METHODS IN DECOMPOSITION ECOLOGY Soil Biolo gy Mo dule Gareth GRIFFI TH [email protected]BS23420 WHAT is measured and HOW 1. WHAT? Inputs into the decomposer subsystem Fluxes (rates of flow o f energy/nutrients) Losses from decom poser subsystem 2. HOW? Decomposer activity (which org anisms) Microbes vs. Animals Primary vs Secondary Decompo sers Active vs. Dormant (or Dead vs. Alive) Polymer Degradatio n (i.e. mineralisation) METHODS IN DECOMPOSITION ECOLOGY
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METHODS IN DECOMPOSITION ECOLOGY Soil Biology Module ... · Mesh size : 7 mm all decomposers Mesh size : 1 mm exclusion of lumbricids Mesh size : 0.5 mm access only to small invertebrates
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Disadvantage :- Over-emphasis on small litter.Difficulty in measuring woody input
B. Decomposition
(i) LITTER BAGS :- measure weight lossMesh size : 7 mm all decomposersMesh size : 1 mm exclusion of lumbricidsMesh size : 0.5 mm access only to small invertebrates (e.g. mites, thrips)
Advantages :- Small litter usually studiedMesh alters micro-environmentMeasures only catabolism and leaching
Disadvantages :-Measure of overall decomposition in situ (esp. removal of polymers)Action of decomposer communities can be studiedCan be used along with biocides
Advantages :-Easy measure of total decomposer activityCombine with biocides to measure contribution of particular taxonomicgroupings (eg. prokaryotes vs. eukaryotes)Use 14C label to estimate catabolic potential (eg. 14C -glucose or 14C -lignin)
Disadvantages :-Disturbance of siteAre biocides 100% effective
Or different Ecological Groupings:- N-free media for Nitrogen fixers Cellulose agar for cellulose-decomposers
Advantages :-Flexibility (a wide range of selective agents are available)
Disadvantages :-No distinction as to source of colonies (spores/hyphae)Bias towards faster-growing microbesNon-culturable microbes will not growDormant stages may be over-represented
Direct measurements of microbial biomassB. Indirect methodsDirect microscopic examination of soil litter
- "Direct count" (devised by Jones & Mollinson)
Homogenise sample‚
Suspend in molten agar (of known volume)‚
Prepare films in haemocytometer‚
Stain and count
Use length of hyphae per unit volume to calculate Biovolume and Biomass
Modifications of this method:- Millipore filter Technique Vital Staining (Fluorescein diacetate-Esterase stain)
Advantages :- Both culturable and non-culturable fungi estimatedCould use antibodies to detect specific cells in soil/litter
Disadvantages :- No count of hyphae attached to soil/root particles.Represents standing crop rather than biomassDifficult to apply to bacteria in soil
Advantages :- Good correlation with biomass levelsDegrade within 14 days of cell death
Disadvantages :-Present in some microalgae/plantsAbsent in pseudofungi (e.g. Pythium, Phytophthora)Variation in concentration at different growth stages
Ergosterol
rDNA genes Do not encode genes only rRNAUniversal -found in all life forms(Ribosome = Cell protein factory)
rRNA gene sequences are highly conservedSequences specific to taxonomic groups
Analysis of 16S sequences in bacteria or 18S/26S sequences in fungi
Use PCR or hybridisation of labelled probes
Can be use to measure microbial biomass(Growth rate α protenin synthsis α rRNA levels)
Advantages: Reveals presence of uncultured microbesInstant taxonomic information
Disadvantages: High tech /costly proceduresNucleic acids adsorbed by soil silicatesProblems with quantification
Ribosomal DNA (rDNA) Sequence analysis
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Organic extraction of lipids
‚Acidic Methylation
‚Column Purification
‚
GC-MS Analysis
Advantages :- Taxon-specific e.g. 17:0, 18:1w7 Bacterial18w6 Fungal
Phospholipid Fatty Acid analysis(PLFA)
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Soil Fumigation
Biomass-C, Biomass-N(Sieve soil to remove soil fauna)
Microbial C or N flush = Fumigated minus non-fumigated
Organic C (Acid dichromate method) x 0.35 = Microbial Biomass-CTotal N (Kjeldahl method) x 0.45 = Microbial Biomass-N
50g soil‚
Chloroform(24 hr / 25 ºC)
‚ Remove CH3Cl
‚
Extract with K2SO4(0.5M/1 hr)
50g soil(control)
Better for qualitative rather than quantitative studies
Antibodies
Monoclonal (specific but expensive)Polyclonal (cheaper but possible undesired cross-specificity)(e.g. Mycena galopus detection in leaf litter by Frankland)
Nucleic acids Permits precise identification of species presentAlso used for genotype analysis and population biology
Hybridisation Radio-labelled probe. Can be quantified.
PCR Highly sensitive and specific.Can be used with tiny amounts of starting materialIdentify microbes non-culturable microbes
Reporter Genes Release of transgenic (Gene-tagged) microbesBUT G.E.M. riske.g. LacZ (E. coli b-galactosidase) + X-gale.g. Lux (Firefly luciferase)