-HQ by
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bacterial culture was examined and presented in the (Table 1). Concentration of 2-HQ had no influence on degradation of 2-HQ by the potential bacterial culture.
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About 94% of added 2-hydroxyquinoxaline disappeared in the culture of Bacillus sp. irrespective of initial concentration within a range of 50-500 ppm in the medium at
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the end of 24 h incubation. Only 1% of degradation was observed in respective of
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uninoculated controls indicating no appreciable level of 2-HQ degradation. Consistent with the results of the present study, degradation of 90% of the added
trichloro-2-pyridinol (TCP), a metabolite of chlorpyrifos by B. pumilus C2A1 in medium at high concentration of 300 µg mL-1 was observed [35]. According to Yang [36] a strain of Alcaligenes faecalis strain DSP3 had capacity to degrade wide range of TCP concentration from 10-800 mg L-1. On other hand, contrasting results on degradation of trichloro-2-pyridinol, a metabolite of chlorpyrifos by other organisms was reported. For instance, Pseudomonas sp. strain ATCC 700113 mineralized TCP in liquid MSM at the concentration of 100-200 mg L-1 was reported [25]. Paracoccus strain TRP degraded 3,5,6-trichloro-2-pyridinol
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(TCP) concentration of TCP 400 mg L-1 within 4 days [37]. Recent report [38] provided an evidence that a bacterial strain, Cupriavidus sp. DT-1 completely degraded TCP upto
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concentration of 50 mg L-1 within 14h. Qiu [21] reported that more than 90% PNP was depleted within 24, 48 and 168 h from the medium with initial concentration of PNP at
S0304-3894(14)00425-7 http://dx.doi.org/doi:10.1016/j.jhazmat.2014.05.080 HAZMAT 15987
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
6-3-2014 16-5-2014 28-5-2014
Please cite this article as: G.V.S. Reddy, B.R. Reddy, M.G. Tlou, Biodegradation of 2-hydroxyquinoxaline (2-HQ) by Bacillus sp., Journal of Hazardous Materials (2014), http://dx.doi.org/10.1016/j.jhazmat.2014.05.080 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
HIGHLIGHTS 2-HQ degrading strain (HQ2) was isolated & identified as Bacillus sp.
•
The generation time of Bacillus sp. in log phase on 2-HQ is 0.79 h or 47.4 min.
•
The sp. degraded 2-HQ under conditions of 1.0 OD inoculum, pH-6-8, & 37-45°C
cr
ip t
•
us
temp.
Bacillus sp. degraded 2-HQ at concentration as high as 500 ppm.
•
Formation of dimers from 2-HQ appears to be initiation of 2-HQ degradation.
te
d
M
an
•
ab*
Ac ce p
Title: Biodegradation of 2-hydroxyquinoxaline (2-HQ) by Bacillus sp. Subba Reddy, G.V., aReddy, B.R. and bTlou, M.G.
a. Dept. of Microbiology, Sri Krishnadevaraya University, Anantapuram – 515 003, A.P., INDIA. b. Present address: Faculty of Science, Dept. of Biochemistry, University of Johannesburg, PO Box-524, APK Campus, Johannesburg 2006, South Africa. *Corresponding author G.V. Subba Reddy Post-Doctoral Research Fellow Dept. of Biochemistry University of Johannesburg, PO Box-524 APK Campus, Johannesburg 2006, South Africa. Email: [email protected] Mobile No. 0027-745235195
Page 1 of 31
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Abstract
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M
an
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te
An aerobic gram +ve bacterial strain capable of utilizing 2-Hydroxyquinoxaline (2-
Ac ce p
HQ) as sole source of carbon and energy was isolated from Chrysanthemum indicum Indian agricultural soil and named as HQ2. On the basis of morphology, physicobiochemical characteristics and 16S rRNA sequence analysis, strain HQ2 was identified as Bacillus sp. The generation time of Bacillus sp. in log phase during growth on 2-HQ is 0.79 h or 47.4 min. The optimal conditions for 2-HQ degradation by Bacillus sp. were inoculum density of 1.0 OD, pH of 6-8, temperature of 37-45°C and 2-HQ concentration of 500ppm. Among the additional carbon and nitrogen sources, carbon sources didn’t influence the degradation rate of 2-HQ, but nitrogen sources – yeast extract marginally enhanced the rate of degradation of 2-HQ. GC-MS analysis of the culture Bacillus sp. grown on 2-HQ indicated the formation of dimers from 2 molecules of 2-
Page 2 of 31
3
hydroxyquinoxaline. The formation of dimer for degradation of 2-HQ by the culture appears to be the first report to our scientific knowledge.
