soil microbial diversity of a managed forest ecosystem and the potential for lignocellulose ...

203 Pages·2013·2.74 MB·English

SOIL MICROBIAL DIVERSITY OF A MANAGED FOREST ECOSYSTEM AND
THE POTENTIAL FOR LIGNOCELLULOSE DEGRADATION AND METAL
BIOACCUMULATION BY WHITE ROT FUNGI
by
FRITZ AKUO NTOKO
A DISSERTATION
Submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy
in the Department of Plant and Soil Science
in the School of Graduate Studies
Alabama A&M University
Normal, Alabama 35762
May 2013
CERTIFICATE OF APPROVAL
Submitted by FRITZ AKUO NTOKO in partial fulfillment of the requirement for
the degree of DOCTOR OF PHILOSOPHY in PLANT AND SOIL SCIENCE.
Accepted on behalf of the Faculty of the Graduate School by the Dissertation
Committee:
Dr. Veronica Acosta-Martinez
Dr. Leopold Nyochembeng
Dr. Elica Moss
Dr. Zachary Senwo Major Advisor
Dean of the Graduate School
Date
ii
Copyright by
FRITZ AKUO NTOKO
2013
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DEDICATION
This work is dedicated to my late sister (Victorine Ebude Ntoko), my sons (Chrys
and Fritz), and all those who have contributed to my academic success.
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SOIL MICROBIAL DIVERSITY OF A MANAGED FOREST ECOSYSTEM AND
THE POTENTIAL FOR LIGNOCELLULOSE DEGRADATION AND METAL
BIOACCUMULATION BY WHITE ROT FUNGI
Fritz Akuo Ntoko, PhD., Alabama A&M University, 2013. 187 pp.
Dissertation Advisor: Dr. Z. N. Senwo, PhD.
ABSTRACT
This study investigated the impact of forest management practices such as prescribed
burning and thinning, on soil microbial community diversity and metabolic function,
using ester-linked fatty acid methyl ester (EL-FAME) analysis and DNA pyrosequencing,
coupled with enzymatic assays. It also explored the potential of white rot fungi in the
forest to degrade plant biomass, as well as bioaccumulate mercury. Results obtained from
EL-FAME analysis suggest that the application of prescribed burning without thinning
resulted in an overall decrease in total microbial population when compared to the
control. However, light thinning with or without prescribed burning resulted in an
increase in total microbial population. The bacterial and fungal species diversities, of the
various treatments were all greater than that of the control, with the highest species
diversities found in the lightly-thinned/9yr burn cycle treatments. The structural and
compositional similarities of bacterial species between the different treatments were
greater than that of the fungi species, as depicted by the Chao-Jaccard-Raw abundance
based similarity indices and shared species. The application of burning without thinning
resulted in the lowest metabolic functions, as observed with the no-thin/3yr burn cycle
treatments. The lightly-thinned 9yr-burn cycle treatments had the highest metabolic
activity based on the geometric mean of enzyme activities (GMea) indices. Evaluation of
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biodegradation of woody (red oak) and non woody plant biomass (corn and wheat) with
two white rot fungi, Pleurotus floridanus and Perenniporia nanlingensis, suggests that P.
floridanus would be a better candidate to pre-treat wheat and wood, while P. nanlingensis
would be better for pre-treating corn. The concentration of Hg in the various fruiting
bodies ranged from 1.37 to 0.03 µg Hg per gram of fungi tissue, and differed
significantly among the species of the various fungi, with the highest bioaccumulation
recorded with Metschnikowia sp. Gerronema strombodes, Boletus sp., and Amanita
alboverrucosa above the EPA acceptable level of 0.3 ppm.

