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Keywords:

  • aerobic granular sludge;
  • biofilm;
  • cation exchange resin;
  • extracellular polymeric substances extraction;
  • extracellular DNA

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  Extracellular polymeric substances (EPS) are an important component of microbial biofilms, and it is becoming increasingly apparent that extracellular DNA (eDNA) has a functional role in EPS. This study characterizes the eDNA extracted from the novel activated sludge biofilm process of aerobic granules.

Methods and Results:  Exposing the sludge to cation exchange resin (CER) was used for the extraction of eDNA and intracellular DNA (iDNA) from aerobic granules. This was optimized for eDNA yield while causing minimal cell lysis. We then compared the DNA composition of these extractions using randomly amplified polymorphic DNA (RAPD) fingerprinting and PCR-based denaturing gradient-gel electrophoresis (DGGE). Upon the analysis of the genomic DNA and the 16S rRNA genes, differences were detected between the sludge biofilm eDNA and iDNA.

Conclusions:  Different bacteria within the biofilm disproportionally release DNA into the EPS matrix of the biofilm.

Significance and Impact of the Study:  The findings further the idea that eDNA has a functional role in the biofilm state, which is an important conceptual information for industrial application of biofilms.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aerobic granular sludge represents an innovative alternative to the conventional floccular activated sludge, which is commonly used in wastewater treatment (Liu and Tay 2004). This novel treatment process relies on the formation of the sludge into biofilms of large granules, typically 1–5 mm in diameter, and these are retained at biomass concentrations 10 times that of floccular systems (Liu and Tay 2004; McSwain et al. 2005). Beneficial properties of aerobic granules include improved sludge settling, higher loading capacity and reduced sludge handling. However, the technology is yet to be implemented on a full-scale basis, and there is continuing interest to improve the understanding of the formation and structure of aerobic granules (Liu and Tay 2004; Zheng et al. 2006).

Extracellular polymeric substances (EPS) play a crucial role in biofilm formation, including flocculation and granulation (Liu and Fang 2002; McSwain et al. 2005). EPS, products of active bacterial excretion, cell lysis and extraneous organic matter (Comte et al. 2006; Tian et al. 2006), are composed of polysaccharides, proteins, nucleic acids and lipids, and form a three-dimensional, gel-like matrix layer (Chen et al. 2007; Flemming et al. 2007). EPS may serve to hold cells together in spatial arrangement, protect cells from harsh environmental conditions and contribute as carbon and energy reserves (Liu and Fang 2002; Flemming et al. 2007).

Extracellular DNA (eDNA) is detected in EPS and is typically considered a remnant of cell lysis, with suggested functions of enhancing gene transfer, a source of nutrients, and a source of nucleotides for DNA synthesis (Finkel and Kolter 2001; Molin and Tolker-Nielsen 2003). However, recently eDNA is hypothesized to be important for biofilm structure (Whitchurch et al. 2002; Böckelmann et al. 2006; Yang et al. 2007) and biofilm development (Steinberger and Holden 2005; Allesen-Holm et al. 2006).

Presently, most studies of eDNA are performed using pure culture biofilms and little is known about the composition and persistence of eDNA in natural and multispecies environments, such as in activated sludge. Palmgren and Nielsen (1996) reported the accumulation of eDNA in EPS of floccular activated sludge. However, to our knowledge, no work has been conducted to examine the composition and potential roles of eDNA in the formation of aerobic granules. The aim of this work was to extract and compare the composition of eDNA and intracellular DNA (iDNA) from aerobic granular sludge.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Acquirement of aerobic granular sludge

The aerobic granular sludges were sampled from a laboratory-scale reactor treating abattoir wastewater, which performed biological nitrogen and phosphorus removal. Details of the reactor operation can be found in Yilmaz et al. (2008). Granular sludge samples (50 ml) with cell dry weight of between 14 and 18 g l−1 [measured as (volatile suspended solids) VSS] were collected from the reactor. After sludge settling at 4°C, the supernatant was discarded, and the sludge was resuspended in 50 ml extraction buffer [2 mmol l−1 Na3PO4, 4 mmol l−1 NaH2PO4, 9 mmol l−1 NaCl and 1 mmol l−1 KCl at pH 7·0 (Frølund et al. 1996)] and homogenized by hand on ice for 30 s with a Potter-Elvehjem polytetrafluoroethylene pestle and glass tube (Lomb Scientific, Taren Point, Australia), after which granules were no longer visible. The resulting slurry was diluted with extraction buffer to 1·5 times the original volume.