ip t
Key words: 2-Hydroxyquinoxaline, Bacillus sp., 16S rRNA sequence analysis, GC-MS analysis.
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1. Introduction
us
Quinalphos [O,O-diethyl-O-(2-quinoxalinyl)-phosphorothioate;Organophosphorous insecticide group, with commercial trade name as Ekalux] is a broad-band based
an
pesticide with insecticidal and acaricidal properties. For this reason, quinalphos is frequently used in many countries over many crops in agriculture for control of insects
M
and represents a source of toxicity to humans and vertebrate animals. A 3-year evaluation (2000-2002) [1] of forensic pesticide and herbicide intoxications in Portugal indicated
d
about 29% of total cases due to quinalphos. Primary toxicity due to acetyl cholinesterase
te
inhibition, is terminated by hydrolysis of quinalphos and its metabolic products
Ac ce p
containing phosphorous detected in human serum and urine [2]. Hydrolysis of the ester bond connecting the aromatic moiety to diethyl phosphorothioate in quinalphos leads to 2-hydroxyquinoxaline (2-HQ), which has also been identified as the main metabolite in soil, in water and on crops [3-6]. In vitro, 2-HQ is capable of photocatalytically destroying antioxidant vitamin C and E and oxidizing various biogenic amines such as serotonin, melatonin, N1-acetyl-5-methoxykynuramine, dopamine, epinephrine and norepinephrine and also anthranilic acid derivatives [7]. 2-HQ is also mutagenic, carcinogenic and genotoxic to organisms [7-9]. 2-Hydroxyquinoxaline appears to be as a secondary source of toxicity, as 2-HQ even in small concentrations leads to growth inhibition, induction of oxidative stress and genotoxicity in test organisms. 2-
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hydroxyquinaxoline is an aromatic and bicyclic compound with fusion of one benzene ring and one pyrazene ring and is getting accumulated in the environment- soil due to its
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slow degradation upon repeated applications of the parent compound – quinalphos [3]. Quinoline, a heterocyclic compound, distinct from 2-HQ, was also detected in the
cr
environment due to production and application of quinoline-based drugs, quinoline-based
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dyes and petroleum products [10]. It is desirable to have microbial/bacterial cultures in our stockpile for mitigation of N- heterocyclic compounds associated toxicity by
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enhancing degradation of N-heterocyclic compounds in the environment. Number of bacterial cultures such as Bacillus sp. [10], Pseudomonas sp. [11, 12], Burkholderia
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pickettii [13] and Rhodococcus sp. [14] with capacity to degrade quinoline were reported. Microbial metabolism of 2-hydroxyquinoxaline, a metabolite formed during degradation
d
of quinalphos received less attention than p-nitrophenol, a metabolite in biodegradation
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of other organophosphates - Methylparathion. In this direction, efforts have been put to
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isolate 2-hydroxyquinoxaline - degrading bacteria from soil by selective enrichment technique method. Focussing on biodegradation of 2-HQ, provides a base of scientific knowledge. This is the first case we are reporting in this article isolation, identification and characterization of bacterium able to degrade 2-HQ as the sole source of carbon and energy source.
