FEMS Microbiology Ecology 43 (2003) 13^19

www.fems-microbiology.org

Zinc contamination decreases the bacterial diversity of

agricultural soil
Bruce F. Mo¡ett a;
, Fiona A. Nicholson b , Nnanna C. Uwakwe a ,
Brian J. Chambers b , James A. Harris a;1 , Tom C.J. Hill a

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a
School of Health and Bioscience, University of East London, Romford Rd., Stratford, London E15 4LZ, UK
b
ADAS Gleadthorpe Research Centre, Meden Vale, Mans¢eld, Nottingham NG20 9PF, UK

Received 5 June 2002 ; received in revised form 17 October 2002; accepted 29 October 2002

First published online 3 December 2002

Abstract

Around half a million tonnes of biosolids (sewage sludge dry solids) are applied to agricultural land in the United Kingdom each year,
and this may increase to 732 000 t by 2005/6. The heavy metals contained in biosolids may permanently degrade the microbial decomposer
communities of agricultural soils. We used amplified ribosomal DNA restriction analysis of the extractable bacterial fraction to compare
the diversity of a zinc-contaminated soil (400 mg kg31 Zn; pH 5.7 and 1.36% Corg ) with that of a control soil (57 mg kg31 Zn; pH 6.2 and
1.40% Corg ) from a long-term sewage sludge experiment established in 1982 at ADAS Gleadthorpe. Comparison of the restriction
fragment length polymorphisms of 236 clones from each soil suggested that the stress caused by zinc toxicity had lowered bacterial
diversity. There were 120 operational taxonomic units (OTUs) in the control soil, but only 90 in the treated soil, a decrease of 25%. While
the control soil had 82 single-occurrence OTUs the contaminated soil had only 52. The fall in diversity was accompanied by a decrease in
evenness. The most abundant OTUs in the contaminated soil (which tended to be common to both soils) accounted for a higher
proportion of clones than in the control. The most dominant OTU, in both soils, belonged to the Rubrobacter radiotolerans group of the
high G+C Gram-positive bacteria. The data was also used to develop efficient sampling strategies.
? 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords : Sewage sludge; Zinc; Heavy metal; Ampli¢ed ribosomal DNA restriction analysis ; Bacterial diversity ; Sampling strategy

1. Introduction tural land [2]. Furthermore, following the ban, at the end
of 1998, on sludge disposal at sea and tighter restrictions
Sewage sludge is a valuable source of plant nutrients on direct discharges to watercourses, re-direction of sludge
and organic matter, but may contain concentrations of to agricultural land in Britain is predicted to increase to
heavy metals which limit its acceptability for application 732 000 t dry solids per year by 2005/06 [2].
to agricultural land. Consequently, maximum permitted Heavy metals impose a chronic stress upon the decom-
metal concentrations in soils receiving sewage sludge poser subsystem, and a variety of experimental systems
have been laid down in European Community Directive and regimes have been investigated. These have included
86/278/EEC [1]. The potential scale of the problem is high- short- versus long-term microcosm incubations and dosing
lighted by the amounts involved. In 1996/97, 520 000 t of with a single metal or a combination of metals with or
sewage sludge dry solids, equivalent to about 50% of UK without the simultaneous addition of organic matter. Dif-
sludge production, were applied to 80 000 ha of agricul- ferences in soil pH, organic matter content, cation ex-
change capacity, temperature and moisture content intro-
duce further variability [3,4]. Giller et al. ([4], see also [5])
caution that it would be foolish to automatically assume
* Corresponding author. Tel. : +44 (208) 223 4061 ; that a simple, negative relationship exists between stress
Fax : +44 (208) 223 4959.
and diversity in microbial communities [6^8]. Even so,
E-mail address : mo¡ett@uel.ac.uk (B.F. Mo¡ett).
studies of the impact of heavy metals upon bacterial di-
1
Present address: Institute of Water and Environment, Cran¢eld versity in soil have shown a uniformly negative in£uence
University, Silsoe, Bedfordshire MK45 4DT, UK. (e.g. [9^13]).
0168-6496 / 02 / $22.00 ? 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII : S 0 1 6 8 - 6 4 9 6 ( 0 2 ) 0 0 4 4 8 - 8