EPS extractions

EPS was extracted by either cation exchange resin (CER) or by formaldehyde/NaOH extraction, based on methods previously described (Frølund et al. 1996; Liu and Fang 2002). For CER extraction, the resin (DOWEX 50 × 8, 20–50 mesh in the sodium form; Sigma-Aldrich, Castle Hill, Australia) was added to homogenized sludge at 70 g g−1 VSS in the absence or presence of 0·1% TritonX-100. The extraction was performed in a shear cell of standard configuration (Holland and Chapman 1966) with an inside diameter of 50 mm and stirred using a rushton impeller (18 mm diameter) at 700 rev min−1 (4°C) for 7 h. The CER/sludge samples were centrifuged (4°C) for 15 min at 15 000 g, and the supernatant and pellet were separately stored at −20°C for subsequent eDNA and iDNA extractions.

For the formaldehyde/NaOH extraction, 1 ml of homogenized sludge was centrifuged (4°C) for 15 min at 15 000 g. After resuspending the sludge pellet in 1 ml of 0·9% (w/v) NaCl solution, 6 μl of 36·5% (v/v) formaldehyde was added and the suspension was incubated at 4°C for 1 h. Then, 400 μl of 1 mol l−1 NaOH was added, and after incubation at 4°C for 3 h the samples were centrifuged (4°C) for 20 min at 15 000 g. The supernatant was separated and stored at −20°C.

DNA extractions

A simple freeze/thaw lysis was performed to obtain a crude DNA extraction for total DNA (tDNA) quantification. The sludge samples were centrifuged (4°C) for 15 min at 15 000 g, resuspended in buffer (200 mmol l−1 NaCl, 200 mmol l−1 Tris, 2 mmol l−1 NaCitrate, 10 mmol l−1 CaCl2 and 50 mmol l−1 EDTA at pH 8·0) with 1% SDS and then subjected to three freeze/thaw cycles (−80°C for 60 min followed by 65°C for 5 min). The samples were then centrifuged (4°C) for 20 min at 15 000 g, and the supernatant, the crude DNA extract, was stored at −20°C.

To obtain DNA of appropriate quality for PCR and molecular analysis, extractions were performed using the FastDNA SPIN kit for Soil (MP Biomedicals, Solon, OH) as per the manufactures instructions. The eDNA and iDNA were extracted from triplicate samples of stored supernatant and pellet (obtained from the CER extraction), respectively. tDNA was extracted directly from the homogenized sludge sample. The purified extracts were stored at −20°C.

Chemical analysis and detection of cell lysis during EPS extraction

DNA quantification was performed on triplicate extracted samples after staining with 4,6-diaminodino-2-phenylindole using a Hitachi F-2000 fluorescence spectrophotometer (Frølund et al. 1996). The extent of cell lysis during EPS extraction was estimated by detection of 2-keto-3-deoxyoctonate (KDO) in the extracts as previously described (Karkhanis et al. 1978). The presence of soluble KDO can be indicative of cell lysis as it is a membrane component of bacteria (Gehrke et al. 1998). Cell lysis during extraction was also monitored during CER extraction by assay for glucose-6-phosphate dehydrogenase activity in the extracted supernatant, as described previously (Lessie and Vander Wyk 1972).

Randomly amplified polymorphic DNA (RAPD) fingerprinting

RAPD fingerprinting of genomic DNA was performed using either primer 1 (5′ -GTAGACCCGT-3′) or primer 2 (5′-AAGAGCCCGT-3′) (Ready-to-Go RAPD Analysis Beads kit; GE Healthcare, Little Chalfont, UK). Twenty-five microlitres of PCR solutions contained 10 ng of template DNA, 25 pmol RAPD primer (1 or 2) and 1× GoTaq Green Master Mix (Promega, Madison, WI). The PCR was subjected to the following conditions: 95°C for 5 min, then 45 cycles of 95°C for 1 min, 36°C for 1 min followed by 72°C for 2 min. 12·5 μl of PCR products were electrophoresed for 90 min at 90 V on a 2% TAE agarose gel containing 0·5 μg ml−1 ethidium bromide.

