A comparison of sediment microbial communities associated with

A comparison of sediment microbial communities associated with

Estumies
Vol.26,
No. 2B, p. 465M74
April 2003
A Comparison of Sediment Microbial Communities Associated with
Phragmites australis and Spartina altemiflora in Two Brackish
Wetlands of New Jersey
BETH R/kvfi "L*, JOAN G. EHRENFEH)L2, and MAX M. HAGGBLOML3
1 Graduate Program i~ E'~vi'ro~me'ntal ,Sciences, 2 D@artment of Ecology, EvoI~utio,, a~d Natural
Resources, s D@art,me~t of Biochemistry and Mi~obioIog3~, Cook College, Rutgers Ur~iversity, New
Bmcnswicl~, New Je'tse~' 08901
ABSTRACT: T h e extensive spread of Phragmites australis throughout brackish marshes on the East Coast of the United
States is a major factor governing management and restoration decisions because it is assumed that biogeochemical
functions are altered by the invasion° Microbial activity is important in providing wetland biogeochemical functions such
as carbon and nitrogen cycling, but there is little known about sediment microbial communities in Phragmites marshes.
Microbial populations associated with invasive Phragmites vegetation and with native salt marsh cordgrass, Sparlirm alg.rnillora , l~lay differ in the relative abundance of microbial taxa (community structure) and in the ability of this biota to
d e c o m p o s e organic substrates (community biogeochemical function). This study compares sediment microbial communities associated with Phragmites and Spartina vegetation in an undisturbed brackish mm~h near Tuckerton, N e w Jersey
(MUL), and in a brackish marsh in the anthropogenically affected Hackensack Meadowlands (SMC). We use phosphofipid
fatty acid (PLFA) analysis and enzymatic activity to profile sediment microbial communities associated with both plants
in each site. Sediment analyses include bulk density, total organic matter, and root biomass. PLFA profiles indicate that
the microbial communities differ between sites with the undisturbed site exhibiting greater fatty acid richness (62 PLFA
recovered from M U L versus 38 from SMC). Activity of the 5 e r w m e s anal3,xed (13-glucosidase, acid phosphatase, chitobiase, and 2 oxidases) was higher in the undisturbed site. Differences between vegetation species as measured by
Principal C o m p o n e n t s Analysis were significantly greater at the undisturbed MUL site than at SMC, and patterns of
enzyme activity and PLFAs did not correspond to patterns of root binmass. We suggest that in natural wetland sediments,
macrophyte rhizosphere effects influence the comammitycomposition of sediment microbial populations. Physical and
chemical site disturbances may impose limits on these rhizosphere effects, decreasing sediment microbial divel~i~- and
potentially, nficrobial biogeochemical fimctions.
nitrogen cycling (Windham in press), a n d the biod e g r a d a t i o n of toxic c o m p o u n d s (Alder et al. 1993;
Adriaens and Vogel 1995; H a g g b l o m et al. 2000).
In this study we c o m p a r e microbial c o m n m n i t y
structure and b i o g e o c h e m i c a l flmctional capacity
in Ph~g'mites and 8])artina aIt~o~,iJlo'ra I,oisel (hereafter 8[artina ) sediments from disturbed and u n d i s t u r b e d sites to d e t e r m i n e w h e t h e r plant species
and a n t h r o p o g e n i c disturbances alter the microbial c o m p o n e n t of the marsh ecosystem. We hypothesized that because the r o o t systems of Phragmites a n d S])arti'~a differ substantially in m o r p h o l o ~ ~ (size a n d relative a b u n d a n c e of rhizomes a n d
roots), total biomass, spatial distribution, a n d the
m e c h a n i s m s of oxygen transport and radial o ~ g e n
loss (Valiela ct al. 1976; Howcs et al. 1981; ~ ' m s t r o n g et al. 1996; Grosse et al. 1996; W i n d h a m
2001), the microbiota associated with their rhizo=
spheres would differ in both c o m m u n i ~ composition and b i o g e o c h e m i c a l flmctions.
Evidence, primarily fl~om stndies of upland soils,
has clearly shown that microbial c o m m u n i l y corn-
Introduction
T h e increasing d o m i n a n c e of Phragmites australis
(Cav.) Trin. ex. Steud. (hereafter Ph'ragmites) in
brackish estuarine marshes has b e e n a cause of maj o r concern, and has sparked extensive and expensive efforts to restore Spard~za species to these
marshes (Meyerson et al. 2000; R o o t h and Stevenson 2000; Rice et al. 2000). Restoration efforts are
ot~en justified in terms of' lost or altered biogeochemical flmctions associated with Photo,mites in=
vasions. While these restoration efforts have stimulated m a n y studies of the comparative ability of
Phra~dtes and 5j)art,ina to s u p p o r t vertebrate a n d
invertebrate fmmas ('vVeinstein a n d Balletto 1999;
Wainwright et al. 2000; }Veis and }Veis 2000), little
attention has been paid to the microbial biota of
the sediments. This biota is critical to the p e r f b >
m a n c e of b i o g e o c h e m i c a l functions, inchtding the
storage or emission of c a r b o n (Brix et al. 1992),
* Correspondit~g m~thor;: tele: 732/932-]051; e-mail: bravit@
eden.rutgers.edu.
© 2003 Estuarine Research Federation
465
466
B. Ravit et al.
position and b i o g e o c h e m i c a l flmctions r e s p o n d to
changes in the p h m t c o m m u n i t y (Wcstover et al.
1997; Grayston ct al. 1998; Ehrenfeld 2001). A
n u m b e r of studies using metabolic profiles, phospholipid fatty acid composition, and DNA analysis
have d e m o n s t r a t e d t h a t m i c r o b i a l c o m m u n i t y
structure differs in rhizosphere soils of different
co-occurring plant species (Borga et al. 1994; Westover et al. 1997; Grayston et al. 1998). }~qfile these
studies used u p l a n d plants, several studies of wetland m a c r o p h y t e s have shown that these species
also at'fi~ct the microbial p o p u l a t i o n s in their rhizospheres ( L u d e m a n n et al. 52000; Piceno and Lovell 2000; Bergholz c t a l . 2001; Lovcll et al. 2001).
