A Novel Approach to Study Response of Activated Sludge to Toxic Shock Loadings

WEFTEC®.06

QUANTIFICATION OF IN SITU GROWTH ACTIVITY: A NOVEL APPROACH TO STUDY RESPONSE OF ACTIVATED SLUDGE TO TOXIC SHOCK LOADINGS

T. Lu,* and D.B. Oerther **

*Department of Civil and Environmental Engineering, University of Cincinnati, Cincinnati, OH 45221 ** Department of Civil and Environmental Engineering, and Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 45221 **Corresponding author. Mailing address: Department of Civil and Environmental Engineering, University of Cincinnati, Box 210071, Cincinnati, OH 45221-0071. Phone: (513) 556-3670. Fax: (513) 556-2599. E-mail: [email protected]

ABSTRACT

A novel denaturing high performance liquid chromatography (DHPLC)-based technique was developed to rapidly separate and identify signature in pre16S rRNA levels among pure cultures of E.coli and A. calcoaceticus as well as activated sludge samples. The chromatography results showed the distinct differences in retention time of the individual species for pre 16S ssDNA, thus providing a qualitative and quantitative characterization of species in a mixture of pre 16S ssDNA. This study is an expansion of previous results reporting the development of a reverse transcription and primer extension assay, and it is the first to document activated sludge response to toxic shock loadings using DHPLC-based quantification of pre16S rRNA levels. The anticipated outcome is to demonstrate the effectiveness of ribosome genesis as a sensitive indicator of toxic loading.

KEYWORDS

DHPLC, pre16S rRNA, ribosome genesis, E.coli, Acinetobacter

INTRODUCTION

The activated sludge system is the most popular form of biological sewage treatment. However, shock loads of toxic chemicals represent a significant problem to activated sludge systems because they disrupt microbial metabolism resulting in a deterioration of process performance (Love et al., 2002). Due to the significance of these disruptions on process performance, a number of research projects have been undertaken to develop upset early warning devices (UEWDs) that can detect the presence of toxic chemicals in sewage (Gutierrez et al., 2002; Aulenta et al., 2002). The current study was guided by the hypothesis that sensitive measurements of ribosome genesis can be used as a quantitative indicator of overall response to toxic loads. Ribosome genesis starts with the transcription of rrn operons to produce a polycistronic transcript, which is then processed in two steps by RNases to produce precursor rRNAs and then mature rRNAs. The rRNAs are finally combined with ribosomal proteins to produce a functional ribosome. It is well documented that high levels of pre16S rRNA correspond to the presence of a toxic inhibition in pure cultures as well as activated sludge (Cangelosi et al., 1997; Licht et al., 1999; Oerther et al., 2000; Oerther et al., 2002).

Recently, a novel reverse transcription and primer extension (RT&PE) approach was developed to investigate the secondary processing of precursor 16S rRNA for pure cultures by determining the ratio of pre16S-5’ rRNA to 16S rRNA (Stroot, 2004). This method was then tested successfully with activated sludge samples. The previous study suggested that the novel RT&PE method could be used to determine whether specific microbial populations are inhibited in activated sludge systems under shock loads of toxic chemicals. To expand this method, the current study examines the use of denaturing high performance liquid chromatography (DHPLC) to rapidly separate and identify signature in pre16S rRNA levels among mixed cultures of environmental bacteria. This approach was used to investigate the secondary processing of the 5’ end of precursor 16S rRNA (pre16S-5’ rRNA) for pure cultures exposed to chloramphenicol. This study represents the first of its kind to document activated sludge response to toxic shock loadings using DHPLC-based quantification of pre16S rRNA levels. This study is significant because it provides us with a better understanding of bacterial growth inhibition under toxic shock loading. In addition, the methods reported in this study could be developed into a standard approach for assaying toxic materials in activated sludge simply on the basis of the DHPLC profile obtained.

RESEATCH OBJECTIVE

The objective of the research is to develop a DHPLC-based approach to monitor the growth physiology of activated sludge exposed to three toxic chemicals, namely: (1) copper (II) (Cu2+), (2) 3,5- dichlorophenol, and (3) 4-nitropheno by applying RT & PE method.

Below is a flow chart summarizing the DHPLC-based approach:

A) Activated sludge collected from WWTP

RNA extraction and purification

pre16S rRNA 16S rRNA 5’ 3’ 5’ 3’

RT&PE primer RT&PE primer (S-D-Bact-0338-a-A-18) (S-D-Bact-0338-a-A-18) pre16S RT&PE product 16S RT&PE product

B) C)

Figure 1 Flow chart for DHPLC/WAVE System-based analysis of the p16S rRNA and 16S rRNA from activated sludge samples. (A) Schematic representation of the RT& PE method. The precursor regions of the pre16S rRNA are shown in orange. The mature regions are shown in black. The primer, S-D-Bact-0338-a-A-18, was chosen because it targets a site that is found in precursor and mature 16S rRNA for all Bacteria. The two RT&PE products from a pure culture were distinguished by differences in length. (B) Ethidium bromide -stained agarose gel electrophoresis of pre 16S rRNA and 16S rRNA from samples. C) DHPLC/ WAVE system based analysis.

