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December 2005
COMPARISON OF GLOMALIN AND HUMIC ACID IN EIGHT NATIVE COMPARISON OF GLOMALIN AND HUMIC ACID IN EIGHT NATIVE
U.S. SOILS U.S. SOILS
K. A. Nichols
USDA-ARS
S. F. Wright
USDA-ARS
Follow this and additional works at: https://digitalcommons.unl.edu/usdaarsfacpub
Part of the Agricultural Science Commons
Nichols, K. A. and Wright, S. F., "COMPARISON OF GLOMALIN AND HUMIC ACID IN EIGHT NATIVE U.S.
SOILS" (2005).
Publications from USDA-ARS / UNL Faculty
. 161.
https://digitalcommons.unl.edu/usdaarsfacpub/161
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0038-W75X/05/17012-985-997
December
2005
Soil
Science
Vol.
170,
No.
12
Copyright
0
2005 by
Lippincott
Williams
&
Wilkins, Inc.
Printed
in
U.S.A.
COMPARISON
OF
GLOIALIN
AND
HUMIC
ACID
IN
EIGHT
NATIVE
U.S.
SOILS
K.
A.
Nichols'
and
S.
F.
Wright
2
Two
important
extractable
fractions
of
soil
organic
matter
(SOM)
are
humic
acid
(HA)
and glomalin-related
soil
protein
(glomalin).
Optimizing
the
purity
of
each
fraction
is
necessary
to
correlate
fraction
quantity
and
molecular
characteristics
with
soil
quality.
Manipulation
of
extraction
sequence
and
controlled
precipitation
of
HA
were
used
to
evaluate
co-extraction
of
HA
and
glomalin.
Eight
bulk
soil
samples
(0
to
10
cm
depth)
were
collected
from
four
U.S.
states
(Colorado,
Nebraska,
Maryland,
and
Georgia).
In
Experiment
1,
glomalin
extraction
(50
mM
citrate,
pH
8.0,
at
121
'C)
was
followed
by
HA
extraction
(0.1
N
NaOH
at
room
temperature),
and
Experiment
2
used
the
reciprocal
sequence.
Experiment
2
HA
was
precipitated
stepwise
at
pH
levels
2.5,
2.0,
and
1.0.
Gravimetric
weight, Bradford-reactive
soil
protein
(BRSP),
and
imnmuno-
reactive
soil
protein
(IRSP),
along
with
percentages
of
C,
N,
H,
and
Fe,
were
used
to
compare
glomalin
and
HA.
The
HA
fraction
from
Experiment
2
contained
2-fold
greater
amounts
of
BRSP
than
HA
from
Experiment
1
and showed
that
pre-extraction
of
glomalin
improved
the
purity
of
HA.
The
glomalin
fraction
from
Experiment
1
contained
1.5
times
the BRSP
of
glomalin
from
Experiment
2
and
was
twice
the
gravimetric
weight.
BRSP
and
gravimetric
weight
were
concentrated
in
HA
that precipitated
at
pH
2.5
or
2.0
and
percentage
IRSP
was
significantly
higher
in
the
pH
2.5
precipitate.
The
results
indicate
that
glomalin
should
be
extracted
first
and
examined
as
a
biomolecule
sepa-
rate
from
the
humic
acid
mixture.
Percentages
C,
H,
N,
and
Fe
in
glomalin
varied
across
soils
and
experiments.
In
seven
soils,
the
changes
in
Fe
percentage
in
glomalin
from
Experiment
1
to
2
were
significantly
correlated
with the
changes
in
glomalin
weight
and
%C.
Iron
in glomalin
from
Experiment
1
was
related
to
soil
pH
and
clay
content,
whereas
soil
organic
C
was
positively
and
significantly
correlated
with Experiment
1
glomalin
BRSP
and
IRSP.
In
Experiment
1,
a
recalcitrant
pool
of
glomalin
was
released
by
treating
soil
with
NaOH,
suggesting
that
a
fraction
of
glomalin
is
difficult
to
remove
from
soil
and
glomalin
extraction
efficiency
could
be improved.
Refinements
to
extraction
and
purification
protocols
such
as
pretreatment
of
soils
with
HCl
and
sequential
extraction
can facilitate
studies
on
organic
matter
structure
and
function.
(Soil
Science
2005;170:985-997)
Key
words:
Organic
matter,
humic
substances,
soil
carbon,
glomalin.