For example, d i a z o t r o p h (nitrogen-fixing) p o p u lations differ in composition a n d a b u n d a n c e between Sparti~ta rhizospheres and the rhizospheres
of o t h e r plants or root-flee bulk soils ( G a n @ and
Yoch ] 988; Piceno et al. 1999). If the sediment microbiota of coastal wetlands are sensitive to the species identity of the m a c r o p h y t e communieT; then
changes in species composition, such as Pltmgmil,es
inw~sions or the reestablishment of Sparti.na in
I)hragmites-dominated sites, may alter this biota potentially atli~cfing the b i o g e o c h e m i c a l fimctions
they p e r f o r m .
Microbial c o m m u n i t i e s are also attL'cted by the
presence of contaminants. Toxic c o m p o u n d s can
affect the relatNe a b u n d a n c e a n d activi V of different microbial species, and the use of these contaminants as c a r b o n sources by c o m p e t e n t microbes
may select for a particular microbial c o m m u n i t y
c o m p o s i t i o n (Wolfaardt et al. 1994; Moiler et al.
1997; Karthikcyan et al. 1999).
This study uses two m e t h o d s to c o m p a r e the biog e o c h e m i c a l flmcfions and c o m m u n i t y structure of
the sediment m i c r o b i o t a u n d e r P;tmgmites and
,Spartina in b o t h c o n t a m i n a t e d and u n c o n t a m i n a t ed brackish marshes. M e a s u r e m e n t s of extracellular enzyme actNi V provide an index of microbial
ability to carry out key" processes that provide biog e o c h e m i c a l flmctions (Sinsabaugh 1994; Sinsab a u g h and M o o r h e a d 1994; Sinsabaugh et al.
1993). This enzyme activity has proven sensitNe to
e n v i r o n m e n t disturbance (Nannipieri et al. 1990;
Dick and Tabatabai 1993). C o m p a r i s o n of enzyme
actNities a m o n g soils is c o m m o n l y used in upland
systems to provide insight into the relative biogeochemical capacity of the soil microbiota to carry
out d e c o m p o s i t i o n a n d n u t r i e n t cycling functions
(Sinsabaugh 1994). Enzyme activities have also
been used in a small n u m b e r of studies to investigate the biodegradative flmction of c o n s t r u c t e d
wet.lands (Kang et al. 1998; Shackle et al. 2000).
T h e structure of a sediment microbial c o m m u nity can be e x a m i n e d by looking at the set of phospholipid fatty acids (PLFAs) in the sediments.
PLFAs are key c o m p o n e n t s of microbial cell membranes, and because different g r o u p s of microorganisms p r o d u c e u n i q u e types and suit:cs of thtty
acids, the structure of the microbial c o m m u n i t y
can be e x a m i n e d by looking at the set o f PI.FAs
extractable from a sediment sample (Borga et al.
1994; \,~fite et al. 1996; H a g g b l o m et al. 2000).
Used in c o n j u n c t i o n with each other, these techniques can provide insight into the eft%cts of diftbrent plant species on sediment microbial communities. We used these techniques to test the hypotheses that sediment microbial c o m m u n i t i e s are
difti~rent below l~hmgmites a n d Sparti~la vegetation,
and these diftbrcnces are consistently observed at
difli~rent sites.
Methods
Adjacent p o p u l a t i o n s of Phragmites and Sparti~la
were located on Saw Mill Creek in the Hackensack
Meadowlands, New Jersey (40°46'N, 74°06'W; referred to as SMC), and on an u n n a m e d tidal creek
of the Mullica Riw~g west of the G a r d e n State Park=
way a b o u t 7 km f r o m the river m o n t h (39°33'N,
74°28'W; r e f e r r e d to as MUL). Saw Mill Creek has
h a d a lengthy history of disturbances from the
presence of a w~riety of industrial and regional con=
taminants such as heax T metals, chlorinated hydrocarbons, polycyclic hydrocarbons, a n d o t h e r toxic
c o m p o u n d s (Kennish 1992), as well as n u m e r o u s
physical disturbances such as ditching a n d tidal obstruction ( Q u i n n 1997). SMC waters are brackish
(salinities o f 12-18%0, Bart personal c o m m u n i c a tion), t{ea~T metal c o n c e n t r a t i o n s in the water colu m n are well above b a c k g r o u n d . Median concentrations m e a s u r e d over a 5-yr p e r i o d (1994--1999)
were 25.9 b~g 1 1 Cd, 18.5 b~g 1 1 C1; 28.9 b~g 1 1 Cu,
and 151 Ixg 1 i Pb (New Jersey Meadowlands Commission u n p u b l i s h e d data). Water c o l u m n metal
concentrations, in turn, affect SMC sediment metal
c o n c e n t r a t i o n s (105 Ixg g 1 Cu, 205 Ixg g 1 Zn, 156
Ixg g 1 C~; 2.8 btg g 1 Hg, and 176 btg g 1 Pb [Weis
personM c o m m u n i c a t i o n ] ) . T h e SMC marsh is located between the eastern a n d western branches
of the New Jersey Turnpike, a n d b o u n d e d on the
n o r t h and south by major r o a d and railroad corridors. In contrast, the MUL site is b o u n d e d on
the inland side by the largest p r o t e c t e d watershed
in the n o r t h e a s t (the Pinelands National Reserw~),
and is part of the Jacques Coustean National Estuarine Research Reserve on the seaward side;
there is virtually no industrial activity within the
watershed. T h e MUI, stu@ area is also brackish,
with sMinities of a b o u t ] 8%o.
