david c. white 1 , aaron peacock 1 ,

Changes in soil viable microbial biomass and composition reflect disturbance impacts and may serve as quantitative

end points for reversibility

David C. White1, Aaron Peacock1, Sareh. J. Macnaughton2, James M. Cantu1, Virginia H. Dale3,

1.Center for Biomarker Analysis, University of Tennessee,Knoxville, TN 37932, 2AEA Technology Environment, Harwell,

Oxon, UK. 3Environmental Sciences Division Oak Ridge National Laboratory, Oak Ridge, TN.

CBA



Microbial community provides multi-species multi-trophic level is analysis >>>

single species for Quantitative Toxicity Assessment

1. Surface Water Pollution Impact quantitatively reflected in the viable biomass and community composition of the periphyrton microbiota*

Parallels Cerodaphnia and Pimephales promelas In acute & chronic testsa) Most sensitive indicator is the increase in filamentous green algae and decrease in diatoms with increasing pollution

Reflected in the phospholipid fatty acid analysis (PLFA)

Green algae 18:19c, 16:43, 18:26, 16:113t with toxicity

Diatoms 22:66, 20:53*, 14:0, 18:26 with toxicity.

b). PHA/PLFA & TG/PLFA [Storage/membrane lipid] with increasing toxic exposure

Not need qualified personnel and tedious microscopic counts *Guckert, J. B., S. C. Nold, H. L. Boston, and D. C. White. 1992. Periphyton response along an industrial

effluent gradient: Lipid-based physiological stress analysis and pattern recognition of microbial community

structure. Canad. J. Fish. Aquat Sci. 49: 2579-2587.




single species for Quantitative Toxicity Assessment (3 dates)

.

Least Impacted Most Impacted

18:19c, 16:43, 18:26, 16:113t

GreenFilamentous Algae

22:66 20:53* 14:0 18:26

Diatoms

Intermediate Impacted

Pollution Impacts in Soils Petroleum Bioremediation of soils at KwajaleinNutrient Amendment and Ex Situ Composting vs Control Showed:

1. VIABLE BIOMASS (PLFA)

2. SHIFT PROPORTIONS: Gram + , Gram -

(Terminal branched PLFA, :: Monoenoic, normal PLFA )

3. Cyclo17:0/16:17c :: Cyclo19:0/18:17c (Stress)

4. = 16:17t/16:7c (Toxicity), [often ]

5. 16:19c/16:17c (Decreased Aerobic Desaturase)

6. % 10Me16:0 & Br17:1 PLFA (Sulfate-reducing bacteria) 7. % 10Me18:0 (Actinomycetes)

8. = PROTOZOA, FUNGI + (Polyenoic PLFA) [ often ]In other studies also usually see:

1. PHA/PLFA (Decreased Unbalanced Growth)

2. RATIO BENZOQUINONE/NAPHTHOQUINONE (Increased Aerobic Metabolism)

DEGREE OF SHIFT IN SIGNATURE LIPID BIOMARKERS PROPORTIONAL TO DEGRADATION





2. Exposure to petroleum hydrocarbons acute & chronic tests Shifts showed reversibility with time and distance plume had migrated

biomass, Gram- negatives, UQ/MK,

Gram- positive, branched PLFA, PHA/PLFA

*Stephen, J. R., Y-J. Chang, Y. D. Gan, A. Peacock, S. M. Pfiffner, M. J. Barcelona, S. M. D. C. White, and S. J. Macnaughton. 1999. Microbial Characterization of JP-4 fuel contaminated-site using a combined lipid biomarker/PCR-DGGE based approach. Environmental Microbiology.

1: 231-241.




single species for Quantitative Toxicity Assessment 4. PHA/PLFA RATIO

Sensitive Measure Of Unbalanced GrowthCarbon Source + Terminal Electron Acceptor but Lacking Essential Nutrient(s)

Necessary For Cell Division

Cells attached to fine rootlets PHA/PLFA <<0.01

Cells in sand away from roots PHA/PLFA > 6




single species for Quantitative Toxicity Assessment 4. PHA/PLFA TOXICITY INCREASES RATIO WITH TREATMENT RATIO

DECREASES

Phytoremediation TCE 7 (2). In the rhizosphere of legume 0.0002 in nonvegetated soil

Subsurface Petroleum and TCE (+ propane & air) Bioremediation ratio between 5 & 35 compared to 0.08-0.2 without active remediation





3. Exposure of pine forest surface soils to vehicular traffic Fort Benning GA Traffic

Reference ~ stands of longleaf pines (Pinus palustris) 28-74 years Light ~ limited to infantry Moderate ~ areas exposed to moderate amounts of tracked and

light vehicle maneuvers Heavy ~ exclusively for heavy wheeled and tracked vehicle exercises Remediated ~ Vehicles excluded & re-vegetated -

Disturbance Intensity Gradient

Heavy Moderate Light Control Remediated

--Tank Maneuvers---Turning in Drive on Neutral Tank Trails

----Target Practice--- Heavy Light Artillery Artillery

---Timber Harvest--- Clear Cut Selective Thinning

---Infantry Training--- Troop Individual Maneuvers Orienteering --Longleaf Pines –

