Front. Environ. Sci. Engin.

China 2010, 4(4): 459465

DOI 10.1007/s11783-010-0249-3

RESEARCH ARTICLE

Development of pretreatment protocol for DNA extraction

from biolm attached to biologic activated carbon (BAC)
granules
Shuting ZHANG, Bo WEI, Xin YU (), Bing LIU, Zhuoying WU, Li GU
Key Laboratory of Urban Environment and Health, Institute of Urban Environment, China Academy of Sciences, Xiamen 361021, China

Higher Education Press and Springer-Verlag Berlin Heidelberg 2010

Abstract The biologic activated carbon (BAC) process

is widely used in drinking water treatments. A comprehensive molecular analysis of the microbial community
structure provides very helpful data to improve the reactor
performance. However, the bottleneck of deoxyribonucleic
acid (DNA) extraction from BAC attached biolm has to
be solved since the conventional procedure was unsuccessful due to rm biomass attachment and adsorption capacity
of the BAC granules. In this study, ve pretreatments were
compared, and adding skim milk followed by ultrasonic
vibration was proven to be the optimal choice. This
protocol was further tested using the vertical BAC samples
from the full-scale biolter of Pinghu Water Plant. The
results showed the DNA yielded a range of 40 g$g1 BAC
(dry weight) to over 100 g$g1 BAC (dry weight), which
were consistent with the biomass distribution. All results
suggested that the nal protocol could produce qualied
genomic DNA as a template from the BAC lter for
downstream molecular biology researches.
Keywords bacterial DNA extraction, biological activated
carbon (BAC), biolm, water treatment, pretreatment
protocol

Introduction

The biologic activated carbon (BAC) or the combining of

ozonation and biologic activated carbon ltration (O3BAC) have been widely used to treat drinking water [13].
Both methods of treatment are efcient in removing
organic contaminants including natural organic matters,
synthesized organic compounds, and other bioavailable
Received December 30, 2009; accepted August 22, 2010
E-mail: xyu@iue.ac.cn

materials, such as NH
4 -N [3,4]. In many cases, O3-BAC
was regarded as the most important barrier for maintaining
chemical and biologic stability and for decreasing the
toxicity and epidemiological risk of drinking water,
especially when the water source was polluted [5]. In
fact, BAC was not only applied in drinking water treatment
but also in wastewater [6,7] and waste gas treatment [8]
because their activated carbon granules were suitable
microbial carriers and affordable.
For most resolvable bioavailable materials, biodegradation acts as the essential mechanism in the contaminant
removal by BAC lters, rather than physical ltration [9
11]. Biolm attached to activated carbon greatly contributes to the biodegradation process [12]. Therefore, it is
important to reveal the microbial community structure and
function of the attached biolm for the purpose in order to
optimize the reactor performance.
Molecular techniques that did not depend on culturing
were researched [13] because drinking water is typically
oligotrophic and most of the microorganisms in this
environment are nonculturable. In fact, polymerase chain
reaction-denaturing gradient gel electrophoresis(PCRDGGE), terminal restriction fragment length polymorphism(T-RFLP), deoxyribonucleic acid (DNA)-sequencing
analysis, etc., have been used successfully in the drinking
water system [14] or similar systems, such as sand and
anthracite biolters [15,16].
However, the stated molecular approaches are confronted with great difculties in DNA extraction when
applied to BAC granules samples. For instance, Liu et al.
[17] attempted to compare the community structure of
bioceramics and BAC biolms using polymerase chain
reaction-single strand conformation polymorphism (PCRSSCP). However, no DNA was obtained even when using
ultrasonic pretreatment. The complication could be due to
two properties of activated carbon granules: the porous

