RESEARCH ARTICLE

Development of Microbial Diversity and Functional

Potential in Bauxite Residue Sand under
Rehabilitation
Natasha C. Banning,1,2 Ian R. Phillips,3 Davey L. Jones,4 and Daniel V. Murphy1
Abstract
Sustainable plant establishment on ore processing residues
requires development of a functional soil, which includes
the introduction of organic matter and reestablishment of
active microbial communities. This study investigated the
development of microbial diversity and function in residue
sand generated from alumina refining of mined bauxite
ore. The residue sand embankments underwent rehabilitation with native vegetation over time, allowing study of
a 3-year chronosequence using space-for-time substitution.
A coastal dune ecosystem was used as a natural alkaline
sand analog with which to compare residue properties.
Microbial biomass carbon in the residue sands were typically well below that of the coastal sand analog (<50 mg
kg1 in residue sand compared to circa 450 mg kg1 in
coastal sand). Although the size of the microbial biomass
appeared to be limited by the low organic matter content of the residue sand, a decline in microbial metabolic

Introduction
Refining bauxite ore to alumina, by digestion with hot concentrated sodium hydroxide, generates approximately 2 tonnes of
residue (nonsoluble ore components) per 3 tonnes of bauxite. World production of this waste has been estimated at
30 million mg per year (Menzies et al. 2004), the majority of which is disposed on land. Establishing a sustainable
vegetation cover on residue storage areas represents a significant challenge to alumina producers globally (Wehr et al.
2006; Courtney et al. 2009). In Western Australia alone, up to
60,000 tonnes of residue is produced per day. The approach
adopted by Alcoa of Australia (Alcoa) is to separate the residue
into coarse (>150 m) and fine (<150 m) fractions. The

1 Soil Biology Group, School of Earth and Environment M087, The University of
Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
2 Address correspondence to N. C. Banning, email natasha.banning@uwa.edu.au
3 Alcoa of Australia, Huntly Mine, PO Box 172, Pinjarra, WA 6208, Australia
4 Environment Centre Wales, School of the Environment and Natural Resources,
Bangor University, Gwynedd LL57 2UW, U.K.

2010 Society for Ecological Restoration International

doi: 10.1111/j.1526-100X.2009.00637.x

Restoration Ecology

quotient indicated a potential alleviation of microbial stress

with rehabilitation age. Despite the low microbial biomass,
the ability of the residue sand microbial community to
function with respect to the metabolism of added amino
acids developed rapidly. Contrary to our original hypothesis, the diversity of the bacterial and fungal community
also developed rapidly, and was similar to, or higher than,
the coastal sand analog in 0.5-year-old rehabilitation. However, the bacterial, and in particular the fungal, community
structure within residue sands were significantly different
to that of the coastal sand analog with shifts in community structure driven, in part, by changing physicochemical
conditions.
Key words: amino acid turnover, ARISA, bauxite residue

sand, chronosequence, microbial biomass, soil organic

matter.

coarse fraction (residue sand) is used to construct embankments, which form storage areas to contain the fine fraction
(residue mud). Alcoas current rehabilitation program focuses
on developing a sustainable vegetation cover on the outer
residue sand embankments with the aim of improving physical stability, controlling dust emissions, minimizing alkalinewater discharge and reducing visual impact on the surrounding
community.
Similarities in general soil physical and chemical characteristics and climates between bauxite residue sand embankments and the Quindalup Dune coastal system of southwest
Western Australia were identified previously, and it was
established that plant species native to the Quindalup Dune
sands were capable of growing in residue sand (Bell et al.
1993; Jasper et al. 2000). To date, approximately 200 ha
of Alcoas residue storage areas in Western Australia have
been rehabilitated using native vegetation, with a projected
1,300 ha requiring rehabilitation by 2030. The Quindalup
Dune system is comprised of calcareous sands resulting in
a free-draining, relatively infertile, naturally alkaline soil
(McArthur 2004) and has been identified as providing a suitable analog for comparing residue rehabilitation performance

Microbial Diversity and Functional Potential in Bauxite

(Jasper et al. 2000). However, residue sand rehabilitation

represents a significant challenge as this material is highly
alkaline (pH 1012; comparatively higher than the coastal
sand pH values of 78), saline, sodic, has poor water
retention and contains virtually no organic matter and has
numerous nutrient deficiencies (Gherardi & Rengel 2001;
Eastham & Morald 2006). To date, rehabilitation performance has been variable and there is limited information on
changes in the key chemical, physical, and microbial characteristics of residue sand following rehabilitation and their
impact on the vegetation cover. Furthermore, there is very
limited knowledge internationally concerning the long-term
growth and sustainability of residue rehabilitation (Courtney
et al. 2009).
Successful rehabilitation must involve the development of
microbially driven organic matter turnover and mineral nutrient cycling for the long-term provision of plant nutrients.
Studies of primary successional ecosystems have suggested
that microorganisms are also critical to early ecosystem development, due to functional abilities such as nitrogen fixation, organic matter turnover, mycorrhizal symbiosis, and
potential facilitation of plant establishment (Chapin et al.
1994; Hodkinson et al. 2002; Walker et al. 2003). Increases
in soil microbial biomass, activity, and bacterial diversity
with successional age have been demonstrated in a variety
of ecosystems during primary succession (Sigler & Zeyer
2002; Nemergut et al. 2007) and restoration of highly disturbed sites (Banning et al. 2008b). We know of no published studies investigating the successional development
of microbial community structure and function in bauxite
residue sand under rehabilitation. Although it is clear that
the geochemical conditions in residue sand will result in
nutrient limitations to plant growth (Eastham et al. 2006),
the extent to which these conditions limit the development of a diverse and functional microbial community is
unknown.
The aim of this study was to characterize the early development of the microbial community in bauxite residue sand from
the time it is initially deposited in a residue storage area, up to
3-year-old residue sand embankments that had undergone rehabilitation. The effect of organic matter addition (composted
manure [CM]) was also investigated in 2-year-old residue rehabilitation. All residue sand properties are compared to that of
a Quindalup Dune coastal sand to provide a natural analog or
target ecosystem for the rehabilitation. We hypothesized that
the residue sand would be initially sterile, but that microbial
biomass, function (respiration, mineralization of added amino
acids), and phylogenetic diversity would increase with rehabilitation age, but remain lower than the coastal sand. These
increases are expected to be driven primarily by decreases in
pH and the input of organic matter through plant growth. We
also hypothesized that a less diverse microbial community with
a unique microbial community structure would develop within
the residue sand in comparison with the natural coastal sand
analog.