EXPERIMENTAL APPROACH

Pure culture

A. calcoaceticus and E. coli, were grown overnight in LB media on a shaker at 35°C. From these cultures, 5 mL were transferred to fresh LB media and incubated for 1 hour on a shaker at 35°C and then exposed to chloramphenicol (20 mg/L) for an additional hour. Similarly, a pure culture of M. mucogenicum was grown over several days in Middlebrook 7H9 media on a shaker at 35°C and 5 mL was transferred to fresh Middlebrook 7H9 media and incubated for 1 hour on a shaker at 35°C prior to exposure to chloramphenicol (20 mg/L) for 24 hours. Samples of A. calcoaceticus and E. coli were removed from the overnight culture and from the fresh culture 15 min after inoculation and 60 min after the addition of chloramphenicol. Samples of M.mucogenicum were removed from the overnight culture and from the fresh culture 60 min after inoculation and 24 hrs after the addition of chloramphenicol due to low growth rate.

Reverse Transcription and Primer Extension (RT & PE)

RNA was extracted from samples by the low pH, hot phenol:chloroform method and further purified with the RNAqueous kit (Ambion, Inc., Austin, TX). DNA was removed from samples by DNaseI treatment (DNA-free™ kit by Ambion, Inc., Austin, TX). The EndoFree RT™ kit by Ambion was used with DNaseI treated RNA as the template and the bacterial probe, S-D-Bact- 0338-a-A-18 (5’ GCTGCCTCCCGTAGGAGT 3’) as the primer. After RT&PE, the RNA was removed from samples by treatment with RNaseA (Sigma, St. Louis, MO). RT &PE products were verified and purified on 2% (wt/vol) agarose gel in 1X TBE buffer with ethidium bromide staining and minielute gel extraction kit (Qiagen, Valencia, CA).

Denaturing high-performance liquid chromatography (WAVE) analysis

The RT & PE product were analyzed using the WAVE System (Transgenomic, Omaha, Neb.). The DNASepTM cartridge which uses alkylated nonporous polysty-rene-divinylbenzene copolymer microspheres for high-performance nucleic acid separations was used in this study. The gradient was formed by buffer A, consisting of 0.1 M triethylammonium acetate (TEAA), pH 7.0, and buffer B, consisting of 0.1 M TEAA and 25% acetonitrile, pH 7.0. Buffer C, consisting of 25% water and 75% acetonitrile, was used for washing the column. The buffers used were obtained from Transgenomic Inc. at analytical grade. The analysis was accomplished with Wave Navigator software version 1.6.1. Different DHPLC gradient and column

temperature were used to optimize the analysis of RT & PE products. Optimized DHPLC gradients for the separation of ssDNA with the WAVE 3500 DNA fragment analysis system is Table 2. The DHPLC optimum running conditions were also used for fraction collection. The WAVE System was provided with the fragment collector FCW 200 which enabled fully automated collection of the peak samples of interest for reamplification and sequencing for verification purpose.

Table 2 Analytical gradient for the separation of ssDNA from at 80.0°C with the WAVE 3500 DNA fragment analysis system.

Step Time Buffer A Buffer B (mins) (%) (%) Loading 0.0 63.0 37.0 Start Gradient 1.0 58.0 42.0 Stop Gradient 17.7 41.3 58.7 Start Clean 17.8 0.0 0.0 Stop Clean 18.8 0.0 0.0 Start Equilibrate 18.9 63.0 37.0 Stop Equilibrate 20.9 63.0 37.0

The flow rate was 0.9 mL/min.

Amplification and determination of the DNA sequence of separated and collect product

The WAVE System was provided with the fragment collector FCW200 which enabled fully automated collection of the peaks of interests for amplification and sequencing. In order to amplify the collected RT&PE products, an aliquot was applied as a template by PCR in 50 µL reaction using a reaction mixture of 1X PCR buffer, 200 µM each deoxynucleoside triphosphate, 1.5 mM MgCl2, 0.025U of Taq DNA polymerase/µl (Takara), and 0.2 µM of each primer. The primers used were specific for conserved bacterial 16S rDNA sequences, S-D-Bact-0011-a-S-17 (5’ GTTTGATCCTGGCTCAG 3’) and S-D-Bact-0338-a-A-18 (5’ GCTGCCTCCCGTAGGAGT 3’) manufactured by the University of Cincinnati DNA Core lab. Amplification of DNA was performed in a Applied Biosystems 2400 Thermal Cycler (Applied Biosystems) by using the following program: an initial denaturing step at 94 ºC for 5 min, followed by 30 cycles of denaturing at 94 ºC for 30 s, annealing at 55 ºC for 30 s, extension at 72 ºC for 30 s, and final extension at 72 ºC for 7 min. The nucleic acid sequences were determined by DNA Sequencing Facility at Children's Hospital (Cincinnati).