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Front. Environ. Sci. Engin. China 2010, 4(4): 459465

structure and the adsorption capacity [18]. The extremely

complicated porous structure not only provided a suitable
dwelling place for the microorganisms but also caused
difculty for the biolm to detach from the granules,
especially when the microorganisms grew inside the
granules or were sheltered by the microenvironment
from shear force. More importantly, the special surface
structures also provided the granules with huge adsorption
capacity [19]. Although most of the active sites in BAC
granules were occupied by the contaminants during the
long-term, the residues were enough to absorb DNA after
cell lysis. The interactions between the organics and the
DNA of the BAC medium also contributed to the very low
or absence of DNA recovery.
These difculties were not only found in DNA
extraction from BAC but also in soil. Soil was another
system with similar properties, such as granular shaped,
biolm attached, and adsorption capacity. Much research
related to soil has already been conducted. In previous
researches, certain materials were used as competitors or
protectors in DNA recovery from clay-rich soil, such as
RNA [20], denhardts solution [21], and skim milk [21
24]. In this study, ve pretreatments were tested and
modied to establish an applicable protocol to extract
DNA from BAC granules. Then, the reliability of the
optical pretreatment was conrmed.

Materials and methods

2.1

Full-scale biologic lters

2.2

Filter samples were collected from six different depths: 0

(media surface), 10, 20, 40, 60, and 80 cm. A sampler for
water treatment lters was used when activated carbon was
collected (Chinese invention patent pending, patent
number: ZL200920162293.8). The sampler was stuck in
the lter until a certain depth (e.g., 20 cm) was reached,
and then, the hollow column at the bottom of the sampler
began its media collection. The length of the sample
column was 4 cm. For each depth, more than 500 g
granules were taken into a sterilized PVC bottle. The
bottles were then quickly transported to the laboratory in
ice bags and stored at 80C until tested. Only the samples
from 0 cm (i.e., the surface granules) were used to nd the
optimal protocol of DNA extraction. All samples were
used to validate the optimal protocol in DNA extraction
efciency from different biomasses.
2.3

Pretreatments of the BAC granules for DNA extraction

Five pretreatment methods were designed to detach biolm

from the granules and prevent the adsorption of DNA.
They were compared in parallel until the optimal method
was selected. In each of the pretreatments, 5 g (wet weight)
granules were rst taken into a 50 mL sterilized centrifuge
tube with 5 mL DNA extraction buffer (50 mM Tris-HCl,
40 mM EDTA-2Na, 0.75 M sucrose, 1.5 mM NaCl, and
pH 9.0) and then mixed vigorously through a vibrator for
1 min. All containers were sterilized by autoclaving.
In pretreatment A, a piece of sterilized gauze was partly
placed into a 50-mL centrifuge tube and formed a funnel.
The mixture was roughly ltrated through the gauze in
order to retain most of the suspended solid. Afterward, the
gauze was scrubbed strongly and carefully. The ltrate was
collected into the centrifuge tube.
In pretreatment B, the samples were grounded in a
mortar. The slurry was transferred into a 50 mL centrifuge
tube [25].
Pretreatment C included both ultrasonic vibration and
mechanical rubbing. The ultrasonic vibration was followed
by a modied method of Buesing et al. [26]. The sample
centrifuge tube was treated in an Ultrasonic Cell Disrupter
System (JY92-II, scientz, NB, China) under 200 W with 40
cycles of 3 s work periods followed by a 10 s breaks. The
total working time of the disrupter was 120 s. After
ultrasonic shaking, the mechanical rubbing as described in
pretreatment A was applied to the samples again in order to
enhance the detachment.

The BAC granules were sampled from the full-scale

BAC-sand dual media lters at Pinghu Drinking Water
Treatment Plant in Zhejiang Province, eastern China. Each
of the downow lters had a size of 6 m
2 m (L
W)
and a ltration velocity of 811 m$h1. The upper layer
media consisted of 70 cm BAC granules, and the lower
layer media consisted of 30 cm regular sand media. The
BAC granules are column-shaped (1.5 mm
1.0 mm) and
coal based. Both media performed different functions,
where BAC was mainly responsible for biodegradation
and nitrication, and sand maintained a low efuent
turbidity. The backwashing time lasted 56 min with
intervals of 24 h, and the lter bed expanded 20% to
30%. These lters were reconstructed into their current
status in 2000 and have since then been steadily running.
The main water quality parameters about the lter
performance during this study can be seen in Table 1.
Table 1

