Professional Documents
Culture Documents
Lionel Ho*, Kalan Braun, Rolando Fabris, Daniel Hoefel, Jim Morran, Paul Monis,
Mary Drikas
Australian Water Quality Centre, SA Water Corporation, 250 Victoria Square, Adelaide, SA 5000, Australia
Article history: Four pilot-scale treatment process streams (Stream 1 e Conventional treatment (coagu-
Received 19 December 2011 lation/flocculation/dual media filtration); Stream 2 e Magnetic ion exchange (MIEX)/
Received in revised form Conventional treatment; Stream 3 e MIEX/Conventional treatment/granular activated
23 April 2012 carbon (GAC) filtration; Stream 4 e Microfiltration/nanofiltration) were commissioned to
Accepted 25 April 2012 compare their effectiveness in producing high quality potable water prior to disinfection.
Available online 4 May 2012 Despite receiving highly variable source water quality throughout the investigation, each
stream consistently reduced colour and turbidity to below Australian Drinking Water
Keywords: Guideline levels, with the exception of Stream 1 which was difficult to manage due to the
Denaturing gradient gel reactive nature of coagulation control. Of particular interest was the bacteriological quality
electrophoresis (DGGE) of the treated waters where flow cytometry was shown to be the superior monitoring tool
Flow cytometry in comparison to the traditional heterotrophic plate count method. Based on removal of
Heterotrophic plate count (HPC) total and active bacteria, the treatment process streams were ranked in the order: Stream 4
Magnetic ion exchange (MIEX) (average log removal of 2.7) > Stream 2 (average log removal of 2.3) > Stream 3 (average log
Photometric dispersion removal of 1.5) > Stream 1 (average log removal of 1.0). The lower removals in Stream 3
analyser (PDA) were attributed to bacteria detaching from the GAC filter. Bacterial community analysis
Water treatment revealed that the treatments affected the bacteria present, with the communities in
streams incorporating conventional treatment clustering with each other, while the
community composition of Stream 4 was very different to those of Streams 1, 2 and 3. MIEX
treatment was shown to enhance removal of bacteria due to more efficient flocculation
which was validated through the novel application of the photometric dispersion analyser.
ª 2012 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: þ61 8 7424 2119; fax: þ61 8 7003 2119.
E-mail address: lionel.ho@sawater.com.au (L. Ho).
0043-1354/$ e see front matter ª 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2012.04.041
w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 3 9 3 4 e3 9 4 2 3935
dose applied during the study was 15 L kL1. A continuous cooled 15 mW argon ion laser, emitting at a fixed wave-
stirred-tank reactor with a cone settler operating at 10% resin length of 488 nm. Fluorescent filters and detectors were all
regeneration was employed for the MIEX DOC process. The standard with green fluorescence collected in the FL1 channel
primary coagulant used was alum as Al2(SO4)3.18H2O; (530 30 nm), orange fluorescence collected in the FL2
however, additional coagulant aids, LT22 and LT425 (BASF channel (585 42 nm) and red fluorescence collected in the
Chemicals, Australia) were also dosed periodically during FL3 channel (>670 nm). Data were analysed using CellQuest
coagulation as required. Due to the ability of the MIEX DOC software (Becton Dickinson, USA). Total numbers of bacteria
process to efficiently remove absorbable organic materials, were enumerated following staining of the bacteria with
the subsequent coagulation treatment is primarily a clarifica- SYTO-9 and the BacLight bacterial viability kit (Molecular
tion step following the main organic carbon removal by the Probes, USA) as described previously (Hoefel et al., 2003).
MIEX resin. As such, the coagulant demand is reduced leading Results for FCM were presented as cells mL1.
to a lower and less variable alum dose range (10e80 mg L1).