Denaturing gradient-gel electrophoresis (DGGE) analysis

DGGE was performed essentially as previously described (Muyzer et al. 1996). The 16S rRNA gene was amplified from triplicate preparations of eDNA, iDNA and tDNA in 50-μl reactions containing 10 ng of template DNA, 2 U FastStart Taq (Roche, Indianapolis, IN), 12·5 pmol each of the bacterial primers 341F (with GC clamp) and 534R (Muyzer et al. 1996), 1·25 mmol l−1 each dNTP and PCR buffer (Roche). The reactions were incubated at 95°C for 5 min, then 5 cycles of 95°C for 30 s, 65°C for 10 s and 72°C for 30 s, followed by 19 cycles of 95°C for 30 s, 65°C for 10 s (0·5°C decrease per cycle), 72°C for 30 s and then 11 cycles of 95°C for 30 s, 55°C for 10 s and 72°C for 30 s with a final extension step at 72°C for 5 min.

The amplified PCR fragments were separated using the DCode™ System (Bio-Rad, Hercules, CA) on gels comprising 8% (w/v) polyacrylamide (37·5 : 1 acrylamide/bisacrylamide) in 0·5× TAE buffer, with a denaturing gradient range of 30–60% (100% denaturing solution contains 7 mol l−1 urea and 40% formamide). Electrophoreses were performed at a constant temperature of 60°C and 100 V for 18 h and silver stained as previously described (Bassam et al. 1991). DGGE band intensities and standard deviations were calculated using GelComparII. For comparison, band intensities were calculated as a percentage of the total intensity for that particular gel lane. Statistical comparison of intensities between each eDNA and iDNA band was performed by the two-tailed Student’s t-test.

DNA sequence analysis

Dominant DGGE bands from the different DNA profiles were excised and used as template for re-amplification using primers 341F and 534R (as earlier). The resultant PCR products were purified (QIAquick PCR purification kit; Qiagen, Valencia, CA) and sequenced (Australian Genome Research Facility, Brisbane, Australia). DNA sequences were edited using ChromasPro1.41, and phylogeny was estimated using the Greengenes database (DeSantis et al. 2006) within ARB (Ludwig et al. 2004). Full-length 16S rRNA gene sequences of bacterial representatives were used to create a phylogenetic tree by the maximum likelihood algorithm, fastDNAml. The short DGGE band sequences were then imported into ARB, aligned and added to the tree by parsimony.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Optimization of eDNA extraction from aerobic granules

eDNA was extracted from EPS of aerobic granular sludge using three different EPS extraction protocols, the results of those were compared to a tDNA extraction (Fig. 1). As expected, the eDNA yields were a fraction of the tDNA extracted. The CER extraction methods yielded around 10 times more eDNA compared to the formaldehyde/NaOH method (Fig. 1) at 3·7 ± 0·4 mg g−1 VSS without Triton X-100 and 6·4 ± 0·7 mg g−1 VSS with Triton X-100. Previously, the CER method has been used for efficient extraction of EPS from activated sludge with minimal cell lysis (Frølund et al. 1996).

image

Figure 1.  DNA (empty bars) and KDO (filled bars) were extracted from aerobic granules by the methods of total extraction, formaldehyde/NaOH and cation exchange resin (CER) in the presence or absence of Triton X-100. Refer to the Materials and methods for details.

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KDO is a cell membrane component of bacteria, and in previous EPS extractions from aerobic granules, KDO concentrations under 0·25 mg g−1 VSS indicate negligible cell lysis (Adav and Lee 2008). During the formaldehyde/NaOH EPS extraction, KDO was not detected. For the CER extractions, the KDO levels were 0·2 ± 0·1 and 0·3 ± 0·1 mg g−1 VSS in the absence and presence of Triton X-100, respectively (Fig. 1). Also, in the CER extracts, no activity of glucose-6-phosphate dehydrogenase was detected (results not shown). Consequently, the CER extraction without Triton X-100 was considered the optimal method in terms of the eDNA yield with minimal cell lysis. The eDNA, obtained by the CER extraction in the absence of Triton X-100, was the subject of further molecular analysis.