LUCIDATING
the
quantities
and compo-
will
help
clarify
processes
involved
in
soil
sitions
of
extractable
soil
organic
fractions
stabilization
and
C
storage.
Humic
substances
(HS),
which
include
humic
acid
(HA), fulvic
acid
'ARS-USDA
Nithern
Great
lains
Reseach
U6cdtos,,
1701
10IM
Ave.
S,
(FA),
and
humin,
are
formed
by the
decompo-
P.O.
13ý
459,
Mnda,
ND
58554.
Dr. Niols
c
e
.
sition
of
plant
and
animal
debris,
microfauna,
E-m•nl:
nkcdhoslmandan.ars.usda.sov
biowastes.
and
other
organic
materials
in
the
soil
2ARS-USDA
Susainale
Agicural
SyL,f.
•at
40,
Roam
140,
(Burdon,
2001;
Hayes
and
Clapp,
2001).
Soil
BARC-Wert
B•sville,
MD
20705.
quality
factors
attributed
to
HS
include
improved
Recemed
Oct.
28,
2004;
&-mepted
July
18,
2005.
buffering
capacity,
increased
moisture
retention,
DOC:
10.1097/01.ss.000019ooo
.06975.3r
and
enhanced
spring
warming.
HS
also
reportedly
985
NICHOLS
AND
WRIGHT
function
as
reservoirs
of
plant-available
micro-
nutrients,
bind
metals
to
alleviate
both
heavy
metal toxicity
and
deficiency,
bind
clays
and
other
small
organic
molecules
to
form
aggregates,
and
act
as
electron
shunts
in
microbial
and abiotic
redox
reactions
(Burdon,
2001; Fan
et
al.,
2000;
Hayes
and
Clapp,
2001.;
MacCarthy,
2001).
Analytical
techniques
such
as
nuclear
mag-
netic
resonance
(NMR),
gas
chromatography-
mass
spectroscopy,
and
thermochemolysis
have
been
applied
to HS to
identify
functional
groups
and
match
function
with
chemistry
(Hatcher
et
al.,
2001).
These
techniques
confirm
that
other
molecules,
such
as
amino
acids,
carbo-
hydrates,
and
lipids
frequently
are
co-extracted
with
HS
(Hatcher
et
al.,
2001,
Kingery
et
al.,
2000;
Simpson,
2001).
These
associated
organic
molecules-proteins,
carbohydrates,
and
lipids-
have
chemical
compositions
that
may
provide
buffering
capacity,
assist
in redox
reactions,
and
bind
organic
matter,
metals,
and
clays-functions
that
have
been attributed
to
HS
(Burdon,
2001).
Humic
acid
is
the
abundant,
alkaline-
soluble,
and acid-insoluble
dark
brown
to
black
extractable
component
of
HS.
Whether
HA
is
a
super-mixture
of
low
molecular
weight
mole-
cules
(Hayes
and
Clapp,
2001,
MacCarthy,
2001)
or
a
high molecular
weight
complex
(Schulten
and
Schnitzer,
1997)
has
not
been
resolved.
Studies
of
HA quantity,
structure,
and
function
begin
with
material
extracted
nonspecifically
from
soil
using
NaOH
or sodium
pyrophos-
phate
at
room
temperature
(RT)
(Swift,
1996).
Glomalin-related
soil
protein,
referred to
in
this
manuscript
as
glomalin,
is a
nonspecifically
extracted,
alkaline-soluble
soil
C
pool
that
is
linked
to
arbuscular
mycorrhizal
(AM)
fungi
(Rdllig,
2004,
Wright
et
al.,
1996).
This
red-
brown
glycoprotein
does
not
have
co-extracted
or
attached
tannins
(Rillig
et
al.,
2001).
Accord-
ing
to
1
H
NMR
experiments,
glomalin
has
a
consistent
organic
structure
across
soils
and
in
sterile
glomalin-free
sand
pot
cultures
that
is
different
from
HA
(Nichols,
2003).
The
gloma-
lin extraction
protocol
consists
of
incubating
soil
in
20
to
50
mM
sodium
citrate
at
an
alkaline
pH
and
121
'C
for
1-hour
intervals
(Wright
and
Upadhyaya,
1996).
This
is
in
contrast
with
RT
extraction
of
HA (Swift,
1996).