2.4. Bacterial community analysis
2.1.3. Stream 3 e MIEX/conventional treatment/GAC
The third treatment stream was comprised of the product The effects of the different treatment processes on the
water from Stream 2 (described above) with the addition of bacteria in the raw water was assessed by profiling the
two parallel pilot-scale granular activated carbon (GAC) filters bacterial community composition of the raw water and
utilising F400 GAC (Calgon Carbon Corporation, USA). F400 is product waters using denaturing gradient gel electrophoresis
a bituminous coal-based GAC with effective granule size (DGGE) analyses. Water samples were analysed by FCM and
0.55e0.75 mm which is commonly applied in water and bacterial numbers adjusted to 2.0 106 cells mL1, with the
wastewater applications for organic contaminant removal. exception of treated water from Stream 4, which could only be
Filtration was achieved using packed bed columns with concentrated to 5.0 105 cells mL1. Duplicate 1 mL samples
gravity fed empty bed contact times (EBCT) of approximately from each water type were concentrated by centrifugation, re-
14 min at 125 L h1 for each column. suspended in 5 mM TriseHCl pH 7.5 and subjected to three
cycles of freeze-thawing (liquid N2 and 100 C). The resultant
2.1.4. Stream 4 e microfiltration/nanofiltration DNA was used as a template for universal 16S rDNA gene-
Dual pilot-scale membrane filtration consisted of micro- directed nested PCR using the primer sets 27F/1492R and
filtration (MF) pre-treatment for particulate removal using 357F-GC/518R, and the products of the reaction analysed by
a single submerged hollow fibre module (Memcor CMF-S DGGE (D-GENE Gel Electrophoresis System, Bio-Rad, USA) as
system, USA) followed by a single FILMTEC NF 270-4040 reported previously (Hoefel et al., 2005). Positive and negative
spiral wound nanofiltration (NF) membrane (DOW Chemical controls used in DGGE were as described by Hoefel et al. (2005).
Company, USA). The MF system was operated at 1000 L h1 The resulting DGGE profiles were analysed using Phoretix
with 75% permeate recovery. The NF system operated in 1D version 11.2 (TotalLab, Newcastle upon Tyne, UK) with the
cross-flow configuration at 43% permeate recovery, producing following settings: lanes were identified automatically (lanes
325 L h1. Nominal pore size for the MF is reported as 0.2 mm controlling controls were excluded from the analysis), back-
with the molecular weight cut-off for the NF being 270 Da. ground subtraction used the rolling ball method with a radius
of 200, bands were manually called, Gaussian peaks were
2.2. Analyses fitted to bands using the advanced fitting option with manual
adjustment as required, bands were aligned using a synthetic
Colour measurements (at 456 nm) were made through a 5 cm reference generated by the software and similarity of profiles
quartz cell using an Evolution 60 Spectrophotometer (Thermo was assessed using the UPGMA option.
Scientific, USA) according to a published method (Bennett and
Drikas, 1993). Results were presented in Hazen units (HU). 2.5. Flocculation index determination
Turbidity measurements were conducted on a 2100AN Labo-
ratory Turbidimeter (Hach, USA) with results expressed in The photometric dispersion analyser (PDA 2000, Rank Bros
nephelometric turbidity units (NTU). Ltd., Cambridge, UK), is a laboratory instrument used for