2. Materials & Methods 2.1. Soil sample
Soil sample [organic matter (%) - 0.42; nitrogen (%) - 1.54; pH - 8.1] was collected from Chrysanthemum indicum agriculture field of Pendlimarry village with prehistory use of quinalphos in YSR Kadapa district, Andhra Pradesh, India.
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2.2. Chemicals 2-Hydroxyquinoxaline was purchased from Sigma-Aldrich Fluka (99%). This 2-HQ
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was used for bacterial growth as sole source of carbon and energy. All other chemicals and solvents used in the present study are of analytical reagent grade/HPLC grade.
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2.3. Culture Medium
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The mineral salts medium(MSM) contained (gL-1) 1.5 NH4NO3, 1.5 K2HPO4. 3H2O, 0.2 MgSO4. 7H2O, 1.0 NaCl and 1 mL of trace elements stock solution. The trace element
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stock solution contained (gL-1): 2.0 CaCl2.2H2O, 0.2 MnSO4.4H2O, 0.1 CuSO4.2H2O, 0.2
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ZnSO4.H2O, 0.02 FeSO4.7H2O, 0.09 CoCl2.6H2O, 0.12 Na2MoO4.2H2O and 0.006 H3BO3.
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2.4. Isolation of bacterial strain
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The enrichment culture technique was set up by inoculating 100 mL of sterile MSM
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containing 0.005% 2-HQ with 5 gram of agricultural soil in 250ml Erlenmeyer flask. The flask was incubated in an orbital shaker (Orbitek LE-IL Model) at 37°C and 175 rpm. After 5 days of incubation period, 5 mL portion of the culture was transferred to fresh medium with high 2-HQ concentration up to 0.05% in Erlenmeyer flasks and the flasks were incubated for 5-days. After 5 more transfers, the culture was purified by serial dilution transfer method and streak plating onto solidified MSM containing 0.005% of 2HQ. Finally, a pure bacterial strain was obtained and designated as HQ2. 2.5. Identification and characterization of bacterial strain 2.5.1. Morphological and biochemical characterization
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Morphological observations of bacterial isolate were made with optical compound microscope. Physiological and biochemical properties of the strain was determined by the
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2.5.2. Sequence analysis and construction of a phylogenetic tree
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procedures as described by Bergey’s Manual of Determinative Bacteriology [15].
The total genomic DNA was extracted from the potential bacterial isolate (HQ2)
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following a standard phenolic extraction procedure [16]. Phylogenetic analysis based on
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16S rRNA gene sequences were made as described by Qin [17]. The 16S rRNA gene sequences of strain HQ2 was amplified on their genomic DNA with universal conserved as
primers
–
AGACTCAGGTTTGATCCTGG-3′
16
forward
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sequence
and
16
primer
reverse
sequence primer sequence
-
5′- 5′-
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ACGGCTACCTTGTTACGACTT-3′. Both forwarded and reverse sequences were
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generated and combined to get assembled partial sequence of 16S rRNA gene. The
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determined sequence was compared with those in the GenBank/EMBL data base using the online tool BLAST program [18] accessible through the web (NCBI). FASTA sequences of the closely related bacterial sp. to the present bacterial isolate was collected and aligned and constructed phylogenetic tree using the Robust Phylogenetic [19, 20] online tool.
