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A novel approach for extraction of PCR-compatible DNA from activated sludge samples collected from different biological effluent treatment plants
Journal of Microbiological Methods 52 (2003) 315 – 323 www.elsevier.com/locate/jmicmeth
A novel approach for extraction of PCR-compatible DNA from activated sludge samples collected from different biological effluent treatment plants Hemant J. Purohit *, Atya Kapley, Aditi A. Moharikar, Gurpreet Narde National Environmental Engineering Research Institute, Nehru Marg, Nagpur 44 0020, India Received 26 March 2002; received in revised form 2 August 2002; accepted 3 September 2002
Abstract This paper describes a method that facilitates the extraction of PCR-compatible DNA from different activated sludge samples. The approach involves a novel preprocessing step in DNA extraction, which removes potential PCR inhibitors. The sludge was washed with different ratios of acetone and petroleum ether after pretreatment with 0.01% Tween-20 at 50 jC. It was observed that an initial washing step with 50 mM Tris – HCl, pH 9.0, before the detergent – solvent step, improved the quality of the extracted DNA. The extraction protocol resulted in amplifiable amounts of DNA when 10 mg of a sludge sample was used, even in the presence of phenol as a sludge contaminant. The usefulness of the extracted template was demonstrated by carrying out different PCR reactions. The random amplified polymorphic DNA (RAPD) patterns demonstrated the diversity of sludge samples. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Activated sludge; DNA extraction; PCR-compatible DNA; RAPD
1. Introduction Even trace levels of polluting chemicals in activated sludge can act as an inhibitor in the enzymatic analysis of DNA. Extraction of nucleic acids from activated sludge has been reported by various groups (Durward and Harris, 1998; Ibrahim and Ahring, 1999; Picard et al., 1992; Porteous et al., 1997; Syn et al., 1999; Tsai and Olson, 1991; Watanabe et al., 1998; Yu and Mohn, 1999). The methods are essentially based on the lysis
* Corresponding author. Tel.: +91-712-226071x251; fax: +91712-222725. E-mail address: [email protected] (H.J. Purohit).
of microbial cells by enzymatic methods, by chemicals, such as detergents and/or alkali, by freezing and thawing, or by a combination of either of these methods. None of the reported methods include removal of the pollutants, which are of main concern, before the DNA extraction procedures. We perform research on the bacteria isolated from different biological effluent treatment systems contaminated with crude oil, phenols, and substituted phenols (Atuanya et al., 2000; Chhatre et al., 1996; Kapley et al., 1999; Kutty et al., 2000; Purohit et al., 1997; Qureshi et al., 2001). The samples, which we collect from these plants, mostly contain amounts of more than 300 mg/l of organic contaminants in the presence of more than 5000 mg/l of colloid solids. To
0167-7012/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 7 0 1 2 ( 0 2 ) 0 0 1 8 5 - 9
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remove organic and inorganic pollutants from the samples, we have developed a protocol, which includes additional wash steps with organic solvents as a prenucleic acid extraction step. The DNA was released from the extracted matrix using straightforward NaOH treatment followed by neutralization. The resultant cell-free system could serve as a source of DNA template suited for PCR. The DNA purified by this protocol would allow for assessment of a microbial DNA profile for the activated sludge sample.
2. Materials and methods 2.1. Activated sludge samples Activated sludge was collected from five different industrial premises (samples S1 –S5), where the effluent has phenol as one of the main organic contaminants. Sample S1 was collected from a waste treatment plant for nitrocresols and nitrotoluene. sludge sample S2 was collected from a pilot plant treating phenol contamination. S3 was collected from a refinery treatment plant. S4 was collected from a unit for removal of chlorinated pesticides. S5 was collected from a biological treatment plant for destruction of toluene, chloronitrobenzene, and nitrophenols. The sludge samples were taken from the reactors using an aerobic mode of waste treatment. The materials were collected from the bioreactors in a 1-l measuring cylinder and were allowed to settle. This leads to thick slurry, which was further concentrated by centrifugation at 2500 g for 10 min. An amount of 10 mg (wet weight) was taken in a 1.5-ml centrifugation tube and washed twice with double-distilled water before the preextraction steps. 2.2. Preextraction steps A preextraction step was included in order to remove the organic pollutants present in the sludge samples. Optimization of the preextraction steps was carried out as follows. The sludge pellets were washed in 200 Al of a hot solution of 0.01% or 0.1% Tween20 in double-distilled water. The samples were incubated at 50 jC for 15 min. The washed samples were harvested at 12,000 g for 5 min and resuspended in an equal volume of the respective solutions. The
