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Journal of Microbiological Methods 66 (2006) 217 – 222

www.elsevier.com/locate/jmicmeth

Removal of humic substances from soil DNA using


aluminium sulfate
Dexian Dong, An Yan, Haiming Liu, Xuehong Zhang, Yuquan Xu *
Laboratory of Molecular Microbiology, College of Life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road,
Shanghai 200240, China
Received 8 November 2005; accepted 11 November 2005
Available online 27 December 2005

Abstract

Direct extraction of soil DNA has become essential for the study of soil microorganisms. Humic substances co-extracted during
DNA retrieval is a big problem because it greatly inhibits the enzymes involved in manipulating DNA. Popular commercial kits
available for soil DNA extraction are unable to overcome this problem. Here we report an effective protocol for the removal of
humic substance from soil DNA. The protocol involves flocculation of the humic substance by excessive Al3+, then removal of
superfluous Al3+ via pH adjustment and finally release of soil microbial DNA by SDS lysis. This technique is superior to that
employed by the UltraClean Soil DNA Kit and can be applied to a wide variety of soils.
D 2005 Elsevier B.V. All rights reserved.

Keywords: DNA extraction; Humic substance; Aluminium sulfate; Extracellular DNA

Direct extraction of soil DNA is becoming a basic these methods also result in a decrease in DNA recov-
technique in soil microbial ecology. However, co- ery (Kuske et al., 1998; More et al., 1994; Zhou et al.,
extracted humic substance is a big problem because it 1995), or even the complete elimination of some DNA
significantly inhibits subsequent PCR and restriction templates of low-abundant microbes.
endonuclease (Wilson, 1997). Many methods have The main reason why humic substances are so dif-
been developed to remove humic substances from soil ficult to remove from soil DNA is probably the similar
DNA, including cesium chloride density centrifugation molecular structure of the two materials. Both are long
(Leff et al., 1995), hexadecyltrimethylammonium bro- chain molecules and carry negative charge. Humic
mide (CTAB; Cho et al., 1996), polyvinylpolypyrroli- substances contain many carboxyl and hydroxyl groups
done (PVPP; Frostegard et al., 1999; Zhou et al., 1995), (Cheng et al., 2004), and their physical–chemical char-
gel electrophoresis (Zhou et al., 1995), and the Sepha- acteristics are similar to the phosphate groups of the
dex G-200 column (Miller et al., 1999). Of these meth- sugar-phosphate backbone of DNA.
ods, some are limited in their application by being The use of multivalent cations to remove suspended
extremely time-consuming or expensive. Most of organic solids is a standard method of purifying drink-
ing water, a process commonly referred to as chemical
flocculation. Baruaha and Upreti (1994) used potentio-
* Corresponding author. Tel.: +86 21 54743347; fax: +86 21
metric titration to examine the ion exchange capacity of
54743348. humic acid extracted from lignite. The retention capac-
E-mail address: xuyq@sjtu.edu.cn (Y. Xu). ity of particular metal ions in the humic acid can be
0167-7012/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2005.11.010
218 D. Dong et al. / Journal of Microbiological Methods 66 (2006) 217–222

