Isolation and assessment of phytate-hydrolysing bacteria from the DelMarVa Peninsula.

Abstract

The Delaware-Maryland-Virginia (DelMarVa) Peninsula, flanking one side of the Chesapeake Bay, is home to a substantial broiler chicken industry. As such, it produces a significant amount of manure that is typically composted and spread onto local croplands as a fertilizer. Phytate (myo inositol hexakisphosphate), the major form of organic phosphorus in the manure, can be hydrolysed by microorganisms to produce orthophosphate. Orthophosphate is a eutrophication agent which can lead to algal blooms, hypoxia and fish kills in the Chesapeake Bay and its tributaries. This transect study reveals a subpopulation of heterotrophic, thiosulfate-utilizing bacteria that can degrade phytate within the watershed as well as its receiving water sediment. Aerobic isolates were typical soil bacteria, e.g. Pseudomonad, Bacillus and Arthrobacter species, as well as a less common Staphylococcus inhabitant. Bacillus pumilus, Staphyloccocus equorum, Arthrobacter bergei and Pseudomonas marginalis strains have not been previously described as phytate-degrading. Each site along the transect - from manure pile to receiving sediment - was host to a population of bacteria that can degrade phytate and hence, each is a possible non-point source of orthophosphate pollution. Each new isolate could provide an enzyme additive for monogastric feed, thus reducing the impact of excessive phytate load on the environment.

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Isolation and assessment of phytate-hydrolysing
bacteria from the DelMarVa Peninsula
Jane E. Hill,1* David Kysela2and
Menachem Elimelech1
1Department of Chemical Engineering, Environmental
Engineering Program, and2Ecology and Evolutionary
Biology, Yale University, New Haven, CT 06520, USA.
Summary
The Delaware–Maryland–Virginia (DelMarVa) Penin-
sula, flanking one side of the Chesapeake Bay, is
home to a substantial broiler chicken industry. As
such, it produces a significant amount of manure that
is typically composted and spread onto local crop-
lands as a fertilizer. Phytate (myo inositol hexak-
isphosphate), the major form of organic phosphorus
in the manure, can be hydrolysed by microorganisms
to produce orthophosphate. Orthophosphate is a
eutrophication agent which can lead to algal blooms,
hypoxia and fish kills in the Chesapeake Bay and its
tributaries. This transect study reveals a subpopula-
tion of heterotrophic, thiosulfate-utilizing bacteria
that can degrade phytate within the watershed as
well as its receiving water sediment. Aerobic isolates
were typicalsoilbacteria,
Bacillus and Arthrobacter species, as well as a
less common Staphylococcus inhabitant. Bacillus
pumilus, Staphyloccocus
bergei and Pseudomonas marginalis strains have
not been previously described as phytate-degrading.
Each site along the transect – from manure pile to
receiving sediment – was host to a population of bac-
teria that can degrade phytate and hence, each is a
possible non-point source of orthophosphate pollu-
tion. Each new isolate could provide an enzyme addi-
tive for monogastric feed, thus reducing the impact
of excessive phytate load on the environment.
e.g. Pseudomonad,
equorum,Arthrobacter
Introduction
Phytate – myo inositol hexakisphosphate – is the main
storage form of phosphorus in plants, accounting for
70–80% of the total phosphorus in the seeds of cereal
and legume crops (Lott et al., 2000). Intensive agricultural
operations, such as pig and poultry feedlots, use grain as
the primary source of macro- and micronutrients. Mono-
gastric animals, however, are unable to degrade phytate
(Harland and Morris, 1995; Whittemore, 1995) and it is
subsequently excreted in the manure (Whittemore, 1995;
Williams et al., 1999; Turner and Leytem, 2004). Addition-
ally, passage through the animal’s gastrointestinal tract
leads directly to nutritional problems. For example, com-
plexation of the compound with metals such as iron can
lead to anaemia (Harland and Morris, 1995), and com-
plexation with proteins can inhibit protein digestion
(Rackis, 1974; Erdman, 1979; Maga, 1982)
The manure from intensive agricultural operations is
commonly composted and then used as a crop fertilizer.
