Transcript Characterization of Cellulolytic and Fermentative Communities in Everglades Soils Ilker Uz Soil and Water Science Department University of Florida.

Characterization of
Cellulolytic and Fermentative
Communities in Everglades
Soils
Ilker Uz
Soil and Water Science Department
University of Florida
Widespread agricultural activity in the northern boundary of Florida
Everglades has resulted in nutrient gradients, causing drastic physicochemical
and ecological changes from the original system
Lake
Okeechobee
WCA -1
WCA -2A
WCA - 3
~ 1500 mgP/kg
Everglades
National Park
F1
Cattail
U3
VEGETATION :
Cattail
~ 500 mgP/kg
Cattail/ sawgrass mix
Sawgrass/slough
0 1 2 3
4 5
Km
Nutrient inputs resulted in
changes in vegetation
Sawgrass
Introduction
Anaerobic Carbon Cycle
 Cellulose degradation
 Fermentation
 Syntrophy
 Methanogenesis
 Homoacetogenesis
 Sulfate Reduction
Plant Detritus
Monomers and Oligomers
Methanotrophs
ANAEROBIC
Fermentative
(propionate, butyrate, etc);
alcohols
H 2 and CO 2
Homo
Bacteria
acetogens
Syntrophic Bacteria
Acetate
Acetate
Sulfate Reducing
Bacteria
H 2 S and CO
2
H 2 and CO 2
Methanogens
CH 4
Acetate
Cellulose Degradation
Aerobic
Anaerobic
Endoglucanase
Glucosidase
Exoglucanase
Lynd et al. 2002. Microbiol.Mol. Biol. Rev. 66(3): 506-577.
Fermentation
An energy-yielding metabolic process in
which an organic compound serves as both
an electron donor and an electron acceptor.
(Madigan et al. 1997. Brock Biology of Microorganisms)
Lactate
Propionate
Ethanol
Glucose
H2
Butanol
CO2
Butyrate
Acetone
Acetate
Syntrophs
Methanogens
Methane
H2 utilizing
bacteria
Genus Clostridium
– Gram positive
– Endospore forming
– Obligate anaerobic
– Contains the majority of anaerobic cellulolytic
bacteria.
– Also contains noncellulolytic fermentation
bacteria.
– Highly diverse in 16S rRNA gene sequence and
divided into 19 clusters.
Cellulose degradation and fermentation are two of
the most studied microbial processes in laboratory
conditions.
However:
• Little is known about the ecology of cellulolytic
and fermentative bacteria.
• Their ecology must be investigated to understand
true microbial nature of the Everglades and
impact of nutrient loading on carbon cycling
mechanism.
Hypothesis
Composition and metabolism of cellulolytic
and fermentative Clostridium group is
function of the nutritional status of the
Everglades soil.
H1: Accumulation of nutrient rich organic material
in impacted site correlates with relatively larger
population size in cellulolytic community.
H2: Nutritional status of soils correlates with the
composition of cellulolytic and fermentative
species.
H3: Impacted soils contain a microbial community
that is poised to respond more rapidly to changes
in nutritional status compared to nonimpacted
soils.
Specific Objectives

Characterization of fermentation processes and
fermentation product pattern under different
carbon sources.

Assessment of cellulolytic and fermentative
bacterial assemblages.

Standardization and application of T-RFLP
method for the Everglades Soils.
Material and Method
Soil Samples
The Everglades WCA-2A
Blue Cypress Marsh
– Impacted (F1) zone
– Impacted zone
– Transition (F4) zone
– Nonimpacted zone
– Nonimpacted (U3) zone
Samples from 0-10 cm depth will be used

Most Probable Number (MPN) Counting
– Anaerobic Cellulolytic Microorganisms
– Fermentative Microorganisms

Molecular analysis of MPN dilutions
– Universal 16S rRNA gene Primers

Isolation of Microorganisms From Soil Samples
– Roll tube method (cellulolytic bacteria)
– Glucose enrichment and glucose agar plate technique
(fermentative bacteria)
Objective 1: Fermentation

Microcosms
– Liquid media with basic nutrients and vitamins
– Soil
– Carbon source
 Glucose
 Cellulose
 Plant material (dried crushed cattail and sawgrass)
 Plant material (no P addition in the media)

Measurement of fermentation products in
microcosms


Acetate, butyrate, propionate, lactate, isobutyrate
Methane
Objective 2: Molecular Ecology
Isolate
DNA
Correct
fragment size
PCR
Soil
Clone
Mixed rDNA
fragment
ATCGATCG
Sequence clones
Transform to E. coli
PCR cloning
vector

Phylogenetic Analysis
Analysis of rRNA gene sequences and determination
of their places in the taxonomy.
 In-silico alignment of sequences
 Creation of phylogenetic tree
Objective 3: T-RFLP Analysis
Isolate
DNA
One primer
labeled
Soil
Sp. A
PCR
Sp. C
Sp. B
Size of labeled fragment
Automatic
sequencer
Detects
labeled fragment
Digest with enzymes
(Mixed template)
Results
Results