analysis of flowing suspensions (Gregory and Nelson, 1984,
2.3. Bacterial enumeration 1986). The method employed was similar to Staaks et al.
(2011) with slight modifications. Briefly, the PDA was con-
Bacterial enumeration was conducted using HPCs and FCM. nected, via flexible tubing, to one jar during jar testing. A
HPCs were performed in accordance with the Australian peristaltic pump circulated the sample water at
Standard AS/NZS 4276.3.1 (Australian Standard, 1995) using 21.6 mL min1. The pump was located after the PDA to avoid
R2A solid media (Oxoid, Australia). Dilutions, when necessary, deterioration of the flocs. A volume of 1 mm3 of the flowing
were performed in maximum recovery buffer (0.1% (w/v) suspension is illuminated by a narrow beam of light from
neutralised bacteriological peptone, 0.85% (w/v) NaCl, pH 7.0). a high intensity light emitting diode at 850 nm wavelength
Incubation was performed using standard conditions of 20 C (Yukselen and Gregory, 2004). The intensity of transmitted
for 72 h. Results for HPC were presented as colony forming light fluctuates concurrently with the number of particles and
units per mL (CFU mL1). is detected by a sensitive photodiode. The optical signal is
FCM analyses were conducted using a FACSCalibur flow converted to a voltage recorded by a computer equipped with
cytometer (Becton Dickinson, USA) equipped with an air- a data logging system. The resultant PDA output is a graph of
w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 3 9 3 4 e3 9 4 2 3937
the flocculation index (FI) as a function of time. The FI is as an aesthetic parameter which can also be used as a surro-
a relative value generated from a ratio of the root mean square gate for organic matter. To put things into perspective, the
(RMS) and direct current (DC) signals and has been used to Australian Drinking Water Guideline levels for turbidity and
compare and characterise flocculation processes (Gregory and colour are 0.5 NTU and 15 HU, respectively.
Nelson, 1984, 1986; Yukselen and Gregory, 2004; Staaks et al., Despite these significant water quality challenges, the
2011). pilot-scale treatment processes were generally efficient in
In our study, three key parameters were extracted from the reducing both the colour and turbidity as shown in Figs. 2 and
FI graphs: the initial floc aggregation (IFA), the relative settling 3, respectively. For example, colour reduction was consis-
factor (RSF) and the variance. The derivation of these param- tently high, especially for the advanced multi-stage processes
eters has been documented previously (Hopkins and Ducosto, (Streams 3 and 4) which averaged greater than 98% reduction
2003; Staaks et al., 2011). The relevance of these parameters over the period. Some difficulty was encountered in main-
will be discussed in the following sections. taining optimum coagulation conditions throughout the
changing water quality periods, especially when rapid
changes occurred, and this is reflected in the poorer removals
in colour and turbidity by conventional treatment (Stream 1).
3. Results and discussion
This was in part due to the reactive nature of coagulation
control where decline of treated water quality dictated the
3.1. Comparison of treatment streams for colour and
operational changes. During these periods, additional chem-
turbidity reduction
icals (including the coagulant aids) were dosed to maintain
target pH and floc settleability for acceptable filter run times
During this study (June 2010eJune 2011) the inlet (raw) water
but only after water quality showed deterioration, resulting in
to Mt. Pleasant WTP and subsequently the pilot-scale
the largest span between maximum and minimum reduction
processes were challenged with water which was out of its
percentages of all the treatments.
usual specification; a consequence of two major water quality
events brought about by large inflows into the MurrayeDarl-
ing Basin from eastern Australia. These flood waters resulted 3.2. Comparison of treatment streams for removal of
in large spikes in turbidity with a maximum of approximately bacteria
190 NTU, followed by periods of high colour with values in
excess of 100 HU. These events followed a period of extended The bacteriological quality of the four treated waters was
drought where river inflows were minimal and source water evaluated using both HPCs and FCM. Results for HPC showed
quality was relatively stable. no clear trends between each of the treatment streams, sug-
From an operational standpoint, the monitoring of these gesting that each of the treatment processes were equally
two water quality parameters (turbidity and colour) are effective in removing bacteria (Fig. 4). Furthermore, large
generally indicative of how well the treatment processes are fluctuations in bacterial numbers in the treated waters were
performing; in addition to conforming to appropriate water evident with a numbers ranging from 2 CFU mL1 up to
quality standards and/or guideline levels. For example, w7 103 CFU mL1.
turbidity has been used as a surrogate for parasites such as In contrast to the HPC data, FCM analyses of the treated
Cryptosporidium and Giardia, while colour is generally regarded waters showed more definitive and stable trends between
Fig. 2 e Colour measurements before (raw) and after the four treatment processes. Dashed line represents Australian
Drinking Water Guideline level of 15 HU.