sludge samples were then extracted with 250, 500 or 1500 Al of acetone and petroleum ether in a 1:4 ratio. The extraction was enhanced by agitation on a rocking platform for 15 min at low speed. Controls contained similar volumes of the mixed solvents to treat the sludge, and were given a temperature treatment without detergent. Similarly, solvent controls were included. Acetone and water mixtures generated a single-phase system, whereas the addition of petroleum ether resulted in two separate phases. The sludge material was pelleted at the bottom of the tube after the acetone/petroleum ether treatment. The sludge samples were pelleted once more at 12,000 g for 15 min and used for DNA extraction. To further improve the method, samples were suspended in 250 Al of 50 mM Tris, pH 9.0, after the initial double-distilled water wash, and were incubated at 50 jC for 15 min. The sludge pellet was recovered by centrifugation at 2500 g for 10 min and subjected to two more washes with double-distilled water. The following variables were also evaluated in a similar manner for optimization of the pretreatment steps. Other detergents, such as NP40, Triton X-100, Tween-80, and SDS, were used instead of Tween-20. However, neither of these detergents yielded PCRcompatible DNA (data not shown). Solvents, such as ethanol, methanol, chloroform, carbon tetrachloride, hexane, and benzene, were also tried, but no advantages were observed. 2.3. Extraction of DNA from the pretreated sludge Two methods were tried for cell lysis. For chemical lysis, pretreated sludge samples were suspended in 50 Al double-distilled water to which 50 Al of 1 N NaOH was added. After mixing, the samples were incubated at room temperature for 30 min. The suspension was neutralized by the addition of 100 Al 1 M Tris buffer, pH 7.5, and the volume was made up to 1 ml with double-distilled water. For enzymatic lysis, the pellets were suspended in 100 Al of 10 mM Tris buffer, pH 8.3, 1 mM EDTA with 100 Ag of lysozyme or proteinase K. The reactions were incubated at 37 jC for 15 min and lysis was stopped by heat inactivation at 98 jC for 5 min. In case of lysozyme-treated samples, 100 Al of 1.0% SDS solution in doubledistilled water was added to completely lyse the cells. The final volume was adjusted to 1 ml by addition of
H.J. Purohit et al. / Journal of Microbiological Methods 52 (2003) 315–323
double-distilled water. In case of proteinase K-treated cells, the volume was directly adjusted to 1 ml with double-distilled water. Prepared lysate (5 Al), equivalent to 10 Ag of activated sludge, was used as template in PCR and RAPD analysis. 2.4. Validation of the extraction protocol PCR amplification of 16S rDNA from the bacterial population in activated sludge has been used to demonstrate PCR compatibility of the DNA. The reaction mix contained 5 Al template solution, 1 PCR buffer (50 mM KCl and 10 mM Tris– HCl, pH 8.3), 200 AM of each of the dNTPs, 3.0 mM MgCl2, 50 pmol of each of the primers, and 2.5 U AmpliTaq DNA polymerase (Perkin Elmer, USA) in a final volume of 50 Al. The thermocycling program used was 35 cycles of 94 jC for 1 min, 60 jC for 1 min, and 72 jC for 1 min. PCR was performed in a Perkin Elmer Gene Amp system 9600. Digestion of the PCR product was performed with frequent-cutting restriction endonucleases: AluI, CfoI, HaeIII, and Sau3AI. To determine that the solvents used in the pre-extraction steps do not harm the bacteria, the sludge samples were spiked with different concentrations of Escherichia coli strain ATCC 35150 (104 and 105 cells per 10 mg sludge) and grown overnight at 37 jC in standard media. These samples were processed for DNA extraction as described above. Recovery of DNA from spiked samples was verified by PCR amplification of the lamB gene as described earlier (Kapley et al., 2000b), using template amounts equivalent to known number of E. coli cells.
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target the genes involved in the utilization of phenols, such as dmpN and pheA, or genes encoding enzymes of the central pathway, mainly catechol dioxygenases, such as dmpB and pheB. Amplification was carried out on a Perkin Elmer 9600 machine, using a modified program that was reported for RAPD (Tcherneva et al., 2000). The modified temperature program consisted of one cycle of 95 jC for 3 min, followed by 10 cycles of 94 jC for 1 min, 35 jC for 2 min, and 72 jC for 1 min. This was followed by 30 cycles of 94 jC for 1 min, 50 jC for 2 min, and 72 jC for 1 min, followed by a final extension step of 72 jC for 4 min. The amplified products of the catabolic genes were subjected to Southern hybridization to confirm identity. Probes used for the dmpN and dmpB loci were generated by PCR amplification using total DNA derived from Pseudomonas CF600, known to harbor the dmp operon (Shingler et al., 1992). Probes generated for pheA and pheB were generated by PCR amplification of plasmid pAT1140, reported to contain the pheA and pheB genes (Kasak et al., 1993). The probes were labeled with the Bioprime Labeling Kit and detection was done with the Photogene Nucleic Acid Detection System Version 2.0 (Gibco BRL, USA). 2.6. Primers The primers used in the study are described in Table 1. All the primers used in the study were synthesized by Microsynth (Switzerland).
3. Results 2.5. Random amplification of template DNA Firstly, a random amplification of polymorphic DNA (RAPD) assay was performed on the DNA extracted from sludge samples by the new method. This assay was carried out to demonstrate the complexity of DNA extracted from the active biomass. This was done by using primer 6 from the ‘Ready-ToGo RAPD Analysis’ kit (Amersham Pharmacia Biotech, USA), as per the instruction of the manufacturers on a Perkin Elmer 480 thermocycler. Secondly, a PCR test was performed to assess the catabolic potential of the active biomass with specific reference to phenol degradation. This was done by using primers that
The protocol was initially developed by using sludge samples collected from the refinery premises (sample S3). Fig. 1 demonstrates the effect of the different preprocessing steps used in extraction of PCR-compatible DNA for the amplification of the 1099-bp 16S rDNA gene product. It can be seen that mild detergent treatment (0.01% Tween-20) with 300:1200 Al of acetone/petroleum ether wash gave the best results (lane 12). Three reported protocols for extraction of DNA from environmental samples (Porteous et al., 1997; Tsai and Olson, 1991; Watanabe et al., 1998) were used as controls in Fig. 1, lanes 2 –4. Lanes 6 – 8 show the amplification pattern when
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Table 1 Primers used in the study Locus
Primer
Product size (bp)
Reference/accession no.