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14 B.F. Mo¡ett et al. / FEMS Microbiology Ecology 43 (2003) 13^19

The principal aim of this work was to test, in accord 2.2. Isolation of extractable bacterial fraction
with Odum’s predictions [14], whether bacterial diversity of
an agricultural soil will decrease and dominance increase The extractable bacterial fraction was obtained from
in response to the stress caused by zinc contamination 50 g of soil using the method of Ste¡an et al. [17], but
associated with sludge applications. The zinc-contaminat- omitting polvinylpolypyrrolidone to increase cell recovery
ed soil formed part of a long-maintained ¢eld experiment rates. In order to remove organic contaminants and extra-
where equilibrium has e¡ectively returned and where var- cellular DNA, the crude bacterial pellet was then twice
iations in soil properties between plots are small. Bacterial suspended in 100 ml of cold 0.1% sodium hexametaphos-
diversity was measured using ampli¢ed ribosomal DNA phate^0.1% sodium pyrophosphate (at pH 8.5 as recom-
restriction analysis (ARDRA) of two large clone libraries. mended by Torsvik et al. [18]). The pellet was also washed
A second aim was to use the clone libraries to develop a twice with Chrombach bu¡er (330 mM Tris^HCl, 1 mM
strategy for optimising sampling. That is, a method of EDTA at pH 8.0), after which it was stored at 320‡C.
using interpolation with preliminary results to predict
the least laborious ¢nal combination of replicates and 2.3. DNA extraction and puri¢cation

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number of clones per replicate likely to show a signi¢cant
di¡erence between treatments. The two data sets have also The method used was based on that of Zhou et al. [19].
been used in a review of the suitability of using diversity Roughly half of each bacterial pellet was ground three
measures with such highly diverse communities [15]. times with an equal volume of ¢ne quartz sand under liq-
Underlying this work is the pressing and practical need uid nitrogen. Each powdered pellet was then immediately
to ascertain whether the present maximum permitted met- added to 13.5 ml autoclaved extraction bu¡er (100 mM
al concentrations in soil, both individually and in combi- Tris^HCl [pH 8.0], 100 mM di-sodium EDTA [pH 8.0],
nation, are safe for the long-term preservation of soil qual- 100 mM sodium phosphate at [pH 8.0], 1.5 M NaCl, 1%
ity and fertility. hexadecylmethylammonium bromide). Proteinase K was
added to 75 Wg ml31 and the mixture incubated at 37‡C
for 30 min while being shaken horizontally at 150 rpm.
2. Materials and methods SDS was then added to a ¢nal concentration of 2% and
each tube incubated at 65‡C in a water bath for 2 h with
2.1. Site history, soils and sampling gentle end-over-end mixing every 15 min. The mix was
then transferred to a centrifuge tube and the debris pel-
The soils were from a long-term sewage sludge ¢eld leted. The supernatant was collected, the pellet extracted
experiment at ADAS Gleadthorpe (Nottinghamshire, twice more and the supernatants combined.
UK). The site was on a loamy sand-textured soil (6% The proteins were denatured by the addition of chloro-
clay) of the Newport association (Typic Quartzipsan- form^isoamyl alcohol and the DNA precipitated in iso-
nents). Pressed sludge cakes enriched with zinc salts, and propanol overnight at room temperature. It was then pel-
an unenriched control sludge, were applied at a rate of leted by centrifugation, washed twice with 5 ml of cold
100 t ha31 dry solids in 1982. In 1986, pressed sludge 70% ethanol, dissolved in 100 Wl sterile, deionised water
cakes naturally rich in Zn were added to the zinc plot. and stored at 4‡C.
A variety of arable and grass crops have been grown on To reduce the likelihood of chimera formation the DNA
the site. Phosphorus and potassium fertiliser additions was then size fractionated by gel electrophoresis and
were applied to all treatments at recommended rates based DNA v 20 kb recovered using a Geneclean Spin kit
on soil analysis. Cereals, but not legumes, received inor- (BIO 101, Carlsbad, CA, USA) according to the manufac-
ganic fertiliser N (NH4 NO3 ) [16]. Elemental lime or sulfur turer’s instructions.
have been applied where necessary to maintain the soil pH
at around 6.5. In the year of sampling the plots were left 2.4. Ampli¢cation of 16S rDNA sequences and cloning
green fallow.
Soil samples taken in 1997 from the control plot con- Approximately 530 bp of the eubacterial 16S rDNA
tained 57 mg Zn kg31 , 13 mg Cu kg31 , 0.3 mg Cd kg31 gene was ampli¢ed using PCR using primers 27f (5P-AG-
and 7 mg Ni kg31 dry soil, Corg and total N contents of AGTTTGATCMTGGCTCAG-3P) and 519r (5P-GWATT-
1.40% and 0.11%, respectively, and a pH of 6.2. The zinc- ACCGCGGCKGCTG-3P). A hot start PCR was followed
contaminated plot had 400 mg Zn kg31 , 22 mg Cu kg31 , by 24 cycles of 94‡C for 1 min, 58‡C for 1 min and 68‡C
0.9 mg Cd kg31 and 6 mg Ni kg31 , Corg and total N for 1 min 30 s. A ¢nal extension at 68‡C for 7 min com-
contents of 1.36% and 0.11%, and a pH of 5.7. For this pleted the programme.
work, samples from two plots were taken in July, 1998. Amplicons, gel-puri¢ed using GenElute1 agarose spin
From each plot, ¢ve randomly-placed soil samples were columns (Supelco, Bellefonte, PA, USA), were cloned us-
taken from the top 10 cm of the pro¢le and thoroughly ing the TOPO TA cloning0 kit using the pCR0 2.1-TO-
mixed immediately before use. PO0 vector (Invitrogen, Groningen, The Netherlands) ac-