ordered in the following sequence: Fe3+Al N Cu N Zn N The results (Fig. 1A) indicate that Al3+ removes part
Ni N Co N Mg. Martyniuk and Wieckowska (2003) used of the DNA and that the loss rate of DNA increased
atomic absorption spectrometry and infrared spectra to gradually with decreasing pH of the Tris buffer from
examine the ion exchange capacity of humic acid 8.1 to 6.0 (1, 2, 3, and 4 in Fig. 1A). Cheng et al. (2004)
extracted from brown coals with respect to 17 metal reported that within the range pH 4.0–9.0, the efficien-
ions; the authors found that the gel form of humic acid cy of aluminium sulfate in removing pure humic acid
is a selective absorber of Cr3+ and Al3+. was highest (about 90%) at pH 6.0; efficiency de-
Braid et al. (2003) reported that the addition of creased gradually to 80% at pH 7.0 and plunged sharply
AlNH4 (SO4)2 during the direct extraction of Soil to 5% at pH 8.0. Kukita et al. (1997) reported that the
DNA via the UltraClean Soil DNA kit significantly depurination of DNA was negligible at pH 6.0 (approx-
reduces the co-purification of PCR inhibitor, with imately 10 5 of total DNA at pH 6.0, 30 8C for 10 h).
only minimal loss of DNA yield. In the current study These two reports indicate that Tris buffer at pH 6.0–
we initially used Al2(SO4)3 to purify the soil DNA 7.0 is a better choice for the removal of humic sub-
extracted by the isopropanol precipitation method stances from soil DNA with aluminium sulfate. Indeed,
(Miller et al., 1999), however, significant soil DNA the brownish hue of precipitate removed at pH 6.0 is
was removed by the Al2(SO4)3, contrary to the findings the most intensive. For a fixed pH of 6.0, the loss rate
of Braid et al. (2003). of DNA decreases gradually with decreasing Al3+ dos-
To confirm that Al3+ removes both humic sub- age (4, 5, 6, and 7 in Fig. 1A). These results strongly
stances and soil DNA, we collected and analyzed a demonstrate that Al3+ acts on both humic substances
compost soil from the rhizosphere of Clivia miniata. and DNA. The use of Al3+ to remove humic substance
The soil was made by the composting of leaves and from a mixture solution of DNA and humic substance is
grasses with soil, and contained abundant humic sub- therefore not recommended. The interaction of Al3+
stances. Debris of decayed leaves and roots were first with humic substance possibly involves complexation,
removed from the soil. We added 0.30 g of soil sample, charge neutralization and precipitation. Al3+ induces a
0.35 g of glass beads (diameter 2.0 mm), and 300 Al of complexation reaction with humic substances and neu-
phosphate buffer (0.1 M NaH2PO4–NaHPO4; pH 8.0) tralizes the negatively charged sites on humic sub-
to a microcentrifuge tube and gently mixed well by stance; this subsequently forms aluminum complexes
vortex. We then added 250 Al of SDS lysis buffer (100 and precipitates (Cheng et al., 2004). DNA has similar
mM NaCl, 500 mM Tris [pH 8.0], 10% [wt./vol.] SDS)
and vortexed horizontally for 10 min at maximum
speed using a Vortex Adapter (Mo Bio Scientific In-
dustries). After centrifuging at 10,000g for 30 s, the
supernatant was transferred into another microcentri-
fuge tube. Protein was removed by adding 250 Al of
chloroform/isoamyl alcohol (24:1), vortexing for 5 s,
incubating at 4 8C for 5 min, and centrifuging at
10,000g for 1 min. The supernatant was divided
into several equimultiples to ensure equal DNA content
in each tube. DNA was precipitated by adding a 0.5
vol. of 7.5 M ammonium acetate and a 1.0 vol. of
isopropanol. After incubation at 20 8C for 15 min, Fig. 1. Interaction of Al3+ with soil DNA. (A) 1, 2, 3, 4 show the
DNA was pelleted at 12,000g for 10 min and washed DNA yields after DNA was dissolved in 10 mM Tris, pH 8.1, 10 mM
three times with 70% ethanol. After being air-dried, Tris, pH 7.4, 10 mM Tris, pH 6.7, and 10 mM Tris, pH 6.0,
pellets were dissolved in 100 Al of 10 mM Tris, pH 8.1, respectively, and purified by 10 Al of 10 mM aluminium sulfate; 5,
6 and 7 show the DNA yields after DNA was dissolved with 10 mM
100 Al of 10 mM Tris, pH 7.4, 100 Al of 10 mM Tris, Tris, pH 6.0 and purified by 7, 3 and 0 Al, respectively, of 10 mM
pH 6.7 and 100 Al of 10 mM Tris, pH 6.0, respectively, aluminium sulfate. (B) 200, 150, 100, and 50 show the DNA yields
and flocculated with 10 mM aluminium sulfate. The when 200, 150, 100, 50 Al, respectively, of 100 mM aluminium
brownish precipitate of humic substances was removed sulfate was added and the pH value of the mixtures were not adjusted.
by centrifuging at 10,000g for 5 min. The supernatant 200a, 150a, 100a, 50a show the DNA yields when 200, 150, 100, 50
Al of 100 mM aluminium sulfate, respectively, was added and the pH
was then ready for PCR. DNA yield was assessed by value of the mixtures was adjusted to 8.0 via 1 M sodium hydroxide.
0.7% agarose gel electrophoresis with ethidium bro- m represents the DNA molecular marker. These profiles were made
mide staining. by 0.7% agarose gel electrophoresis with ethidium bromide staining.
D. Dong et al. / Journal of Microbiological Methods 66 (2006) 217–222 219