16S rDNA
F 5VAGAGTTTGTCCTGGCTCA 3V R 5VGCTCGTTGCGGGACTTAACC 3V F 5VAAATGCATGCTTGGCGCTGATGGTGC 3V F 5VCGGATGCATATGGATTGCATCACCGGC 3V F 5VTGGGAATTCATCACAACGACAA 3V R 5VGTGCCGGATCCCTGACTTTCTT 3V F 5VCATGACTTCGCCCATATGTACGACC 3V R 5VGTATTTCGGCGGCCGCATGCCATAGC 3V F 5VCGACCTGATCTCCATGACCGA 3V R 5VTCAGGTCA GCACGGTCA 3V F 5VCTGATCGAATGGCTGCCAGGCTCC 3V R 5VCAACCAGACGATAGTTATCACGCA 3V 5Vd[CCCGTCAGCA] 3V
1099
Amann et al. (1995), Kapley et al. (2001) Kapley and Purohit (2001) M57500
pheA pheB dmpN dmpB lamB No. 6 (RAPD)
different volumes of buffer with 0.1% Tween-20 and the acetone/petroleum were used. Only the protocol described by Watanabe et al., 1998 gave the expected 1099-bp product albeit along with products from nonspecific priming. No amplification products were observed when detergents and solvents were changed.
Fig. 1. Demonstration of preprocessing steps in extraction of PCRcompatible DNA using eubacterial primers. Lanes 1, 5, 9, 13 show standard 1-kb ladder from Gibco BRL. Lanes 2 – 4 use DNA extraction protocol as described by Porteous et al. (1997), Tsai and Olson (1991), and Watanabe et al. (1998), respectively. Lanes 6 – 8 and 10 – 12 show PCR amplification of the 1099-bp eubacterial product using extraction protocol with acetone and petroleum ether in the ratios of 50:200, 100:400, and 300:1200 Al, respectively. Lanes 6 – 8 use 0.1% Tween-20, while lanes 10 – 12 use 0.01% Tween-20.
400 1004 483 238
Kapley and Purohit (2001) Mesarch et al. (2000)
309
Kapley et al. (2000b) Available from Amersham
The actual pre-extraction approach (Fig. 1, lane 12) i.e. using 0.01% Tween-20 and 300:1200 ml of acetone: petroleum ether was tried on the other sludge samples. Fig. 2 shows the expected product with the desired efficiency of amplification (lanes 2 – 6). The quality of the DNA was further improved by a wash step included before the detergent– solvent treatment step. The samples that were brought to pH 9.0 were
Fig. 2. Amplification of eubacterial product using the biomass collected at different industrial premises. PCR amplification of the 1099-bp eubacterial product using template DNA derived from sludge samples S1 to S5 as described in Materials and methods. Lanes 1, 7, 13 show 1-kb ladder from Gibco BRL. Lanes 2 – 6 use the DNA extraction protocol with 0.01% Tween-20 and 300:1200 Al of acetone/petroleum ether. Lanes 8 – 12 use the extraction protocol with the Tris, pH 9.0, wash step included.
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pelleted and washed with the detergent– solvent system, followed by a wash with double-distilled water before the DNA could be extracted by NaOH-lysis protocol. The template obtained with this revised approach was amplified even more efficiently (Fig. 2, lanes 8– 12). Acidic and neutral pH or Tris wash steps did not further improve the results (data not shown). The final DNA extraction protocol is summarized as a flow chart in Fig. 3. This protocol was used to extract total DNA from activated sludge samples spiked with different concentrations of E.
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coli. For sludge samples containing 50 or more E. coli cells, a positive PCR signal was obtained (data not shown). The amplification of the 1099-bp product of the 16S rDNA fragment demonstrates the PCR-compatible nature of the DNA extracted from sludge. However, since the PCR template harbors a heterogeneous mixture of DNA species, it is expected that the 1099bp product represent different bacterial species. To corroborate this, the amplification product was gelpurified and subjected to restriction digestion. Fig. 4
Fig. 3. Flow sheet depicting the final DNA extraction protocol.
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Fig. 4. Restriction digestion of the 1099-bp 16S rDNA product. This shows the digestion pattern of sludge samples S1 to S5 in lanes 2 – 5 using restriction endonucleases, AluI, CfoI, HaeIII, and Sau3AI, respectively. Lane 1 in all four gel pictures shows the standard 100-bp ladder from Gibco BRL.
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shows the digestion patterns obtained with AluI, CfoI, HaeIII, and Sau3AI, respectively. The variable band patterns corroborate the microbial diversity within the samples. To confirm the diversity of the eubacterial population and assess catabolic potential, DNA was used for PCR-based random amplification analysis (Tcherneva et al., 2000). Fig. 5 (lanes 8– 12) shows the variable banding patterns obtained with the commercially available RAPD kit. Fig. 5 (lanes 2– 6) shows the patterns generated for samples S1 – S5 using primers targeted towards catabolic genes. As expected, the
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amplification patterns vary, indicating the diversity of phenol or catechol-utilizing genotypes. The band patterns generated by using primers targeting catabolic genes show some product sizes that do not match the expected size. To demonstrate the presence of a homologous gene, Southern hybridization was done using dmpN, dmpB, pheA, and pheB probes. Phenol monooxygenase, represented by pheA and catechol 1,2 dioxygenase represented by pheB, did not hybridize to any amplification product, suggesting that these genes are not present in sludge samples. It was demonstrated that that the dmpN probe binds more strongly than the dmpB probe to samples S1 and S5. All four probes were tested positive with the control DNA and did not show any nonspecific binding to RAPD-generated fragments (data not shown).
