SUPPORTING INFORMATION PARAGRAPH
Supplementary Material A
Scheme of the well fields and filters at Oasen WTP Lekkerkerk
Non-subsurface aerated groundwater filter system
Non-subsurface aerated wells
Non-subsurface aerated filters
Subsurface aerated groundwater filter system
Tap water
Subsurface
aerated well
Non-subsurface
aerated wells
Subsurface aerated filters
Supplementary Material B
Gradient tube enrichment, DNA extraction and DGGE
Gradient tubes were used to enrich for FeOB from groundwater and filter wash water to
check for other culturable FeOB than Gallionella spp. in the original samples. Gradient tubes
are generally applied for the culturing of microorganisms that prefer growth at an oxic-anoxic
interface, like Gallionella spp.. The gradient tubes were prepared according to the protocol of
Emerson and Floyd (2005) with slight modifications. The gradient tubes consisted of a 1%
high melting agarose bottom layer with freshly prepared FeS and a 0.15% low melting
agarose top layer with 5mM sodium bicarbonate. Both layers contained Modified Wolfe's
Minimal Medium and the headspace contained sterilized air. A sterile vitamin and trace
element solution was added after autoclaving. The gradient tubes were inoculated aseptically,
in duplicate per sample, and stored at room temperature in the dark. After three weeks,
biomass from one tube per sample was transferred aseptically into the new gradient tubes in
triplicate. One of these three tubes was autoclaved and used as a negative control. This
enrichment was repeated twice.
The growth of FeOB in the inoculated tubes is visible from the formation of a sharp band
(Figure B.1).
Inoculated
Inoculated
Neg. control
Blank
Figure B.1: Gradient tubes with FeS at the bottom and oxygen at the top; left to right: blank (no bacterial
cells), negative control (autoclaved), inoculated with subsurface aerated groundwater (sampled 24 hours
after start of abstraction) in duplicate
DNA extraction
DNA from the gradient tube samples was extracted by bead beating using a Ultraclean™
Microbial DNA isolation kit (MO BIO Laboratories) after centrifuging.
DGGE
The 16S rRNA bacterial genes were amplified using general bacterial 16S-rRNA primers
(Bac341f+GC and Bac907rM (rA+rC); Schäfer and Muyzer 2001 and references therein).
Amplification was performed by initial denaturation for 5 min at 94°C, followed by 30 cycles
of amplification (30 s denaturation at 95°C; 40 s annealing at 57°C; 40 s elongation at 72°C),
and 30 min at 72°C to complete elongation. The DGGE was performed according to Schäfer
and Muyzer (Ibid.) using a 20%-70% gradient of urea and formamide.
References
Emerson D, Floyd MM. 2005. Enrichment and isolation of iron-oxidizing bacteria at
neutral pH. Methods in Enzymology 397:112-123.
Schäfer H, Muyzer G. 2001. Denaturing gradient gel electrophoresis in marine microbial
ecology. Methods in Microbiology. p 425-468.
Supplementary Material C
Overview of all qPCR results
The balance calculation for Gallionella spp. is schematized in Figure C.1.
Inoculation by raw water
te . f .
x
in ,t
* Q * t
ts . f .
Measured: xin,t (qPCR), Q
Washout to backwash water
dXbw = xbw * Vbw
Measured: xbw (qPCR), Vbw
Washout to filtrate water
te . f .
x
out ,t
* Q * t
ts . f .
Measured: xout,t (qPCR), Q
FigureC.1: Balance calculation of Gallionella spp. in groundwater filters
Where
Q
t
V
x
X
flow (m3 h-1)
time (h)
volume (m3)
cell concentration (cells m-3)
total cell number (cells)
Indices
bw
in, out
t
s.f., e.f.
backwash
in influent, effluent
time (h)
start, end of filter runtime
The individual results for the balance terms of the WTP Lekkerkerk trickling filter are
summarized in Figure C.1 and C.2 for the non-subsurface aerated and the subsurface aerated
filter, respectively. For both figures, bar graphs in the left column show the cumulative cell
numbers per filter run time of 48 hours for the influent (top), backwash water (middle) and
effluent (bottom); these cumulative balance terms are shown in the manuscript. Scatter plots
in the right column present the actual Gallionella cell numbers in the influent (top), backwash
water(middle) and effluent (bottom) samples; the curved arrow indicate the direction of the
backwash flow;
Non-subsurface aerated filter
Groundwater - Influent
15
1·10
1·1013
1·1009
7
1·10
5
N mL-1
1·103
3·1011
11
1·10
1·10
1·101
Backwash water
3x1015
1015
13
10
1011
1009
Filtrate - Effluent
1·1015
1·1011
1·1009
1.4·1014
1·10
N mL-1
1·107
13
1·105
1·103
1·101
0
12
24
36
Filter run time (h)
48
Figure C.1: Gallionella balances for the WTP Lekkerkerk non-subsurface aerated trickling filter by
qPCR over nine months after external washing; all data points in the scatter plots represent the singular
qPCR result for one sample, except for the control backwash water samples, which were taken and
measured in quintuple
Subsurface aerated filter
1·1015
Groundwater - Influent
1·107
1·1013
N mL-1
1·105
9·1013
1·103
1·1011
1·101
0
14
28
42
Days from start of extraction
1·1009
Backwash water
1015
13
1011
9x1013
10
1009
1·1015
Filtrate - Effluent
N mL-1
1·107
9·1013
1·1013
1·105
1·103
1·1011
1·101
0
12
24
36
Filter run time (h)
48
1·1009
Figure C.2: Gallionella balances for the WTP Lekkerkerk subsurface aerated trickling filter by qPCR
over nine months after external washingall data points in the scatter plots represent the singular qPCR
result for one sample, except for the control backwash water samples, which were taken and measured in
duplicate