3938 w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 3 9 3 4 e3 9 4 2
Fig. 3 e Turbidity measurements before (raw) and after the four treatment processes. Dashed line represents Australian
Drinking Water Guideline level of 0.5 NTU.
each of the treatment processes, as shown in Fig. 5. This results are on average two orders of magnitude lower than
highlights the shortcomings of utilising HPCs for monitoring bacterial enumeration by FCM (Siebel et al., 2008). This in part
bacteriological quality, a finding supported by others is due to the nutrient concentrations on conventional HPC
(Hammes and Egli, 2005; Berney et al., 2008; Hammes et al., agar plates which can be between 800 and 1000 times higher
2008; Siebel et al., 2008). Many of the authors ascribe the than the concentrations detected in drinking water (Berney
deficiency of HPCs to human error. For example, the statistical et al., 2008; Hammes et al., 2008). The large discrepancy
accuracy of the plating method is dependent upon colonies between HPC and FCM results has led some to suggest for
being counted between 30 and 300 per plate, and this is a reconsideration of existing drinking water guidelines and
dependent upon the appropriate dilution factor. Hammes legislation (Berney et al., 2008).
et al. (2008) documented that the standard error of HPC The raw water total bacterial count averaged
results was >30% compared with FCM results which were 1.8 107 cells mL1 (minimum ¼ 8.5 106 cells mL1,
<5%. Another deficiency and perhaps the biggest drawback of maximum ¼ 3.2 107 cells mL1) during the study period, of
the HPC method is its selectivity as it is unable to enumerate which 55% were shown to be active, as determined by FCM.
viable, non-culturable bacteria, which explains why HPC This number is relatively high in comparison to other water
Fig. 4 e Heterotrophic plate counts (HPC) before (raw) and after the four treatment processes.
w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 3 9 3 4 e3 9 4 2 3939
Fig. 5 e Bacterial enumeration by flow cytometry (FCM) before (raw) and after the four treatment processes.
sources and may be attributed to the water being sourced contamination or re-growth after the membrane during
from the River Murray via the Mannum to Adelaide pipeline. sampling. The latter is possible since the sampling point for
The residence time in this non-disinfected pipeline is between the NF effluent is located on a stainless steel pipe approxi-
2 and 3 d prior to the Mt. Pleasant WTP, which subjects the mately 2 m after the NF module.
pipeline to sloughing of biofilm and consequently higher Comparison of the bacterial diversity in the raw and
numbers of bacteria entering the WTP. treated waters by DGGE (Fig. 6) showed that Streams 1, 2 and 3
The order of effectiveness of the processes based on had similar profiles to the raw water, with some minor band
removal of total and active bacteria followed the trend: differences between these samples and a dominant band
Stream 4 > Stream 2 > Stream 3 > Stream 1 (see Table 1). As apparent in the treated samples and not detected in the raw
expected, the advanced multi-stage process of MF/NF was the water. However, the profile from Stream 4 was noticeably
superior treatment stream due to its size-exclusion nature different to the raw water or the other treated waters, with
(2.7-log removal of both total and active bacteria). However, an only a few bands in common (Fig. 6A), suggesting that this
average number of 4.5 104 cells mL1 community is different to the communities in the other
(minimum ¼ 8.3 10 3
cells mL1, samples. This result suggests that either Stream 4 treatment
5 1
maximum ¼ 2.0 10 cells mL ) was still detected in the NF was allowing particular bacterial species to breakthrough
treated water, even though the nominal molecular weight cut- (that are not dominant in the raw water and consequently not
off of the membrane is quoted as 270 Da; approximately detected), or that bacteria colonised the pipe post-NF and
100e10,000 times smaller than bacterial cells (between 0.5 and these were being detected in the Stream 4 sample. Consid-
10 mm in size). The limit of detection of the FCM method in this ering that there were a few bands in common between Stream
study is 5.0 103 cells mL1 (unpublished work), suggesting 4 and the raw water or other streams, a combination of some
that either some bacteria were breaking through the breakthrough of bacteria from the raw water and biofilm from
membrane or that there was possibly some form of the post-NF pipe would also be consistent with this result.