2.6. Growth of bacteria on 2-HQ For growth of the bacterial isolate on 2-hydroxyquinoxaline, 50 mL of sterile MSM in sterile 250 mL Erlenmeyer flasks was spiked with 2-hydroxyquinoxaline at concentration of 50 µg mL-1. Meanwhile, potential bacterial isolate inoculum was prepared by growing the bacterial strain in 50ml of MSM with yeast extract (0.1%) and
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supplemented with 50 ppm of 2-HQ per ml of MSM and incubated in an orbital shaker at 175 rpm at 37°C for overnight. The overnight culture was harvested aseptically (8000g,
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15min, 4°C) and thoroughly washed with MSM and suspended in sterile MSM to get suspension with the desired OD. Flasks with test chemical - 2-hydroxyquinoxaline in
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MSM and inoculated with the potential bacterial culture at the final OD of 1.0 per ml of MSM. Uninoculated flasks with fortified medium served as control. All flasks were
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incubated in an orbital shaker at 175 rpm at 37°C for 24 h. Five milliliter aliquots from
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growing culture broth were withdrawn after every 6 h for determining OD value at wavelength 600nm in UV-Visible spectrophotometer (Chemito-UV-2600) and total
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number of viable bacterial colony forming units by serial dilution technique method on nutrient agar medium plates. The specific growth rate of Bacillus sp. was calculated in
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d
the logarithmic phase. 2.7. Biodegradation of 2-HQ
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Experiments for biodegradation of 2-hydroxyquinoxaline by the bacterial isolate
were carried out in 250-mL Erlenmeyer flasks in the same manner as done for growth experiments (2.6). At regular intervals of 24 hrs, 10mL of culture filtrate was aseptically withdrawn from flasks for growth measurement and residue analysis. Culture/medium from both uninoculated and inoculated flasks was centrifuged at 8000g for 15 minutes in a refrigerated centrifuge. Supernatants collected in this fashion were thrice extracted with dichloromethane with equal volume of supernatant. The extracts were pooled together dried over anhydrous (NH4)2SO4, filtered, and evaporated to dryness at room temperature. The residue was dissolved in methanol for HPLC and GC-MS analysis. 2.7.1. Factors influencing on biodegradation of 2-HQ
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Appropriate modifications in growth conditions of the bacterial culture on 2-HQ were made in order to assess the effect of various factors on degradation of 2-HQ by the
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potential bacterial isolate. For this purpose, MSM spiked with 2-HQ at 50 mg L-1 was distributed in 250-mL flasks at the rate of 100 ml per flask. Additional carbon source
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(glucose/sodium acetate) or additional nitrogen source NH4Cl, (NH4)2SO4, urea or yeast
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extract was included in certain flasks at 0.01% concentration. Flasks were inoculated with bacterial suspension to get OD of 1.0 in the inoculated media. Flasks with/without
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supplementation, devoid of inoculums were maintained as appropriate controls. After incubation of all flasks at 37°C and 175 rpm in a shaker, flasks were withdrawn at regular
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intervals after extraction with dichloromethane for residue analysis. Influence of concentration of 2-HQ on its degradation was assessed by growing the bacterial isolate
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on 2-HQ in MSM at different concentrations (50-500 ppm). In another experiment, flasks
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containing 2-HQ at 50 mg L-1 in MSM at pH 7.0 and bacterial cell suspension at initial
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OD of 1.0 were incubated in a shaker at 175 rpm and at different temperatures - 30-45°C in order to study influence of temperature on degradation of 2-HQ. Flasks with MSM after adjustment to different pH and spiked with 2-HQ at 50 mg L-1 was incubated with cell suspension of bacterial isolate at initial OD of 1.0 in a shaker at speed of 175 rpm and 37°C in order to find out influence of pH on degradation of 2-HQ. 2.8. Analytical methods
2.8.1. 2-HQ residue analysis by High Performance Liquid Chromatography (HPLC) Residue of 2-HQ extracted and finally dissolved in methanol in different experiments was analyzed by HPLC (Shimadzu, Japan) equipped with a ternary gradient pump, programmable variable-wave length UV detector, column oven, and electric
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sample valve and ODS-2, C18, reverse phase column (4.6mm×250mm×5μm). The 2-HQ residue analysis was conducted by using an isocratic mobile phase of methanol. Sample
1
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injection volume was 20 µL, the mobile phase was programmed at flow rate of 1 mL minand 2-hydroxyquinoxaline was detected at 345 nm wave length with retention time of
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0.833 minutes.