4. Discussion
Fig. 5. PCR-based random amplification of sludge samples. Lanes 1, 7, and 13 show the standard 1-kb ladder from Gibco BRL. Lanes 2 – 6 show the band pattern generated by random amplification of sludge samples S1 to S5 using primers that target the catabolic genes described in the text. Lanes 8 – 12 show the band pattern generated using commercially available RAPD kit from sludge samples S1 to S5, respectively.
Chemically contaminated DNA is frequently not suited for enzymatic analysis. Therefore, poor DNA quality could lead to misinterpretation of PCRdependent analysis. The treatment plants at refineries receive effluents including a wide variety of pollutants, ranging from aliphatic, aromatic, and phenolic compounds. Also, the biological treatment process generates a variety of oxidized intermediates. Hence, the sludge samples collected from refinery premises were used as a model sample during this study. There are many reported protocols for DNA extraction from chemically contaminated activated sludge. We have compared our protocol with three reported procedures (Porteous et al., 1997; Tsai and Olson, 1991; Watanabe et al., 1998) and our protocol appeared to be more efficient. Another reported and frequently used protocol described by Yu and Mohn (1999) uses solid beads to disintegrate the sludge. The inclusion of this step increases the extraction efficiency and has been demonstrated useful for simultaneous extraction of DNA and RNA from activated biomass. The protocol has been reported to be very rapid. However, this protocol has only been used for processing samples from laboratory-scale reactors treating the pulp or paper mill waste, not from the industrial effluent derived from treatment plants. We have not tried this protocol, since we were targeting
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the organics associated with the activated biomass. Hence, we only used buffer and detergent washes, followed by extraction with organic solvents to remove any organic molecules. Before the solvent wash step, treatment of the sample with Tris, pH 9.0, at 50 jC, was also found to be crucial in the removal of contaminants. This could probably be due to the dissociation of membrane bound contaminants by Trizma base at higher pH. The removal of inorganic and organic contaminants present in these samples are prerequisite to purify PCR-compatible DNA, and the combination of wash steps used in the protocol efficiently eliminates these contaminants. The washed and extracted biomass then releases the total DNA by simple alkaline treatment (Kapley et al., 2000a). We have selected this cell lysis approach because it dissolves both genomic and plasmid DNA. Sludge samples spiked with the E. coli strain also showed amplification of target loci. This proves that the solvent treatment does not hamper or modify the target template. Samples that contained 103 cells and below, did not show amplification of the lamB product. This is probably due to the fact that the DNA extracted from 10 mg spiked sludge is finally diluted to 1 ml and only 5 Al of this is used as template in a PCR. This equals five bacterial cells, which is below the sensitivity of the detection protocol (data not shown). The DNA extracted by the developed protocol can be used to check the diversity by effective RAPD analysis. The primer combinations used do not attempt to completely characterize the activated sludge samples, but demonstrate the usefulness of the novel DNA extraction protocol. We have further corroborated this by using primers designed to target the genes involved in the various degradation pathways. The target loci were selected based on the fact that the sludge samples do contain different amounts of phenol and that these primers were already validated in different systems. The current protocol can be used to efficiently monitor the presence of microorganisms in sludge from effluent treatment plants. The method is simple, rapid, and efficient, since PCR-compatible DNA can be extracted from as little a sludge quantity as 10 mg. With various PCR tests, we have demonstrated that DNA can be enzymatically amplified.
Acknowledgements The authors would like to thank Dr. Victoria Shingler, Umea˚ University, Sweden, for the gift of the strain Pseudomonas CF600 that contains the plasmid coding for the dmp operon, and Dr. Maia Kivisaar, Estonian Biocenter, Estonian SSR, USSR, for her gift of plasmid pAT1140 containing the pheAB operon. References Amann, R.I., Ludwig, W., Schleifer, K.-H., 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59, 143 – 169. Atuanya, E.I., Purohit, H.J., Chakrabarti, T., 2000. Anaerobic and aerobic biodegradation of chlorophenols using UASB and ASG bioreactors. World J. Microbiol. Biotechnol. 16, 95 – 98. Chhatre, S.A., Purohit, H.J., Shanker, R., Chakrabarti, T., Khanna, P., 1996. Bacterial consortia for crude oil spill remediation. Water Sci. Technol. 34, 187 – 193. Durward, E., Harris, W.J., 1998. Colorimetric method for detecting amplified nucleic acids. BioTechniques 25, 608 – 614. Ibrahim, A., Ahring, B.K., 1999. Extraction of intact ribosomal RNA from anaerobic bioreactor samples for molecular ecological studies. BioTechniques 27, 1132 – 1138. Kapley, A., Purohit, H.J., 2001. Tracking of phenol degrading genotype. Environ. Sci. Pollut. Res. 8, 89 – 90. Kapley, A., Purohit, H.J., Chhatre, S., Shanker, R., Chakrabarti, T., Khanna, P., 1999. Osmotolerance and hydrocarbon degradation by genetically engineered bacterial consortium. Bioresour. Technol. 67, 241 – 245. Kapley, A., Lampel, K., Purohit, H.J., 2000a. Development of duplex PCR for the detection of Salmonella and Vibrio in drinking water. World J. Microbiol. Biotechnol. 16, 457 – 458. Kapley, A., Lampel, K., Purohit, H.J., 2000b. Thermo cycling steps and optimization of multiplex PCR. Biotechnol. Lett. 22, 1913 – 1918. Kapley, A., Lampel, K., Purohit, H.J., 2001. Rapid detection of Salmonella in water by multiplex polymerase reaction. Water Environ. Res. 73, 461 – 465. Kasak, L., Horak, R., Nurk, A., Talvik, K., Kivisaar, M., 1993. Regulation of the catechol 1,2-dioyxygenase and phenol monooxygenase-encoding pheBA operon in Pseudomonas putida PaW85. J. Bacteriol. 175, 8038 – 8042. Kutty, R., Purohit, H.J., Khanna, P., 2000. Isolation and characterization of a Pseudomonas sp. strain PH1 utilizing meta-aminophenol. Can. J. Microbiol. 46, 211 – 217. Mesarch, M.B, Nakatsu, C.H., Nies, L., 2000. Development of catechol 2,3-dioxygenase-specific primers for monitoring bioremediation by competitive quantitative PCR. Appl. Environ. Microbiol. 66, 678 – 683. Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Appl. Environ. Microbiol. 58, 2717 – 2722.