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cording to the manufacturer’s guidelines, except that in the distribution of bacterial groups recovered using in-
transformants were selected with 25 Wg ml31 of both am- direct versus direct extraction, apart from the former pos-
picillin and methicillin. sessing about twice the relative abundance of Q-Proteobac-
teria, while Frostegafird et al. [23] showed that during direct
2.5. Digestion and sequencing of clones extraction at least 75^90% of added DNA remained in the
soil, with the larger fraction the worst a¡ected. PCR-based
Cloned sequences were ampli¢ed with vector-speci¢c methods contribute several potential sources of error,
primers M13f-40 (5P-GTTTTCCCAGTCACGAC-3P) and some of which are unquanti¢able. These include contam-
M13r (5P-GGAAACAGCTATGACCATG-3P). The cy- ination from DNA originating from the reagents [24],
cling conditions used were 3 min incubation at 96‡C fol- PCR ampli¢cation bias [25], chimera formation [26] and
lowed by 35 cycles of 94‡C for 1 min, 57‡C for 1 min and variation in 16S rDNA copy number between bacterial
68‡C for 1 min 30 s. A ¢nal 2 min extension at 68‡C species [27]. The physico-chemical similarity of the sam-
completed the programme. ples, combined with their parallel processing, would have
To ensure symmetrical excision of inserts all restriction limited the e¡ects of some of the systematic sources of