On the Delaware–Maryland–Virginia (DelMarVa) Penin-
sula, an overapplication of phosphorus from poultry farms
is typical (Sharpley, 2000; Sims et al., 2000; Boesch et al.,
2001) and the phytate in the manure (Turner and Leytem,
2004) is not directly bioavailable to most plants (Jackman
and Black, 1951; Findenegg and Nelemans, 1993;
Cakmak et al., 1999; Richardson et al., 2000). Excess
phosphorusloading from
sources into freshwater bodies via erosion and surface
run-offcauseseutrophication. Algal
harmful), hypoxia, occasional anoxia and catastrophic fish
kills are all hallmarks of such excess nutrient addition
(Burkholder and Glasgow, 1997; Sharpley, 2000; Sims
et al., 2000; Boesch et al., 2001).
The hydrolysis of phytate to orthophosphate and lower
substituted inositol phosphates is achieved enzymatically
with phytase. Organisms producing phytase have been
obtained from such diverse locations as soil (Cosgrove
et al., 1970; Richardson and Hadobas, 1997), cattle
rumen (Lan et al., 2002), cattle shed floor (Kim et al.,
2002), soybean mash (Choi et al., 2001), seawater (Kim
et al., 2003), culture collection repositories (Berka et al.,
1998; Casey and Walsh, 2004) and plant seeds (Nakano
et al., 2000; Greiner, 2004; Greiner and Egli, 2003),
suggesting that the ability to degrade phytate might be
widely distributed in a variety of ecosystems. Known
phytate-degrading organisms include aerobic bacteria
[e.g. Pseudomonas spp. (Richardson and Hadobas,
1997; Kim et al., 2002), Bacillus subtilis (Shimizu, 1992)
and Klebsiella spp. (Greiner et al., 1997)], anaerobic
bacteria [e.g. Escherichia coli (Greiner et al., 1993) and
Mitsuokella spp. (Lan et al., 2002)], fungi [e.g. Aspergillus
thesenon-pointpollution
blooms (some
Received
correspondence. E-mail jane.hill@uvm.edu; Tel. (+1) 802 656 3826;
Fax (+1) 802 656 3358.
13March,2007; accepted7 July,2007. *For
Environmental Microbiology (2007) 9(12), 3100–3107doi:10.1111/j.1462-2920.2007.01420.x
© 2007 The Authors
Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd

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spp. (Ullah, 1988; Shimizu, 1992) and Penicillium spp.
(Tseng et al., 2000)] and plants [e.g. barley (Greiner and
Egli, 2003) and wheat (Nakano et al., 2000; Greiner and
Egli, 2003)]. However, no study has investigated the
prevalence of phytate-degrading organisms across a
spatial gradient on monogastric farms, where manure
laden with phytate is abundant.
In this study, a spatial transect at a poultry manure-
enriched region in the Chesapeake Bay provides samples
from which phytate-degrading, aerobic bacteria have
been isolated. Pseudomonad, Arthrobacter, Staphylococ-
cus and Bacillus species were identified and their phytate-
degrading activity was assessed.
Results
Enrichment and isolation
Samples collected from each site [1 – chicken shed floor;
2 – from the culvert next to chicken shed (composite
sediment/water); 3 – manure composting pile; 4 – settling
pond (composite sediment/water); 5 – Pocomoke River
site A (composite sediment/water); and 6 – Pocomoke
River site B (composite sediment/water)] were initially
enriched on heterotrophic, thiosulfate agar plates with
orthophosphate as a source of phosphorus (M1). Forty-
eight isolates were purified by streaking to isolation. Iso-
lates from the original enrichment plate were chosen
based on their colony colour and morphology differences
(relative to each other). The purified isolates maintained
their original colour and morphology with each subse-
quent subculture. Three isolates were chosen randomly
from each sampling location and their identity, phylogeny
and phytate-degrading behaviour were explored.
Identification and phylogenetic context of
phytate-utilizing isolates
After DNA extraction, each isolate responded to universal
16S rDNA probes. Phylogenetic analysis of the se-
quenced amplicons provided taxonomic information for
each strain. A phylogenetic tree bearing each isolate is
shown in Fig. 1. The first number of each isolate repre-
sents the site number from which the isolate was first
obtained and the letter is an identification code. Isolates
from the genera Bacillus, Pseudomonas, Staphylococcus
and Arthrobacter were identified.