24-74 years Vehicles & Infantry

Excluded

Intensity of Disturbance

Hierarchical Time Overlap of Ecological Disturbance Indicators

Centuries Decades Years Days HoursSpatial Distribution of Cover Plants Age Distribution of Trees

Composition & Distribution ofUnderstory Vegetation

MacroinvertebrateComposition

Stream MetabolismStorm ConcentrationMacroinvertebrate Populations

-------SOIL MICROORGANISMS------






disturbance viable biomass (PLFA) 18:0, 20:0, Me Br saturated mono and poly unsaturated, 14:0, 15:0, 16:0

with disturbance in actinomycetes & spore-former Gram positives

in gram-negative bacteria and microeukaryotes

RECOVERY APPROACHES REFERENCE






disturbance ~ changes in grasses, trees, bushes & stream properties correlate with usage but requires Biological expertise to differentiate.

PLANT COMMUNITIES & STREAM ECOLOGY PARALLEL MICROBES

disturbance in actinomycetes & spore-former Gram positives

in gram-negative bacteria and microeukaryotes Requires chemistry ~ following protocol. for analysis of lipid biomarkers.

RECOVERY APPROACHES REFERENCE

Tree Diagram for 28 PLFA Variables

Ward`s method

1-Pearson r

Linkage Distance

20:018:017:0

a17:012Me18:0i10Me16:0

i17:0br16:0a

10Me16:0i17:1w7c

i16:0cy19:0

poly20a20sat17:1

poly20b20:3w3

18:1w5c18:1w9c18:2w6br16:0b

15:116:1w5c

16:016:1w7c

15:014:0i14:0

0 2 4 6 8 10

Eukaryote and Gram-negative Bacterial PLFA

Actinomycete Type PLFA

Tree Diagram for 28 PLFA Variables

Ward`s method

1-Pearson r

Linkage Distance

20:018:017:0

a17:012Me18:0i10Me16:0

i17:0br16:0a

10Me16:0i17:1w7c

i16:0cy19:0

poly20a20sat17:1

poly20b20:3w3

18:1w5c18:1w9c18:2w6br16:0b

15:116:1w5c

16:016:1w7c

15:014:0i14:0

0 2 4 6 8 10

Eukaryote and Gram-negative Bacterial PLFA

Actinomycete, Gram-positive Type PLFA

Two clades of microbes disturbance in actinomycetes & spore-former

Gram positives, in Gram-negative bacteria and microeukaryotes

in Gram-negative bacteria and microeukaryotes

in actinomycetes & spore-forming bacteria

0 5 40 80

Reference

Light Moderate Heavy

Generalized Squared Distances Between Groups

0 5 40 80

Reference

Light Moderate Heavy

Generalized Squared Distances Between Groups

Linear Discriminant analysis showed that the reference and light transects were very similar while the moderate and heavy transects greatly differed in regards to the microbial community structure.

a15:0 i17:0 18:1w9c

i16:0 a17:0 18:0

16:1w7c Cy17:0 10Me18:0

i17:1w7c 17:0 Cy19:0

10Me16:0 i10Me16:0 20’s sat

18:2w6

PLFA used in Discriminant Analysis

Median Neural Network

61 Inputs (PLFA)5 Hidden Nodes4 OutputsR2=0.97

0.00%

1.00%

2.00%

3.00%

4.00%

5.00%

6.00%

7.00%

8.00%

9.00%

10.00%

16:1

w7c17

:0

18:1

w9c

16:0

18:0

i16:

0i1

7:1

a15:

0

12m

e16:

0

br19:

1a

18:1

w7t

18:1

w5c17

:1a1

7:0

15:0

poly20

b

i16:

1

Variables with ANN Sensitivity Values over 2%

Gram-Negative, Microeukaryotes, Gram-positive, Actinomycetes

ANN Analysis

• Was able to correctly predict classification 66% of the time (25% chance only)

• Allowed inspection of novelty indexes which showed that remediated transects are very different from all other treatments

HYSTERESES OF RECOVERY

Predictive Analysis of disturbance using the soil microbial community

• TWO APPROACHES:

• Linear Discriminant model using 17 PLFA predictor variables

• Two groups disturbance in actinomycetes & spore-former

Gram positive bacteria, in gram-negative bacteria and microeukaryotes

• Non-linear Artificial Neural Network Analysis using all 60 PLFAs and microbial biomass

• Predict classification 66% of time (Chance = 25%)

Hysteresis in recovery from sensitivity

Soil viable microbial biomass and composition reflect disturbance impacts and may serve as quantitative end

points for reversibility

Rational (Defensible) End Point[Multi species, multiple tropic level assessments [vs single species toxicity

assessment ]

Recovered ƒ Reversibility of Microbial Community Composition When uncontaminated soil, periphyton has same, or is approaching the same type of community composition as treated sediment

SURFACE WATER

1. Biofilms for run-off Diatoms Filamentous Algae (pollution) SOIL

2. Petroleum Hydrocarbon Contamination Gram-negative, Biomasss Gram-positive

reversed with recovery

3. PHA/PLFA with pollution recovery

4. Disturbance (traffic) disturbance in actinomycetes & spore-former Gram

positives, in gram-negative bacteria and microeukaryotes reversed with recovery

david c. white 1 , aaron peacock 1 ,

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