Methods
Site Description and Sampling

Residue sand was sampled from the Kwinana Residue Storage

Area (32 12 S, 115 49 E) and coastal sand of the Quindalup
Dune system was sampled from the Port Kennedy Scientific
Park (32 22 S, 115 44 E) approximately 40 km and 57 km,
respectively, south of Perth, Western Australia. The climate
of the region is of mediterranean-type with a mean annual
rainfall of 760 mm, which mainly falls during the winter
months, and a mean annual temperature range of 1029 C.
The vegetation of the sampled area of the Quindalup Dune
coastal sand is characterized by Open Heath (1.7 m in
height) of Olearia axillaris, Melaleuca systena, and Spyridium
globulosum over open mixed herbs and sedges (Outback
Ecology 2006). Samples were collected in 2006 during winter
(July), the time of year that rehabilitation of residue sand
embankments is carried out.
Fresh residue sand (0-year-old) was sampled directly from
the pipeline being used to hydraulically construct an embankment, in order to analyze pre-amendment residue characteristics. Residue sand embankments that had undergone Alcoas
standard rehabilitation prescription 0.1, 0.5, 2, and 3 years
previously were also sampled. The standard prescription
involves incorporation of phospho-gypsum (a phosphate-rich
by-product of superphosphate fertilizer manufacturing) at 225 t
ha1 to approximately 1,500 mm depth, to reduce pH, incorporation of di-ammonium phosphate fertilizer (2,751 kg ha1 )
to approximately 200-mm depth followed by broadcasting of
seed. The seed mix contains 55 plant species native to the
coastal, predominantly the Quindalup Dune, ecosystems of
Western Australia. A 30-mm depth of woody mulch is then
applied to the sand surface as a dust suppressant. Seedlings of
species that cannot establish from seed are planted by hand.
In addition to residue treated with the standard prescription,
2-year-old residue rehabilitation plots that had been amended
with CM at 100 m3 ha1 were sampled to determine the effect
of organic matter addition on the residue sand. The chemical
composition of the CM is given in Eastham et al. (2006). A
comparison of vegetation characteristics of 2-year-old residue
and the coastal sand analog is given in Table 1.
With the exception of 0-year-old residue (for which three
samples were collected from the one available pipeline), a
composite sample from randomly located cores was collected
from 0 to 50 mm depth, excluding surface woody mulch or
litter layers at all sites. Three replicate sites were sampled per
treatment, at a minimum distance of 25 m apart. Samples were
stored at 4 or 20 C (for DNA extraction) prior to analysis.
pH, Plant Available Water and Particle Size Analysis

The pH of all samples was determined on a saturated paste

using deionized water as the background solution. Soil moisture content was determined gravimetrically by oven drying
10 g subsamples at 105 C for 24 hours. Soil water holding
capacity was determined at field capacity (5 kPa) and permanent wilting point (1500 kPa), as described by Rayment

Restoration Ecology

Microbial Diversity and Functional Potential in Bauxite

Table 1. Vegetation characteristics of 2-year-old residue rehabilitation

and coastal sand analog site within the Quindalup Dune System. Mean
values (n = 3) are shown.
Vegetation Characteristicsa
Cover
Density
(Plants m2) (%)

Bauxite residue sandb

2-year-old; standard amendment
2-year-old; composted manure
Coastal sand analogc

1.3
1.7
10

Richness

59
50

19 (/36 m2 )
18 (/36 m2 )

63

18 (/50 m2 )

a Data for bauxite residue sand rehabilitation are for native plant species only, weed
species present are not included. Vegetation plots were monitored 4 months after soil
sampling.
b Standard amendment and composted manure prescriptions are described in
Methods.
c Data from Outback Ecology (2006).

and Higginson (1992), and results expressed as plant available water (water content at 5 kPa minus water content at
1500 kPa). Particle size analysis was undertaken as outlined
in Rayment and Higginson (1992).
C, N, and P Pools

Total organic C and total N were determined on oven-dried,

finely ground soil by wet oxidation using the Walkley-Black
method (Nelson & Sommers 1996) and by total combustion
using a CHN analyser (Elementar, Analysensysteme, GmbH,
Hanau, Germany), respectively. Potentially mineralizable N
(PMN) was determined in triplicate by the accumulation
of NH+
4 N during 7 days anaerobic incubation (10 g dry
weight equivalent: 40 mL H2 O) at 40 C (Keeney & Bremner
1966). Inorganic N concentrations were determined colorimetrically by automated flow injection analysis (Skalar Analytical
B.V., Breda, The Netherlands). Bicarbonate-extractable (Colwell) P was determined spectrophotometrically (Rayment &
Higginson 1992).
Microbial Biomass C and Respiration

Microbial biomass C was determined by chloroform-fumigation and K2 SO4 extraction in 0.5 M K2 SO4 (Vance et al.
1987). Oxidizable-C in the soil extracts was measured using a
Shimadzu 5000A Total Oxidisable C analyzer with an acidified
sparging step to remove inorganic carbon (Shimadzu, Corp.,
Kyoto, Japan). Microbial biomass was calculated from the
difference between fumigated and nonfumigated C using a kEC
value of 0.45 (Jenkinson et al. 2004). Microbial quotients are
expressed as the ratio of microbial biomass C to total soil
organic C.
Microbial respiration was determined by incubation of 50 g
(dry weight equivalent) of sample adjusted to 40% water holding capacity at 15 C. Headspace CO2 C was measured after 5
and 10 days using a Series 225 infrared gas analyzer (IRGA;
The Analytical Development Co. Ltd, Hoddeston, U.K.) by
comparison with a known standard (4.94 0.1% CO2 in He;
Restoration Ecology