physical–chemical characteristics to humic substances, 8.0 via the formation of aluminum hydroxide (Huang
and therefore probably also interacts with Al3+ and and Shiu, 1996); these precipitates are very stable
precipitates in a similar manner. (Huang and Shiu, 1996). Once humic substances and
To take advantage of the strong flocculation effect of superfluous Al3+ are removed by precipitation, these
Al3+ on humic substances and yet avoid its negative substances are unable to act on the DNA released by
effect on DNA, we designed the following protocol for subsequent SDS lysis; a higher DNA yield is therefore
the direct extraction of soil DNA. Before DNA was obtained. These experiments indicate that our protocol
lysed out of soil microbial cells, humic substance with- design is feasible and that removing superfluous Al3+
in the soil was flocculated by excessive Al3+ under the via pH adjustment is both effective and necessary.
buffer condition of pH 6.6. We then adjusted the pH to To determine the ideal dosage of Al3+, the experi-
8.0 and precipitated superfluous Al3+. DNA within soil ments with pH adjustment described above were
microbial cells was then released by SDS lysis. repeated three times. DNA purity was evaluated by
To test this protocol design, we mixed 0.30 g of the the absorbance ratio at 260:340 nm via a WFZ UV-
compost soil from the rhizosphere of Clivia miniata 2000 spectrophotometer (UNICO, Shanghai), as the
with 300 Al of phosphate buffer (0.1 M NaH2PO4– humic substance was absorbed at 340 nm and the
NaHPO4; pH 6.6), respectively, in eight microcentri- DNA absorbed at 260 nm (Howeler et al., 2003;
fuge tubes. These tubes were divided into two groups. Yeates et al., 1998). Results (Fig. 2) indicate that as
We then added 50, 100, 150, 200 Al of 100 mM the dosage of Al3+ is increased, the optical density of
aluminium sulfate, respectively, to each group and the humic substance at 340 nm decreased, and the
complemented their volume with 150, 100, 50, 0 Al, ratio of DNA to humic substance (260:340 nm)
respectively, of phosphate buffer (pH 6.6), vortexed for increased. When up to 200 Al of 100 mM aluminium
2 min. The pH of one group was adjusted to 8.0 by sulfate was added to the dosage of Al3+, the optical
adding 150 Al of 1 M sodium hydroxide but the pH of density at 340 nm only was 0.055 and the ratio of
another group was not adjusted. DNA was then 260:340 nm was 13.19. This indicates minimal or no
extracted according to the steps described above (SDS humic substance in this treatment, and that 200 Al of
lysis, bead-beating, protein removal, isopropanol pre- 100 mM aluminium sulfate is enough to remove the
cipitation and ethanol washing) and dissolved with 100 humic substance.
Al of 10 mM Tris buffer of pH 7.6. Commercial kits such as the UltraClean Soil DNA
The results (Fig. 1B) indicate that DNA yield Kit and FastDNA Spin Kit are popular methods for the
obtained without pH adjustment decreased with in- direct extraction of soil DNA. To compare the superi-
creasing Al3+ dosage (200, 150 100, and 50 in Fig. ority between our protocol and the commercial kits, we
1B); even 50 Al of 100 mM aluminium sulfate removes chose the UltraClean Soil DNA Kit and RISA (ribo-
some of the DNA (compare 50 and 50a in Fig. 1B). somal intergenic spacer analysis) method to compare
Interaction occurred between Al3+ and DNA in the
treatments without pH adjustment. The results shown
in Fig. 1A reveal that just 1 or 2 Al of 100 mM (10 or 20
Al of 10 mM) aluminium sulfate is enough to remove
the humic substance from the soil sample. Superfluous
Al3+ almost certainly exists in these treatments. The
superfluous Al3+ was probably absorbed via cation
exchange onto the surfaces of soil particles that absorb
all kinds of metal ions. The higher the dosage of
aluminium sulfate, the greater the amount of Al3+
absorbed onto the surface of soil particles. After cell
lysis, the Al3+ absorbed onto the surface of soil particles
would be released into the solution by ion exchange
and precipitate the DNA. However, in the treatments
with pH adjustment, DNA yield was not effected by the
dosage of aluminium sulfate (200a, 150a 100a, 50a in Fig. 2. Dosage effect of Al3+ on the removal of humic substances.
bOD of HS (340 nm)Q represents the optical density of humic sub-
Fig. 1B). This indicates that the superfluous Al3+, stances under 340 nm wavelength. bDNA/HA (260/340 nm)Q repre-
including that absorbed onto the surface of soil parti- sents the absorbance ratio of DNA under 260 nm and humic substance
cles, precipitates intensively under the condition of pH under 340 nm.
220 D. Dong et al. / Journal of Microbiological Methods 66 (2006) 217–222