Most Probable Number (MPN):
Soil
Cellulose
Fermentation
F1
2.39x105
5.42x106
F4
3.47x105
9.17x106
U3
2.43x104
1.72x106
Soil
Cellulose
Fermentation
Impacted
5.42x105
5.42x106
Nonimpacted
2.11x104
2.21x106
Everglades
Blue Cypress
Glucose Microcosms
50
1000
Everglades-Impacted
Acetate
Butyrate
Propionate
Methane
40
800
30
mM
600
20
400
10
200
0
0
0
20
50
40
60
80
Everglades-Nonimpacted
1000
40
800
30
mM
mmole/g methane
600
20
400
10
200
0
0
0
20
40
Time (day)
60
80
mmole/g methane
Acetate
Butyrate
Blue Cypress-Impacted
40
600
30
mM
400
20
Propionate
Methane
mmole/g methane
200
10
0
0
0
20
40
60
80
Blue Cypress-Nonimpacted
40
600
30
mM
400 mmole/g methane
20
200
10
0
0
0
20
40
Time (day)
60
80
0.1 substitutions/site
100
77
99
Clostridium cellulolyticum
Clostridium josui
F2
U8
U27
F1
F8
U19
72
T26
T14
99
T8
F14
F7
Bacteroides cellulosolvens
U1
Acetivibrio cellulolyticus
100
100
Acetivibrio cellulolyticus
Clostridium aldrichii
U2
71
T3
U33
Clostridium stercorarium
T25
U4
U11
92
F10
U16
Clostridium thermocellum
T11
F3
Clostridium acetobutylicum
100
100
Clostridium butyricum
Clostridium tetanomorphum
Clostridium glycolicum
100
Clostridium bifermentans
100
Clostridium ghonii
86
Clostridium sordellii
Rhodococcus opacus
100
89
100
U3
F4
100
Clostridium termitidis
Clostridium cellobioparum
Clostridium papyrosolvens
Fig. Phylogenetic tree of
Clostridium cluster III 16S rRNA
gene clone sequences obtained
from soil samples from F1 (F), F4
(T), U3 (U).
Cluster III
Cluster I
Cluster XI
78
T10
T4
Fig. Phylogenetic tree of Clostridium
cluster I 16S rRNA gene clone sequences
obtained from soil samples from F1 (F),
F4 (T), U3 (U).
F1
Clostridium quinii
T21
65 T6
83 T5
63
Clostridium disporicum
T1
95
T30
Clostridium paraputrificum
F15
Clostridium chromoreductans
T29
Clostridium butyricum
62
Clostridium favososporum
U28
T36
Clostridium acetobutylicum
T41
T16
93
Clostridium saccharobutylicum
Clostridium saccharoperbutylacetonicum
Clostridium cellulovorans
68
100
Sarcina ventriculi
Sarcina maxima
86
Clostridium fallax
F18
86
Clostridium bowmanii
60
T2
72
Clostridium tunisiense
94
Clostridium argentinense
Clostridium tetanomorphum
100
Clostridium pasteurianum
Clostridium acidisoli
U44
Clostridium ragsdalei
Clostridium
carboxidivorans
60
76
T24
79
Clostridium magnum
T26
65
F1, F4 and U3
0.1 substitutions/site
Only F1 and U3
95
U3-9
100
F1-19
F1-13
F1-26
F1-8
F1-2
U3-33
U3-22
100 U3-1
100
U3-20
U15
100
F1-25
99
F1-20
F3
U3-7
U3-19
100
U5
U3-30
62
92
100
T12
F17
T3
Clostridium glycolicum
Clostridium mangenotii
80
Clostridium bifermentans
100
96
Clostridium ghonii
Clostridium sordellii
Clostridium thermocellum
Clostridium cellulolyticum
100
Clostridium termitidis
Clostridium papyrosolvens
100
100
100
100
Rhodococcus opacus
Cluster
XI
Cluster
III
Summary

Impacted soils shows higher and faster
metabolic activity.
– Fermentation process seems to be similar in
impacted and nonimpacted soil microcosms.
(Based on glucose depletion and acetate production trend data)
– Difference in fatty acid accumulation and
depletion pattern may be more dependent of
syntrophic activity rather than type of
fermentation bacteria.
Summary
Based on microcosm studies, type of plant
material as carbon and nutrient source

does not appear to be important in the Everglades
soils.
 created significant difference in Blue Cypress
Marsh.
Summary

Microbial community structure is affected
by the nutrient loading.
– It is a possibility that the composition of
fermentation bacteria depend on activity of
higher trophic bacterial groups.
– Differences observed in phylogenetic analysis
may be used as indicator to monitor bacterial
changes.
Summary

Impacted sites contain larger celluloytic
community.
THANK YOU