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digests included EcoRI. Products were digested ¢rst with error upon our results. We also took other precautions,
HpaII and those, which contained no sites for this enzyme, such as gel puri¢cation of genomic DNA to reduce the
were given a second double digest with MboI. Fragments incidence of chimeras and minimising the number of
were separated in 3% NuSieve1 GTG1 (FMC Bioprod- PCR cycles to limit ampli¢cation bias.
ucts, Rockland, ME, USA) agarose gels and sizes were
determined using Kodak Digital Science1 1D Analysis 3.2. Coverage
software (Kodak, Rochester, NY, USA) using Hinf-di-
gested xX174 as the molecular size marker. The e⁄ciency of coverage of each sample can be esti-
Clones from the most dominant operational taxonomic mated using the equation for binomial sampling [28]. For
unit (OTU) were sequenced and a distance matrix ob- an OTU with a population density of p, the probability of
tained using ClustalW [20]. They were then taxonomically it being detected r times in a sample of N clones is
assigned using BLAST (NCBI) [21] with the GenBank, N!
EMBL, DDBJ and PDB databases. pr ð13pÞn3r :
ðN3rÞ!r!
2.6. Data analysis When r is set to zero, in order to calculate the chance of
not detecting an OTU, the equation simpli¢es to (13p)N .
The sum of each clone’s fragments was used as a means Thus, the chance of not detecting an OTU that had a true
of quality control; those with signi¢cant discrepancies relative abundance of, for example, 1/100 in a sample of
(more than T 70 bp from the expected total) were re-ana- 236 clones, was (131/100)236 or 9.3%. For OTUs with true
lysed and validated, corrected or discarded. Also, the ac- abundances of 1/250, 2/250 and 3/250 the chances of miss-
curacy of grouping was improved by using the relative ing them were 39%, 15% and 5.8%, respectively. Hence,
mobility of each fragment in relation to two common the sample size was large enough to detect essentially all
marker fragments (at 81 bp and 125 bp produced by OTUs whose true occurrence was just over 1% of the bac-
EcoRI cleavage of additional lengths of vector DNA).
Fragment patterns were sorted into OTUs according to
the number and sizes of fragments. Sorting was conserva-
tive : fragment patterns were assumed to be the same un-
Control
less distinctly di¡erent. 100
Cumulative number of OTUs

Statistica 5.1 (StatSoft Inc., Tulsa, OK, USA) was used

to calculate non-parametric and parametric tests to com-
pare the data sets. The chi-square test was limited to only Zinc-contaminated
comparing groups with expected frequencies greater than
50
¢ve.

3. Results and discussion

0
0 50 100 150 200
3.1. Limitations of the methods
Number of clones analysed
Fig. 1. Collector’s curves of OTUs of bacteria in zinc-contaminated (400
We chose to extract the bacterial fraction before lysis in mg Zn kg31 dry soil) and control (57 mg Zn kg31 dry soil) soils. In¢n-
order to minimise the amount of extracellular DNA ite diversity is indicated by the dashed line. Each mean curve was ob-
present. Courtois et al. [22] found no signi¢cant di¡erence tained by randomising the input order of clones 100 times.

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16 B.F. Mo¡ett et al. / FEMS Microbiology Ecology 43 (2003) 13^19

terial population in each sample. Of course, this ignores respectively, which was 40^60% higher than the unsludged
biases in the methods or di¡erences in gene copy number. control. However, a previous study found that the amount
of zinc released by a weak extractant, 0.1 M CaCl2 , was
3.3. Comparison of clone library diversity in control and 11.5 times higher [29]. Sandaa et al. [9] recorded a marked
zinc-contaminated soils reduction in bacterial diversity in a clay loam containing
Zn, Cu, Ni and Cd at 102, 21, 11 and 0.6 mg kg31 dry soil,
Collector’s curves of the OTUs identi¢ed in the two respectively, and Huysman et al. [30] found that in copper-
soils are shown in Fig. 1. Out of the 236 clones analysed contaminated loamy sands and sandy loams resistance to
in each soil, 120 OTUs were identi¢ed in the sludged con- metals among aerobic bacteria was discernible at only 20
trol and 90 in the zinc-amended and sludged soil, a de- mg Cu kg31 , one seventh the current EU limit.
crease of 25%. The decrease in diversity was accompanied The eight sequenced clones from the most abundant
by a decrease in evenness. While the control soil was pre- OTU, common to both soils, possessed a mean sequence
dominantly composed of rare OTUs (86% of them were similarity of 89%. All were high G+C Gram-positive bac-
represented by only one or two clones) the polluted soil teria of the Rubrobacter radiotolerans group and most