H.J. Purohit et al. / Journal of Microbiological Methods 52 (2003) 315–323 Porteous, L.A., Seidler, R.J., Watrud, L.S., 1997. An improved method for purifying DNA from soil for polymerase chain reaction amplification and molecular ecology applications. Mol. Ecol. 6, 787 – 791. Purohit, H.J., Chhatre, S.A., Kapley, A., Khanna, P., 1997. Microbial consortia: an approach for management of oil spill/pollution. ENFO News, 2 – 3 (March). Qureshi, A., Prabu, S.K., Purohit, H.J., 2001. Isolation and characterization of Pseudomonas strains for utilization of p-nitrophenol. Microbes Environ. 16, 49 – 52. Shingler, V., Powlowski, J., Marklund, U., 1992. Nucleotide sequence and functional analysis of the complete phenol/3,4-dimethly phenol catabolic pathway of Pseudomonas sp. strain CF600. J. Bacteriol. 174, 711 – 724. Syn, C.K.C., Teo, W.L., Swarup, S., 1999. Three-detergent method
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for the extraction of RNA from several bacteria. BioTechniques 27, 1140 – 1145. Tcherneva, E., Rijpens, N., Jersek, B., Herman, L.M., 2000. Differentiation of Brucella species by random amplified polymorphic DNA analysis. J. Appl. Microbiol. 88, 69 – 80. Tsai, Y.-L., Olson, B.H., 1991. Rapid method for direct extraction of DNA from soil and sediments. Appl. Environ. Microbiol. 57, 1070 – 1074. Watanabe, K., Yamamoto, S., Hino, S., Harayama, S., 1998. Population dynamics of phenol-degrading bacteria in activated sludge determined by gyrB-targeted quantitative PCR. Appl. Environ. Microbiol. 64, 1203 – 1209. Yu, Z., Mohn, W.W., 1999. Killing two birds with one stone: simultaneous extraction of DNA and RNA from activated sludge biomass. Can. J. Microbiol. 45, 269 – 272.
A novel approach for extraction of PCR-compatible DNA from activated sludge samples collected from different biological effluent treatment plants Hemant J. Purohit *, Atya Kapley, Aditi A. Moharikar, Gurpreet Narde National Environmental Engineering Research Institute, Nehru Marg, Nagpur 44 0020, India Received 26 March 2002; received in revised form 2 August 2002; accepted 3 September 2002
Abstract This paper describes a method that facilitates the extraction of PCR-compatible DNA from different activated sludge samples. The approach involves a novel preprocessing step in DNA extraction, which removes potential PCR inhibitors. The sludge was washed with different ratios of acetone and petroleum ether after pretreatment with 0.01% Tween-20 at 50 jC. It was observed that an initial washing step with 50 mM Tris – HCl, pH 9.0, before the detergent – solvent step, improved the quality of the extracted DNA. The extraction protocol resulted in amplifiable amounts of DNA when 10 mg of a sludge sample was used, even in the presence of phenol as a sludge contaminant. The usefulness of the extracted template was demonstrated by carrying out different PCR reactions. The random amplified polymorphic DNA (RAPD) patterns demonstrated the diversity of sludge samples. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Activated sludge; DNA extraction; PCR-compatible DNA; RAPD
1. Introduction Even trace levels of polluting chemicals in activated sludge can act as an inhibitor in the enzymatic analysis of DNA. Extraction of nucleic acids from activated sludge has been reported by various groups (Durward and Harris, 1998; Ibrahim and Ahring, 1999; Picard et al., 1992; Porteous et al., 1997; Syn et al., 1999; Tsai and Olson, 1991; Watanabe et al., 1998; Yu and Mohn, 1999). The methods are essentially based on the lysis
* Corresponding author. Tel.: +91-712-226071x251; fax: +91712-222725. E-mail address: [email protected] (H.J. Purohit).
of microbial cells by enzymatic methods, by chemicals, such as detergents and/or alkali, by freezing and thawing, or by a combination of either of these methods. None of the reported methods include removal of the pollutants, which are of main concern, before the DNA extraction procedures. We perform research on the bacteria isolated from different biological effluent treatment systems contaminated with crude oil, phenols, and substituted phenols (Atuanya et al., 2000; Chhatre et al., 1996; Kapley et al., 1999; Kutty et al., 2000; Purohit et al., 1997; Qureshi et al., 2001). The samples, which we collect from these plants, mostly contain amounts of more than 300 mg/l of organic contaminants in the presence of more than 5000 mg/l of colloid solids. To
0167-7012/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 7 0 1 2 ( 0 2 ) 0 0 1 8 5 - 9
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remove organic and inorganic pollutants from the samples, we have developed a protocol, which includes additional wash steps with organic solvents as a prenucleic acid extraction step. The DNA was released from the extracted matrix using straightforward NaOH treatment followed by neutralization. The resultant cell-free system could serve as a source of DNA template suited for PCR. The DNA purified by this protocol would allow for assessment of a microbial DNA profile for the activated sludge sample.