At b o t h sites, transects 12 m in length, parallel
to the main surface water channel, were established starting at a r a n d o m l y chosen p o i n t within
monospecific p o p u l a t i o n s of each species. T h r e e
Brackish Sediment Microbial Communities
plots, each 4 m 2, were m a r k e d at 2-m interwfls
along the transect. T h r e e replicate sediment cores
e x t e n d i n g to 70-cm d e p t h were r a n d o m l y extract,
ed within each plot using a Russian peat corer
(Aquatic Research Instruments) d u r i n g July-August 2001. All cores were extracted within 2 h of
low tide, and w r a p p e d in gas-impermeable Saran
wrap for transport to tile laboratory, where they
were divided into 10-cm subsections. Each o f these
sections was subsampled for moisture c o n t e n t
( g r a v i m e t r i c analysis), o r g a n i c m a t t e r c o n t e n t
(loss-on-ignition at 500°C), and bulk density (using
a 1 cm deep X 1 cm diam ring p u s h e d into the
core section, dried, and then weighed). 'i\vo additional sets of subsamples were taken f r o m the 0 10, 30-40, a n d 60-70 cm subsections. Subsamples
were processed immediately for enzyme activities
or frozen at - 2 0 ° C for later PLFA analysis as described below.
Root and rhizome biomass was d e t e r m i n e d from
samples o b t a i n e d by excavating soil blocks within
each of the plots. This m e t h o d was used because
preliminary sampling at SMC suggested that: the
peat corer was n o t adequately sampling larger rhizomes. At SMC, rectangular blocks (15 × 44 × 10
cm) were excaw~ted using a metal fl-ame to define
the block. A b o v e g r o u n d vegetation was (:lipped to
the marsh surface, a n d the f r a m e inserted into the
sediments to a depth of 10 cm using a sharp knife
and a shovel to cut the r o o t mat, and then to remove the intact block. At MUL a circular metal
frame (8.'5 X 11 cm) was used. Blocks were tran>
p o r t e d to the laboratory where sediments were
washed fl~om the roots and rhizomes. Because of
the large mass of material and the interwoven nature of diffL'rent r o o t sizes a n d status, no a t t e m p t
was m a d e to separate live and dead roots or rhizomes. Washed biomass was dried at: 70°C to constant weight.
PttOSPtIOLIPH) FxI"I'Y ACID ANALYSIS
T h e m e t h o d o f PLFA analysis described by White
et al. (1979) was slightly modified. Samples were
thawed and dewatered by centrifiaging, and duplicate subsamples were r u n for each extraction. Fatty
acids were extracted using a single-phase chlorot b r m : m c t h a n o l : 0 . 0 5 M p h o s p h a t e buftbr (pH 7.5)
solvent. T h e c o n c e n t r a t e d c h l o r o f o r m extract was
separated into lipid fl'acfions on a silicic acid colu m n using the m e t h o d of King et al. (1977). T h e
p h o s p h o l i p i d flmcdon was eluted with methanol,
saponified, and methylated a c c o r d i n g to protocols
for the Microbial Identification System (MIDI
1995). T h e MIDI Sherlock Microbial Identification
System (MIS, Microbial ID, Newark, Delaware) was
used to identi~ ~ individual fatty acid methyl esters
based on their gas c h r o m a t o g r a p h (GC) retention
467
time. Individual fatb r acids were quantified as a per(enrage of the total fatty acids recovered f r o m the
sample. Fatty acids c o n t r i b u t i n g < 1% of the total
a m o u n t extracted f r o m each sample or observed
in only o n e sample were eliminated from the data
set, leaving 41 fatty acids for statistical analysis.
ENZ~'.~E ANAIXSIS
Using the m e t h o d s of Sinsabaugh et al. (1993),
the activity o f 5 extracellular enzymes related to
c a r b o n (C), nitrogen (N), p h o s p h o r u s (P), a n d
soil organic matter (SOM) cycling were measured.
~-glucosidase (E.C. 3.2.1.21 ), an enzyme i m p o r t a n t
in degradation of cm'bohydrates, was used as an
indicator of C cycling potential. ~-N-acetylglucosaminidase (E.C. 3.2.1.30), c o m m o n l y known as chitobiase, is involved in d e g r a d a t i o n of the N-con=
raining fungal cell wall c o m p o u n d acetylglucosamine, and was used as the N-related enzyme. Acid
p h o s p h a t a s e (E.C. 3.1.3.2), an enzyme necessary
for the d e g r a d a t i o n of P c o n t a i n i n g organic molecules, was used as an indicator of P cycling potentiM. 'I~'o enzymes active in the d e g r a d a t i o n of comp l e x o r g a n i c substrates, p h e n o l o x i d a s e (E.C.
1.10.3.2 and E.C. 1.14.18.1) and pcroxidase (E.C.
1.11.1.7) were used as indicators of SOM cycling.
Ttle substrates used for ~-glucosidase, chitobiase,
and acid p h o s p h a t a s e were p-nitrophenol (pNP)
derivatives t h a t r e l e a s e p N P w h e n h y d r o l y z e d ,
which is then analyzed spectrophotometrically.
Substrates (10 raM) were each dissolw~d in 50 mM
acetate bufIbr (pH 5) a n d a d d e d to a sediment
slurry. Two ml of slurry were incubated with 2 ml
of each substrate at 20°C for the time specified for
each enzyme. Enzyme and sediment controls were
r u n simultaneously. After incubation the samples
were centrifitged a n d 1 ml of' the s u p e r n a t a n t was
then a d d e d to 0.2 ml o f I N N a O H followed by
the addition of deionized HeO. Absorbance was
m e a s u r e d on a s p c c t r o p h o t o m e t e r (Spectronic Instruments) at 410 n m and enzyme activity expressed as b~M substrate g 1 h 1. T h e same procedure was used to m e a s u r e p h e n o l oxidase a n d peroxidase activity using L-DOPA as the substrate. Two
ml of sediment slurry were mixed with 2 ml o f
substrate (plus 0.2 ml of' 0.03% H202 fbr pcroxidase) a n d incubated 30 rain at 20°C. Enzyme a n d
sediment controls were run simultaneousl> Samples were c e m r i f u g e d at 2,500 RPM for 10 min a n d
the s u p e r n a t a n t directly used to measure absorb a n c e at 460 ran. Enzyme activity is expressed as
b~M substratc g 1 h i T h e peroxidasc assay includes the addition of }I202, a n d measures the sum
of p h e n o l and peroxidase activity. Peroxidase activit)' alone was obt~fined by subtracting p h e n o l oxidase results f r o m the peroxidase total results.
