Isolation and characterisation of endocrine disruptor nonylphenol-using bacteria from South Africa

South African Journal of Science http://www.sajs.co.za Volume 113 | Number 5/6 May/June 2017 © 2017. The Author(s). Published under a Creative Commons Attribution Licence. Isolation and characterisation of endocrine disruptor nonylphenol-using bacteria from South Africa AUTHORS: Lehlohonolo B. Qhanya1 Ntsane T. Mthakathi1 Charlotte E. Boucher2 Samson S. Mashele1 Chrispian W. Theron2 Khajamohiddin Syed1


Introduction
Endocrine disruptors or endocrine disrupting chemicals (EDCs) are chemicals that can alter the functioning of endocrine systems in humans and other animals including wildlife, and can thus cause cancerous tumour development, birth defects and other developmental disorders. 1,2Many chemicals have been identified as EDCs, and many are used in the formulation of various pharmaceutical products, pesticides, industrial chemicals, heavy metals, persistent organochlorines and other organohalogens, alkylphenols, and synthetic and natural hormones. 2,3,hese environmental pollutants mimic natural hormones of the endocrine system and display either oestrogenic or androgenic activities. 1,2,4They can thus have adverse effects by either unnaturally inhibiting or stimulating the endocrine system and/or hormonal production. 1,2,4Exposure to EDCs increases the chance of physiological abnormalities and alters cognitive function in animals, including humans. 1,2Physiological abnormalities include low sperm count and decreased sperm quality 5 , as well as premature puberty in both girls 6 and boys 7 .Several other metabolic disorders have been reported, including different types of cancers and thyroid-related problems including obesity. 1,2,8vestigations have also shown that these types of chemicals also affect other animals.Effects of EDCs on aquatic species have been well documented. 9EDCs have been reported to have adverse effects on invertebrates and wildlife populations. 10Female snails exposed to tributylin exhibited masculinisation (a disorder called imposex in which female snails develop a male sex organ, including a penis and vas deferens), which in turn led to a decline in the population. 11Alligators of Lake Apopka (Florida, USA) were reported to have impaired sexual development and function as a result of exposure to dichlorodiphenyltrichloroethane (DDT). 12Exposure to dichlorodiphenyldichloroethylene (DDE) resulted in a decline in numbers of bald eagles in Europe and North America. 13 date, information concerning EDCs has been primarily derived from studies conducted in developed countries. 2uch information is still, however, lacking from large parts of Africa, Asia and Central and South America. 2 Studies on EDCs from South Africa in particular are very scarce.A report presented by the Water Research Commission of South Africa revealed the presence of EDCs in South African water. 14In addition to this report, studies conducted in a few places within South Africa have also revealed the presence of EDCs.DDT, DDE and phthalate esters have been found in Limpopo [15][16][17] ; oestrone, oestradiol and oestriol (steroids hormones) in the Western Cape 18 and in KwaZulu-Natal 19 ; p-nonylphenol, diethylhexyl phthalate and dibutyl phthalate in Gauteng 20 ; and lastly DDT, chlordane, hexachlorobenzene, heptachlor and endosulfan in the Eastern Cape 21 .In addition, a large number of EDCs was found in upstream and downstream sections of wastewater treatment plants. 22,23 a result of their adverse effects on humans and wildlife, EDCs are considered to be priority pollutants, and worldwide research is ongoing to develop remediation strategies to remove these chemicals from the environment.
Strategies for removal -including advanced oxidation processes 24 , electrochemical separation and degradation technologies 25 and bioremediation and combinatorial techniques 26,27 -have been extensively investigated.Bioremediation is a particularly attractive approach, as it represents natural and economically feasible processes for detoxification of environmental pollutants under environmental conditions.An understanding of indigenous microorganisms is therefore important to facilitate the design of efficient bioremediation strategies.However, to date, studies on the analysis of the capabilities of microorganisms to utilise/degrade EDCs have not been reported from South Africa.This study is the first of its kind on the enrichment, isolation, identification and further assessment of the EDC-degradation capability of bacteria from South African soils.

Soil sample collection and preparation
Soil samples were aseptically collected from soil at different coal-fired power stations in and around the Mpumalanga Province, South Africa.The selected sampling areas are represented in a schematic diagram with GPS coordinates (Figure 1).Soil samples (5 g) were re-suspended in 30 mL of DNase-free and RNase-free water. 28,29The samples were vigorously vortexed for 5 min, followed by incubation on a rotary shaker for 1 h at room temperature at 100 rpm. 28,29After incubation, the soil was allowed to settle out of solution (30 min), and the supernatants were collected and immediately used for isolation of microorganisms.