Primers based on published Bacillus species phytase
genes (Kerovuo et al., 1998; Tye et al., 2002) were devel-
oped and used to interrogate the Bacillus species isolated
from the DelMarVa Peninsula. Positive controls were
Fig. 1. Phylogenetic analysis of DelMarVa
isolates based on 16S rDNA sequences. The
tree was constructed using the ARB software
package to align sequences of 1087 positions
that were then analysed using PAUP* to infer a
maximum likelihood phylogeny using a
Hasegawa–Kishino–Yano DNA substitution
model including invariant sites and
gamma-distribution rate variation.
Neighbour-joining trees from 10 000 bootstrap
data sets provide the support values. Isolate
number indicates original source of isolate;
letter indicates isolate identifier.
Phytate-degrading strains are indicated by an
asterisk.
0.10
1a
4a
Pseudomonas putida
Pseudomonas umsongensis
4b
6b*
4c*
5c*
Pseudomonas marginalis
1b*
3b*
5a*
Pseudomonas syringae
Escherichia coli
Vibrio fischeri
Neisseria gonorrhoeae
Thiobacillus denitrificans
Nitrosomonas oligotropha
Thiobacillus baregensis
Rhodobacter sphaeroides
Thiobacillus sp.
Campylobacter jejuni
1c*
2a*
3a*
Bacillus pumilus
2b
Bacillus subtilis
Bacillus licheniformis
5b
6a
6c
Bacillus cereus
Bacillus anthracis
3c
uncultured Bacillus sp. AY082370
2c*
Staphylococcus equorum
Lactobacillus acidophilus
Clostridium botulinum
Arthrobacter bergeri
Streptomyces nodosus
Chlamydia trachomatis
Synechocystis sp.
100
100
100
84
100
100
100
98
81
74
γ - Proteobacteria
High G+C
Gram-positive
98
98
Isolation and assessment of phytate-hydrolysing bacteria3101
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successful, but no fragments were amplified from any
isolate using these primers through the polymerase chain
reaction.
Sample screening
The 48 isolates were screened via a replica plate tech-
nique for their potential to degrade phytate. Seventy per
cent of the isolates were capable of using M2 (phytate as
sole source of P) for growth. For this plate-based assay,
Ca-phytate, a white precipitate, was the only source of
phosphorus. Supplementary evidence in the form of
zones of clearing on the plates immediately adjacent
to the biomass indicated utilization of the Ca-phytate by
the isolate via extracellular or outer membrane-bound
enzyme(s). The addition of an acid indicator (methyl red)
showed that this zone of clearing was acidic in nature. Ten
(69%) of the 18 isolates chosen for 16S rRNA determina-
tion were able to grow and produce a zone of clearing on
M2, indicating potential phytate-degrading ability. Phytate
utilization by the 10 isolates was further supported by their
growth in liquid culture (M2) at 28°C in scrupulously clean,
acid-washed glassware and via the use of phosphate-free
media components. Phytate-degrading strains are indi-
cated in Fig. 1 via an asterisk. Negative controls showed
no significant growth for isolates grown on media without
any phytate present.
Replica plates and liquid cultures were used to probe
sulfur use by the isolates. For those isolates that were
designated potential phytate degraders, replica plates with
fourfold isolate representation showed growth on sulfate
but not on tetrathionate after a 24 h incubation at 28°C.
This result was confirmed in liquid culture as is shown for
a representative phytate-utilizing isolate in Fig. 2.
Phytase assessment
Each isolate was tested for phytate-degrading activity.
Liquid cultures were grown for 21 h (stationary phase) in
M2 at 28°C. An E. coli (known phytate degrader) culture
grown to stationary phase in Luria–Bertani broth was
always grown in tandem as a positive control. Growth in
medium M1, where phosphate is present, produced an
overwhelming absorbance signal with the molybdate
assay, making the detection of phytase activity difficult.
Thus, all results presented here (Table 1) represent those
of pure cultures grown in the phosphate-free, phytate
medium M2.