468
B. R a v i t e t al.
14,000~
(a) M o i s t u r e C o n t e n t (%)
100
5o
150
200
250
o
-g
12,000
lO
SITE
•
0-10 cm
[]
[]
10-20cm
20-30m
10,000
2O
g
8,000
~- 30
6,000
40
INT
'
' •
50
INT
'
~
60
INT
' "~
~c
'
, ~
o
•
4,000
27
2,000
70
o
O.OO
g
0.10
0.30
INT ~
0.20
10
20
NS
NS
30
NS
0.40
0.50
0.60
~
4o
50
NS : . . . . . . . . . . .
60
70
(c) O r g a n i c C a r b o n (LOI)
0
g
o
5
10 .
.
.
.
15
20
10
20
~0
" ~
-
SMC S
b) B u l k D e n s i t y (g c m -3)
INT
40
50
INT
60
INT
J- S M C Spartina
- - , , I - " S M C Phragmites
.L
~
MUL
° MUL
Spartina
Phragmites
Fig. 1. Sediment characteristics through the profile. (A)
gravimetric moisture content, (B) bulk density, and (C) organic
matter (as p e r c e n t loss-on-ignition). The results of/wo-way analyses of ~ariance are given as the name of the significant £tctor
in the ANOVA (Site, Species, or Int for interaction).
STATIS I'ICAL ANAL'~'ISIS
S e d i m e n t c h a r a c t e r i s t i c s , i n c l u d i n g r o o t biomass, w e r e a n a l y z e d using two-fhctor analyses of'
w~riance 0~NOVA; SITE factor: M U L versus SMC,
a n d SPECIES fhctor: Photo, mites and Spartit~a, n =
3 fbr e a c h site-species c o m b i n a t i o n ) . Two-way ANOVAs were also a p p l i e d to t h e d a t a fl~om e a c h enz y m e a n d each fatty acid. C o n t r a s t analysis was
u s e d to c o m p a r e v e g e t a t i o n effects within e a c h
site. Principal c o m p o n e n t s analyses w e r e u s e d to
e x a m i n e p a t t e r n s a m o n g the f o u r site-species c o m b i n a t i o n s b a s e d o n the multivariate sets o f e n z y m e
activities a n d fatty- acids.
Results
S e d i m e n t characteristics w e r e d i f f e r e n t b e t w e e n
the sites (Fig. 1), a l t h o u g h t h e quantitative differe n c e s a m o n g t h e site-species c o m b i n a t i o n s for all
o f the variables w e r e small. I n m o s t cases, A N O V A
.
SMC PH
.
MUL S
.
.
.
.
.
MUL PH
Fig. 2. Total root and rhizome biomass in excavated sedim e n t blocks for the top 30 cm o f sediment.
rcw~'aled significant i n t e r a c t i o n s bct~vecn sites a n d
species, a n d c o n t r a s t analyses d e m o n s { r a t e d differe n t r e l a t i o n s h i p b e t w e e n t h e species at the two
sites. S e d i m e n t m o i s t u r e b e n e a t h Ph'rag~mi~es was
h i g h e r t h a n b e n e a t h S/~artirza in SMC, b u t the opposite p a t t e r n was f o u n d at M U I , (Fig. l a). T h e
b u l k density o f surface s e d i m e n t s u n d e r Spartirea
was lower t h a n u n d e r Ph~g~mites in M U L b u t n o t
SMC, b u t few o t h e r d i f f e r e n c e s w e r e f o u n d lower
in t h e profile (Fig. l b). O r g a n i c m a t t e r (Fig. l c )
was consistently h i g h e r u n d e r 5~art~i~,a t h a n u n d e r
PhraUtdtes at M U L , b u t t h e o p p o s i t e was o b s e r v e d
at ~he SMC site. T h e a b s o l m e difli~rence b e t w e e n
the sites is again relatively small (partictflarly f o r
the two Ph'rag~mi~es p o p u l a t i o n s ) .
Total r o o t a n d r h i z o m e b i o m a s s in the top 10
c m was h i g h e r u n d e r S/Jarti,tta titan u n d e r Ph~(g'mites at b o t h sites, a n d r o o t biomass was g e n e r a l l y
h i g h e r at t h e d i s t u r b e d SMC site t h a n t h e undist u r b e d M U L site (Fig. 2; species f a c t o r in two-way
A N O V A F1,~1 = 9.407, p < 0.01, site f a c t o r F1,~1 =
11.11, p < 0.01 ). N o significant d i f f e r e n c e s in r o o t
b i o m a s s were f o u n d f o r the lower layers.
P a t t e r n s o f extracellular e n z y m e ac{ivity w~ried
with b o t h t h e species a n d t h e site (Fig. 3). In m o s t
cases two-way A N O ' V ~ s h o w e d t h a t the p a t t e r n o f
diftL'rences b e t w e e n t h e species d e p e n d e d o n the
6O
• SMC Spartina
[ ] SMC Phragmites
[ ] MUL Spartina [ ] MUL Phragmites
40
3O"
2O
10
0
Phenol
Oxidase
Peroxidase
Total
Oxidase
Chitobiase
E3-Glucosidase
Acid
Phosphatase
Enzyme
Fig. 3. Activities (b~M g soil i h 1) of extrace]hflar enzymes
of tile two species at each site, for three sediment depths.
