Presentation on theme: "Characterization of Cellulolytic and Fermentative Communities in Everglades Soils Ilker Uz Soil and Water Science Department University of Florida."— Presentation transcript:
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 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
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
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)
H 2 utilizing bacteria Butyrate Methanogens Syntrophs Methane Butanol Acetone Lactate Propionate Ethanol Acetate Glucose H2H2 CO 2
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.
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.
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 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
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 Soil Isolate DNA PCR Correct fragment size Transform to E. coli ATCGATCG Sequence clonesClone PCR cloning vector Mixed rDNA fragment
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 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)
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.