After 21 h, culture supernatants were probed for the
presence of orthophosphate. Controls with and without
inocula showed negligible levels of phosphate in the
medium thus, any phosphate measured in the supernate
was the result of bacterial activity, most of which can be
attributed to the hydrolysis of phytate. Supernate concen-
trations of orthophosphate ranged from 41 mmol ml-1to
105 mmol ml-1. Of the three highest liberators, two were
phylogenetically affiliated with Pseudomonad marginalis
(3-b: 93 mmol ml-1and 5-c: 104 mmol ml-1) and one with
Bacillus pumilus (1-c: 105 mmol ml-1), even though these
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0
Time (h)
OD600
2
1
3
10
20
3040
Fig. 2. Representative growth curve for isolate 1-c (Pseudomonas
sp.) on different sulfur sources in M2 medium over 48 h. Series 1 is
growth on thiosulfate, series 2 is growth on sulfate and series 3 is
growth on tetrathionate.
Table 1. Phytase activity of DelMarVa isolates: phosphate released by each culture after 21 h as well as the orthophosphate released by
supernate enzymes incubated for 7 h.
Isolate SpeciesSupernate Pia(mmol ml-1)Enzyme activityb(mmol ml-1min-1)Phytase localizationc
1-bd
1-cd
2-ad
2-cd
3-ad
3-bd
4-cd
5-ad
5-cd
6-bd
Pseudomonas marginalis
Bacillus pumilis
Bacillus pumilis
Staphyloccocus equorum
Bacillus pumilis
Pseudomonas marginalis
Pseudomonas umsongensis
Pseudomonas marginalis
Pseudomonas marginalis
Pseudomonas umsongensis
70
105
56
41
67
93
61
52
104
62
0.04
n.m.
0.04
0.04
0.06
0.03
n.m.
n.m.
n.m.
0.05
EX
CA
EX
EX
EX
EX
CA
CA
CA
EX
a. Orthophosphate release after 21 h incubation with crude culture supernatant.
b. Orthophosphate released from culture supernate activity at 37°C.
c. Enzyme activity was either extracellular (EX) or cell associated (CA).
d. Phytate-degrading strain.
n.m., not measurable.
3102 J. E. Hill, D. Kysela and M. Elimelech
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Page 4

isolates did not have the highest optical densities at
600 nm after 21 h (data not shown). The theoretical
maximum orthophosphate concentration derived from
phytate is 190 mmol ml-1.
Discussion
We isolated aerobic bacteria from diverse sampling loca-
tions along a hypothetical phytate fate pathway at an
active poultry operation on the DelMarVa Peninsula. A
subpopulation of bacteria that seemed particularly adept
at degrading phytate was isolated on plates using an
enrichment combination of thiosulfate, glucose and
orthophosphate. On average, 70% of these isolated bac-
teria were able to grow on phytate and produce an acidic
zone of clearing on plates containing calcium phytate as
the sole source of phosphorus (Richardson and Hadobas,
1997; Hill and Richardson, 2006).
The Bacillus strains isolated from the DelMarVa
Peninsula can be segregated into four clades based on
their 16S rDNA sequences: Bacillus cereus/anthracis,
B. subtilis/lichiniformis, B. pumilus, and one close to an
uncultured Bacillus species. The isolated B. pumilus
species all produced a positive response to the plate-
clearing assay; however, the B. subtilis clade isolate did
not grow on a calcium phytate agar plate, nor did the
B. anthracis isolates or the isolate aligned with an uncul-
tured strain. This lack of growth could be a reflection of the
isolates’ inability to degrade phytate or perhaps, merely
an indication of unfavourable conditions for phytase
production. Several phytate-degrading Bacillus species
have been published previously, including Bacillus amy-
loliquefaciens (Kerovuo et al., 1998), Bacillus sp. DS11
(Kim et al., 1998) and B. subtilis strains (Powar and
Jagannathan, 1982; Shimizu, 1992), with each producing
a positive response to the plate-clearing as well as an
enzyme activity assay. Growth on plates is usually easier
than in a liquid medium for phytate-degrading bacteria
(Choi et al., 2001; Hill and Richardson, 2006), hence, no
further assessment was conducted on strains that could
not grow on plate systems. B. pumilus strains were
probed with phytase gene primers known to work on other
phytase-producing Bacillus species (Kerovuo et al., 1998;
Tye et al., 2002). These strains did not produce an ampli-
fication product; however, they are likely to host a version
of the known beta propeller phytase family.