RAPD fingerprinting of the granular eDNA and iDNA

RAPD analysis was performed for comparison of eDNA and iDNA isolated from granular sludge. The amplification profiles were reproducible (from triplicate analyses, not shown) and revealed both similarities and differences in the banding patterns (Fig. 2). The profile of iDNA using primer 1 (lane 4) included strong bands of around 1000 and 900 bp in size, and weaker bands of around 700 and 450 and 250 bp in length. However, for eDNA using primer 1 (lane 3), several weak bands at around 1000, 400 and 250 bp and a single strong band at 700 bp were detected. Likewise, for primer 2, an additional strong band (1100 bp, lane 7) was detected from eDNA in comparison to the iDNA profile. From these profiles, it was evident that some RAPD bands were specific for eDNA, indicating differences in the DNA compositions of EPS compared to the intracellular fraction.

image

Figure 2.  Comparative RAPD analysis of extracellular DNA (eDNA) and intracellular DNA (iDNA) extracted from aerobic granules using two different RAPD primers. Lanes 1 and 5, λ DNA ladder (EcoRI & HindIII; Promega); lanes 2 and 6, 100-bp DNA ladder (GeneRuler; Fermentas, Scoresby, Australia); DNA size markers. Lane 3, eDNA; lane 4, iDNA; RAPD profiles using primer 1. Lane 7, eDNA; lane 8, iDNA; RAPD profiles using primer 2. Bands more prominent in eDNA are indicated by arrows.

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Analysis of the 16S rRNA genes

The PCR-DGGE analysis of the aerobic granular eDNA, iDNA and tDNA revealed complex banding patterns with major similarities (Fig. 3). This is probably expected, as the majority of the DNA in the sludge would be emanating from the cells in the granules. Cluster analysis of the DGGE banding patterns show that in general the replicate analyses for the specific extraction types clustered together; evidence that differences between the DNA in EPS and the cells extracts can be reproducibly detected. The intensity of many common bands, shared between the three DNA types, was similar; however, several bands detected in the iDNA profiles were absent in the eDNA band profiles and certain eDNA bands had higher intensity than the corresponding iDNA band (Table 1). A number of bands were selected for sequencing. For some bands, the intensity varied across the different DNA types, and for others, the intensity was relatively constant (Fig. 3). The chosen bands were excised and amplified for DNA sequencing, and the short (c. 160 bp) DNA sequences were added to an existing phylogenetic tree (not shown, see Materials and methods for description), to affiliate those with certain genera (Table 1).

image

Figure 3.  Image of denaturing gradient-gel electrophoresis profiles (performed in triplicate) obtained from PCR products of the 16S rRNA gene amplified from extracellular DNA (lanes A–C), intracellular DNA (lanes D–F) and total DNA (lanes G–I). Cluster analysis (Pearson correlation) performed on the lane banding patterns is shown to the left of the gel image. Numbered bands shown at the base of the figure indicate the position of the bands that were excised from various lanes for DNA sequencing.

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Table 1.   Analysis of DGGE bands chosen from the aerobic granular eDNA, iDNA and tDNA, showing intensity values (with standard deviation) and phylogenetic affiliation of the 13 bands
Band numberIntensity value (% of total intensity)Putative affiliation
eDNAiDNAtDNA
  1. DGGE, denaturing gradient-gel electrophoresis; eDNA, extracellular DNA; iDNA, intracellular DNA; tDNA, total DNA.

  2. *Intensity value is significantly different between eDNA and iDNA by the Student’s t-test, < 0·01.

 *13·2 ± 0·36·1 ± 0·14·6 ± 0·1Rhodocyclus, Nitrosomonas
 *23·6 ± 0·36·5 ± 0·25·3 ± 0·1Rhodocyclus, Nitrosomonas
  34·5 ± 0·24·7 ± 0·34·8 ± 0·2Not determined
  44·9 ± 0·24·3 ± 0·44·4 ± 0·2Azoarcus
 *51·7 ± 0·23·8 ± 0·12·6 ± 0·1Nitrosomonas
  62·8 ± 0·12·7 ± 0·03·1 ± 0·1Not determined
 *73·5 ± 0·12·9 ± 0·12·6 ± 0·1Nitrosomonas
 *87·5 ± 0·55·0 ± 0·64·3 ± 0·1Rhodocyclus
 *93·0 ± 0·21·6 ± 0·12·8 ± 0·1Rhodocyclus
 103·8 ± 0·14·1 ± 0·24·8 ± 0·1Thiomonas
*112·1 ± 0·11·2 ± 0·11·7 ± 0·2Rhodocyclus
*123·3 ± 0·12·0 ± 0·22·0 ± 0·0Alphaproteobacteria
*130·7 ± 0·10·05 ± 0·05Not determined