Preparation of BAC biolm samples

Inuent and efuent of the BAC biolter (2008.7.162009.5.15)

DO/(mg$L1)

CODMn/(mg$L1)

pH

1
NH
4 -N=mg$L

NO2 -N=mg$L 1

NO3 -N=mg$L 1

inuent

6.813.2

3.987.58

6.87.1

1.234.61

0.010.53

0.936.86

efuent

0.88.7

3.026.92

6.97.2

0.083.37

0.000.35

1.888.29

parameters

Shuting ZHANG et al. Development of protocol for DNA extraction from biollm on BAC granules

In pretreatment D, only ultrasonic vibration described

above was performed.
In pretreatment E, skim milk was introduced based on a
modied method used by Hoshino et al. [21]. The skim
milk was bought from a local grocery store. Three doses of
skim milk, 0.1, 0.5, and 1.0 g, were added to the sample
centrifuge tubes, which were designated as E1, E2, and E3,
respectively. The nal concentration of E1, E2, and E3
were 0.02 g$mL1, 0.1 g$mL1, and 0.2 g$mL1. Afterward,
a vigorous vortex was performed to dissolve the powder.
The mixture was then ultrasonic vibrated, following the
procedure of pretreatment C.
2.4

DNA extraction and purication

After pretreatment, the microbial cells collected were lysed

with three cycles of freezing thawing. Then, 1 mL
suspension was transferred into a new 2 mL centrifuge
tube for the following steps. DNA extraction and chloroform purication were performed using modied methods
[27,28]. The puried DNA were dissolved in 40 L of
sterile TE buffer (10 mM Tris-HCl, 1 mM EDTA, and pH
8.0) and stored at 4C for further manipulation or at 20C
for long-term storage. Each extraction process was
repeated three times.
To test for DNA contaminants in the skim milk, the skim
milk was dissolved at concentrations of 0.001, 0.01, 0.1,
and 1 g$mL1. Then, the milk solutions were lysed using
three cycles of freezing thawing and stored at 20C for
PCR amplication.
2.5

Quantitative determination of extracted DNA

The concentration of the extracted DNA was determined

by the NanoDrop Spectrophotometer (ND-1000 V3.5.2,
NanoDrop Technology, Wilmington, DE, USA) with a
minimum limit of detection for 2 ng$L1 (dsDNA) and
maximum concentration of detection for 15000 ng/L
(dsDNA). The puried DNA was further analyzed by 1%
agarose gel electrophoresis. SYBR GreenII(Sorlabio),
amounting to 1.5 L, was added and mixed into a 1 mL
6
loading buffer(TaKaRa). Then, 2 L of puried DNA
were mixed with the same volume of the above loading
buffer containing SYBR GreenII. The gel image was
captured by the GelDoc XR system (Bio-Rad Laboratories,
Hercules, California).
2.6

461

performed on the skim milk solutions prepared in Section

2.4.
2.7

Determination of Lipid-P biomass

The samples of biomass were determined using the lipid-P

method [30] to validate the DNA extraction protocol. In
this method, the phospholipid content of the samples was
extracted by a mixture of chloroform, methanol, and water.
The inorganic phosphorus was then released by digestion
and measured as the biomass indicator. In the determination, 30-mm cuvettes were used, and the methods
detection limit was 1 nmol phosphorus. The equivalent
of 1 nmol phosphorus was 108 bacteria cells, which is
equal to the size of E. coli [31]. The determinations were
performed three times for each sample.