Nine pseudomonads were isolated in this study, with
five showing phytate-degrading potential via growth and
production of a zone of clearing on agar media containing
calcium phytate. Pseudomonad production of phytase
has been described previously. For example, Richardson
and Hadobas (1997) identified four Pseudomonas iso-
lates, two that cluster in the Pseudomonas putida clade
and two that cluster in the Pseudomonas mendocina
clade. A Pseudomonas fragi (strain Y9451) isolate has
been identified as producing a phytase enzyme (In et al.,
2004), and Kim and colleagues (2003) also identified a
pseudomonad, but not to the species level. The isolates
from this survey cluster in two clades, P. putida/
umsongensis and P. marginalis. Three out of five in the
former and all of the latter showed phytate-degrading
potential. The lack of activity in two of five Pseudomonad
isolates of the P. putida clade could be due to a loss of trait
in the strain, or perhaps the culture conditions used in this
study were not effective in inducing the production of
phytase in these particular isolates. It is unclear at this
point whether phytate hydrolysis is a common trait within
the genus Pseudomonas.
Bacteria from the genera Staphylococcus and Arthro-
bacter have not been shown previously to degrade
phytate. Species of the genus Staphylococcus are Gram-
positive, facultative anaerobes often associated with
warm-blooded animals in a pathogenic context. The
species in this survey correlates most closely with Sta-
phylococcus equorum, which is normally associated with
food products like meat and dairy (Ghosh et al., 2006). It
is the first Staphylococcus isolate reported to show defini-
tive evidence of phytate-degrading activity. Species of the
genus Arthrobacter are Gram-positive, aerobic bacteria
widely distributed in soil environments. The species iso-
lated in this survey has Arthrobacter bergei as the nearest
relative based on 16S rDNA sequence identity. While
amplification of the 16S rRNA gene beyond 500 bp was
not possible for the Arthrobacter isolate under the primer
conditions applied in this study, the high sequence identity
is significant enough to confidently place the isolate in the
Arthrobactor clade. Arthrobacter was shown to produce
low levels of phytase activity in the culture supernate after
21 h of growth on phytate (J.E. Hill, unpublished).
However, most of the activity appeared locked in the cell
debris and thus it could not be consistently quantified.
This study presents the first Arthrobacter isolate reported
to show phytate-degrading activity.
For the phytate-degrading bacteria isolated in this
study, orthophosphate liberation was generated in most
cases by an extracellular phytase enzyme. This was con-
firmed through the measurements of crude and semi-
purified cell extracts. Higher yields of orthophosphate
from the action of a phytate-degrading enzyme may be
possible after more purification, which would eliminate
possible suppression of enzyme activity caused by ortho-
phosphate in the medium (Touati and Danchin, 1987;
Greiner et al., 1993) as well as decrease the presence of
organic material, which is known to bind both phytate
(Borie et al., 1989; Nanny and Minear, 1994) and molyb-
date (Turner et al., 2006). Especially in the cases of cell-
bound activity, a qualitative confirmation – through the
observation of a substantial yellow-coloured precipitate
Isolation and assessment of phytate-hydrolysing bacteria3103
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collected upon sample centrifugation – indicates that a
greater activity was present than was reflected in the
absorbance reading. The sonication of whole-cell culture
suspensions with isolates known to have extracellular
activity produced similar results, confirming the likely pro-
duction of cell debris-associated, molybdate-complexed
phosphate.
The significance of metabolism of both thiosulfate and
phytate is not yet fully understood; however, some
hypotheses from this work can be generated. We have
shown here that the cultures isolated from this study will
grow on sulfate and thiosulfate, but not on tetrathionate,
a polymer of thiosulfate (S2O62-). In addition we have
shown that growth on thiosulfate leads to the production
of acid. Coupled with some preliminary results from the
amplification of the soxB gene (J.E. Hill, unpublished)
from several isolates, this work suggests that the isolates
are utilizing the Paraccocus pathway, one of the two major
pathways for the oxidation of thiosulfate (Kelly et al.,
1997). It could be argued that the increase in energy
generated by use of the Paracoccus pathway, along with
the production of acid, makes phytate more soluble and
therefore more bioavailable (Evans and Martin, 1987;
1988a,b; Turner et al., 2002). This could confer a selec-
tive advantage on organisms in microaerophilic niches in
a variety of soil and terrestrial environments. The link
between organic phosphorus cycling and sulfur oxidation
warrants further research.