BOC Gases Ltd, Sydney, Australia). A control treatment without soil was used to adjust for atmospheric CO2 concentration.
A subset of residue and coastal sands were sterilized (121 C
for 30 minutes) and incubated under the conditions described
above to correct for any abiotic CO2 evolution, of which none
was recorded. Metabolic quotients are expressed as the rate of
microbial respiration per unit of microbial biomass C.
Amino Acid Turnover

To determine the rate of amino acid turnover in soil, a mixture

of 18 uniformly 14 C-labeled amino acids was added to each
sample and the subsequent evolution of 14 CO2 measured
over 7 days as described in Jones et al. (2005b). Briefly, 100
L of a mixture containing equimolar concentrations of 18
14 C-labeled l-isomeric amino acids (ICN Pharmaceuticals,
Inc., CA, U.S.A.), at a total amino acid concentration of
10 mM and specific activity of 1.3 kBq ml1 , was added
dropwise to 5 g of sample contained in a polypropylene tube.
This addition rate is equivalent to 3.36 mg N kg1 of soil.
A 1 M NaOH trap was suspended inside the tube to absorb
evolved 14 CO2 . The NaOH trap was then replaced following
0.75, 1.5, 3, 6, and 24 hours and the 14 CO2 contained in
the traps determined by liquid scintillation counting (Wallac
1404 EG&G Ltd, Milton Keynes, U.K.). The half-life of
the amino acid in soil was calculated by fitting a double
first order exponential decay equation to the 14 CO2 evolution
data as described in Boddy et al. (2007). Kinetic analysis
was undertaken with the software package Sigmaplot 8.0 for
Windows (SPSS, Inc., Chicago, IL, U.S.A.).
Microbial Community Profiling by Automated Ribosomal
Intergenic Spacer Analysis (ARISA)

Total soil DNA was extracted from a 0.8 g sample using

the UltraClean soil DNA isolation kit (Mo Bio Laboratories,
Inc., Carlsbad, CA, U.S.A.) with cell lysis performed using a
Mini Bead Beater (BioSpec Products, Inc., Bartlesville, OK,
U.S.A.) at 2,500 rpm for 2 minutes. The bacterial intergenic
spacer region between the small and large subunits of rRNA
genes was amplified using primers S-D-Bact-1522-b-S-20
(5 -TGCGGCTGGATCCCCTCCTT) and L-D-Bact-132-a-A18 (5 -CCGGGTTTCCCCATTCGG; Normand et al. 1996).
Amplified sequences contained the spacer region plus approximately 150 bp of the 23S rRNA gene (Kennedy et al. 2005).
The two fungal intergenic spacer regions spanning the 5.8S
rRNA gene were amplified using primers ITS1-F (5 -CTTGGT
CATTTAGAGGAAGTAA; Gardes v & Bruns 1993) and
ITS4 (5 -TCCTCCGCTTATTGATATGC; White et al. 1990).
Amplified sequences contained the two spacer regions, the
5.8S gene and a section of the 28S rRNA gene (Gleeson
et al. 2005).
Polymerase chain reaction (PCR) mixtures contained 1 reaction buffer (Bioline Aust Pty Ltd., Alexandria NSW, Australia), 15 pmol of each primer (GeneWorks, Hindmarsh SA,
Australia), 0.25 mM each dNTP, 2.5 mM MgCl2 , 1.25 U
Taq DNA polymerase (Bioline), 10 g bovine serum albumen

Microbial Diversity and Functional Potential in Bauxite

(Ambion, Austin, TX, U.S.A.) and between 1.0 and 5.0 L

of template DNA (diluted by 1 in 10 where necessary for
improved amplification) made up to 50 L with molecular
grade water. In both reactions, the forward primer was 5
labeled with 6-carboxyfluorescein (FAM). Thermocycling conditions were as follows: 94 C for 1 minute followed by 30
cycles of 94 C for 1 minute, 55 C (or 53 C for fungal PCRs)
for 30 seconds, 72 C for 1 minute with a final extension step
of 72 C for 5 minutes. Duplicate PCR amplifications were
pooled and cleaned with Wizard PCR Preps DNA purification
system (Promega Corporation, Alexandria NSW, Australia).
Intergenic spacer lengths were determined by electrophoresis using an ABI 3730 automated sequencer with GeneScan
1200 LIZ (Applied Biosystems, Foster City, CA, U.S.A.) size
standards containing 72 fragments ranging from 20 to 1200 bp
at intervals of approximately 20 bp. Electropherogram analysis was performed using GeneMapper v4.0 software (Applied
Biosystems). Fragments smaller than 200 bp and larger than
1200 bp were excluded from the profiles. Profiles of ribotype
abundances (based on peak heights) were created using the
program RiboSort (Scallan et al. 2008), within the statistical
package R version 2.6.0 (Chambers 2008). Fragment sizes that
differ by less than 0.5 bp were considered to be identical ribotypes. Only fragments with fluorescence greater than 1% of the
total fluorescence summed across all samples were included.
Final profiles contained a total number of fragments across all
samples of 538 (bacterial) and 220 (fungal).
Statistical Analyses

Significant differences in measured properties between bauxite

residue and coastal analog sand samples were determined by
one-way analysis of variance (ANOVA), using Tukeys multiple pairwise comparisons using GenStat v9.2 (VSN International Ltd, Hemel Hempstead, U.K.) at a 5% significance level.
The Shannon diversity index (H) was calculated for the
ARISA profiles according to the equation: H =  pi ln (pi );
where pi is the DNA fragment abundance as a proportion of the
total of all DNA fragments for a sample. Multivariate analyses
of ARISA profiles were based on Bray-Curtis dissimilarities
on log (x + 1) transformed data, standardized by sample sum.
Bray-Curtis was chosen as it is unchanged by inclusion or
exclusion of individual variables (i.e. DNA fragments) which

are jointly absent between samples. Tests of the null hypothesis

that there are no differences among a priori defined groups
were performed by permutational multivariate analysis of
variance (PERMANOVA) using 999 permutations (Anderson
2001). Differences were interpreted as significant at p values
greater than 0.05.
Relationships between ARISA profiles and soil properties were analyzed using distance-based multivariate multiple
regression (DistLM) using a forward selection procedure to
take into account covariance between properties. The fitted
model was visualized using distance-based redundancy analysis (dbRDA). All multivariate statistical routines were carried
out using PRIMER 6 & PERMANOVA+(Primer-E Ltd., Plymouth, U.K.) as described in Clarke and Gorley (2006).