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2.8.2. Identification of 2-hydroxyquinoxaline metabolites by GC-MS analysis MSM was spiked with 2-hydroxyquinoxaline at 50 µg mL-1 in the same manner as
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mentioned earlier in section 2.6. The flasks were incubated under optimal conditions at 37°C and 175 rpm for 5-days. At regular intervals 10 mL aliquots of culture was
M
withdrawn aseptically and extraction with dichloromethane for metabolites identification
d
as in the same manner as specified in section 2.7. Dried residues were subjected to Gas
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Chromatography-Mass Spectrometry (GC-MS) analysis. Residues finally recovered in methanol from control and bacterial cultures were analyzed in GC-MS-QP-5050
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chromatograph (M/s. Shimadzu Instruments, Japan). Initially the residues extracted from the controls were analyzed. The data obtained on GC-MS from control was taken as reference for the identification of any new peaks found in the sample recovered from bacterial culture grown on 2-hydroxyquinoxaline. 3. Results & Discussion
3.1. Isolation and identification of a 2-HQ degrading bacterium A soil bacterial strain capable of utilizing 2-HQ as its sole carbon and energy source was isolated (HQ2) by an enrichment technique. Cell morphology of HQ2 is gram-
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positive and its colonies are opaque, glistering and powdery undulate. Biochemical characteristics of (HQ2) such as IMVIC tests, glucose and lactose fermentations, catalase
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activity, casein hydrolysis and gelatin liquefaction whereas urease, nitrate reducatse and starch hydrolysis are positive. Morphological and biochemical characteristics suggest that
cr
the strain HQ2 falls in to Bacillaceae group.
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3.2. Phylogenetic analysis
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Analysis of 16S rRNA sequence, using Robust Phylogenetic tree [19, 20] online tool, the neighbor-joining dendrogram was constructed and shown in the (Fig. 1). Based
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on the dendrogram, along with morphological and biochemical characteristics potential bacterial strain (HQ2) was 69% identical with Bacillus firmus and is tentatively identified
d
as Bacillus sp. The nucleotide sequence coding for 16S rRNA of potential strain HQ2
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(1476 bases) were deposited in the GenBank database with accession number
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Bankit1538194 Seq1JX239694.
3.3. Growth of bacteria on 2-hydroxyquinoxaline Growth experiment was performed in MSM fortified with concentration of 50 µg
mL-1 of 2-HQ (2.6). Viable cell count in growing culture of bacteria was measured at regular intervals (supplementary table 1). At beginning (0 time) immediately after inoculation the viable cell count of 28×109 CFU/mL was recorded in Bacillus sp. After incubation for 6 h, initial total viable cell count increased to 50×109 CFU/ml in the culture of Bacillus sp. During this log phase of bacteria growth rate and generation time calculated as per the equation - K = log Nt - log No/log 2×t. Doubling time of Bacillus sp. at log phase is 0.79 h or 47.4min/generation.
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Growth of Arthrobacter sp. HY2 was observed after incubation in MSM containing 50 mg L-1 PNP in MSM after 6 h with concomitant decrease in PNP and was further
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increased by two folds within 12 h [21]. Similarly, commencement of growth of Arthrobacter protophormiae RKJ100 on PNP was noticeable after 2 h and reached
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maximum on 12-16 h [22, 23]. Optimum growth of Serratia sp. DS001 was found at a
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concentration of 0.3mM PNP with doubling time of 13.8 h [24]. However, growth of potential culture in the present study was different on 2-hydroxyquinoxaline and had
an
longer generation time.
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3.4. Biodegradation of 2-hydroxyquinoxaline (2-HQ) by Bacillus sp. Capacity of the biodegradation of 2-HQ by bacterial isolate - Bacillus sp. was
te
d
assessed under different conditions.
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3.4.1. Influence of additional carbon source on biodegradation of 2-HQ Biodegradation of 2-HQ by the potential bacterial isolate - Bacillus sp. in the
presence or absence of glucose or sodium acetate in MSM was compared. The extent of 2-hydroxyquinoxaline disappeared in the culture - Bacillus sp. was determined and is presented in (Fig. 2a).