Brackish Sediment Microbial Communities
469
' I A B L E 1. P a t t e r n s o f s i g n i f i c a n c e in /wo-way a n a l y s e s o f v a r i a n c e a n d 1 d f c o n t r a s t s o f site a n d s p e c i e s efti-cts o n e x t r a c e l l u l a r e n z y m e
activities, f o r e a c h o f t h r e e s e d i m e n t d e p t h s ( T o p = 0 - 1 0 c m , M i d d l e = 3 0 - 4 0 c m , B o t t o m = 6 0 - 7 0 c m ) . P = PhfrGmites, S = Spartina.
L e t t e r s (a,b,c) i n d i c a t e s i g n i f i c a n c e level o f t h e overall 2-way A N O \ ~ M a n d n u m b e r s (1,2,3) i n d i c a t e s i g n i f i c a n c e o f t d f c o n t r a s t s
c o m p a r i n g species w i t h i n e a c h site. i n t = i n t e r a c t i o n f h c t o r ~,~ p < 0.05, u,e p = 0.01, ~,~; = p < 0.001; " P = S " i n d i c a t e s n o s i g n i f i c a n t
d i f f e r e n c e in 1 d f c o n t r a s t s .
Enzyme
Site
Phenoxidase
MUL
SMC
Peroxidase
MUL
SMC
Chitobiase
MUI,
SMC
{3-Glncosidase
MUI,
SMC
Acid
phosphatase
MUL
SMC
Top
Middle
Bottom
lnt ~
Int ~
Int ~
P > Ss
Int°
S > ps
P > S~
S > P:~
Sitec
S= P
P > Ss
S = P
Intb
P > Ss
Int~
P > Ss
P = S
Site~ ( M > S)
P = S
In£ °
P = S
P = S
Siteb ( M
P > Ss
P = S
S > ps
S= P
P > Ss
S > ps
Intb
S > ps
P > Ss
Intb
P'> S3
P > S3
Site~
S > p3
S= P
Site b (M > S)
Site b (M > S)
P = S
P = S
P > S~
P > S~
S = P
site ( T a b l e 1). P h e n o l o x i d a s e activities w e r e h i g h er t r a d e r Phragmil~es t h a n Sparl,ina al M U L , b u t t h e
o p p o s i t e p a t t e r n was obserw~'d at SMC. Conversely,
p e r o x i d a s e , the o t h e r e n z y m e i m p l i c a t e d i n t h e
d e g r a d a t i o n of lignin, hmnics, and other c o m p l e x
substra~es, was h i g h e r t r a d e r Spartina t h a n Phragmites at M U L . C h i t o b i a s e h a d low levels o f activity
at all sites, b u t s i g n i f i c a n t l y d i f f e r e n t p a t t e r n s at t h e
two sites w e r e f o u n d (Phragmites s e d i m e n t s s h o w e d
g r e a t e r activi V t h a n d i d ,S~)artinas e d i m e n t s at M U I ,
b u t n o t SMC). ~-glucosidase s h o w e d h i g h e r surface s e d i m e n t activiO" u n d e r b o t h species at M U L
t h a n at SMC, b u t h i g h e r activi~" u n d e r S/)arti~la
t h a n t r a d e r PhraFmites l o w e r i n t h e profile. Acid
p h o s p h a t a s e activity was especially h i g h i n s u r f a c e
s e d i m e n t s u n d e r Phf~gmites at M U L , a n d all sedim e n t s a m p l e s fi~om M U L e x h i b i I e d h i g h e r activity
t h a n t h e c o r r e s p o n d i n g s e d i m e n t s f r o m SMC (Fig.
3). C o n t r a s t analyses ( T a b l e 1) s h o w e d t h a t diffi~'re n c e s b e t w e e n t h e species w e r e c o m m o n l y obs e r v e d for m o s t e n z y m e s at m o s t d e p t h s in M U I ,
b u t m o r e rarely i n SMC.
P r i n c i p a l c o m p o n e n t s analysis u s i n g t h e 5 enzymes at t h e 4 site-species c o m b i n a t i o n s (Fig. 4a)
i n d i c a t e d t h a t w h i l e t h e two species w e r e s e p a r a M e
at: e a c h site, this d i f f e r e n c e was g r e a t e r at M U L
t h a n at SMC. A t SMC b o t h species c l u s t e r e d close
t o g e t h e r ; at M U L t h e Iwo sets o f s a m p l e s c l u s t e r e d
f a r t h e r apart. T h e r e was n o t e n d e n c y f b r t h e two
p o p u l a t i o n s of' e a c h species to b e f o u n d i n adjac e n t areas of' t h e o r d i n a t i o n space.
T h e d i s t r i b u t i o n o f p h o s p h o f i p i d fatD T acids for
each o f t h e site-species c o m b i n a t i o n s (Fig. 5) suggests small d i f f e r e n c e s b e t w e e n species a n d sites.
O f t h e 41 fatty acids r e t a i n e d for analysis, 7 w e r e
f o u n d o n l y at M U I , b u t o n l y o n e was f f m n d exclusively at SMC. At M U L , 7 fatty- acids w e r e f o u n d
> S)
o n l y u n d e r Ph~tgmitss b u t n o t S/;arti~ta, w h e r e a s at
SMC, only o n e fatty acid was r e s t r i c t e d to Phragmites s e d i m e n t s . U b i q u i t o u s thtty acids c o n t r i b u t e d
a h i g h e r p e r c e n t a g e to t h e total fhtty acids extract,
ed t i ' o m SMC s e d i m e n t s t h a n f r o m M U L sedim e n t s . T h e s e p a t t e r n s s u g g e s t a less diverse m i c r o bial p o p u l a t i o n with less d i f f e r e n c e b e t w e e n t h e
species at SMC t h a n at M U L . Two-way ANOVAs for
each o f t h e PLFAs ( T a b l e 2) show t h a t for a b o u t
h a l f t h e fat~" acids t h e r e w e r e n o s i g n i f i c a n t differe n c e s a m o n g samples; f o r t h e r e m a i n i n g f a t ~ acids, i n t e r a c t i o n effects w e r e p r e d o m i n a n t . I n o n l y
a few cases, i n s u r f a c e a n d d e e p s e d i m e n t layers,
w e r e d i f f e r e n c e s a m o n g species u n i t b r m l y s e e n at
the two sites. C o n t r a s t analysis i n d i c a t e s species diff e r e n c e s are s i g n i f i c a n t at MUL, b u t n o t at SMC.