Results and discussion

3.1

DNA extraction pretreatments

In previous studies, DNA extraction was conducted

without any pretreatment. However, a direct extraction of
DNA from BAC biolms was difcult to produce using the
common extraction method mentioned in Section 2.4 or
commercial kit (D5625-01, Soil DNA Kit, Omega). No
visible DNA bands via gel electrophoresis or detectable
DNA traces via spectrophotometer were obtained, which
suggested that the adsorption capacity of BAC had
strongly inuenced DNA extraction, thus necessitating
desorption pretreatment. Fig. 1 shows the results of the ve
pretreatments of samples, followed by the DNA extraction
and purication. Only pretreatment E2, which mixed 0.5 g
skim milk with 5 g sample, had obtained a visible and clear
electrophoretic band in 1% agarose gel. Pretreatments A
and C also showed visible bands but were both too sheared
to be applicable. Pretreatments B and D, however, did not
produce any visible bands.
Consistent with the gel electrophoresis, the
spectrophotometer results also showed that pretreatment

PCR amplication of SSU rDNA gene

Further verication of the developed protocol was done by

PCR. Small subunits of rDNA (SSU) fragments were
amplied with the universal bacterial primer pairs (27F:5AGAGTITGATCCTGGCTCAG-3; 1492R:5-TACGGCTACCTTGTTACGACTT-3) using the extracted DNA as
templates [29]. In addition, PCR amplications were also

Fig. 1 1% agarose gel electrophoresis results of ve different

pretreatments. M: DL2000 DNA marker (TaKaRa)

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Front. Environ. Sci. Engin. China 2010, 4(4): 459465

E2 produced maximum DNA of about 36.3 g$g1 sample,

as shown in Fig. 2. While other pretreatments produced
much lower DNA, about 3.3, 1.4, 4.2, and 2.3 g$g1 DNA
were obtained by pretreatments A, B, C, and D,
respectively (Fig. 2). Pretreatments A and C showed
higher DNA production than pretreatments B and D, which
suggests that the simplest treatment, mechanical rubbing in
both methods, could cause the bacterial cells to detach at a
low efciency, and the ultrasonic vibration (in pretreatment
C) could promote the detachment. However, the use of
ultrasonic vibration was unreliable since pretreatment D
had only received about 1/10 concentration of the highest
DNA yield. In pretreatment B, the activated carbon
granules were grounded into ne particles, resulting in a
great augment for the specic surface area and an increase
in adsorption capacity, which could explain the lowest
DNA yield by this method.

Fig. 3 Results of PCR tests of recovered DNA extracted by ve

pretreatments. M: DL2000 DNA marker

Fig. 4 1% agarose gel electrophoresis results of adding different

doses of skim milk in DNA extraction M: DL2000 DNA marker

Fig. 2 Results of recovered DNA concentration (g$g1 [wet

wt]) by spectrophotometer

Moreover, the DNA yielded by pretreatments AD were

so minute and sheared that when the DNA were used as a
template, no successful SSU rDNA PCR results could be
obtained (Fig. 3). However, the extraction of DNA using
pretreatment E2 could easily lead to an ideal PCR result
(Fig. 3). The PCR results of skim milk solutions were all
negative (data not shown), showing no DNA contaminants
in milk powder, therefore, the DNA extracts in pretreatment E2 was from the biolm and not from milk powder.
This conclusion agreed with Hoshino et al. [23] but
contrasted with Ikeda et al. [24]. A possible explanation for
this could be the different brands of skim milk that were
chosen, or experimental error.
Furthermore, pretreatments adding 0.5 g$mL1 (E2),
0.1 g$mL1 (E1) and 1.0 g$mL1 (E3) skim milk were
also tested (Fig. 4). Pretreatment E2 obtained the brightest
bands in 1% agarose gel, thus, the nal adopted protocol
was the combination of sample pretreatment with E2
followed by DNA extraction and purication, as described
in Section 2.4.