the population composition of soil bacteria and fungi by electrophoretic buffer system was 0.5 TBE (45 mM
the number and intensity of PCR bands of the ribosom- Tris, 44 mM boric acid, 1 mM EDTA, pH 8.9). The
al internal transcribed spacer (ITS; Kent and Triplett, gel was made by 7% polyacrylamide (29:1) and sized
2002). to 8.3  7.3  0.1 cm3. Silver staining was performed
We collected four soil samples: compost soil from according to the procedure we reported previously
the rhizosphere of Clivia miniata (no. 1), sediment (Dong et al., 2005).
soil from the rhizosphere of rice (no. 2), surface soil Fig. 3A and B indicate that the DNA yields obtained
under the pine tree (no. 3), and compost soil from the by our protocol are higher than that by the UltraClean
rhizosphere of Petunia Hybrida (no. 4). These soils Soil DNA Kit: the band intensity and optical density at
were pestled and homogeneously mixed and accurate- 260 nm in our protocol is higher (compare 1 and k1, 2
ly weighed to 0.30 g. Soil DNA was extracted accord- and k2, 3 and k3, 4 and k4 in Fig. 3A and the hollow
ing to the instruction of the UltraClean Soil DNA Kit; columns in Fig. 3B). In terms of optical density, DNA
and our protocol that humic substance was removed yields recovered by the UltraClean Soil DNA Kit were
prior to cell lysis. The ITS fragment between 16S and 70.4%, 46.3%, 63.5%, and 53.0%, in soil order, respec-
23S of soil bacteria was amplified using the primers tively, of that achieved by our protocol. Fig. 3B also
G1: (5V–GAAGTCGTAACAAGG–3V) and L1: (5V– reveals that the humic substances co-extracted by the
CAAGGCATCCACCGT–3V; Jensen et al., 1993). UltraClean Soil DNA Kit were 4.1, 3.2, 4.2, and 3.5
The ITS fragment between 18S and 28S (including times greater, respectively, than that co-extracted by our
ITS1 + ITS2 + 5.8S) of soil fungi was amplified using protocol (compare the solid columns of 1 and k1, 2 and
the PCR primers ITS4: (5V–TCCTCCGCTTATTGA- k2, 3 and k3, 4 and k4 in Fig. 3B). The DNAs from the
TATGC–3V) and ITS5 (5V–GGAAGTAAAAGTCG- UltraClean Soil DNA Kit could not be amplified unless
TAACAAGG–3V; White et al., 1990). PCR was they were diluted. Their PCR products were obtained
carried out according to the method we reported pre- by 20 times dilution.
viously (Dong et al., 2005). Polyacrylamide gel elec- Figs. 3C and D respectively show a comparison of
trophoresis was carried out at constant 80 V for 2.5 soil bacterial and fungal population compositions be-
h at 16 8C with an electrophoretic apparatus (Mini- tween the two methods. The number of ITS PCR bands
PROTEAN II System, Bio-Rad Laboratories, USA). produced by our protocol is more than that of the
Two Al of PCR products was added to each lane. The UltraClean Soil DNA Kit (compare 1 and k1, 2 and