PLFA p r i n c i p a l c o m p o n e n t s analysis (Fig. 4b) indicates o r d i n a t i o n s e p a r a t i o n is m u c h g r e a t e r bet w e e n species at M U L t h a n at SMC, a s i m i l a r findi n g to t h a t o b s e r v e d w'ittl e n z y m e activities. O n l y
in t h e MUL-Ph~g'mites s a m p l e s w e r e t h e r e signific a n t d i f f e r e n c e s with d e p t h ( t h e m i d - l a y e r s a m p l e s
d i f f e r e d K o m t h e t o p a n d b o t t o m layers), so t h e
s a m p l e s f r o m t h e t h r e e d e p t h s w e r e l u m p e d tog e t h e r in d i e anMysis. T h e Phragmit(,~sa n d Spartina
s a m p l e s f r o m t h e SMC s a m p l e s c o - o c c u r i n t h e ord i n a t i o n space; i n c o n t r a s t , l h e species ficom t h e
MUL samples are clearly separated, primarily
a l o n g t h e first o r d i n a t i o n axis. I n t e r e s t i n g l y , t h e
Spartina s a m p l e s fi~om M U L o v e r l a p t h e a r e a o f t h e
o r d i n a t i o n space o c c u p i e d by t h e SMC samples.
Discussion
T h e d a t a s u g g e s t t h a t t h e d i f f e r e n t i a t i o n o f sedi m e n t m i c r o b i a l c o m m u n i t i e s b e n e a t h p l a n t species in b r a c k i s h m a r s h e s is c o n t i n g e n t o n q u a l i t i e s
a s s o c i a t e d with t h e i n d i v i d u a l sites. P a t t e r n s o f dif-
470
B. Ravit et al.
(a) Enzymes
2
SMC-Phragmites
MU L-Phragmites
• SMC.Spartina
• MUL.Spartina
m
O
u~
1.5
O3
143
1
0.5
II
[]
(-~
0
[]
-0.5
X
-1
-1.5
-2
-1
0
-0.5
0.5
1
1.5
PCA AXIS 1
2
2.5
3
3.5
Z=2.73, 54.7%
(b) PLFAs
&&
CO
~i
r~
II
Cq
m
2
1
[]
x
0
.;
[]
•
(J
•
Q.
•
Jail
[]
•
",
-1
-2
-2
-1.5
-1
-0.5
0
PCA Axis 1
0.5
1
1.5
2
2.5
X= 9.24, 22%
Fig. 4. Principal compouents ordiuatior~ diagrams for (a) enzyme activities and (b) aggregate PLEA variables. For each diagram,
the eigenvalue and the percent of ~ariation explained are given along each axis.
ference between descriptors of the m i c r o b i o t a ob=
served at the u n d i s t u r b e d MUL site were m o r e fi~equently statistically significant than at: SMC, a n d
were often opposite in direction to those o c c u r r i n g
at the heavily disturbed SMC site. These patterns
of difference bet~veen species within each site do
not clearly c o r r e s p o n d to the relatively small differences in physical conditions between sites.
T h e u n d i s t u r b e d MUI, site generally h a d h i g h e r
levels of enzyme activib~ with greater variation in
activity between plant species tfian did tfie SMC
marsh. Soil enzyme activities have b e e n interpreted
to be indices of' microbial biomass, microbial activit}', and the ecological health of' soils and sediments
(Nannipieri et al. 1990; Dick and Tabatabai 1993;
Ajwa et al. 1999). A l t h o u g h individual enzyme activities may not always correlate with o t h e r measures of soil qualiO ~ or microbial activity (Frankenb e r g e r a n d Dick 1983), assessments based on the
aggregate activities of several enzymes have proven
471
Brackish Sediment Microbial Communities
T A B L E 2. N u m b e r of" s i g n i f i c a n t f a c t o r s of" e a c h i a c t o r i n 2-way
a n a l y s e s o f v a r i a n c e o f site a n d species effects for t h e p h o s p h o l i p i d fittty a c i d s in t h e s n r f a c e s e d i m e n t s .
20
0
;& ~ , ~
o
25
~°
-
,
o
,~,
~
,~,
o
2
,
og
. r ~
~
o
o
=~
aa>{
~
o
2 ga
,
Depttt
Site
Species
I~',teracfion
No~e
0-10 cm
3 0 - 4 0 cm
60-70 cm
6
7
8
3
0
,i
13
7
8
18
23
19
g~
"<~<~,
S a w Mil~ Creek
[~
~gFe
Fatty Acid
I ~s~cs~o,,°°
~s~,~,.,o~
I
Fig. 5. R e l a t i v e a m o u n t s (as p e r c e n t a g e s o f t h e t o t a l Iht/y
a c i d e x t r a c t ) o f 25 m o s t c o m m o n PLFAs i s o l a t e d f r o m snrt~ace
s e d i m e m s in t h e f o u r sets o f s a m p l e s .
usefifl in c o m p a r i n g sites with d i f f e r e n t l a n d uses
a n d h i s t o r i e s of' d i s t u r b a n c e ( F r a n k e n b e r g c r a n d
D i c k 1983; N a n n i p i e r i e t al. 1990). T h e c o n s i s t e n t
p a t t e r n o f l o w e r e n z y m e activities at SMC, f o u n d
a c r o s s m a n y o f file e n z y m e assays (5 e n z y m e s at
each of 3 depths under each species), suggests that
m i c r o b i a l f l m c t i o n in g e n e r a l m a y b e l o w e r at t h e
H a c k e n s a c k site t h a n in t h e u n d i s t u r b e d M U L site.