Given that the laboratory-scale reactor was operated for biological phosphorous removal (Yilmaz et al. 2008), the multiple detection of Rhodocyclus-affiliated species may represent strains of CandidatusAccumulibacter phosphatis’, a poly-phosphate-accumulating organism that often dominates laboratory-scale, phosphorus removal reactors (Slater et al. 2010). The reactor was also performing nitrification, and Nitrosomonas-related sequences were prominent in the DGGE bands. On occasion, more than one species was identified from a band in a shared position but across different lanes (see bands 1 and 2, Table 1), and likely this is because of the co-migration of the different PCR fragments. Additionally, multiple detection of related organisms was obtained from different DGGE bands. On those instances, the DNA sequences were different, and this represents detection of related species not resolved by our phylogenetic analysis.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

There is an increasing interest to better understand the role of EPS in biofilm structure, mass transfer, heterogeneity, function and cell communication (Flemming et al. 2007). It is crucial that appropriate techniques are applied to study EPS components. An optimal eDNA extraction method should aim to maximize eDNA yield with minimal cell lysis. Previously, high yields of EPS are extracted from floccular sludge using a formaldehyde/NaOH method (Liu and Fang 2002; Comte et al. 2006). However, we found the method yielded low amounts of eDNA from the granular sludge, even after biofilm homogenization was included to improve extractions (McSwain et al. 2005). Consequently, the CER extraction obtained c. 10 times more DNA than the formaldehyde/NaOH method. Our DNA yields from the CER extractions (3·7 and 6·4 mg g−1 VSS) are comparable and in the low end of the spectrum of eDNA yields extracted previously from activated sludges, which range from 4 to 52 mg g−1 VSS depending on the wastewater treatment plant (Palmgren and Nielsen 1996; Dominiak et al. 2011). CER is reported to be a mild extraction procedure (Frølund et al. 1996), and we detected minimal cell lysis, thus minimizing contamination by iDNA; consequently, we determined this as an effective method for extraction of eDNA from aerobic granules.

Our main focus was comparison of DNA composition from the extracellular and intracellular fractions of the biofilm by RAPD profiling and DGGE analysis. Similarities in the banding patterns of eDNA and iDNA were evident, suggesting that the eDNA is derived from cells within the sludge, either from naturally occurring lysed cells or from excreted DNA. Additionally, we cannot completely rule out the occurrence of some cell lysis during the CER extraction and consequent contamination of eDNA with some iDNA. This occurrence would cause some apparent similarities in the respective DGGE profiles. However, importantly in this mixed culture biofilm, differences in eDNA and iDNA composition were detected. Based on the comparison of DGGE profiles, bands 7, 8, 9, 11 and 12 had higher eDNA intensity values compared to iDNA values (significantly different) (Table 1). Evidence that species-specific differences of these DNA proportions were detected, and consequently, suggesting these particular bacteria (three of the bands were affiliated with Rhodocyclus) release DNA into the granular EPS at different levels.

Some interesting hypotheses could explain this occurrence. One possibility is that different types of cells have different dynamics of cell turnover. There is also the possibility that certain cells actively excrete DNA. Elsewhere, similar findings were made from analysis of a constructed multispecies biofilm where the composition of eDNA was significantly different to that of the cellular DNA (Steinberger and Holden 2005). They concluded that the production of eDNA in those biofilms was species dependent. This was also the finding of a recent study of activated sludge biofilms where they detected differing amounts of eDNA was localized with certain bacterial types (Dominiak et al. 2011). Supporting our results, they also found that Accumulibacter and Nitrosomonas, among other bacteria, are producers of eDNA. Such activity may relate to suggestions that eDNA has a structural role in biofilms (Whitchurch et al. 2002; Böckelmann et al. 2006; Yang et al. 2007). Consequently, from the differences detected in our sludge biofilm, there is the possibility that specific bacterial species are excreting DNA that may contribute to granule structure. An improved understanding of this phenomenon can potentially contribute towards improved structural stability of granules in these novel wastewater treatment systems.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by Environmental Biotechnology Cooperative Research Centre, Waste Technologies of Australia, The University of Queensland, and by the Smart State Fellowship Program (QLD Government).

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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