Results
Physical and Chemical Characteristics

Both the residue and coastal sands contain approximately 93%

sand (502000 m) with, on average, 5% silt (250 m)
and 2% clay (<2 m) in residue sand, and 2% silt and 4%
clay in coastal sand, as determined by particle size analysis.
Field bulk densities of the two sands were similar (mean of
1.25 g cm3 ). Plant available water, was similar in all residue
sands but less than half that of the coastal sand (Table 2).
Bauxite residue sand is typically highly alkaline due to the
presence of a range of constituents including free caustic
(Snars et al. 2004). Residue sand pH decreased following
gypsum incorporation and with age of rehabilitation (Table 2),
which is attributable to the precipitation of carbonate and
leaching of soluble alkalinity, respectively. The residue sand
pH of 3-year-old rehabilitation was equivalent to the pH of
the coastal sand analog (Table 2). The total organic C content
of all residue sands was substantially lower than the coastal
sand analog. Furthermore, concentrations of total N and the
organic N pool that are potentially mineralizable (PMN) were
also much lower in all residue sand samples compared with
the coastal sand. However, amendment with CM increased the
organic C, total N and PMN quantities compared with residue
amended with inorganic fertilizer in 2-year-old rehabilitation
(Table 2). The concentration of available P in residue sand

Table 2. Physical and chemical properties of bauxite residue sand and coastal sand analog. Mean values (n = 3) with the same letter are not significantly
different (p < 0.05).
Site

Bauxite residue sand

0
0.1
0.5
2
2 CM
3
Coastal sand analog

Available Water (gwater gsand 1 )

pH

Organic C (%)

Total N (%)

Available-P (mg kg1 )

PMN

0.21a
0.22a
0.23a
0.21a
0.23a
0.21a
0.48b

10.04a
9.91a
7.74b
7.22bc
7.65b
7.56bc
7.00c

0.12a
0.11a
0.20a
0.16a
0.56b
0.22a
0.94c

0.01a
0.03ab
0.03ab
0.02a
0.07b
0.01a
0.17c

3.63a
4.56ab
48.6c
39.5bc
71.9c
3.77a
0.79a

0.03a
0.75ab
0.70ab
0.27a
6.89b
0.81ab
32.9c

CM, composted manure; PMN, potentially mineralizable nitrogen.

Restoration Ecology

Microbial Diversity and Functional Potential in Bauxite

was also low initially, and similar to the coastal sand, but
10- to 20-fold higher in 0.5- and 2-year-old embankments.
The type of fertilization (inorganic vs. compost) did not affect
P concentrations (Table 2).

Amino Acid Mineralization

The biodegradation of added amino acids in all the samples

followed typical biphasic mineralization kinetics with an
initial rapid rate phase of 14 CO2 evolution followed by a
slower secondary phase (Fig. 2; Jones et al. 2005b). The only
exception was the 0-year-old residue in which almost zero
breakdown of the amino acids to 14 CO2 was recorded. After
24 hours, 2730% of the added 14 C label was recovered as
14 CO in the residue sand samples and 36% in the coastal
2
sand. After fitting a double first order kinetic equation to the
mineralization rates, the half-life of the amino acid pool was
found to range between 6.2 1 hour (0.1-year-old) and 3.8
0.1 hour (2-year-old) in the residue sand samples. Differences
in amino acid half-life between residue samples were not
significant, but all were lower than the coastal sand which had
a mean half-life of 1.3 0.1 hour. Amino acid half-life was
weakly negatively correlated to the differences in microbial
biomass (r 2 = 0.51; p < 0.05), to a lesser extent microbial
basal respiration (r 2 = 0.25; p < 0.05) and weakly positively
correlated to pH (r 2 = 0.47; p < 0.05).

Microbial Biomass, Respiration, and Quotients

Bacterial and Fungal Diversity and Community Structure

Despite the extremely low microbial biomass C of the 0- and

0.1-year-old residue sands, amplifiable DNA was extracted
from all samples. Diversity of the bacterial community in
residue sand increased rapidly with rehabilitation age, and was

(a)

600

(b)

30

-1

Microbial biomass C (mg kg )

The microbial biomass C, microbial quotient and respiration

of all residue sands were substantially lower than the coastal
sand analog (Figs. 1ac). The observed trend in microbial
biomass C of residue sand, both in absolute mass and as a quotient, was an increase with rehabilitation age. Linear regression
analysis showed a strong positive correlation between microbial biomass C and organic C (r 2 = 0.85; p < 0.001), total
N (r 2 = 0.82; p < 0.001) and a weak negative correlation to
pH (r 2 = 0.22; p = 0.03). The values for microbial biomass
and microbial quotient of 2-year-old CM amended residue
sand were higher on average than those for 2-year-old standard amended residue sand, but variable and the differences
were not significant. However, the microbial respiration rate
was higher in the CM amended residue sand compared to the
standard amendment. The metabolic quotient of residue sand
showed a decreasing trend with rehabilitation age (Fig. 1d),
although the microbial biomass of 0- and 0.1-year-old residue
sand was very low and close to detection limits, resulting in
highly variable metabolic quotients. The metabolic quotient of
2- and 3-year-old residue was similar to the coastal sand.

25
Respiration
-1 -1
(mgCO 2-C kg d )

500
400
300
200
100
0

15
10
5
0

(c)

6.0
5.0

Metabolic quotient

Microbial quotient (%)

20

4.0
3.0
2.0
1.0
0.0
0

Rehabilitation age (y)

(d)

1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0

Rehabilitation age (y)

Figure 1. Microbial characteristics of bauxite residue sand sampled from embankments of a residue storage area with different ages of rehabilitation
following a standard amendment procedure ( ) or a composted manure treatment ( ), in comparison with a coastal sand analog (). Symbols or lines
represent means SE (n = 3). Metabolic quotient is in mg CO2 -C mg microbial-C1 d1 .