RESULTS AND DISCUSSION

RT & PE Analysis

Samples were collected from overnight, fresh growth media and fresh growth media with chloramphenicol which correspond to three distinct types of cells based on pre16S rRNA levels (Stroot et al., 2003). RT & PE products from three cultures of A. calcoaceticus and Type III cells of A. calcoaceticus, E. coli, and M. mucogenicum were shown in Figure 1.

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A.calcoaceticus Type III Type I Type II Pre 16S ssDNA Pre 16S ssDNA 16S ssDNA 16S ssDNA

E.coli M. mucogenicum

Figure 1 Agarose gel electrophoresis analysis of RT&PE products derived from the S-D-Bact- 0338-a-A-18 primer and RNA extracted from A) three A. calcoaceticus cultures that contained Type I, Type II, or Type III cells. B) Type III cells from A. calcoaceticus, E. coli, and M. mucogenicum

The result of Figure 1A clearly shows that pre 16S ssDNA and 16S ssDNA were present as two prominent bands [400 and 550 nts]. To better estimate the ratio of the two bands, a 10-fold dilution of RT&PE products are shown in next lanes. The larger RT&PE band (pre 16S rRNA) increased substantially in the chloramphenicol treated samples (Type III cells). This result conforms that under inhibition of bacterial growth, bacterial has higher levels of pre 16S rRNA. On Figure 1B, the pre16S-5’ and 16S RT&PE products of type III cells derived from each culture are easily distinguished from lane 2-6. The 10-fold dilution is shown on lane 7-11 as the same order. While the ratio of the heights of the RT&PE products (pre16S-5’/16S) exceeds unity for A. calcoaceticus (lane 2 and 3) and E. coli (lane 4 and 5), M. mucogenicum (lane 6) has an extremely low ratio. This poor response to chloramphenicol may be explained by the low growth rate of M. mucogenicum and the corresponding low rate of ribosome genesis. This difference in chloramphenicol sensitivity needs to be taken into account when using the RT&PE method for environmental samples with slow-growing bacteria.

DHPLC Analysis

Although two distinct bands from the RT&PE product could be distinguished by agarose gel electrophoresis, DHPLC-based approach involving the WAVE system is used to achieve rapid, reliable and reproducible analysis of their exact size and abundance. In the current study the sensitivity and specificity of DHPLC for the detection of RT & PE products was evaluated using five different methods (not shown) to predict the optimal conditions for analysis. The result produce by optimized method was shown as Figure 2. The pre 16S ssDNA and 16S ssDNA that were derived from pure cultures of E. coli and A. calcoaceticus were independently analyzed on the WAVE System with the optimized gradient (Table 2). The height of each particular peak was specified as the absorbance in mV and depended on the volume and the DNA concentration injected. Both species exhibited distinct peak profiles consisting of one mature 16S peak and one

pre 16S peak with characteristic retention times. It also shows the distinct differences in retention time of the individual species for pre 16S ssDNA, thus providing a relative qualitative and quantitative characterization of species in a pre 16S complex.

16 16 2 E.coli 1 A.calcoaceticus ) 12 ) 12 1 8 8 2

Absorbance (mV Absorbance (mV 4 4

0 0 345678 345678 Time (min) Time (min)

Figure 2 Distinct peak profiles of E. coli and A. calcoaceticus was analyzed with the optimized DHPLC conditions. 16S ssDNA (peak 1) and pre 16S ssDNA (peak 2).

To address this further, equimolar amounts of RT & PE products from E.coli and A. calcoaceticus were artificially mixed and analyzed using the WAVE System (Figure 3). Both pre 16S ssDNA exhibited distinct peak with characteristic retention times. The analysis showed that the peaks of the examined species were eluted from the column with highly reproducible retention times. The gradient was capable of separating two species. The identities of the species correlated with their characteristic retention times and were confirmed by identification based on sequence analysis.

) 12

4 Absorbance (mV

0 3456 Time (min)

Figure 3 Chromatogram representing the analysis of two bacterial species using the optimized DHPLC conditions. Peaks shown from left to right direction represent 16S ssDNA for E.coli and A. calcoaceticus, pre 16S ssDNA for A. calcoaceticus and pre 16S ssDNA for E.coli.

CONCLUSIONS

This study has successfully demonstrated that the products from the RT&PE assay be quantified using DHPLC to quantify pre 16S rRNA levels. Future work will evaluate if the new method can successfully discriminate between changes in pre 16S rRNA levels in a mixed microbial community collected from a full-scale activated sludge sewage treatment plant and exposed to toxic loading with copper and organic electrophiles.

It is expected that the approach reported here can be used to establish a specialized database of bacterial phylogeny and to detect bacteria by the combination of distinct retention profiles of RT & PE product. It is envisioned that this method will be used to understand how bacterial communities respond to toxic shock loadings. The results reported in this study encourage future efforts to develop an improved method using the WAVE system to quantify the results of the RT & PE assay. In summary, the RT & PE method, combined with DHPLC, is a potentially powerful tool to study the physiology of activated sludge response to toxic shock loadings.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Peter G. Stroot (University of South Florida) for helpful discussions about this project. The authors are also grateful to specialists from Transgenomic Inc. for helpful discussions.

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