2. Materials and methods 2.1. Activated sludge samples Activated sludge was collected from five different industrial premises (samples S1 –S5), where the effluent has phenol as one of the main organic contaminants. Sample S1 was collected from a waste treatment plant for nitrocresols and nitrotoluene. sludge sample S2 was collected from a pilot plant treating phenol contamination. S3 was collected from a refinery treatment plant. S4 was collected from a unit for removal of chlorinated pesticides. S5 was collected from a biological treatment plant for destruction of toluene, chloronitrobenzene, and nitrophenols. The sludge samples were taken from the reactors using an aerobic mode of waste treatment. The materials were collected from the bioreactors in a 1-l measuring cylinder and were allowed to settle. This leads to thick slurry, which was further concentrated by centrifugation at 2500 g for 10 min. An amount of 10 mg (wet weight) was taken in a 1.5-ml centrifugation tube and washed twice with double-distilled water before the preextraction steps. 2.2. Preextraction steps A preextraction step was included in order to remove the organic pollutants present in the sludge samples. Optimization of the preextraction steps was carried out as follows. The sludge pellets were washed in 200 Al of a hot solution of 0.01% or 0.1% Tween20 in double-distilled water. The samples were incubated at 50 jC for 15 min. The washed samples were harvested at 12,000 g for 5 min and resuspended in an equal volume of the respective solutions. The
sludge samples were then extracted with 250, 500 or 1500 Al of acetone and petroleum ether in a 1:4 ratio. The extraction was enhanced by agitation on a rocking platform for 15 min at low speed. Controls contained similar volumes of the mixed solvents to treat the sludge, and were given a temperature treatment without detergent. Similarly, solvent controls were included. Acetone and water mixtures generated a single-phase system, whereas the addition of petroleum ether resulted in two separate phases. The sludge material was pelleted at the bottom of the tube after the acetone/petroleum ether treatment. The sludge samples were pelleted once more at 12,000 g for 15 min and used for DNA extraction. To further improve the method, samples were suspended in 250 Al of 50 mM Tris, pH 9.0, after the initial double-distilled water wash, and were incubated at 50 jC for 15 min. The sludge pellet was recovered by centrifugation at 2500 g for 10 min and subjected to two more washes with double-distilled water. The following variables were also evaluated in a similar manner for optimization of the pretreatment steps. Other detergents, such as NP40, Triton X-100, Tween-80, and SDS, were used instead of Tween-20. However, neither of these detergents yielded PCRcompatible DNA (data not shown). Solvents, such as ethanol, methanol, chloroform, carbon tetrachloride, hexane, and benzene, were also tried, but no advantages were observed. 2.3. Extraction of DNA from the pretreated sludge Two methods were tried for cell lysis. For chemical lysis, pretreated sludge samples were suspended in 50 Al double-distilled water to which 50 Al of 1 N NaOH was added. After mixing, the samples were incubated at room temperature for 30 min. The suspension was neutralized by the addition of 100 Al 1 M Tris buffer, pH 7.5, and the volume was made up to 1 ml with double-distilled water. For enzymatic lysis, the pellets were suspended in 100 Al of 10 mM Tris buffer, pH 8.3, 1 mM EDTA with 100 Ag of lysozyme or proteinase K. The reactions were incubated at 37 jC for 15 min and lysis was stopped by heat inactivation at 98 jC for 5 min. In case of lysozyme-treated samples, 100 Al of 1.0% SDS solution in doubledistilled water was added to completely lyse the cells. The final volume was adjusted to 1 ml by addition of
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double-distilled water. In case of proteinase K-treated cells, the volume was directly adjusted to 1 ml with double-distilled water. Prepared lysate (5 Al), equivalent to 10 Ag of activated sludge, was used as template in PCR and RAPD analysis. 2.4. Validation of the extraction protocol PCR amplification of 16S rDNA from the bacterial population in activated sludge has been used to demonstrate PCR compatibility of the DNA. The reaction mix contained 5 Al template solution, 1 PCR buffer (50 mM KCl and 10 mM Tris– HCl, pH 8.3), 200 AM of each of the dNTPs, 3.0 mM MgCl2, 50 pmol of each of the primers, and 2.5 U AmpliTaq DNA polymerase (Perkin Elmer, USA) in a final volume of 50 Al. The thermocycling program used was 35 cycles of 94 jC for 1 min, 60 jC for 1 min, and 72 jC for 1 min. PCR was performed in a Perkin Elmer Gene Amp system 9600. Digestion of the PCR product was performed with frequent-cutting restriction endonucleases: AluI, CfoI, HaeIII, and Sau3AI. To determine that the solvents used in the pre-extraction steps do not harm the bacteria, the sludge samples were spiked with different concentrations of Escherichia coli strain ATCC 35150 (104 and 105 cells per 10 mg sludge) and grown overnight at 37 jC in standard media. These samples were processed for DNA extraction as described above. Recovery of DNA from spiked samples was verified by PCR amplification of the lamB gene as described earlier (Kapley et al., 2000b), using template amounts equivalent to known number of E. coli cells.
