An exploration into the chemical makeup of soil and its influence on microbial soil profiles.

How does this affect the validity of using decomposition microbes found in soil, as forensic tools?

Catherine L. Aindow : 200805506

Project Supervisor: Dr L.

Aims of this presentation

Pesticide application Carbon: Nitrogen cycle Vegetation type and density Effects upon microbial soil populations Can microbial soil profiles be linked to sites of cadaver decomposition? Could these variables render hypothesis untrue?

Soil Composition and Variables

Soil is a complex matrix of inorganic and organic compounds. Supports the growth of a variety of microorganisms. Variables that affect chemical and microbial composition within soil. Influences the carbon to nitrogen biocycle within soil.

Carbon : Nitrogen Cycle

Climate
Temperature, Soil Fertility, Water Content. Etc.

Plant growth high Labile litter and root fluids low C:N ratio
low Recalcitrant litter high C:N ratio

Carbon allocation to plant roots

N (air)

Bacteria
(sacrophytic)

Fungi
(sacrophytic)

EM, ECM fungi

(symbiotic)

AM fungi
(symbiotic)

Bacteria
(symbiotic)

N, P (mineral)

N, P (organic)

Cadaver Decomposition
Cadaver decomposition alters natural bio-cycle within soil. Release of lipids, proteins and amino acids into soil. Influences the growth of specific microbes. Microbial profiling may distinguish sites of

Effects of Pesticide Application upon Soil Communities

Study conducted to show the effects of common pesticide Carbendazim upon chemical makeup of soil. A range of organisms were examined including nematode populations. Employed use of microcosmic conditions.

Methodology (Microcosm)

6 samples (30 kg each) Polyethylene pipes Nylon mesh for leachate collection. Addition of 3 earthworms per sample Sown with approx. 10 wheat seeds Soil-pesticide mixtures thoroughly mixed before packed.

Addition of artifical rainwater 12 hour light-dark cycle maintined.

Did not account for daylight saving Method does not correctly imitate the way in which pesticide would settle upon the soil.

Methodology (The Process)

Dose samples labelled T0 up to T5 T0 Ionized water T1 0.76 T2 2.28 T3 6.84 T4 20.52 T5 61.56 (concs: a.i. kg-1 soil dry mass) 400 ml sprayed

Nitrogen and ammonium concs measured before treatment and after 7,14,28 and 56 days. Extraction Colorimetric method used for quantification (650 nm).

Nitrate Concentrations after Treatment

Dose levels T4 & T5 increase nitrate concentrations significantly in comparison to other dose levels. Soil chemistry able to recover after 56 days. Recovery may be due to shoot growth.

Nitrate concentration after treatment with known concentrations of Carbendazim

Vegetation growth and Nitrate Concentrations

Shoot growth increases dramatically after 56 days of treatment with all concentrations of Carbendazim. Increase in shoot growth is also seen in control. Therefore pesticide application does not seem to hinder vegetation growth. Correlation between vegetation growth and nitrate concentrations. State of homeostasis achieved.

Shoot growth (grams) after treatment with known concentrations of Carbendazim

Conclusion from experiment

Pesticide application does alter chemical makeup of soil. Increases nitrate concentrations. Potentially would influence microbial populations that thrive in low C:N environments. Would render influence of cadaver decomposition upon soil profiles irrelevant. Vegetation growth may combat adverse effects.

The effects of vegetation type and density upon microbial soil populations

Study took place within a forest in Sweden (Betsele). 3 transects of land: 90m long & between 25m and 70m apart. Dwarf shrub (DS), short herb (SH) and tall herb (TH) vegetation types were analysed.

DS E.g. species: Vaccinium myrtillus Open and of low productivity.

SH E.g. species: Oxalic acetosella

Characterized by several short herbs

TH E.g. species: Actae spicata Dominated by pineas abies forest & has high soil pH and nitrogen.