Glomalin
precipitates
from
solution
at
pH
2.5
to
2.0
(Nichols,
2003),
a
pH
level
where
a
high
protein
fraction
of
HA
has
been
isolated
(Hayes
and
Clapp,
2001; Hayes
and
Graham, 2000).
Glomalin
appears
to
have
properties and
func-
tions similar
to
fungal
hydrophobins
(Nichols,
2003;
Nichols
and
Wright,
2004;
Wright
and
Upadhyaya,
1996),
which
are small,
self-
aggregating,
hydrophobic
proteins
found
on
hyphae
of
many
types
of
fungi,
including
ectomycorrhizal
fungi
(Wessels,
1997).
Hydro-
phobins
protect
hyphae
from
solute
loss,
allow
hyphae
to
grow
through
soil
and
maintain
turgor
pressure
while
crossing
air-
or
water-filled
pores
(Wessels,
1997).
Glomalin
sloughs
from
hyphae
(Wright,
2000)
and
accumulates
in
soils
(Rillig
et
al.,
2001;
Wright
and
Upadhyaya,
1996).
Accumu-
lation
is
thought
to
result
from
the
insolubility,
hydrophobicity
and
high
Fe
content
of
the
molecule.
Iron
concentrations
of
0.8
to
8.8%
(Wright
and
Upadhyaya,
1998),
may
protect
glomalin
from
degradation
by
as
proposed
for
the
role
of
Fe
in
organic
matter
(Hassink
and
Whitmore,
1997)
and
may
increase
the thermal
stability
and
antimicrobial
properties
of
glomalin
(Paulsson
et
al.,
1993).
The
objectives
of
the
current
work
were
to
(1)
determine
if
extraction-protocol-defined
fractions
of
both
glomalin
and
HA
can
be
iso-
lated
from
the
same
soil sample,
and
if
so,
can
they
be
isolated
separately
or
are
they
co-
extracted;
(2)
test
sequential
extraction
protocols
and
stepwise
precipitation
to
isolate
glomalin
and
HA
if
co-extraction
occurs;
(3)
compare
C,
N,
H,
Fe,
and
protein
concentrations
of
glomalin
and
HA;
and
(4)
compare
amounts
of
giomalin
and
HA
to
soil
properties,
such organic
C,
clay,
P,
and Fe
concentrations
that
may
affect
glomalin
and
HA
accumulation
and
function.
MATERIALS
AND
METHODS
Soils
Bulk
soil
samples
(0
to
10
cm
depth)
were
collected
with
a
shovel
at
two
sites
in
each
of
four
states:
Maryland
(MD),
Nebraska
(NE),
Colorado
(CO),
and
Georgia
(GA)
(Table
1).
Soil
was
freshly
collected
at
each
site
except
for
the
NE
soils
that
had
been
stored
at
RT
for
3
years.
Each
site
had
native
vegetation.
Soils
were
air-dried
and
sieved
to
collect
material
<2
mm.
Selected
soil
characteristics
are
shown
in
Table
1.
Cation
exchange
capacity,
pH, P.
sand,
and
clay
concentrations
were
measured
by
the
Soil
Testing
Laboratory
at
the
University
of
Maryland.
Phosphorus
concentra-
tion
was
measured
with
a
colorimetric
assay
on
solution
extracted
from
soil
tising
the
Mehlich
I
method
(Mehlich,
1953).
Soil
pH
was
measured
SOIL
SCIENCE
986
VOL.
170
-
No.
12
HA
AND
GLOMALIN
EXTRACTION
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ci
.a
0.
o
t
CO
E
- 2,
2,
in
1:1
(wt/vol)
0.01
iV
CaCl,
solution.
Soil
texture
was
measured using
the
pipette
method
(Day,
1965).
Total
organic
C,
N,
and
H
was
measured by
combustion
with
a
Perkin-Elmer
Series II
C,
H,
N,
S/O
2400 Analyzer
(Shelton,
CT) on
soil
treated
with
0.1
N
HC1.
Iron
was
extracted
from
soil
by
a
modified
Aqua
Regia
(McGrath
and
Cunliffe,
1985)
procedure
and
quantified
by
AA
(atomic
absorp-
tion).
Briefly,
concentrated
HNO
3
was
added
to
the
sample
and
heated to
85
to
90
'C
(a
tempera-
ture
high
enough
to
cause
evaporation
but
not
boiling)
for
2
hours.
