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

2. 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 Nutrient inputs resulted in changes in vegetation VEGETATION: U3 Lake Okeechobee WCA-1 -3 Everglades Agricultural Area WCA-2A 012345 Km Everglades National Park Cattail Sawgrass/slough Cattail/sawgrass mix F1 ~ 1500mgP/kg ~ 500mgP/kg Cattail Sawgrass

3. Introduction Anaerobic Carbon Cycle Cellulose degradation Fermentation Syntrophy Methanogenesis Homoacetogenesis Sulfate Reduction ANAEROBIC Plant DetritusMonomers and Oligomers (propionate, butyrate, etc); alcohols H 2 and CO 2 Acetate H 2 and CO 2 CH 4 H 2 S and CO 2 Acetate Fermentative Bacteria Homo acetogens Syntrophic Bacteria Sulfate Reducing Bacteria Methanogens Methanotrophs

4. AerobicAnaerobic Lynd et al. 2002. Microbiol.Mol. Biol. Rev. 66(3): 506-577. Endoglucanase Exoglucanase Glucosidase Cellulose Degradation

5. 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)

6. H 2 utilizing bacteria Butyrate Methanogens Syntrophs Methane Butanol Acetone Lactate Propionate Ethanol Acetate Glucose H2H2 CO 2

7. 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.

8. 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. Cellulose degradation and fermentation are two of the most studied microbial processes in laboratory conditions.

9. Hypothesis Composition and metabolism of cellulolytic and fermentative Clostridium group is function of the nutritional status of the Everglades soil.

10. 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.

11. 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.

12. Material and Method The Everglades WCA-2A – Impacted (F1) zone – Transition (F4) zone – Nonimpacted (U3) zone Blue Cypress Marsh – Impacted zone – Nonimpacted zone Soil Samples Samples from 0-10 cm depth will be used

13. 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)

14. 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

15. Objective 2: Molecular Ecology Soil Isolate DNA PCR Correct fragment size Transform to E. coli ATCGATCG Sequence clonesClone PCR cloning vector Mixed rDNA fragment

16. Phylogenetic Analysis Analysis of rRNA gene sequences and determination of their places in the taxonomy. In-silico alignment of sequences Creation of phylogenetic tree

17. Objective 3: T-RFLP Analysis Soil Isolate DNA PCR One primer labeled Automatic sequencer Detects labeled fragment Size of labeled fragment Sp. A Sp. B Sp. C Digest with enzymes (Mixed template)

18. Results

19. Results Most Probable Number (MPN): SoilCelluloseFermentation F12.39x10 5 5.42x10 6 F43.47x10 5 9.17x10 6 U32.43x10 4 1.72x10 6 SoilCelluloseFermentation Impacted5.42x10 5 5.42x10 6 Nonimpacted2.11x10 4 2.21x10 6 Everglades Blue Cypress

20. Glucose Microcosms  mole/g methane Everglades-Nonimpacted Everglades-Impacted mM

21.  mole/g methane mM Blue Cypress-Impacted Blue Cypress-Nonimpacted

22. 0.1 substitutions/site Rhodococcus opacus F3 T11 F14 U1 U33 U3 Clostridium glycolicum F1 U11 F4 F2 Clostridium bifermentans Clostridium tetanomorphum Clostridium aldrichii U4 F8 Clostridium papyrosolvens F10 Clostridium termitidis Clostridium cellobioparum Clostridium cellulolyticum Clostridium josui U8 U27 U19 T26 T14 T8 F7 Bacteroides cellulosolvens Acetivibrio cellulolyticus U2 T3 Clostridium stercorarium T25 U16 Clostridium thermocellum Clostridium acetobutylicum Clostridium butyricum Clostridium ghonii Clostridium sordellii 100 92 99 100 77 100 89 72 99 100 71 100 86 Cluster I Cluster XI Cluster III Fig. Phylogenetic tree of Clostridium cluster III 16S rRNA gene clone sequences obtained from soil samples from F1 (F), F4 (T), U3 (U).

23. 0.1 substitutions/site Clostridium saccharoperbutylacetonicum Clostridium cellulovorans F15 Clostridium tetanomorphum Clostridium ragsdalei Clostridium argentinense Clostridium carboxidivorans T6 T41 T24 Clostridium tunisiense U44 T5 F1 T16 Clostridium butyricum T10 T4 Clostridium quinii T21 Clostridium disporicum T1 T30 Clostridium paraputrificum Clostridium chromoreductans T29 Clostridium favososporum U28 T36 Clostridium acetobutylicum Clostridium saccharobutylicum Sarcina ventriculi Sarcina maxima Clostridium fallax F18 Clostridium bowmanii T2 Clostridium pasteurianum Clostridium acidisoli Clostridium magnum T26 62 65 94 72 60 65 79 60 83 68 78 63 95 93 100 86 100 Fig. Phylogenetic tree of Clostridium cluster I 16S rRNA gene clone sequences obtained from soil samples from F1 (F), F4 (T), U3 (U). Rhodococcus opacus T3 U3-22 Clostridium glycolicum F1-8 Clostridium mangenotii F1-26 Clostridium thermocellum F3 U15 Clostridium bifermentans Clostridium cellulolyticum F1-13 U3-9 F1-19 F1-2 U3-33 U3-1 U3-20 F1-25 F1-20 U3-7 U3-19 U5 U3-30 T12 F17 Clostridium ghonii Clostridium sordellii Clostridium termitidis Clostridium papyrosolvens 100 76 92 62 100 80 100 99 100 95 100 96 100 Cluster XI Cluster III Only F1 and U3 F1, F4 and U3

24. 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.

25. 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.

26. 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.

27. Summary Impacted sites contain larger celluloytic community.

28. THANK YOU