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target the genes involved in the utilization of phenols, such as dmpN and pheA, or genes encoding enzymes of the central pathway, mainly catechol dioxygenases, such as dmpB and pheB. Amplification was carried out on a Perkin Elmer 9600 machine, using a modified program that was reported for RAPD (Tcherneva et al., 2000). The modified temperature program consisted of one cycle of 95 jC for 3 min, followed by 10 cycles of 94 jC for 1 min, 35 jC for 2 min, and 72 jC for 1 min. This was followed by 30 cycles of 94 jC for 1 min, 50 jC for 2 min, and 72 jC for 1 min, followed by a final extension step of 72 jC for 4 min. The amplified products of the catabolic genes were subjected to Southern hybridization to confirm identity. Probes used for the dmpN and dmpB loci were generated by PCR amplification using total DNA derived from Pseudomonas CF600, known to harbor the dmp operon (Shingler et al., 1992). Probes generated for pheA and pheB were generated by PCR amplification of plasmid pAT1140, reported to contain the pheA and pheB genes (Kasak et al., 1993). The probes were labeled with the Bioprime Labeling Kit and detection was done with the Photogene Nucleic Acid Detection System Version 2.0 (Gibco BRL, USA). 2.6. Primers The primers used in the study are described in Table 1. All the primers used in the study were synthesized by Microsynth (Switzerland).
3. Results 2.5. Random amplification of template DNA Firstly, a random amplification of polymorphic DNA (RAPD) assay was performed on the DNA extracted from sludge samples by the new method. This assay was carried out to demonstrate the complexity of DNA extracted from the active biomass. This was done by using primer 6 from the ‘Ready-ToGo RAPD Analysis’ kit (Amersham Pharmacia Biotech, USA), as per the instruction of the manufacturers on a Perkin Elmer 480 thermocycler. Secondly, a PCR test was performed to assess the catabolic potential of the active biomass with specific reference to phenol degradation. This was done by using primers that
The protocol was initially developed by using sludge samples collected from the refinery premises (sample S3). Fig. 1 demonstrates the effect of the different preprocessing steps used in extraction of PCR-compatible DNA for the amplification of the 1099-bp 16S rDNA gene product. It can be seen that mild detergent treatment (0.01% Tween-20) with 300:1200 Al of acetone/petroleum ether wash gave the best results (lane 12). Three reported protocols for extraction of DNA from environmental samples (Porteous et al., 1997; Tsai and Olson, 1991; Watanabe et al., 1998) were used as controls in Fig. 1, lanes 2 –4. Lanes 6 – 8 show the amplification pattern when
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Table 1 Primers used in the study Locus
Primer
Product size (bp)
Reference/accession no.
16S rDNA
F 5VAGAGTTTGTCCTGGCTCA 3V R 5VGCTCGTTGCGGGACTTAACC 3V F 5VAAATGCATGCTTGGCGCTGATGGTGC 3V F 5VCGGATGCATATGGATTGCATCACCGGC 3V F 5VTGGGAATTCATCACAACGACAA 3V R 5VGTGCCGGATCCCTGACTTTCTT 3V F 5VCATGACTTCGCCCATATGTACGACC 3V R 5VGTATTTCGGCGGCCGCATGCCATAGC 3V F 5VCGACCTGATCTCCATGACCGA 3V R 5VTCAGGTCA GCACGGTCA 3V F 5VCTGATCGAATGGCTGCCAGGCTCC 3V R 5VCAACCAGACGATAGTTATCACGCA 3V 5Vd[CCCGTCAGCA] 3V
1099
Amann et al. (1995), Kapley et al. (2001) Kapley and Purohit (2001) M57500
pheA pheB dmpN dmpB lamB No. 6 (RAPD)
different volumes of buffer with 0.1% Tween-20 and the acetone/petroleum were used. Only the protocol described by Watanabe et al., 1998 gave the expected 1099-bp product albeit along with products from nonspecific priming. No amplification products were observed when detergents and solvents were changed.
Fig. 1. Demonstration of preprocessing steps in extraction of PCRcompatible DNA using eubacterial primers. Lanes 1, 5, 9, 13 show standard 1-kb ladder from Gibco BRL. Lanes 2 – 4 use DNA extraction protocol as described by Porteous et al. (1997), Tsai and Olson (1991), and Watanabe et al. (1998), respectively. Lanes 6 – 8 and 10 – 12 show PCR amplification of the 1099-bp eubacterial product using extraction protocol with acetone and petroleum ether in the ratios of 50:200, 100:400, and 300:1200 Al, respectively. Lanes 6 – 8 use 0.1% Tween-20, while lanes 10 – 12 use 0.01% Tween-20.
400 1004 483 238
Kapley and Purohit (2001) Mesarch et al. (2000)
309
Kapley et al. (2000b) Available from Amersham
The actual pre-extraction approach (Fig. 1, lane 12) i.e. using 0.01% Tween-20 and 300:1200 ml of acetone: petroleum ether was tried on the other sludge samples. Fig. 2 shows the expected product with the desired efficiency of amplification (lanes 2 – 6). The quality of the DNA was further improved by a wash step included before the detergent– solvent treatment step. The samples that were brought to pH 9.0 were
Fig. 2. Amplification of eubacterial product using the biomass collected at different industrial premises. PCR amplification of the 1099-bp eubacterial product using template DNA derived from sludge samples S1 to S5 as described in Materials and methods. Lanes 1, 7, 13 show 1-kb ladder from Gibco BRL. Lanes 2 – 6 use the DNA extraction protocol with 0.01% Tween-20 and 300:1200 Al of acetone/petroleum ether. Lanes 8 – 12 use the extraction protocol with the Tris, pH 9.0, wash step included.
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pelleted and washed with the detergent– solvent system, followed by a wash with double-distilled water before the DNA could be extracted by NaOH-lysis protocol. The template obtained with this revised approach was amplified even more efficiently (Fig. 2, lanes 8– 12). Acidic and neutral pH or Tris wash steps did not further improve the results (data not shown). The final DNA extraction protocol is summarized as a flow chart in Fig. 3. This protocol was used to extract total DNA from activated sludge samples spiked with different concentrations of E.