Restoration Ecology

(a)

5.0

0
50

0.1

Diversity index (H')

0.5
2

40

3
2CM
Coastal sand

30

20

4.8
4.6
4.4
4.2
4.0
0

10

(b)

4.0
0
0

12

18

24

Incubation time (h)

Figure 2. Cumulative 14 CO2 production arising from the mineralization

of 14 C-labeled amino acid in bauxite residue sand under different ages of
rehabilitation, and a coastal sand analog. Symbols represent means SE
(n = 3).

Diversity index (H')

14

CO2 evolution (% of total

14

C-amino acid added)

Microbial Diversity and Functional Potential in Bauxite

3.0

2.0

1.0

equivalent to that of the coastal sand by 0.5 years (Fig. 3a).

However, there were significant differences in community
structure between all pairwise comparisons, with the exception
of 0.1- and 0.5-year-old residue (p = 0.06), 0.5- and 3year-old residue (p = 0.06) and 2- and 3-year-old residue
(p = 0.07). Differences in bacterial community structure were
correlated to all of the measured soil properties, as shown in
Figure 4a. Multivariate multiple regression using a forward
selection model to take into account co-correlation among
soil properties suggested that six variables (pH, organic C,
respiration, available water, available P, microbial quotient)
best explained the variation in bacterial community structure,
cumulatively accounting for 57% of variation. Individually,
pH explained the highest proportion of variation at 14%.
Fungal community diversity also increased rapidly with
residue age, such that residue sand 0.5-year-old and older had
higher values for diversity than the coastal sand (Fig. 3b). All
residue samples had a significantly different fungal community
structure from the coastal sand; however, there were no
differences in fungal community structure between residue
samples, with the exception of 2-year-old CM amended
residue and 0- and 0.1-year-old residue. Differences in fungal
community structure were correlated to all of the measured
soil properties (Fig. 4b). Multivariate multiple regression using
a forward selection model suggested that three variables
(available water, pH, and available P) best explained the
variation, cumulatively accounting for 31% of variation.

Discussion
A potential limiting factor in successful residue rehabilitation,
and one not easy to directly manipulate, is a limitation in the
type and number of microbial species from the surrounding
environment that are able to colonize and grow within the

Rehabilitation age (y)

Figure 3. Shannon diversity (H) of bacteria (a) and fungal
(b) communities based on ARISA profiles of bauxite residue sand
following standard amendment ( ) or amendment with composted
manure ( ) in comparison with the coastal sand analog (). Note
different y-axis scales used in (a) and (b).

unique geochemical conditions of residue sand. However,

contrary to our initial hypothesis, we demonstrated a rapid
increase in bacterial and fungal diversity (between 2 and
6 months) in residue sand to a level comparable to the
coastal sand analog. Although microbial community profiling
techniques, including ARISA, do not provide an accurate
assessment of true diversity, as many rare species will not
be detected, comparative interpretations can be made (Bent &
Forney 2008). As a DNA-based profiling technique was used,
we cannot know whether all the species detected were living
or functionally active. Nevertheless, the rapid development of
a diverse microbial community suggests that rehabilitation of
residue sand embankments is unlikely to be constrained by a
lack of microorganisms able to colonize and survive within
the residue sand.
Increases in the diversity of bacterial communities with
successional development have been demonstrated previously
in deglaciated soil chronosequences (Nemergut et al. 2007),
and this pattern mimics plant dynamics found during primary
succession (del Moral & Wood 1993). Laboratory studies
investigating the recolonization of sterilized soil, have also
demonstrated that recovery of microbial diversity can be
extremely rapid, in the order of 23 days (Marschner &
Rumberger 2004; Wertz et al. 2007). The development of
the microbial community structure is likely to be strongly

Restoration Ecology

Microbial Diversity and Functional Potential in Bauxite

RDA2 (22.2% of fitted, 15.4% of total variation)

40

Rehabilitation age (y)

(a)

0
0.1
0.5
2 CM
2
3
Coastal sand

20
Microbial biomass
Organic C
Avail-P

Microbial quotient

Microbial resp

-20

pH
Avail-water

-40

-60
-40

-20
0
20
40
RDA1 (24.4% of fitted, 16.9% of total variation)

60

40

RDA2 (17.6% of fitted, 11.4% of total variation)

(b)

20
Microbial biomass
Microbial resp
Microbial quotient

PMN

Avail-P

Metabolic quotient

-20

-40

pH

-60
-40

-20
0
20
40
RDA1 (23.2% of fitted, 15% of total variation)

60

Figure 4. Ordination plot illustrating the relationships between (a) bacterial and (b) fungal community structures, determined by ARISA, and residue and
coastal sand properties tested using dbRDA. Vectors show properties with a correlation greater than 0.2.

regulated by the availability of limiting resources (Bardgett

et al. 2005) and other edaphic factors (Fierer & Jackson
2006). In this study, all of the measured residue and coastal
sand properties were found to explain a significant proportion

Restoration Ecology

of variation in bacterial and fungal community structures.

Redundancy analysis (dbRDA) indicated that changes in pH
were particularly important in separating the bacterial and
fungal community structures in 0- and 0.1-year-old residue