S o m e s t u d i e s h a v e f o u n d t h a t e n z y m e activities
a r e c o r r e l a t e d w i t h soil o r g a n i c m a t t e r c o n t e n t
(~Valdrop e t al. 2000), b u t o t h e r s t u d i e s h a v e n o t
f o u n d this to b e case (Eiva~i a n d B a y a n 1996). T h e
p a t t e r n o f e n z y m e activities we o b s e r v e d d i d n o t
n e c e s s a r i l y c o r r e s p o n d to t h e p a t t e r n s o f organic:
m a t t e r in t h e s e d i m e n t s a m p l e s . F o r e x a m p l e , p e r o x i d a s e activities at M U L a r e h i g h e r u n d e r S/}arl&la
t h a n Phra~mites ( T a b l e 1), d e s p i t e t h e o p p o s i t e patt e r n o f organic: m a t t e r c o n t e n t (Fig. l c ) . T h e d i f
f e r e n c e s w e o b s e r v e d in S M C m a y also r e f l e c t t h e
d i s t u r b e d a n d c o n t a m i n a t e d n a t u r e o f t h e sedim e n t s . I n a v a r i e t y o f studies, D i c k a n d T a b a t a b a i
(1993) f o u n d t h a t e n z y m e activities a r e sensitive to
metal contamination;
Kuperman and Carreiro
(1997) f o u n d a 10-to-50-fold r e d u c t i o n in e n z y m e
activity, w h i c h p a r a l l e l e d i n c r e a s e s in heax T m e t a l
concentrations.
T h e r e s u l t s o f this study w e r e c o n t r a r y to e x p e c t a t i o n in sew~'ral ways. A t SMC, t h e d i s t u r b e d site,
the root biomasses were considerably higher than
t h o s e r e p o r t e d in t h e l i t e r a t u r e (Spardtea > 10,000
g m 2 v e r s u s 3 , 3 0 0 - 5 , 0 0 0 g m 2 [Valiela e t al.
197(5]; Phragmites .~6,500 g m 2 v e r s u s 1,400 g m 2
[ W i n d h a m 2 0 0 1 ] ) ; in c o n t r a s t , t h e b i o m a s s e s rec o r d e d at t h e M U L site w e r e w i t h i n t h e s e r e p o r t e d
r a n g e s f r o m o t h e r a r e a s (S])arti~a ~ 6 , 5 0 0 g m 2,
Phra~mites .-~1,600 g m 2). W e d o n o t k n o w if this
f i n d i n g is a r e s u l t of' o u r s a m p l i n g a l a r g e s e d i m e n t
b l o c k r a t h e r t h a n s m a l l e r s e d i m e n t c o r e s o r a result o f m o r p h o l o g i c a l e t f c c t s o f a n t h r o p o g c n i c impacts. W h i l e t h e i n d i v i d u a l r h i z o m e s o f Phragmi~es
a r e m u c h l a r g e r t h a n t h e Spartina r h i z o m e s , e a c h
s u p p o r t s a s m a l l e r n u m b e r o f r o o t s , a n d t h e rtftz o m e s a r e less d e n s e l y p a c k e d w i t h i n t h e s u r f a c e
s e d i m e n t s t h a n a r e t h e Spartina r o o b r h i z o m e syst e m s . T h i s p a t t e r n was p a r t i c u l a r l y m a r k e d at SMC,
w h e r e file S/Jarti,tta o c c u r r e d as t u s s o c k s s e p a r a t e d
by s p a r s e l y v e g e t a t e d o r b a r e m u d ; at M U L , t h e
Spartina f o r m e d a m o r e typical u n i f o r m lawn. T h e
tussocks had notably dense root,rhizome mats,
compared with the other sampling locations.
A n o t h e r s u r p r i s i n g f i n d was t h a t t h e c o m p a r i s o n
o f m i c r o b i a l s t r u c t u r e a n d f l m c t i o n d i d n o t corr e s p o n d to t h e a m o u n t s o f r o o t b i o m a s s in t h e sedi m e n t s . A l t h o u g h e n z y m e activities w e r e h i g h e r in
m o s t cases at M U L , r o o t b i o m a s s e s w e r e l o w e r at
this site, p a r t i c u l a r l y in t h e s u r f a c e h o r i z o n s . In add i t i o n , t h e r e was a l a r g e r c o n t r a s t in r o o t b i o m a s s
b e t w e e n S'/~artina a n d Phragmites at M U L t h a n at
SMC, b u t t h e p a t t e r n s o f e n z y m e activity a n d P L F A
o c c u r r e n c e s (lid n o t c o r r e s p o n d to t h e s e p a t t e r n s
o f root: b i o m a s s . T h e s e restflts s u g g e s t t h a t m i c r o b i a l c o m m u n i t y s t r u c t u r e a n d f u n c t i o n a r e n o t simp l y a n d d i r e c t l y d r i w : n by t h e q u a n t i t i e s o f r o o t s in
the sediments.
Difl~wcnccs in microbial communities were
f o u n d to b e m a r k e d l y diflL'rent b e t w e e n t h e two
sites. W e h a d e x p e c t e d t h a t t h e d i f f e r e n c e s in oxyg e n a t i o n m e c h a n i s m s e m p l o y e d by file tsvo p l a n t
s p e c i e s ( V e n t u r i - e n h a n c e d c o n v e c t i v e a i r f l o w in
Ph~ztgmites [ A r m s t r o n g e t al. 1996] v e r s u s c o n v e c t i o n - e n h a n c e d diftilsive flow in Sparti~,a [ t I w a n g
a n d M o r r i s 1991; t { o w e s a n d Teal 19941) w o u l d
r e s u l t in d i f l c r e n t levels of' r h i z o s p h e r e o x y g e n a ,
t i o n , p r o v i d i n g c o n d i t i o n s t h a t select: t b r d i f l b r e n t
rhizosphere microbial communities. Microbial
populations were significantly different between
p h m t s p e c i e s at t h e M U L site b u t n o t at: t h e S M C
site.