Based on the above results, the protocol with E2 proved

to be the optimal choice as evidenced by the result of triple
validation (i.e., electrophoretic image, DNA concentration
determination, and PCR). In E2, skim milk was introduced,
and the optimal dose was also selected, which was an
important factor in DNA yielding. Skim milk has been
reported to be effective in releasing DNA from different
samples [2124]. Skim milk was proposed as a competitor
for the adsorption sites with DNA molecules, prevention of
DNA degradation [21], or obstruction of the chemical
groups on activated carbon granules. The situation in soil
was similar, where DNA recovery could be also lowered
by the adsorption onto clay [23,32,33]. In addition, other
molecules were reported to compete with DNA for the
adsorption sites on carbon, and thus, the DNA could be
released to the solution [23,24]. Additives, such as RNA
[20] and denhardts solution [21,34] are known to
effectively increase DNA yields or enhance the stability
of DNA molecules. RNA could compete with DNA for the
adsorption site, and denhardts solution was used to protect
DNA from degradation and enhance the efciency of PCR
amplication [20,21]. However, RNA was proven to only
partly compete for the adsorption sites with DNA and was
too expensive to be used in routine laboratory work [20].
Factors including the amount of skim milk, ultrasonic
vibration time, and frequency could also inuence the
experimental result. Small doses of skim milk (E1) may be
inadequate to saturate the adsorption sites on activated

Shuting ZHANG et al. Development of protocol for DNA extraction from biollm on BAC granules

463

carbon. However, excess skim milk (E3) may inhibit DNA

extraction. This conclusion was agreed with Hoshino et al.
[23], and it was speculated that the protein in the excess
skim milk may interfere with DNA extraction and
quantization. The present settings of ultrasonic vibration
time and frequency were determined based on several
pretests and were shown to have adequate strength and
prevent the damage of cells.
3.2 DNA yields of samples from six different depths of the
biolter

The nal protocol with pretreatment E2 was used to extract

genome DNA from samples of six different depths of the
biolter to conrm the reliability of this method. The clear
bands on the agarose gel electrophoresis demonstrated that
all the extractions were successful (Fig. 5). DNA
concentrations were determined by a spectrophotometer,
as shown in Fig. 6. The concentrations of DNA extracted
from 0 cm and 10 cm samples were both greater than
100 g$g1 activated carbon (dry weight). The samples
from the deeper layers of 20 cm and 40 cm generated lower
DNA concentrations of about 60 g$g1 activated carbon
(dry weight). Samples collected from 60 cm and 80 cm
only yielded around 40 g$g1 media (the 80 cm sample
was sand granules, not BAC) (dry weight), which were at
the lowest.

Fig. 6 DNA yields (g$g1 [dry wt]) determined by spectrophotometer and biomass determined using lipid-P method of sixdepth samples. (a) DNA yields; (b) biomass

the DNA bands on the agarose gel electrophoresis (Fig. 5)

was inconsistent with DNA concentration (Fig. 6). It could
be attributed to the fact that the upper samples contained
more contaminants, such as humic substances, which could
interfere with DNA purication and quench the uorescence intensity during the agarose gel electrophoresis.

Fig. 5 Agarose gel electrophoresis bands of DNA extracted

from samples of different depth in BAC lter. M: DL2000
DNA marker

Theoretically, the DNA yield should be proportional with

the biomass, where more biomass provides greater DNA
production. Figure 6 shows the distribution of the biomass
determined using the lipid-P method along the depths.
Because the organic matters and other nutrients would be
utilized by the microorganisms along the depths, the
biomass had the same tendency as the substrate, which
dropped from about 400 nmol P$g1 at the top media to
about 80 nmol P$g1 at a depth of 80 cm. The intensity of

Conclusion

A DNA pretreatment protocol that introduced skim milk

into the procedure was developed to obtain genomic DNA
from biolm attached to BAC granules. Moreover, the
method proved to be effective in extracting DNA from
BAC biolm samples with different biomass that were
collected from full-scale drinking water plant. Since the
DNA extraction used to be the bottleneck of microbial
ecology study of BAC biolm, this method may support
further research in optimizing the treatment methods of
drinking water, wastewater, or waste gas that involves
BAC.

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Front. Environ. Sci. Engin. China 2010, 4(4): 459465

Acknowledgements The nancial support of this study was provided by

the National Natural Science Foundation of China (Grant No. 50678080),
Key Project of the Knowledge Innovation Program, Chinese Academy of
Science (Grant No. KZCX2-YW-452), and the 100 Talents Program, Chinese
Academy of Sciences.

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