Medium preparation
All chemicals and reagents used in this study were purchased from Sigma-Aldrich (Johannesburg, South Africa), unless otherwise stated.Minimal medium 28,29 with added trace element solution 30 was used for isolation of microorganisms.The minimal medium consisted of 8.5 g/L Na 2 HPO 4 .2H 2 O, 3.0 g/L KH 2 PO 4 , 0.5 g/L NaCl, 1.0 g/L NH 4 Cl, 0.5 g/L MgSO 4 .7H 2 O, 14.2 mg/L CaCl 2 and 0.15 g/L KCL.The minimal medium was supplemented with 10 mL of trace element solution 30

Enrichment procedure
Supernatant (1 mL) from the soil samples was used to inoculate 100 mL of minimal medium in a 500-mL conical flask, supplemented with nonylphenol as the sole carbon source.A control was set up to contain medium and nonylphenol, without inoculation of soil samples.After 4 weeks of incubation at 37 °C at 100 rpm, 1 mL of culture was used to inoculate fresh minimal medium (100 mL) with nonylphenol as the sole carbon source.This serial enrichment of bacterial isolates was repeated until a single, homogenous culture was obtained.Aliquots (100 µL) of cultures were spread on minimal medium agar plates with nonylphenol (5 mM) as the sole carbon source, to monitor the growth of microorganisms at 37 °C.The minimal medium plates with nonylphenol were prepared as described elsewhere. 31Bacterial growth was also analysed by measuring the absorbance at 600 nm.

Isolation of genomic DNA and amplification of 16S rRNA gene
Genomic DNA (gDNA) from bacterial isolates was extracted using the ZR Fungal/Bacterial DNA MiniPrep kit (catalogue number D6005, Inqaba Biotec, Pretoria, South Africa) according to the manufacturer's protocol.

Research Article
Nonylphenol-using bacteria from South Africa Page 2 of 7 The gDNA was visualised using agarose gel electrophoresis, and gDNA concentration was measured using a SimpliNano microvolume spectrophotometer (catalogue number GE29-0617-12, Sigma-Aldrich, St. Louis, MO, USA).The isolated gDNA was used for amplification of the 16S rRNA gene.The 16S rRNA gene was amplified by polymerase chain reaction (PCR) using primers 63f and 1387r as described elsewhere. 32A KAPA HiFi HotStart PCR kit (catalogue number KK2501, KAPA Biosystems, Wilmington, MA, USA) was used to amplify the 16S rRNA gene according to manufacturer's instructions.The PCR products were run on a 0.8% agarose gel and were purified using the Wizard ® SV Gel and PCR Clean-Up System (catalogue number A9281, Promega, Madison, WI, USA).

16S rRNA gene sequencing
Samples were prepared for sequencing using the BigDye™ Terminator V3.1 Cycle Sequencing Kit (catalogue number 4337455, Thermo Fischer Scientific, Waltham, MA, USA).The aforementioned primers 63f and 1387r 32 were used for sequencing.The sequencing reactions were performed according to the parameters described by the manufacturer.
Sequencing reactions were purified using the EDTA-ethanol method described by the manufacturer, and submitted for sequencing using a 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).Consensus sequences were derived from the sequences obtained from the forward and reverse primer reactions for each product, using Geneious ® R9 9.1.2.software.

Phylogenetic analysis
16S rRNA gene sequences of bacterial isolates were subjected to BLAST analysis at NCBI (the US National Center for Biotechnology Information) against 16S ribosomal RNA sequences (Bacteria and Archaea) to identify the closest homologs.Among the resulting hits, the 16S rRNA sequences with 100% or 99% identity homologs were selected.Based on the obtained bacterial species, the type strains belonging to each species were selected, and the 16S rRNA sequences were retrieved from elsewhere (http://www.bacterio.net/).The Escherichia coli ATCC 11775 type strain 16S rRNA gene sequence (also retrieved from http://www.bacterio.net/)was used as an out-group.Phylogenetic analysis was carried out using the maximum likelihood method based on the Tamura-Nei model. 33Initial tree(s) for the heuristic search were obtained by applying the neighbourjoining method to a matrix of pairwise distances estimated using the maximum composite likelihood approach.All positions containing gaps and missing data were eliminated.Evolutionary analyses were conducted in MEGA5. 34Phylogenetic analysis included the isolate 16S rRNA gene sequence, hit homologs and type strain 16S rRNA gene sequences.The phylogenetic tree was presented with branch lengths, and the bacterial isolates identified in this study are highlighted in bold font.