KEY WORDS: metabolic activity, mercury, white rot fungi, pyrosequencing, fatty acid
analysis.
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TABLE OF CONTENTS

CERTIFICATE OF APPROVAL ................................................................................... ii
DEDICATION .............................................................................................................. iv
ABSTRACT ....................................................................................................................v
LIST OF TABLES ........................................................................................................ ix
LIST OF FIGURES ....................................................................................................... xi
ACKNOWLEDGMENTS ........................................................................................... xiii
INTRODUCTION AND REVIEW OF LITERATURE ...................................................1
1.1 Introduction .......................................................................................................1
1.2 Soil microbial diversities and functions in soils .................................................7
1.2.1 Drivers and concept of diversity ............................................................... 10
1.2.2 Ecological importance of microbial biodiversity ....................................... 14
1.3 Measurement of microbial diversity ................................................................. 17
1.3.1 Dilution plating and culturing methods ..................................................... 18
1.3.2 Community-level physiological profiles .................................................. 20
1.3.3 Fatty acid analysis .................................................................................... 21
1.3.4 Protein based methods ............................................................................. 26
1.3.5 Nucleic acid analyses for soil ecology studies .......................................... 28
1.3.6 Soil microorganisms ................................................................................. 36
1.4 Forest management and soil ecology ............................................................... 43
1.4.1 Effects of thinning and fire on soil properties ........................................... 48
1.5 Rationale and research objectives .................................................................... 51
METHODOLOGY ........................................................................................................ 56
2.1 Study site description....................................................................................... 56
2.2 Soil sampling and analyses .............................................................................. 60
2.3 Microbial communities sizes and structures ..................................................... 60
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2.4 Bacterial and fungal diversity by pyrosequencing techniques ........................... 62
2.5 Metabolic capacity of the forest ecosystem via enzyme activities..................... 65
2.6 Collection and screening and identification of fungi fruiting bodies ................. 66
2.7 Molecular identification of fungi fruiting bodies .............................................. 67
2.8 Characterization of biomass and compositional analysis of biomass ................ 68
2.9 Pretreatment of substrate and estimation of enzyme production ....................... 69
2.10 Analysis of mercury in fungi tissue .................................................................. 70
2.11 Statistical analyses ........................................................................................... 71
RESULTS AND DISCUSSION .................................................................................... 73
3.1 Microbial community size and structure by EL-FAME .................................... 73
3.2 Microbial community diversity, and richness, according to pyrosequencing .... 83
3.2.1 Bacterial community richness and diversity .............................................. 83
3.2.2 Shared species richness and similarity in the bacterial community
structure ................................................................................................... 98
3.2.3 Fungal community richness and diversity ............................................... 101
3.2.5 Shared species richness and similarity in the fungal community
structure ................................................................................................. 117
3.3 Metabolic capacity of the forest ecosystem by enzymatic assays .................... 121
3.4 Additional soil properties............................................................................... 132
3.5 Evaluating biodegradation of plant biomass ............................................ 136
3.6 Bioaccumulation of mercury in fungi tissue ............................................ 154
CONCLUSION ........................................................................................................... 158
REFERENCES ............................................................................................................ 163
APPENDIX ................................................................................................................. 176
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LIST OF TABLES
Table Page
2 1. Treatment applications at the Bankhead National Forest ............................... 58
3.1. Principal Component Analysis (PCA) factor loadings and percent
contributions of variables to relationships between soil microbial
community compositions, and enzymatic activities with treatments, at
0 - 10cm soil depth. ...................................................................................... 78
3.2. Changes in microbial community compositions and size at 0 - 10
depth, relative to the control site. .................................................................. 81
3.3. Analysis of variance for abundance of microbial groups and biomass
of the different treatments at 0 - 10 cm, and with depth (0 - 10cm and
10 – 20cm). .................................................................................................. 82
3.4. Analysis of variance for relative abundance of bacterial classes with
different treatments. ..................................................................................... 93
3.5. Bacterial species diversity indices of various treatments. .............................. 95
3.6. Pearson’s correlation matrix between bacterial classes and soil
properties. .................................................................................................... 96
3.7. Pearson’s correlation matrix between bacterial classes and soil
enzymatic activities. ..................................................................................... 97
3.8 Observed and estimated shared bacterial species between treatments. ........... 99
3.9. Chao-Jaccard-Raw abundance-based similarities between pairs of
treatments, calculated from shared OTUs. .................................................. 100
3.10. Analysis of variance for relative abundance of fungal phyla with
different treatments. ................................................................................... 109
3.11 Analysis of variance for relative abundance of fungal classes with
respect to thinning, burning, and combined treatments................................ 110
3.12. Diversity indices of fungal species in the different treatments. .................... 114
3.13. Pearson’s correlation matrix between fungal classes and soil
properties. .................................................................................................. 115
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