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coli. For sludge samples containing 50 or more E. coli cells, a positive PCR signal was obtained (data not shown). The amplification of the 1099-bp product of the 16S rDNA fragment demonstrates the PCR-compatible nature of the DNA extracted from sludge. However, since the PCR template harbors a heterogeneous mixture of DNA species, it is expected that the 1099bp product represent different bacterial species. To corroborate this, the amplification product was gelpurified and subjected to restriction digestion. Fig. 4
Fig. 3. Flow sheet depicting the final DNA extraction protocol.
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Fig. 4. Restriction digestion of the 1099-bp 16S rDNA product. This shows the digestion pattern of sludge samples S1 to S5 in lanes 2 – 5 using restriction endonucleases, AluI, CfoI, HaeIII, and Sau3AI, respectively. Lane 1 in all four gel pictures shows the standard 100-bp ladder from Gibco BRL.
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shows the digestion patterns obtained with AluI, CfoI, HaeIII, and Sau3AI, respectively. The variable band patterns corroborate the microbial diversity within the samples. To confirm the diversity of the eubacterial population and assess catabolic potential, DNA was used for PCR-based random amplification analysis (Tcherneva et al., 2000). Fig. 5 (lanes 8– 12) shows the variable banding patterns obtained with the commercially available RAPD kit. Fig. 5 (lanes 2– 6) shows the patterns generated for samples S1 – S5 using primers targeted towards catabolic genes. As expected, the
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amplification patterns vary, indicating the diversity of phenol or catechol-utilizing genotypes. The band patterns generated by using primers targeting catabolic genes show some product sizes that do not match the expected size. To demonstrate the presence of a homologous gene, Southern hybridization was done using dmpN, dmpB, pheA, and pheB probes. Phenol monooxygenase, represented by pheA and catechol 1,2 dioxygenase represented by pheB, did not hybridize to any amplification product, suggesting that these genes are not present in sludge samples. It was demonstrated that that the dmpN probe binds more strongly than the dmpB probe to samples S1 and S5. All four probes were tested positive with the control DNA and did not show any nonspecific binding to RAPD-generated fragments (data not shown).
4. Discussion
Fig. 5. PCR-based random amplification of sludge samples. Lanes 1, 7, and 13 show the standard 1-kb ladder from Gibco BRL. Lanes 2 – 6 show the band pattern generated by random amplification of sludge samples S1 to S5 using primers that target the catabolic genes described in the text. Lanes 8 – 12 show the band pattern generated using commercially available RAPD kit from sludge samples S1 to S5, respectively.
Chemically contaminated DNA is frequently not suited for enzymatic analysis. Therefore, poor DNA quality could lead to misinterpretation of PCRdependent analysis. The treatment plants at refineries receive effluents including a wide variety of pollutants, ranging from aliphatic, aromatic, and phenolic compounds. Also, the biological treatment process generates a variety of oxidized intermediates. Hence, the sludge samples collected from refinery premises were used as a model sample during this study. There are many reported protocols for DNA extraction from chemically contaminated activated sludge. We have compared our protocol with three reported procedures (Porteous et al., 1997; Tsai and Olson, 1991; Watanabe et al., 1998) and our protocol appeared to be more efficient. Another reported and frequently used protocol described by Yu and Mohn (1999) uses solid beads to disintegrate the sludge. The inclusion of this step increases the extraction efficiency and has been demonstrated useful for simultaneous extraction of DNA and RNA from activated biomass. The protocol has been reported to be very rapid. However, this protocol has only been used for processing samples from laboratory-scale reactors treating the pulp or paper mill waste, not from the industrial effluent derived from treatment plants. We have not tried this protocol, since we were targeting
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the organics associated with the activated biomass. Hence, we only used buffer and detergent washes, followed by extraction with organic solvents to remove any organic molecules. Before the solvent wash step, treatment of the sample with Tris, pH 9.0, at 50 jC, was also found to be crucial in the removal of contaminants. This could probably be due to the dissociation of membrane bound contaminants by Trizma base at higher pH. The removal of inorganic and organic contaminants present in these samples are prerequisite to purify PCR-compatible DNA, and the combination of wash steps used in the protocol efficiently eliminates these contaminants. The washed and extracted biomass then releases the total DNA by simple alkaline treatment (Kapley et al., 2000a). We have selected this cell lysis approach because it dissolves both genomic and plasmid DNA. Sludge samples spiked with the E. coli strain also showed amplification of target loci. This proves that the solvent treatment does not hamper or modify the target template. Samples that contained 103 cells and below, did not show amplification of the lamB product. This is probably due to the fact that the DNA extracted from 10 mg spiked sludge is finally diluted to 1 ml and only 5 Al of this is used as template in a PCR. This equals five bacterial cells, which is below the sensitivity of the detection protocol (data not shown). The DNA extracted by the developed protocol can be used to check the diversity by effective RAPD analysis. The primer combinations used do not attempt to completely characterize the activated sludge samples, but demonstrate the usefulness of the novel DNA extraction protocol. We have further corroborated this by using primers designed to target the genes involved in the various degradation pathways. The target loci were selected based on the fact that the sludge samples do contain different amounts of phenol and that these primers were already validated in different systems. The current protocol can be used to efficiently monitor the presence of microorganisms in sludge from effluent treatment plants. The method is simple, rapid, and efficient, since PCR-compatible DNA can be extracted from as little a sludge quantity as 10 mg. With various PCR tests, we have demonstrated that DNA can be enzymatically amplified.
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