Microbial Diversity and Functional Potential in Bauxite

sand from all other samples. Many previous studies have

demonstrated the importance of pH as a driver of bacterial
community structure and diversity, at local scales as well
as continental scales (Fierer & Jackson 2006). Extremes
of pH can restrict microbial community diversity directly
by imposing stress on colonizing microorganisms, as well
as indirectly through the regulation of dissolved organic
matter availability (Cookson et al. 2007). Thus, management
practices aimed at reducing pH are an important factor
in altering microbial community structure and increasing
diversity in residue sand.
Fertilization practices resulted in a large increase in available P concentrations in residue sand, although some contribution from the gypsum (phospho-gypsum) cannot be discounted.
Although excess available P may inhibit arbuscular mycorrhizal fungi (Marschner & Dell 1994), P fertilizer addition
has been found recently to increase fungal phylotype richness in Australian pasture soils (Wakelin et al. 2009). The
ITS1-F primer used in this study was designed to target basiodiomycetes and ascomycetes (Gardes & Bruns 1993), which
are the main fungal partners in ectomycorrhizal associations.
Thus, the large increase in available P may have contributed
to the greater fungal diversity found in older residue sand
rehabilitation compared with the coastal sand analog.
The microbial community within residue sand also demonstrated the functional potential to rapidly turnover a readily
degradable nutrient source (amino acids). The uptake and
turnover of amino acids by microorganisms is a key component
of terrestrial N cycling. As in other N-limited environments,
the residue and coastal sands in this study contained low levels
of inorganic N (<5 mg kg1 ), and therefore strong competition for organic N between microorganisms, and between
microorganisms and plants, is likely to exist (Bardgett et al.
2003; Jones et al. 2005a). Although the decomposition rate of
amino acids can differ between soil types, it is generally very
rapid in surface soils (Jones 1999). The average half-life of
the soil amino acid pool across a global soil transect (n = 40)
was 1.8 0.1 hour (Jones et al. 2009).
The amino acid half-life within residue sands was similarly
rapid (with the exception of 0-year-old residue) demonstrating
rapid development of mineralization capacity in the microbial
community. Furthermore, it indicates that utilization of amino
acids would not be a rate-limiting step in the development of
N cycling processes within the residue (although the amount
of amino acid available may be). The differences in amino
acid half-lives were not correlated to the changes in bacterial
or fungal community structure, most likely a consequence
of substantial functional redundancy among heterotrophic
microorganisms (Jones et al. 2005b).
The rapid development of a microbial community within
residue sand may have a role in facilitating early establishment
of the native vegetation. However, the microbial biomass and
in situ activity is most likely constrained by the lack of organic
matter, and as such will be dependent on plant net primary
production and the input of organic matter through root
exudation, root biomass turnover, and litterfall. As expected,
microbial biomass C in the fresh residue sand from the

discharge pipe was close to zero. Despite little change in

total organic C or N contents, microbial biomass C increased
with rehabilitation age, which may be an early indicator of
longer term positive trends in the total organic C (Sparling
1992). The microbial quotient in residue sand also displayed
an increasing trend with rehabilitation age, although all values
were below 2.0, proposed by Anderson (2003) to be critically
low in soils at neutral pH. As hypothesized, the microbial
biomass, respiration, and microbial quotient in residue sand
rehabilitation remained significantly lower than the coastal
sand analog.
In contrast, the metabolic quotient in residue sand recovered
rapidly to that of the coastal sand analog (0.057 mg CO2 C
mg microbial-C1 d1 ), which was similar to that of forest
soils within the same climactic zone (Banning et al. 2008b).
Metabolic quotients can be influenced by the cellular efficiency
of C utilization, which in turn is influenced by pH or
other stress factors, and the extent of substrate limitation of
the microbial biomass as a whole (Wardle & Ghani 1995).
A microbial cell under pH stress will partition more C into
the respiration pathway, rather than making new biomass,
resulting in increased metabolic quotients (Anderson 2003).
The rapid decline in metabolic quotient between 0.1- and 0.5year-old residue sand is most likely a consequence of the
rapid decline in pH (from 9.9 to 7.7), a result of gypsum
incorporation (Courtney & Timpson 2004) and most likely
leaching over time, and therefore indicates a declining level
of microbial stress.
The use of organic fertilizer in residue sand rehabilitation
was found to promote the development of an active microbial
biomass. The compost may have also provided an inoculum of
microbial species better able to grow in residue sand. However,
the compost treatment did not provide any clear benefit to plant
establishment (i.e. plant density, cover, or richness) which is
in agreement with a previous study (Eastham et al. 2006).
This may be the result of competitive interactions between the
microorganisms and plants for the added nutrients, particularly
during the early establishment phase. Microorganisms, particularly within the rhizosphere, can alter rates of nutrient supply
that may benefit plant diversity and productivity or may be
detrimental, for example, by increasing competition for N in
already nutrient-limited systems (van der Heijden et al. 2008).
During succession or active ecosystem restoration, natural
and disturbed ecosystems do not necessarily converge with
time (Harris et al. 2005). In this study, the analog ecosystem does not represent a pre-disturbance state, as the residue
sand is a nascent substrate, but provides a natural analog in
terms of plant species composition, soil textural properties, and
alkalinity (Jasper et al. 2000). The finding that certain microbial characteristics of the residue sand, including microbial
and metabolic quotients, amino acid mineralization potential
and bacterial diversity, become more similar to the coastal
sand with rehabilitation age suggest the rehabilitation is on a
desired trajectory. The shifts in microbial community structure in residue sand suggested an increasing similarity to the
coastal sand with rehabilitation age with respect to bacteria
(although maximum similarity to the coastal sand was still

Restoration Ecology

Microbial Diversity and Functional Potential in Bauxite

only 28%) but not fungi. Studies of rehabilitation chronosequences in other ecosystems (e.g. forests, grasslands) have
also identified shifts in microbial community structure toward
their respective reference sites (McKinley et al. 2005; Banning
et al. 2008a; Glen et al. 2008). However, given inherent differences between residue and coastal sands (e.g. geographic location, mineralogy) the microbial community structure in residue
sand under rehabilitation is perhaps unlikely to converge with
the coastal sand analog in the future and such convergence may
not be necessary for the development of a functional ecosystem. Further exploration of functional attributes of the residue
sand microbial community would benefit the assessment of
rehabilitation sustainability.
Implications for Practice
Rehabilitation strategies for bauxite residue sand used

by Alcoa of Australia, as described in this study, are

sufficient to allow colonization by a diverse range of
microorganisms.
The microbial biomass supported in residue sand is
strongly limited by the low levels of organic matter.
However, improved understanding of the competition
between microorganisms and plants for nutrients is
required to determine whether organic matter addition
(e.g. compost) is beneficial to residue sand rehabilitation.
Broad-scale heterotrophic functions of microorganisms
(e.g. amino acid utilization) are unlikely to be ratelimiting steps in the establishment of nutrient cycling
processes in residue sand under rehabilitation.
As residue sand is a nascent substrate, not all characteristics of residue sand under rehabilitation are likely to converge with any chosen natural analog ecosystem and thus
criteria for rehabilitation success will also depend on a
general understanding of functional ecosystem attributes

Acknowledgments
This research was supported under Australian Research
Councils Linkage Projects funding scheme (project number
LP0776593) with grant partner Alcoa of Australia. We thank
Ms M. LeRoy, Ms A. Byrne, Dr Y. Sawada, and Mr M. Smirk
for valuable technical support. Fragment analysis for ARISA
profiling was carried out by the Australian Genome Research
Facility. The Soil Biology Group forms part of the Terrestrial
Ecosystems Research Initiative at The University of Western
Australia.
LITERATURE CITED
Anderson, M. J. 2001. A new method for non-parametric multivariate analysis
of variance. Austral Ecology 26:3246.
Anderson, T. H. 2003. Microbial eco-physiological indicators to assess soil
quality. Agriculture Ecosystems & Environment 98:285293.
Banning, N. C., C. D. Grant, A. Grigg, E. L. Brodie, G. L. Andersen, D. B.
Gleeson, and D. V. Murphy. 2008a. Successional changes in soil microbial communities in post-mining rehabilitation forest ecosystems. 12th
International Symposium on Microbial Ecology. Cairns, Australia.