Disappearance of 2-hydroxyquinoxaline in uninoculated medium with/without
additional carbon was not appreciable and didn’t exceed 1% of added 2hydroxyquinoxaline even at the end of 1-day incubation. But about 90% of added 2hydroxyquinoxaline disappeared in culture of Bacillus sp. (inoculated) in the presence of glucose or sodium acetate at end of 1-day incubation. There was no significant difference
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in figures of % of degradation of 2-hydroxyquinoxaline caused by Bacillus sp. culture in the presence or absence of additional carbon. It is clear from the results of the present
ip t
study that the degradation of 2-HQ by the potential bacterial strain was not influenced by the additional carbon.
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In contrast, Feng [25] reported that Pseudomonas sp. strain ATCC 700113
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stimulated mineralization of 3,5,6-trichloro-2-pyridinol (TCP) in MSM at the concentration of 100 mg L-1 by the addition of carbon sources such as glucose, maleic
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acid and succinic acid. Similarly, addition of glucose (100 mg L-1) as co-substrate for EBN-12 mutant strain resulted in complete degradation of 100 mg L-1 of p-nitrophenol
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(PNP), a metabolite in degradation of parathion/methyl parathion in 20 h rather than in 24
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h in its absence [26]. Addition of glucose at low concentration (0.1-0.5%) enhanced the
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degradation of PNP by Pseudomonas sp. [27] and Arthrobacter sp. HY2 [21]. In the present study inclusion of glucose at low concentration in MSM had no influence on
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degradation of 2-hydroxyquinoxaline by Bacillus sp. 3.4.2. Influence of additional nitrogen source on biodegradation of 2-HQ In order to find out a suitable nitrogen source from - inorganic forms - ammonium
chloride and ammonium sulphate, organic forms - urea and yeast extract
for
biodegradation of 2-HQ by the potential bacterial isolate - Bacillus sp. was grown on 2HQ in MSM amended with the additional nitrogen source. Appropriate controls - MSM devoid of both the additional nitrogen source and inoculum, MSM devoid of the additional nitrogen source with receipt of inoculum and inoculated MSM amended with additional nitrogen source were used.
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Disappearance of 2-hydroxyquinoxaline in uninoculated medium irrespective of the nitrogen source was limited to only 1% even at the end of 1-day incubation. But
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degradation of 2-hydroxyquinoxaline occurred to the extent of 90-96% in the culture of Bacillus sp. at the end of 1-day incubation (Fig. 2b). Growth of Bacillus sp. on MSM
cr
without additional nitrogen caused 90% degradation of 2-HQ as against 90-96% by the
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same culture on MSM with different additional nitrogen source. Among the additional nitrogen sources used in the present study, yeast extract marginally enhanced the
an
degradation of 2-HQ. On contrary, addition of ammonium ion at 3.8mM did not affect TCP degradation by the resting cells of Pseudomonas sp. strain ATCC 700113 but a
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concentration of 15mM had slightly inhibitory effect on TCP degradation [25]. Qiu [28] reported that addition of ammonium chloride and ammonium sulphate (1g L-1) did not
d
favor the growth of Ochrobactrum sp. B2 or degradation of parathion/methyl parathion
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metabolite - PNP. Addition of sodium nitrate, ammonium sulphate (0.04%) did not exert
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significant effect on PNP degradation by P. putida [29]. Srilatha [30], reported that additional nitrogen source did not enhance the degradation rate of PNP by the bacterial cultures Arthrobacter sp. and Nocardioides sp. Quinoline is a bicyclic aromatic compound and is structurally similar to 2-HQ due to presence of one benzene ring and one heterocyclic ring but differs from 2-HQ with one nitrogen in heterocyclic ring. Zhu [14] reported that additional nitrogen source in particular (NH4)2SO4 enhanced the growth and degradation of quinoline by Rhodococcus sp. QL2. In the present study, supplementation of additional nitrogen in the form of yeast extract marginally improved degradation of 2-hydroxyquinoxaline by Bacillus sp. 3.4.3. Influence of inoculum density on biodegradation of 2-hydroxyquinoxaline
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In order to find out the optimal size of inoculum density for degradation of 2-HQ by the potential bacterial isolate - Bacillus sp. was cultivated on MSM after inoculation with
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the three different sizes of inoculum densities. The cell suspension of culture was adjusted to desired cell densities and added to MSM fortified with 2-HQ to provide initial
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cell densities at 0.5, 0.75 and 1.0 OD at A600 in inoculated medium.