Nonylphenol degradation
A degradation study using whole cells was carried out as described elsewhere 35,36 to assess the capabilities of the bacterial isolates to degrade nonylphenol.A single colony of isolates from minimal medium plate containing nonylphenol as the carbon source was used to inoculate 5 mL of Luria-Bertani broth, which was then cultured overnight at 150 rpm at 37 °C.The growth of the isolates was measured at 600 nm after diluting the culture in Luria-Bertani broth.The cultures were then washed twice with saline (0.9% sodium chloride solution), followed by inoculation with an equal amount of each overnight bacterial culture for all six isolates onto separate, fresh minimal media (5 mL) containing nonylphenol (2.5 mM) as a carbon source in 50-mL glass tubes (test cultures).The test cultures were incubated for 12 h at 37 °C at 150 rpm.After incubation, 5 mL of ethyl acetate was added to the test cultures, which were then vortexed for 5 min at maximum speed, followed by centrifugation for 5 min at 2500 g at room temperature.After centrifugation, two distinct fractions were separated by a thin middle layer composed of bacterial cell debris.The upper organic fraction containing nonylphenol was removed from the lower aqueous fraction into a fresh glass tube.The extraction was repeated twice, followed by evaporation of the organic fraction.The remaining residue was re-suspended in 200 µL of HPLC-grade methanol.Minimal medium with nonylphenol but without culture was used as a control and treated the same as the test culture.
HPLC analysis of nonylphenol was carried out following the method described elsewhere, with modifications. 35,36Briefly, the abovementioned methanol samples were filtered through 0.45-µm glass fibre filters and analysed using a Shimadzu Prominence instrument (Shimadzu, Roodepoort, South Africa) equipped with a C18 analytical column (4.6 mm×250 mm; particle size 5 µm from Sigma-Aldrich, South Africa) and with a dual wavelength UV/Vis detector.Separation was achieved using a 22.5-min linear gradient of acetonitrile in water (50% to 96.5%, and then re-equilibrated for 10 min at 50% acetonitrile at a flow rate of 1.25 mL/min).A volume of 5 µL of sample was injected for analysis.
Nonylphenol was detected at 277 nm, and the percentage degradation of nonylphenol by test cultures was related to the control nonylphenol, which was taken as 100%.

Statistical analysis
All experiments were carried out in triplicate and results were subjected to statistical analysis as described elsewhere. 35,36The activities, in terms of percentage degradation, of the different bacterial isolates were analysed for means and standard deviations and compared for statistical differences using a Student's t-test on GraphPad QuickCalcs software package (GraphPad Software Inc., CA, USA).

Enrichment and isolation of nonylphenol-utilising bacteria
The sampling areas selected for this study (represented in Figure 1) have been reported to harbour polycyclic aromatic hydrocarbons (PAHs). 37AHs are hydrophobic compounds well known for their carcinogenicity and mutagenicity towards humans. 38,39In this study, we aimed to test the ability of bacterial species growing in the presence of PAHs to degrade EDCs, as these chemicals are also hydrophobic and aromatic in nature.To isolate microorganisms capable of utilising nonylphenol as a sole source of carbon, we followed a standard enrichment method.Soil samples collected from six different places (Figure 1) were inoculated into minimal medium supplemented with nonylphenol as a carbon source.After 4 weeks of incubation, growth of bacteria was observed on minimal medium plates supplemented with nonylphenol as a carbon source, as well as assessed through spectrophotometry.The initial bacterial growth on plates was non-homogenous, suggesting the presence of more than one type of species.After three successive serial cultures, a homogenous population of bacteria was observed on minimal medium plates, indicating that successive serial culturing resulted in the enrichment of a single type of bacteria that are capable of utilising nonylphenol as a sole source of carbon.In this study, six bacteria were isolated from the six different soil samples.

Identification of bacterial isolates
In order to identify the enriched bacterial isolates, 16S rRNA gene sequence-based phylogenetic analysis was carried out.The 16S rRNA genes from the gDNA of bacterial isolates were PCR amplified using the 63f and 1387r primer set as described elsewhere. 32Analysis of the PCR amplified products on agarose gel showed prominent DNA bands with approximate sizes of ≥1200 base pairs (Figure 2).This analysis indicates specific amplification of the 16S rRNA gene.The amplified 16S rRNA gene was gel purified and subjected to sequence analysis using the same primers used for its amplification.Sequence analysis was performed using both forward and reverse primers, yielding a consensus sequence of 300-500 overlapping base pairs between the sequences.The sizes of the 16S rRNA sequences obtained for each of the bacterial isolates are presented in  1).Isolate 3 showed 99% identity to Stenotrophomonas spp.and Isolate 4 showed 99% identity to Enterobacter spp.This indicates that most of the isolates belong to Pseudomonas (Table 1).Phylogenetic analysis of isolates based on 16S rRNA gene sequences compared to the 16S rRNA gene sequences of hit species, highlighted the differential alignment of bacterial isolates with different species (Figure 3).Based on the phylogenetic alignment, the six bacterial isolates were named as shown in Table 1.Furthermore, homology analysis (per cent identity) of 16S rRNA gene sequences among bacterial isolates (Table 2) revealed that Isolates 3 and 4 have low per cent identity compared with that of the other isolates, clearly reinforcing that they in fact belong to different bacterial genera.Species assigned to Pseudomonas on the other hand showed high per cent identity (Table 2), demonstrating that they belong to the same genus.