Restoration Ecology

Banning, N. C., C. D. Grant, D. L. Jones, and D. V. Murphy. 2008b. Recovery

of soil organic matter, organic matter turnover and nitrogen cycling
in a post-mining forest rehabilitation chronosequence. Soil Biology &
Biochemistry 40:20212031.
Bardgett, R. D., T. C. Streeter, and R. Bol. 2003. Soil microbes compete effectively with plants for organic-nitrogen inputs to temperate grasslands.
Ecology 84:12771287.
Bardgett, R. D., G. W. Yeates, and J. M. Anderson. 2005. Patterns and determinants of soil biological diversity. Pages 100119 in R. D. Bardgett,
M. B. Usher, and D. W. Hopkins, editors. Biological diversity and function in soils. Cambridge University Press, Cambridge, United Kingdom.
Bell, D. T., C. F. Wilkins, P. G. Moezel, and S. C. Ward. 1993. Alkalinity
tolerance of woody species used in bauxite waste rehabilitation, Western
Australia. Restoration Ecology 1:5158.
Bent, S. J., and L. J. Forney. 2008. The tragedy of the uncommon: understanding limitations in the analysis of microbial diversity. ISME J 2:689695.
Boddy, E., P. W. Hill, J. Farrar, and D. L. Jones. 2007. Fast turnover of
low molecular weight components of the dissolved organic carbon
pool of temperate grassland field soils. Soil Biology & Biochemistry
39:827835.
Chambers, J. M. 2008. Software for data analysis: Programming with R.
Springer, New York.
Chapin, F. S., L. R. Walker, C. L. Fastie, and L. C. Sharman. 1994. Mechanisms of primary succession following deglaciation at Glacier Bay,
Alaska. Ecological Monographs 64:149175.
Clarke, K. R., and R. N. Gorley. 2006. PRIMER v6: user manual/tutorial.
PRIMER-E Ltd., Plymouth, United Kingdom.
Cookson, W. R., M. Osman, P. Marschner, D. A. Abaye, I. Clark, D. V.
Murphy, E. A. Stockdale, and C. A. Watson. 2007. Controls on soil
nitrogen cycling and microbial community composition across land use
and incubation temperature. Soil Biology & Biochemistry 39:744756.
Courtney, R., G. Mullen, and T. Harrington. 2009. An evaluation of revegetation success on bauxite residue. Restoration Ecology 17:350358.
Courtney, R. G., and J. P. Timpson. 2004. Nutrient status of vegetation grown
in alkaline bauxite processing residue amended with gypsum and
thermally dried sewage sludgea two year field study. Plant and Soil
266:187194.
Del Moral, R., and D. M. Wood. 1993. Early primary succession on the
volcano Mount St. Helens. Journal of Vegetation Science 4:223234.
Eastham, J., and T. Morald. 2006. Effective nutrient sources for plant growth
on bauxite residue: II. Evaluating the response to inorganic fertilizers.
Water Air and Soil Pollution 171:315331.
Eastham, J., T. Morald, and P. Aylmore. 2006. Effective nutrient sources for
plant growth on bauxite residue. Water, Air, & Soil Pollution 176:519.
Fierer, N., and R. B. Jackson. 2006. The diversity and biogeography of soil
bacterial communities. Proceedings of the National Academy of Sciences
of the United States of America 103:626631.
Gardes, M., and T. D. Bruns. 1993. ITS primers with enhanced specificity for
Basidiomycetesapplication to the identification of mycorrhizae and
rusts. Molecular Ecology 2:113118.
Gherardi, M. J., and Z. Rengel. 2001. Bauxite residue sand has the capacity
to rapidly decrease availability of added manganese. Plant and Soil
234:143151.
Gleeson, D., N. Clipson, K. Melville, G. Gadd, and F. McDermott. 2005. Characterization of fungal community structure on a weathered pegmatitic
granite. Microbial Ecology 50:360368.
Glen, M., N. L. Bougher, I. J. Colquhoun, S. Vlahos, W. A. Loneragan, P. A.
OBrien, and G. E. S. J. Hardy. 2008. Ectomycorrhizal fungal communities of rehabilitated bauxite mines and adjacent, natural jarrah forest in
Western Australia. Forest Ecology and Management 255:214225.
Harris, J. A., P. Grogan, and R. J. Hobbs. 2005. Restoration ecology and the
role of soil biodiversity. Pages 319342 in R. D. Bardgett, M. B. Usher,
and D. W. Hopkins, editors. Biological diversity and function in soils.
Cambridge University Press, Cambridge.