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The corresponding figures of the 2-HQ degradation by Bacillus sp. upon growth with inoculum of 0.5, 0.75 and 1.0 OD sizes were 80.38, 86.5 and 90.18%, respectively at
an
the end of 24 h incubation (Fig. 3). Thus, the grown bacterial culture with highest initial cell density, i.e. 1.0 OD supported the maximum degradation of 2-HQ indicating
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inoculum of 1.0 OD optimal.
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d
Labana [31] examined influence of inoculum size 2×105, 2×106, 2×107, 2×108 and 2×109 cells/g soil on degradation of methyl parathion metabolite - PNP by Arthrobacter
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protophormiae RKJ100 in soils. Among these inoculum densities, 2×105, 2×106 cells/g soil depleted PNP after 10 days of incubation whereas 2×109 cells were able to degrade PNP after 5 days of incubation. The optimum inoculum size was found to be 2×108 which achieved 98% depletion in just 2 days. Ramadan [32] reported that Pseudomonas cepacia did not mineralize p-nitrophenol at a density less than 104 cell/ml of lake water. In general, longer lag phase before onset of rapid degradation of xenobiotic by microbial cultures with a smaller inoculum density is noticeable. It has been suggested that the longer lag phase seems for multiplication of small active population of xenobiotic/pesticide-degrading bacteria to certain level so as to display degradation of the
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xenobiotic/metabolite [33]. This type of correlation between the length of acclimation period and size of inoculum density in liquid cultures may also be applicable to microbial
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degradation of metabolites of pesticides [34].
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3.4.4. Influence of concentration of 2-hydroxyquinoxaline on its biodegradation
The potential bacterial isolate - Bacillus sp. was grown on 2-HQ at different
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concentrations of 50, 100, 200 and 500 mg L-1 in MSM. Degradation of 2-HQ by
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bacterial culture was examined and presented in the (Table 1). Concentration of 2-HQ had no influence on degradation of 2-HQ by the potential bacterial culture.
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About 94% of added 2-hydroxyquinoxaline disappeared in the culture of Bacillus sp. irrespective of initial concentration within a range of 50-500 ppm in the medium at
d
the end of 24 h incubation. Only 1% of degradation was observed in respective of
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te
uninoculated controls indicating no appreciable level of 2-HQ degradation. Consistent with the results of the present study, degradation of 90% of the added
trichloro-2-pyridinol (TCP), a metabolite of chlorpyrifos by B. pumilus C2A1 in medium at high concentration of 300 µg mL-1 was observed [35]. According to Yang [36] a strain of Alcaligenes faecalis strain DSP3 had capacity to degrade wide range of TCP concentration from 10-800 mg L-1. On other hand, contrasting results on degradation of trichloro-2-pyridinol, a metabolite of chlorpyrifos by other organisms was reported. For instance, Pseudomonas sp. strain ATCC 700113 mineralized TCP in liquid MSM at the concentration of 100-200 mg L-1 was reported [25]. Paracoccus strain TRP degraded 3,5,6-trichloro-2-pyridinol
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(TCP) concentration of TCP 400 mg L-1 within 4 days [37]. Recent report [38] provided an evidence that a bacterial strain, Cupriavidus sp. DT-1 completely degraded TCP upto
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concentration of 50 mg L-1 within 14h. Qiu [21] reported that more than 90% PNP was depleted within 24, 48 and 168 h from the medium with initial concentration of PNP at