Degradation of nonylphenol by bacterial species
Whole-cell nonylphenol degradation experiments were carried out to assess the nonylphenol degradation capability of each bacterial isolate.As shown in Figure 4, all bacterial isolates showed degradation of nonylphenol.The degradation of nonylphenol by bacterial isolates ranged from 41% to 46% (Figure 4).However, the difference in percentage of nonylphenol degradation by all six bacterial species was considered to be the same, because the percentage differences among the isolates was not statistically significant (0.2<p<0.7).Nonylphenol degradation by the bacterial species identified in this study is reinforced by the literature.Species belonging to the genus Pseudomonas have been shown to degrade EDCs such as di-n-butyl phthalate 40 , p-nonylphenol 41 and polyethoxylated nonylphenols 42,43 .Bacterial species belonging to Stenotrophomonas were previously found to be capable of using either nonylphenol or octylphenol as a sole carbon source. 44For species belonging to the well-known human-pathogenic and plant association Enterobacter, degradation of EDCs has been reported particularly for bisphenol A 45 , polychlorinated biphenyls 46 , endosulfan 47 , dibutyl phthalate 48 and nonylphenol 49 .
All of the bacterial species isolated in this study also have the capability to degrade PAHs.1][52][53][54] Degradation of PAHs using Stenotrophomonas 55 , in particular Stenotrophomonas maltophilia [56][57][58] , has been investigated.Hydrocarbon degradation capabilities for some of these species have also been demonstrated with aliphatic 59 and aromatic hydrocarbons 60 .This suggests that the soil samples used in this study, from areas where PAHs were reported to be present, harbour bacterial species that are capable of degrading both classes of xenobiotics, PAHs and EDCs.

Conclusion
The distribution of EDCs, their effects towards living organisms and microorganisms capable of degrading ECDs, and the mechanisms of EDC degradation have been thoroughly documented by the developed world.Information on these matters is, however, lacking from Africa, Asia and Central and South America.Percentage degradation of nonylphenol by bacterial isolates was related to the control nonylphenol, which was taken as 100% as described elsewhere 35,36 .The values represent mean±s.d. for three biological replicates.Percentage degradation among different bacterial isolates was found to be not statistically significant (0.2<p<0.7).
Our study is thus the first of its kind from South Africa, in which we successfully enriched, isolated, identified and demonstrated nonylphenol degradation capabilities of indigenous bacterial strains.The areas from which soil samples were collected were previously reported to be polluted with PAHs, and their selection resulted in the isolation of bacterial species capable of degrading EDC nonylphenol, suggesting that these organisms have the capability to degrade a variety of xenobiotic chemicals.Further investigations on the capacity of the isolates to degrade different EDCs and PAHs are currently underway.The results presented in this study will lead to the isolation and characterisation of microorganisms from different parts of South Africa that are capable of degrading different EDCs, and will thus enrich EDC-related information from Africa.

Figure 1 :
Figure 1: Schematic representation of soil sample collection areas in Mpumalanga, South Africa.The numbers 1 to 6 in stars indicate the areas from which the soil samples were collected.The GPS coordinates of the sampling areas are given in the figure.

Figure 2 :
Figure 2: Agarose gel electrophoresis analysis of 16S rRNA genes amplified from six bacterial isolates.PCR amplified products were run on 1% agarose gel.Lane M indicates the DNA ladder (O'GeneRuler DNA Ladder Mix 100-10 000 base pair, catalogue number SM1173, ThermoFisher).Markers with high intensity were indicated by their size.Lanes 1 to 6 indicate the PCR amplified 16S rRNA gene of the respective bacterial isolates.

Figure 3 :
Figure 3: Phylogenetic analysis of the 16S rRNA gene sequences of the bacterial isolates.16S rRNA gene sequences of the type strains belonging to the same genus and an out-group bacterial species (E.coli) were also included in the analysis.Superscript letter 'T' next to strain name indicates the type strain.Each bacterial isolate was named based on its alignment to the homolog bacterial species.Branch lengths are also shown in the tree.Bacterial species isolated and named in this study are highlighted in bold font.

Figure 4 :
Figure 4: Analysis of nonylphenol degradation by bacterial isolates.Percentage degradation of nonylphenol by bacterial isolates was related to the control nonylphenol, which was taken as 100% as described elsewhere35,36 .The values represent mean±s.d. for three biological replicates.Percentage degradation among different bacterial isolates was found to be not statistically significant (0.2<p<0.7).

Table 2 :
Homology (percentage identity) analysis of 16S rRNA gene sequences of bacterial isolates