Next,
concentrated HCl
(1:3
HNO
3
:HCI)
was
added
followed by
incu-
bation
at
60
'C
for
1
hour.
After
hydrolysis,
sam-
ples
were
decanted
through
a
Whatman
1
filter
into
a
volumetric
flask
and
brought
to
volume
with
deionized
water
(dH
2
O). Iron concentra-
tion
was
measured
with
a
Varian
atomic
absorption
spectrometer
(AA-400,
Palo
Alto,
CA)
with
deuterium
background
correction.
Extraction
of
Gloinalin
Glomalin
was
extracted
with
50
mM
sodium
citrate,
pH
8.0,
at
121
'C
for
I
hour
(Wright
and
Upadhyaya,
1999).
Samples
were
centrifuged
and the supematant
was
decanted
and
saved.
The
procedure
was
repeated
until
the
supernatant
was
straw-colored
(up
to
3
more
times).
Supernatants
from
each
1-hour
extraction
cycle
were
combined
and
centrifuged.
A
1-mL
subsample
was
removed
for
protein
assays
(see
below),
and
the remainder
was
flocculated
at
pH
2.0
to
2.5
by
slowly
adding
1 N
HC1,
the
solution
was
placed
on
ice
for
45
minutes,
and the precipitate
was
pelleted
by
centrifuging.
The
pellet
was
dissolved
in
a
minimum amount
of
0.1
Al
NaOH
and
imme-
diately
dialyzed
against
dH
2
O
in
dialysis
tubing
with
molecular
weight cutoff
of
8,000
to
12,000
Daltons
(D).
Proteolysis
of
glomalin
by exposure
to
0.1
N
NaOH
was
not
detected
by
ninhydrin
(unpublished
data).
Water
in the
dialysis
cham-
ber
was
changed
at
least
5
times
with
8-
to
12-hour
incubation
periods
each
time.
Dialyzed
material
was
centrifuged
and the
supernatant
was
collected
and
freeze
dried.
All
centrifuging
was
carried
out
at
6
,850g
for
10
minutes.
Extraction
of
Huinic
and
Fulvic Acids
The
International
Humic
Substances
So-
ciety
method
described
bySwift
(1996)
was
used
to extract HA
and
FA.
Modifications
to the
method
were
in
sample
size
(2
g
instead
of
50
g)
and
in the
purification
steps.
Briefly,
soil
was
987
, 0
2,.
6"0
• "6
'-I
I-
NICHOLS
AND
WRIGHT
pre-incubated
in
1 N HC1
followed
by
a
multi-
step
extraction
procedure:
(i)
extraction
with
0.1
N
NaOH
at
RT
under
N
2
overnight;
(ii)
centrifuging
to
collect
the
supernatant;
(iii)
acidification
of
the supernatant;
(iv)
precipita-
tion
of
HA
overnight;
and (v) separation
of
HA
(precipitate)
from
FA
(supernatant)
by
centrifuging.
The
NaOH
extraction
followed
by
acidic
separation
was
repeated
until
the
solution
was
almost
clear
(two
more
times)
to
assure
that
all
humic
and
fulvic
acids
were
extracted.
Protein
was
not
detected
in
FA
by
the
Bradford
soil
protein
assay
(see
below),
so
there
was
no
further
analysis
of
this
fraction.
All
centrifuging
was
carried
out
at
6
,850g
for
10
minutes.
Insoluble
solid particles
were
removed
from
HA
by
redissolution
in
a
minimum
volume
of
KOH
under
N2,
addition
of
KCI
(until
[K+]
>_
3
M),
and
centrifuging
at 10,844g
to
remove
suspended
solids.
HA
was
precipitated
with
HCL.
After
settling
overnight,
samples
were
centrifuged and
the
supernatant
was
discarded.
Precipitated
HA
was
suspended
in
an
HC1/HF
solution, incubated
overnight,
and
collected
by
centrifuging
at
6,850g.
The
supematant
was
discarded.
The
HCl/HF
treatment
was
repeated
twice.
Residual
acid
was
removed
by
repeatedly
washing
the
precipitate
with
dH
2
0
and
centri-
fuging
at 10,844g
for
3
minutes.
Precipitated
HA
was
redissolved
in
a
mini-
mal
measured
volume
of
0.1
N
NaOH.