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had less rare OTUs (67% were ‘singletons’ and ‘double- closely matched environmental clones obtained from agri-
tons’) and signi¢cantly more clones in its two most dom- cultural soils [31,32]. The four clones from the zinc-con-
inant OTUs. A review of the suitability of indices for taminated soil were more tightly clustered than the four
quantifying diversity and evenness in these samples is giv- from the control soil.
en in Hill et al. [15]. OTUs unique to either soil (Table 1) accounted for 75%
More detail of each dataset is shown in Fig. 2 and Table of the total. The zinc-contaminated soil contained 48 while
1. The histogram, representing the 42 OTUs common to the control had 78, a signi¢cantly higher number
both soils, reveals that while 19 increased in relative abun- (P = 0.041 for z = 2.05 using the Wilcoxon test for two
dance in response to zinc-contamination only seven de- matched samples). The di¡erence was most pronounced
creased. This di¡erence was signi¢cant using the non-para- for OTUs represented by a single clone: while the polluted
metric sign test for matched pairs on the 26 groups soil had 35 unique singletons the control had 61. These
(P = 0.031 for z = 2.16) but not when the relative abundan- two main trends, a concentration of already abundant
ces were compared using either a paired t-test or a chi- OTUs and a dilution of rare OTUs, may be connected.
square test. This increased relative abundance of many This is because a sizeable change in absolute abundances
OTUs that were already relatively abundant in the control of one major subset of OTUs will automatically force a
soil may have occurred because the same, possibly r-se- compensatory change in relative abundances of the re-
lected, characteristics that favoured them in the disturbed mainder. Hence, both trends could be explained by one
environment of agricultural soil also bene¢tted them when action ^ either metal toxicity favoured common OTUs or
an additional stress was imposed. Alternatively, metals it disfavoured uncommon OTUs ^ or a mix of both.
naturally present in the sludge applied to the control
may have already selected for metal tolerance to some 3.4. Ecological implications
degree, and in the zinc treatment the e¡ect was simply
more pronounced. The sludged control contained 57, 13, In accord with Odum’s [14] predictions, both richness
7 and 0.3 mg kg31 dry soil of total Zn, Cu, Ni and Cd, and evenness of the bacterial population were lower in the

35

30
Clones per OTU

25

20

15

10

0
OTUs common to both soils
Fig. 2. Distribution of clones among 42 OTUs common to both the zinc-contaminated (dark bars) and the control soils (light bars).

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 B.F. Mo¡ett et al. / FEMS Microbiology Ecology 43 (2003) 13^19 17

zinc-contaminated soil than in the control. Chander and 120 300

Total number of clones
Brookes, using the same ¢eld experiment, found that zinc required for p = 0.05
toxicity had decreased total microbial biomass by 41% and 100 250
the ratio of microbial biomass to organic carbon by 53%

Clones per replicate

when compared with the sludged control. At the time, 80 200

Total no. of clones

however, the zinc concentration was 705 mg kg31 dry
soil whereas when we sampled, 8 years later, it had fallen 60 150
to 400 mg kg31 dry soil. In 1990, one of the lower-dose
treatments had a more comparable zinc level of 375 mg 40 100
Combination of replicates
kg31 dry soil and it showed a more modest impact: 6.0% and clones per replicate to
achieve p = 0.05
less microbial biomass and a 15% smaller microbial bio-
20 50
mass to soil organic carbon ratio.
Heavy metals a¡ect the decomposer community directly
0 0
and indirectly. Direct toxicity decreases the e⁄ciency of
0 5 10 15 20

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conversion of organic matter into new microbial biomass, Number of replicates
while the inhibition of plant growth and, hence, organic Fig. 3. The relationship between the number of replicate soil samples
matter inputs lowers microbial biomass further [33,34]. and the number of clones per replicate predicted to conclude, using a
The 1994 pea and 1995 spring barley crops can be used t-test, that a signi¢cant di¡erence in OTU diversity exists between con-
trol and zinc-contaminated soils. Rarefaction was used with data from
to illustrate the latter. For both, establishment was even
each soil to estimate the mean and variance of OTU diversity for repli-
but as they developed the plants on the high-zinc plots cates of di¡erent sizes. The number of replicates was than varied until
became yellow and stunted, leading to pea seed and barley the critical value of t (P = 0.05) was reached. The least laborious (in
grain yields that were s 2 t ha31 less than the untreated terms of total number of clones analysed) combination was predicted to
controls (P 6 0.01) which themselves had yields of approx- be four replicates of 47 clones in each treatment.