Microbial Diversity and Functional Potential in Bauxite

Hodkinson, I. D., N. R. Webb, and S. J. Coulson. 2002. Primary community

assembly on land- the missing stages: why are the heterotrophic
organisms always there first? Journal of Ecology 90:569577.
Jasper, D., I. Lockley, S. Ward, and A. White. 2000. Current approaches and
future challenges for rehabilitation of bauxite residue. Pages 7172
in R. W. Bell and A. Brion, editors. Proceedings of Remade Lands
2000. International Conference on the Remediation and Management of
Degraded Lands, 30th Nov.-2nd Dec., Fremantle, Australia. Promaco
Conventions, Canning Bridge.
Jenkinson, D. S., P. C. Brookes, and D. S. Powlson 2004. Measuring soil
microbial biomass. Soil Biology & Biochemistry 36:57.
Jones, D. L. 1999. Amino acid biodegradation and its potential effects on
organic nitrogen capture by plants. Soil Biology & Biochemistry
31:613622.
Jones, D. L., J. R. Healey, V. B. Willett, J. F. Farrar, and A. Hodge. 2005a.
Dissolved organic nitrogen uptake by plants-an important N uptake
pathway? Soil Biology & Biochemistry 37:413423.
Jones, D. L., S. J. Kemmitt, D. Wright, S. P. Cuttle, R. Bol, and A. C. Edwards.
2005b. Rapid intrinsic rates of amino acid biodegradation in soils
are unaffected by agricultural management strategy. Soil Biology &
Biochemistry 37:12671275.
Jones, D. L., K. Kielland, F. L. Sinclair, R. A. Dahlgren, K. K. Newsham, J. F.
Farrar, and D. V. Murphy. 2009. Soil organic nitrogen mineralization
across a global latitudinal gradient. Global Biogeochemical Cycles
23:GB1016.
Keeney, D. R., and J. M. Bremner. 1966. Comparison and evaluation of
laboratory methods of obtaining an index of soil nitrogen availability.
Agronomy Journal 58:498503.
Kennedy, N. M., D. E. Gleeson, J. Connolly, and N. J. W. Clipson. 2005.
Seasonal and management influences on bacterial community structure
in an upland grassland soil. FEMS Microbiology Ecology 53:329337.
Marschner, H., and B. Dell. 1994. Nutrient uptake in mycorrhizal symbiosis.
Plant and Soil 159:89102.
Marschner, P., and A. Rumberger. 2004. Rapid changes in the rhizosphere
bacterial community structure during re-colonization of sterilized soil.
Biology and Fertility of Soils 40:16.
McArthur, W. M. 2004. Reference soils of south-western Australia. Department of Agriculture, Perth, Western Australia.
McKinley, V. L., A. D. Peacock, and D. C. White. 2005. Microbial community
PLFA and PHB responses to ecosystem restoration in tallgrass prairie
soils. Soil Biology & Biochemistry 37:19461958.
Menzies, N. W., I. M. Fulton, and W. J. Morrell. 2004. Seawater neutralization
of alkaline bauxite residue and implications for revegetation. Journal of
Environmental Quality 33:18771884.
Nelson, D. W., and L. E. Sommers 1996. Total carbon, organic carbon and
organic matter. Pages 9611010 in D. L. Sparks, editor. Methods of Soil
Analysis Part 3: Chemical Methods. Soil Science Society of America Inc,
Wisconsin.
Nemergut, D. R., S. P. Anderson, C. C. Cleveland, A. P. Martin, A. E.
Miller, A. Seimon, and S. K. Schmidt. 2007. Microbial community

10

succession in an unvegetated, recently deglaciated soil. Microbial Ecology 53:110122.

Normand, P., C. Ponsonnet, X. Nesme, M. Neyra, and P. Simonet. 1996. ITS
analysis of prokaryotes. Pages 112 in A. D. Akkermans, J. D. Van
Elsas, and E. I. Debruijn, editors. Molecular microbial ecology manual.
Kluwer Academic Press, Amsterdam.
Outback Ecology 2006. Coastal soils and vegetation communities as analogues
for bauxite residue rehabilitation. Unpublished report for Alcoa of
Australia, March 2006.
Rayment, G., and F. Higginson. 1992. Australian laboratory handbook of soil
and water chemical methods. Inkata Press, Melbourne.
Scallan, U., A. Liliensiek, N. Clipson, and J. Connolly. 2008. Ribosort: a program for automated data preparation and exploratory analysis of microbial community fingerprints. Molecular Ecology Resources 8:9598.
Sigler, W. V., and J. Zeyer. 2002. Microbial diversity and activity along the
forefields of two receding glaciers. Microbial Ecology 43:397407.
Snars, K. E., R. J. Gilkes, and M. T. F. Wong. 2004. The liming effect of
bauxite processing residue (red mud) on sandy soils. Australian Journal
of Soil Research 42:321328.
Sparling, G. P. 1992. Ratio of microbial biomass carbon to soil organic-carbon
as a sensitive indicator of changes in soil organic-matter. Australian
Journal of Soil Research 30:195207.
Van Der Heijden, M. G. A., R. D. Bardgett, and N. M. Van Straalen. 2008.
The unseen majority: soil microbes as drivers of plant diversity and
productivity in terrestrial ecosystems. Ecology Letters 11:296310.
Vance, E. D., P. C. Brookes, and D. S. Jenkinson 1987. An extraction method
for measuring soil microbial biomass-C. Soil Biology & Biochemistry
19:703707.
Wakelin, S. A., A. L. Gregg, R. J. Simpson, G. D. Li, I. T. Riley, and
A. C. McKay. 2009. Pasture management clearly affects soil microbial community structure and N-cycling bacteria. Pedobiologia 52:
237251.
Walker, L. R., B. D. Clarkson, W. B. Silvester, and B. R. Clarkson. 2003.
Colonization dynamics and facilitative impacts of nitrogen-fixing shrub
in primary succession. Journal of Vegetation Science 14:277290.
Wardle, D. A., and A. Ghani. 1995. A critique of the microbial metabolic quotient (qCO2 ) as a bioindicator of disturbance and ecosystem development.
Soil Biology & Biochemistry 27:16011610.
Wehr, J. B., I. Fulton, and N. W. Menzies. 2006. Revegetation strategies for
bauxite refinery residue: a case study of Alcan Gove in Northern
Territory, Australia. Environmental Management 37:297306.
Wertz, S., S. Czarnes, F. Bartoli, P. Renault, C. Commeaux, N. Guillaumaud,
and A. Clays-Josserand. 2007. Early-stage bacterial colonization between
a sterilized remoulded soil clod and natural soil aggregates of the same
soil. Soil Biology & Biochemistry 39:31273137.
White, T. J., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics. Pages
315322 in M. A. Innis, D. H. Gelfand, J. J. Sninsky and T. J. White,
editors. PCR protocols: a guide to methods and applications. Academic
Press, New York.

Restoration Ecology