Fig. 3. Comparison of the superiority between our protocol and the UltraClean Soil DNA Kit. 1, 2, 3 and 4 indicate the soil numbers 1, 2, 3 and 4
with our protocol. k1, k2, k3 and k4 indicate the soil numbers 1, 2, 3 and 4 with the UltraClean Soil DNA Kit. m is DNA molecular marker. (A)
DNA yields by 0.7% agarose gel electrophoresis with ethidium bromide staining. (B) Optical density of DNAs at 260 nm wavelength (hollow
columns) and the optical density of humic substances at 340 nm wavelength (solid columns). (C and D) Population compositions of soil bacteria
and fungi, respectively, by polyacrylamide gel electrophoresis of the ribosomal internal transcribed spacer (ITS) PCR fragment with silver staining.
D. Dong et al. / Journal of Microbiological Methods 66 (2006) 217–222 221

k2, 3 and k3, 4 and k4 in Figs. 3C and D). This


demonstrates that some bacterial and fungal populations
that were not detected by the UltraClean Soil DNA Kit
were detected by our protocol. Some band intensities at
the same location differed (compare 1 and k1, 2 and k2,
3 and k3, 4 and k4 in Figs. 3C and D). This phenom-
enon had been reported previously (Krsek and Welling-
ton, 1999; Maarit Niemi et al., 2001; Martin-Laurent et
al., 2001), and may reflect the different recovery rate of
DNA resulting from different extraction methods. The
quantitative superiority of either method needs to be
further tested. In any case, the DNA yields and number
of ITS PCR bands indicate that our protocol is superior
to that of the UltraClean Soil DNA Kit.
To further test if our protocol is appropriate for
various soils, we collected eight soils of various origins.
Their DNAs were extracted by our protocol and ana-
lyzed by RISA. These soils included commercial green-
house soil from the rhizosphere of watermelon at
Sanduan, Shanghai (no. 5), vegetable garden soil
Fig. 4. Further applicability of our protocol. 5–12 indicate soil numb-
from the rhizosphere of lettuce (no. 6), commercial ers 5–12. m is DNA molecular marker. (A) DNA yields by 0.7%
greenhouse soil from the rhizosphere of watermelon agarose gel electrophoresis with ethidium bromide staining. (B and C)
at Wanxiang, Shanghai (no. 7), border soil from a Population compositions of soil bacteria and fungi, respectively, by
sewage ditch (no. 8), surface soil under a fir tree (no. polyacrylamide gel electrophoresis of the ribosomal internal tran-
9), garden soil from the rhizosphere of green pepper at scribed spacer (ITS) PCR fragment with silver staining.
Suqian, Jiangshu Province (no. 10), undisturbed soil
from the forest at Donghai, Shanghai (no. 11), and be applied into the direct extraction process of soil
garden soil from the rhizosphere of red beet (no. 12). RNA. As both humic substances and superfluous Al3+
The results shown in Fig. 4 prove that our protocol is have been removed prior to the disruption of soil
suitable for these soils. Figs. 4A, B and C show DNA microbial cells, no humic substances or superfluous
yields, and bacterial and fungal population composi- Al3+ are able to act on the RNA released by subsequent
tions respectively. Including the four soils tested for cell disruption (Sessitsch et al., 2002).
superiority, twelve soils have now been tested by our
protocol. We therefore conclude that our protocol is
Acknowledgement
applicable to a wide variety of soils.
Our method has two other potential advantages over
This work was supported by the Grants from the
existing methods, although these advantages have yet to
10th Five-year Programs of Chinese National Science
be tested. One is that our method could remove extra-
(No. 2004BA308A21-6) and from the National Natural
cellular DNA within soil. Research had shown that
Science Foundation of China (No. 30370041).
microbial DNA released by cell death persists in the
soil environment for some time (England et al., 2004).
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