Table 1 e Average bacterial numbers (total and active) in the effluent of the treatment processes and log removal values of
bacteria (total and active) by each of the treatment processes from July 2010 to June 2011.
Treatment process Average total numbers Average active numbers Log removal Log removal
in effluent (cells mL1) in effluent (cells mL1) (total) (active)
DGGE analysis identified only a few novel amplicons (rep- Hammes, F.A., Egli, T., 2005. New method for assimilable organic
resenting 1 or 2 bacterial species) associated with the GAC carbon determination using flow-cytometric enumeration and
treated stream; a natural microbial consortium as inoculum. Environmental
Science and Technology 39, 3289e3294.
overall, the community analysis suggested that the treat-
Hammes, F., Berney, M., Wang, Y., Vital, M., Köster, O., Egli, T.,
ments affected the bacteria present, with the communities 2008. Flow-cytometric total bacterial cell counts as
in streams incorporating conventional treatment clustering a descriptive microbiological parameter for drinking water
with each other, and the streams with MIEX being the most treatment processes. Water Research 42, 269e277.
similar of the communities compared; Haznedaroglu, B.Z., Bolster, C.H., Walker, S.L., 2008. The role of
verification that MIEX treatment enhanced removal of starvation on Escherichia coli adhesion and transport in
saturated porous media. Water Research 42, 1547e1554.
bacteria through more efficient coagulation by the novel
Hoefel, D., Grooby, W.L., Monis, P.T., Andrews, S., Saint, C.P., 2003.
application of the PDA (eg. greater rate of floc formation,
Enumeration of water-borne bacteria using viability assays
better floc settling performance and larger, more compact and flow cytometry: a comparison to culture-based
and stronger flocs). techniques. Journal of Microbiological Methods 55, 585e597.
Hoefel, D., Monis, P.T., Grooby, W.L., Andrews, S., Saint, C.P., 2005.
Negligible differences were observed between the removal Profiling bacterial survival through a water treatment process
of active bacteria cells compared with total bacteria cells by and subsequent distribution system. Journal of Applied
Microbiology 99, 175e186.
the treatment processes with log removals ranging from
Hopkins, D.C., Ducosto, J.J., 2003. Characterizing flocculation
0.9 0.3 to 2.7 0.4.
under heterogeneous turbulence. Journal of Colloid and
Interface Science 264, 184e194.
Jarvis, P., Mergen, M., Banks, J., McIntosh, B., Parson, S.A.,
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Acknowledgements
coagulation with magnetic resin plus coagulation systems.
Environmental Science and Technology 42, 1276e1282.
This project was supported by Water Quality Research Liikanen, R., Miettinen, I., Laukkanen, R., 2003. Selection of NF
Australia, South Australian Water Corporation, United Water membrane to improve quality of chemically treated surface
International, Grampians Wimmera Mallee Water, Water water. Water Research 37, 864e872.
Corporation, Delft University of Technology, DCM Process Lovins, W.A., Taylor, J.S., Hong, S.K., 2002. Micro-organism
rejection by membrane systems. Environmental Engineering
Control and Orica Watercare. The assistance of Jasper Ver-
Science 19, 453e465.
berk, Paul Colby, Renae Phillips and Nic Reid are duly
Lebaron, P., Parthuisot, N., Catala, P., 1998. Comparison of blue
acknowledged. nucleic acid dyes for flow cytometric enumeration of bacteria
in aquatic systems. Applied and Environmental Microbiology
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