Methodology

Aimed to identify chemical and microbiological differences between biomes. Soil samples taken in triplicate on 18th August 2004 Samples were taken via a 0.15m auger and then incorporated into one bulk sample for each transect. Samples were sifted through sieve Chemical analysis PLFA analysis Sample storage

NOTE
After sifting samples were weighed out into appropriate amounts no value was stated. Extraction method Bligh & Dyer method PLFA analysis silica gel columns, then eluted in sequence with chloroform acetone and methanol. Specific method of chemical testing was also not stated.

Soil chemistry observed in Betsele sites

Site Forest Type Treatment pH C to N Ratio NH4-N (g g-1 o.m.) NO3-N (g g-1 o.m.)

Results (Chemistry)
Betsele

DS SH

4.0 4.6

38.1 22.9

4.6 5.2

0.9 0.7

TH

5.3

14.9

15.9

3.4
0.003

Pvalue

<0.001 <0.001 0.012

All forest types display similar pH Carbon to nitrogen ratio decreases with increasing vegetation density Nitrogen levels increase as

vegetation density increases. All p-values less than 0.05 significant.

PLFA Analysis

No significant difference within total PLFA mass between biomes Fungal bacteria mass decreased as vegetation thickened

ratio decreased as vegetation thickened.

Distinctive microbial profiling between different biomes.

Total amount of bacteria increases as vegetation thickened.

Due to an increase in gram negative bacterial levels.

Combination of high pH and high nitrogen levels favours the growth of gram negative bacteria.

Gram positive Deters growth of bacterial levels did fungi. not alter significantly.

Fungi to bacteria

Effects of nitrogen loading upon carbon: nitrogen ratio

Site in Sweden (Norrliden) All sites DS Total of 15 plots investigated (4 concs - 3 replicates for each). 4 concentrations of nitrogen (in form of NH4NO4) 1 control (given a concentration of 3kg N ha-1 year-1) N3 was discontinued at an earlier date to see how soil chemistry would recover from loading

N0 control N1 34 kg N ha-1 year-1 N2 68 kg N ha-1 year-1 N3 108 kg N ha-1 year-1 N1 & N2 treated from 19712004 N2 treated from 1971 1990 Samples were taken on 25th August 2004

NORRLIDEN RESULTS (CHEMISTRY)

Site Forest Type DS Norrliden DS DS Treatment pH C to N ratio 37.5 31.1 27.7 NH4-N (g g-1 o.m.) 0.5 39.9 88.4 NO3-N (g g-1 o.m.) 0.7 1.5 7.3 N0 N1 N2 4.1 4.1 4.2

DS
-

N3
-

4.1
<0.001

27.2
<0.001

3.3
0.012

0.6
0.003

P-Value

All sites have similar pH Carbon to nitrogen ratio decreases as concentrations of nitrogen loading increases. Nitrogen levels within soil increase with high levels of nitrogen loading however seem to recover after time.

Carbon to nitrogen ratio does not seem to recover even after discontinuation of nitrogen loading.

Nitrogen loading PLFA results (Norrliden site) Total PLFA content gram positive
decreases with high levels of nitrogen loading. biomarkers as nitrogen levels increase. Fungal biomass also Fungi to bacteria decreases with the ratio decreases as addition of nitrogen fungi biomarker however appears to levels drop and gram recover after positive bacteria treatment biomarkers increase discontinuation. (slightly). No significant change is seen within the bacterial populations, though there is a slight increase in

Discussion

Many similarities between microbial communities that have same biome. Gram positive markers able to withstand nitrogen loading and therefore would withstand changes made by pesticides.

Gram positive bacteria biomarker levels did not differ across different biomes Further study conducted showed no change in gram positive PLFA biomarkers across a tree-girdling experiment. (Not affected by water content). Since gram positive bacteria do not change across biomes and across different chemical makeups, could be a good candidate for a decomposition biomarker.

How would it work?

Decomposition of cadaver would release intestinal bacteria. Would also alter chemical makeup of soil. Which would influence changes within soil profiles

Increase in gram positive bacteria within soil samples could only then be attributed to cadaver association May also indicate where body has been moved from.

Problems
No concrete the same. ground for proving Just too much to this hypothesis. consider to rely on More work would decomposition need to be carried microbes as a out. forensic tools. Too many variables and influences. No soil profile is

Any Questions?