This study reveals that in every sample, there are
common aerobic bacteria that can degrade phytate. Thio-
sulfate oxidation by these organisms – whether purpose-
ful or incidental – will produce acidic conditions, which are
likely to enhance the hydrolysis of phytate by organisms in
that niche. The subsequent production of orthophosphate
by these cell-bound or extracellular enzymes could there-
fore be creating multiple sources of relatively mobile,
non-point phosphorus pollutant – orthophosphate. This
suggests that point-source pollution control strategies,
such as phytase addition to animal feed, provide a sen-
sible approach to managing phosphate contamination.
The release of orthophosphate by manure and soil popu-
lations also hints that, given the right circumstances,
native bacterial communities can degrade phytate. The
resulting release of orthophosphate, which is more bio-
available to plant communities, could thus enhance the
benefits of manure-derived fertilizer.
Experimental procedures
Enrichment inocula source
Composite samples were collected aseptically from six
locations on the DelMarVa Peninsula, spanning the spatial
gradient from a commercial chicken shed to the closest,
down-gradient receiving water body (Pocomoke River, MD),
relative to the shed. Specifically, the locations were as
follows: 1 – chicken shed floor; 2 – from the culvert next to
chicken shed (composite sediment/water); 3 – manure com-
posting pile; 4 – settling pond (composite sediment/water);
5 – Pocomoke River site A (composite sediment/water); and
6 – Pocomoke River site B (composite sediment/water).
Samples were stored at 0°C for 24–36 h before plating.
Culture media
Enrichment media varying in phosphorus, carbon and sulfur
source were used to culture isolates from each sampling
location. The combination producing the highest proportion
of isolates exhibiting potential phytate hydrolysis was found
using an enrichment medium containing orthophosphate,
thiosulfate and glucose. These organisms and conditions of
enrichment were studied further.
Three media recipes were used to isolate bacteria and
assess their potential to degrade phytate. Where relevant, all
glassware was acid-washed and all medium components
were orthophosphate-free. Per litre, each medium contained:
10.0 g of Na2S2O3·5H2O, 8.0 g of glucose, 1 g of MgCl2·6H2O,
0.30 g of NH4Cl, 0.04 g of FeCl3·6H2O and 1.0 ml of trace
element solution. Medium 1 (M1) also contained (per litre)
1.5 g of K2HPO4, 1.5 g of KH2PO4 and 0.01 g of biotin.
Medium 2 (M2) also contained (per litre) 3.0 g of sodium
phytate, 5 g of CaCl2·2H2O and 0.01 g of biotin. Filtered
(0.22 mm) sodium phytate was added after autoclaving
other ingredients. The pH of each solution was adjusted
to 7.0 using 25% HCl. Where relevant, 20 g l-1agar was
added. All media ingredients were purchased from Sigma
(ultrapure). Two additional media components, sulfate and
tetrathionate, were used to assess the growth of the bacterial
isolates on additional sulfur sources. These media were
based on M2 with an equimolar substitution used for each
sulfur source tested.
Isolation and purification
Five grams of each DelMarVa sample was suspended in
20 ml of sterile 10 mM KCl and placed on a rotary shaker
(5 r.p.m.) at 4°C for 24 h before addition to culture media. A
small aliquot (20 ml) of the suspension was spread on an M1
agar plate and incubated at 28°C until growth occurred. Sub-
cultures from the enrichment plate, chosen for their unique
colony morphology, were streaked onto fresh agar plates of
the same composition. Each subculture was streaked to an
individual colony at least three times to ensure purity. Stock
cultures were stored in glycerol at -70°C.
DNA extraction, sequencing and phylogenetic analysis
Pure cultures were grown in M1 broth overnight. DNA extrac-
tion from 1 ml of pure culture (approximately 108cells ml-1)
was achieved with Qiagen’s DNAeasy Tissue Culture kit
(Qiagen). 16S rRNA genes were amplified with universal
primers 27f (AGA GTT TGA TCM TGG CTC AG), 516f (TGC
CAG CAG CCG CGG TAA), 1046r (GACAGC CAT GCAVCA
CCT) and 1492r (CGG YTA CCT TGT TAC GAC TT) (Weis-
burg et al., 1991) using Amplitaq® polymerase (Applied
3104 J. E. Hill, D. Kysela and M. Elimelech
© 2007 The Authors
Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 3100–3107