imately 4 and 5 t ha31 , respectively. Even so, Chander and

Brookes [34] concluded that direct e¡ects of toxicity were
the more important. Lawler et al. [35] found a signi¢cant labour involved in achieving some semblance of coverage,
correlation between total zinc or copper concentration and ARDRA is rarely replicated. This is a major unaddressed
inhibition of bioluminescence in an engineered strain of problem associated with these ¢ne-scale methods. We sug-
Pseudomonas £uorescens in metal-contaminated arable gest deciding upon an achievable upper limit of total
soils receiving regular applications of sewage sludge. Di- clones to be processed (perhaps 500^1000) and then, using
rect impact would range from direct killing of susceptible preliminary data, determine the most statistically powerful
species through to more subtle pressures such as changes combination of replicate samples and clones per replicate
in competitive ability [4], and since the stress is chronic it to use.
would continue to modify the decomposer community un- If a t-test is used to compare sites (i.e., two independent
til a new equilibrium was reached. Thereafter it would samples) any increase in replication above two per treat-
dynamically maintain this altered state. ment is especially bene¢cial due to its large dual in£uence
upon both the calculated value of t and the critical thresh-
3.5. Sampling strategies old value of t that must be exceeded in order to conclude
that a signi¢cant di¡erence exists. For two replicates per
Proving that a signi¢cant di¡erence exists between treat- treatment the critical threshold of t at the P = 0.05 level
ments using any chosen diversity measure requires repli- (two-tailed test) is 4.3, while for three replicates it is 2.8,
cation. However, due to the practical constraints of the 35% less. At the same time this increase in replication will,
in the absence of any other changes, raise the calculated
value of t by 22%. For four replicates these values are a
43% reduction in the required threshold combined with a
Table 1
Distribution of clones unique to either the zinc-contaminated or the 41% increase in calculated t, respectively. Clearly, any in-
control soils (n = 236 clones for each) crease in replication above the minimum will pay hand-
Clones per OTU Number of OTUs some dividends.
If OTU richness, S, is the test variable, rarefaction [36]
control zinc
can be used with the data from the control and zinc-pol-
1 61 35
luted soils to estimate, for each treatment, the mean OTU
2 12 8
3 4 3 diversity per replicate for a chosen number of clones ana-
4 1 2 lysed per replicate. These values can then be used in the
The greater diversity in the contaminated soil is highlighted in particular
formula for t, and the total number of replicates adjusted
by the greater number of OTUs which are represented by a single until t reaches the 0.05 level of signi¢cance. Thus, the total
clone. number of clones required to be processed can be esti-

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18 B.F. Mo¡ett et al. / FEMS Microbiology Ecology 43 (2003) 13^19

mated for di¡erent combinations of replicate samples and Acknowledgements

clones per replicate (Fig. 3). The equation for t for equal
numbers of replicates per treatment is This work was supported by The Nu⁄eld Foundation
E C 3E Zn which funded an Undergraduate Research Bursary (N.U.).
t ¼ rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi; We also sincerely thank Professor John McGinnety for
c 2C þ c 2Zn
granting a sabbatical period (B.M.) and Professor Shawn
K
Doonan for support during manuscript preparation
where E is the expected mean number of OTUs predicted (T.H.). We thank Kerry Walsh for method development
by rarefaction to be found in each replicate of n clones in and GRI, Braintree, Essex for sequence data.
the control (C) and zinc-contaminated (Zn) soils, c2 is the
variance of E (also predicted by rarefaction), and K the
number of replicates in each treatment. Degrees of free-
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