In u p l a n d soils, d i f f e r e n c e s in m i c r o b i a l c o m m u n i ~ ~s t r u c t u r e a n d b i o g e o c h e m i c a l f u n c t i o n are
f r e q u e n t l y o b s e r v e d b e n e a t h d i f f e r e n t p l a n t species ( G r a y s t o n et al. 1998; ~Valdrop e t al. 2000;
K o u r t e v e t al. in p r e s s ) . T h e s e d i f f e r e n c e s a r e
t h o u g h t to c o r r e s p o n d to d i f f e r e n c e s in r o o t bio-
472
B. Ravit et al.
mass, root distribution,
root exndation,
phmt
growth characteristics, and litter chemistry (Hobb i e 19{12; B i n k l e y a n d G i a r d i n a 1998; E h r e n f e l d
2 0 0 1 ) . It is s u r p r i s i n g to f i n d t h a t s i g n i f i c a n t diff e r e n c e s i n e n z y m e activi~" a n d fatD ~ a c i d p r o f i l e s
w e r e f o u n d at t h e M U L site b u t n o t at t h e S M C
site. ~ : e o f f e r f o u r p o s s i b l e n o n - m u t u M l y e x c l u s i v e
e x p l a n a t i o n s f o r t h e s p e c i e s - s p e c i f i c m i c r o b i a l eff e c t s o b s e r v e d in s e d i m e n t s at t h e s e 15vo sites.
First, it m a y b e t h a t t h e s t r e s s f l d a n d s p e c i a l i z e d
c o n d i I i o n s o f h i g h l y d i s t u r b e d a n a e r o b i c soils i m pose controls on microbial community structure
and biogeochemical function that make the more
subtle effects of variations in plant species relatiwdy u n i m p o r ~ a n L S e c o n d , d e s p i t e t h e d i f f e r e n c e
in r o o t o ~ g e n a t i o n
mechanism,
there may be
e q u a l l y l o w o x y g e n loss to t h e s e d i m e n t s f r o m t h e
r o o t s , i f m o s t o r all o f t h e t r a n s p o r t e d o x y g e n is
u s e d by t h e r o o t t i s s u e s ( H o w e s a n d T e a l 1 9 9 4 ;
B r i x e t al. 1 9 9 6 ; A r m s t r o n g e t al. 2 0 0 0 ) . I f t h e r e
is n o d i f f e r e n c e in t h e r e d o x s t a t u s o f t h e S M C
rhizoplanes and rhizospheres of the two species,
d i f f e r e n c e s i n t h e m i c r o b i o t a w o u l d b e less l i k e l y
to o c c u r . T h i r d , r o o t , d e r i v e d C is t h o u g h t to b e
the major feature generating differences in the
m i c r o b i o I a , as s u g g e s t e d f o r u p l a n d s o i l s ( G a r l a n d
1 9 9 6 ; G r a y s t o n e t al. 1{198); h o w e v e r , i n t h e m a r s h
p e a t s , t h e r e is a l a r g e a m o u n t o f C a v a i l a b l e in t h e
salt m a r s h s e d i m e n t s , a n d t h i s C m a y r e n d e r t h e
plant-derived C of minor importance to the microbiota.
Finally, the higher enzyme activities and larger
n u m b e r o f fat D" a c i d s r e c o v e r e d f r o m t h e M U L
s e d i m e n t s ( t o t a l o f 62) v e r s u s t h e S M C s e d i m e n t s
( t o t a l o f 38) s u g g e s t t h a t t h e h i s t o r y o f c o n t a m ination and physical disturbance
in the Meadowlands has had a direct etlix:t on microbiallymediated
biogeochemical
processes through
a
d i m i n u t i o n o f m i c r o b i a l a b u n d a n c e , activity, a n d
diversity. Without more specific data on contaminant loads and comparative studies within the
Meadowlands
district, this remains speculation.
There are abundant data in the microbiological
literatm'e showing that stress due to contamination and disturbance can reduce enzyme activity
and microbial
diversity (Dick and Tabatabai
1993; Kuperman
and Carreiro 1997)o The large
difference in enviromnemal
hislory of the two
s i t e s m a y haw~" o v e r w h e h n e d
the effects of the
two plant species, contributing to the conclusion
that brackish marsh microbial communities
are
p r i m a r i l y a f f e c t e d by t h e n a t u r e o f t h e site. I n
highly disturbed
systems, site-specific variables
may be of greater importance in determining microbial community
composition
than the macr o p h y t e s s p e c i e s f o r m i n g t h e p l a n t c o v e r . O u r resuits indicate that root-mediated
effects on mi-
crobial communities
appear to be impacted in
w e t l a n d s e d i m e n t s o f d i s t u r b e d s y s t e m s . T h e r e is
a n o t a b l e l a c k ot' s t u d i e s u s i n g t h e s e a n a l y t i c a l
t e c h n i q u e s i n o t h e r w e t l a n d soils, a n d so t h e r e is
a c l e a r n e e d f o r a d d i t i o n a l w o r k t o t e s t t h e s e hypotheses.
ACKNOWLEDGMENTS
We thank the Meadowlands Environmental Research Institute, Educational Foundation of America, and National Science
Foundation Grant LMG0120616 for the flmding that supported
this research. We are deeply grateflfl to Oscar Abelleira, Michael
Ovadia, Megi Kourteva, Tish Robertson, Kvitee, Dr. Jean-Christophe Clement, Dr Peter Kourtev, and Dr Elisha ~Ihy-Or t~r
their unflinching assistance in the field and the laboratory. We
also thank Ed Konsevick and .Joe Sarnoski Ii'om New .Jersey
Meadowlands Commissions tier providing boat transportation to
the SMC site.
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