A
sub-
sample
(0.5
ml)
was
removed
for
protein
assays
(see
below),
and the
remaining
solution
was
acidified
rapidly
to
precipitate
HA.
Acid
was
removed
from
the precipitate by
centrifuging
at
10,844g
and
washing
with
dH
2
O.
The
precipi-
tate
was
freeze-dried.
Experiment
1:
Citrate
Extraction
Followed
by
Sodiumn
Hydroxide
Extraction
Five
2
-g
samples
per
soil
were
citrate-
extracted
for
glomalin
followed
by
NaOH
extrac-
tion
of
HA.
Residual
soil
(soil
remaining
after
sequential
extraction
of
glomalin
followed
by
HA)
from
all
soils
except
Pawnee
was
re-
extracted
with
citrate
to
determine
whether
the
NaOH
treatment to
extract
HA
facilitated
the
re-
lease
ofa
recalcitrant
pool
ofglomalin
(R.
glomalin).
Experiment
2;
Sodium
Hydroxide
Extraction
Followed
by
Citrate
Extraction
Humic
acid
was
extracted
from
five,
2-g
samples
per
soil,
and
the remaining
soil
was
extracted
with
citrate.
Stepwise
Precipitation
of
Glomalin
in
.HA
A subsample
of
the purified
and
freeze-dried
HA
from
each
soil
from
Experiment
2
was
extracted
with
citrate
to
assess
co-extraction
of
glomalin.
Trace
amounts
of
citrate-insoluble
material
were collected
by
centrifuging
at
10,844g,
and
the supernatant
was
titrated
in
steps:
(1)
pH
2.5
(HA2.5),
(2)
pH
2.0
(HA2.0),
and
(3)
pH
1.0
(HAL.0).
At
each
step,
the
precipitate
was
collected
by
centrifuging
at
6
,850g
after
a
30-minute
incubation
on
ice.
After
step
3,
the
supernatant
was
discarded.
Each
precipitate
was
redissolved
in
0.1
N
NaOH
and
dialyzed
against
water
in
dialysis
tubing
with
molecular
weight
cutoff
of
500
D.
After
dialysis
and
centrifuging
at
6,850g
for
10
minutes,
the
supernatant
was
collected
and
freeze-dried.
A
subsample
was
collected
for
the
protein
assays
(see
below)
by
dissolving
the freeze-dried
fractions
in
dH'O
at
neutral
pH.
Protein
Assays
Bradford
reactive
soil
protein
(1BRSP)
and
immunoreactive
soil
protein
(IRSP)
(Rillig,
2004)
concentrations
were
measured
on
sub-
samples
(collected
as
described above)
of
glo-
malin
and
HA from Experiments
1
and
2,
R.
glomalin
from
Experiment
1,
and the
three
fractions
of
HA
collected
at
different
pH
levels
in
Experiment
2.
A
modified Bradford
protein
assay
(Wright
6t
al.,
1996)
was
used
to
measure
BRSP
con-
centration
using
the
Bio-Rad
Protein
Assay
(Bio-Rad,
Hercules,
CA),
which
detects
pro-
teins
>3,000
to 5,000
D
(Bio-Rad
Protein
Assay,
LIT33
Rev
C).
IRSP
was
measured
by
enzyme-linked
immunosorbent
assay
(ELISA),
as
described
by
Wright
and Upadhyaya
(1998)
with
modifications
in
the
enzyme
and
color
developer.
ExtrAvidin
(Sigma-Aldrich,
Inc.)
phosphatase
was
used instead
of
peroxidase.
Wells
were
rinsed
with
Tris
[Tris
(hydroxy-
methyl) aminomethane]-buffered
saline
with
Tween
20
(polyoxyethylenesorbitan
monolau-
rate)
before
adding the
color
developer,
p-
nitrophenyl
phosphate
in
diethanolamine
buffer
(Wright,
1994).
Absorbance
was
read
at 405
nm
after
15
minutes.
Test
samples
were
compared
with
a
standard
curve
produced
by
dilutions
of
highly
irmmunoreactive
glomalin
extracted
from
a
temperate
soil
under
native
grasses.
Percent
imnunoreactivity
was
calculated
as
amount
of
IRSP
divided by
amount
of
BRSP
multiplied
by
100.
988
SOIL
SCIENCE