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METHODS IN DECOMPOSITION ECOLOGY

Soil Biology Module

Gareth GRIFFITH

GWG@aber.ac.uk

BS23420

WHAT is measured and HOW

1. WHAT?

Inputs into the decomposer subsystem

Fluxes (rates of flow of energy/nutrients)

Losses from decomposer subsystem

2. HOW?

Decomposer activity (which organisms)

Microbes vs. Animals

Primary vs Secondary Decomposers

Active vs. Dormant (or Dead vs. Alive)

Polymer Degradation (i.e. mineralisation)

METHODS IN DECOMPOSITION ECOLOGY

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ThroughputA. Deposition

Litter nets etc.Input t/ha/yr g/m2/yr

e.g. 6.0 60 Willow20.0 200 Oak

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

Cotton Strip AssayBury in soil or litter

Retrieve after several months/years

Measure a. Loss in tensile strength

b. Loss in dry weight

c. Extent and nature of microbial colonisation

Advantages :-

Strips represent a model resource

Double up as method for soil baiting

International unit of decomposition studies (IBP)

Disadvantages :-

Cellulose only (no lignin, uniform structure)

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Community Activity

Microbial Activity

Respirometry

Rate of Catabolism (output of CO2 and uptake of O2)

Techniques:- Gilson Respirometry - Soil samplesLitter Bags

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

Examples of Respirometry

1. Sydney Island, Antarctica (Wynne-Williams)

2. Aspen Forest, Canada (Parkinson)

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Indirect

Measure:

1. Fumigation (Biomass-C or -N)

2. Enzymes

3. ATP/ Ergosterol

4. Cell Wall Polymers

Direct

1. Plate / Particle counts

2. Hyphal length

3. "Molecular" methods (Nucleic acids, Antibodies)

4. Enrichment (high temp for thermophiles)(Cotton strips for cellulose degraders)= Soil baiting

Methods for measuring microbial biomass

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Analysis of Microbial Communities

1. What’s there? (species, kingdom)Numbers (hard to define foir fungi)

Biomass (Biovolume; live vs dead)

2. What’s it doing?Activity (respiration / cellmatabolism)

Active vs dormant states (mycelium vs spores)

3. Where exactly and when precisely is it thereSpatio-temporal variation

Fine-scale studies (% AMF infection etc)

‘Look-See’ Methods

1. Soil Smear (H.J. Conn, 1981)

2. Buried slide (Cholodni-Rossi. 1930)

3. Enrichment cultureSoil baiting - (add nutrient, then isolate)

4. Particle platingGood for slow-growing fungi

Semi-quantitative

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Pasteur Koch

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Sampling from Nature(representative??)

Sample Processing(losses???)

Sample Analysis(accuracy???)

‘Spiked’ samples (internal standards)Methodology is often limited by technology

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Direct measurements of microbial biomassUse of nutrient agar media

Dilution Plating - "Propagules"Particle Plating - % Infection (e.g. Roots, leaves)

Plus use of Selective Media

For different taxonomic groups:-

i) Fungi Low pH (5.0-6.0), Bacteriostats (e.g. Streptomycin, Penicillin)

i) Bacteria Neutral pH (7.0), Cycloheximide (inhibits fungi + yeasts)

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

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Use Calcofluor plusepifluorescencemicroscopy tovisualise hyphae onmembrane filters

Fluorescein-tagged monoclonal antibody

clampconnectionstypical ofbasidiomycetefungi

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Cell Wall PolymersDirect assay of insoluble and specific components of microbial biomass.

e.g. N-acetyl glucosamine / Chitin (fungi) N-acetyl muramic acid / Peptidoglycan, (bacteria)

Extract soil/litter(e.g. acid hydrolysis, lyases and other CW degrading enzymes)

‚Purify by chromatography to separate polymer subunits

‚Estimate concentration of subunits

‚Extrapolate to dry weight of cells in soil/litter

(by comparison with cells grown in pure culture)

Advantages : Specific identification of microbial components of soil/litter

Disadvantages : Dead cell wall included in estimate(i.e. Represents standing crop rather than biomass)

Extract soil/litter with buffer/EDTA or - use enzymes to break open ells (eg. Lysozyme)

Filter separate protein fraction and resuspend in buffer

Conduct assay (standard kit)

Estimate activity per gram soil or litter

EXAMPLES :- DehydrogenasesEsterases CellPhosphatases Activity

ChitinaseCellulase Functionalnitrogenase Groupings

Advantages :- Reliable measure of cellular activity (ie. of living cells)

Disadvantages :- Extraction procedureDo all cells have these enzymes at comparable levels

Enzymes

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BIOLOG plates

http://www.biolog.com/

Microbial Community-Level Physiological Profiles (CLPP)

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Short term energy store of cells (energy stored in high-energy phosphate bonds)

Extract soil and litter (known weight)‚

Filter and remove inhibitors of assay (e.g. phenolics)‚

Estimate ATP content via Luciferin and Luciferaseusing a Photometer

‚Calculate number of living cells

[ATP] x k ≈ Biomass-C (k = 120-160 in soil, 250 in water)

Adenylate energy charge = ATP + 0.5 ADPATP+ADP+AMP

AEC usually ≈ 0.8 - 0.95 (for active cells)BUT = 0.1-0.3 for dormant cells

Advantages:- Accurate measure of living (and therefore active) cells

Disadvantages:- Extraction procedureInhibition by soil phenolicsDo all cells have the same ATP levels?(i.e. resting vs. active)

ATP Content

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ATP/ml

Viable cells/ml

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Fungal specific STEROL (present in cell membranes)(hence use as EBI fungicides)

‚Level correlates well with fungal biomass

‚Extract mycelium

‚Purify hydrophobic component

‚Analyze concentration

[HPLC - highperformance liquid chromatography][UV Spectrometry]

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

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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

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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)

Molecular Methods