Effects of Drought on Microbes Colonizing Lignocellulose

Whitney Walker, Robert Burgess, Mary Beth Leigh

UAF, Fairbanks Alaska

INTRODUCTION RESULTS LITERATURE CITED

Much of the global carbon pool is tied up in soil
organic compounds from plant litter, such as lignin,
hemicelluloses, and celluloses (6;1). Litter
decomposition is important to global carbon cycling,
and may be impacted by climate change.
Extracellular enzymes produced by microbes such
as fungi and bacteria can break down lignocellulose
to soluble subunits (1) which are then mineralized
to CO2. Basidiomycetes are largely responsible for
the breakdown of lignin (10), especially white rot
and some brown rot fungi (3). Bacteria are known to
play a more minor role in the breakdown of lignin.
Two-sample t-tests were run to compare the control & drought treatments for each type of
gene (laccase, bacterial 16S, fungal ITS, and bacterial:fungal ratio). None of these results were • Amatangelo, Raab, Stewart, Waldrop, Neff,
Vitousek. (in process). Plant lignin influences
significant (16S p = 0.8; ITS p = 0.6; laccase p = 0.35; B:F p=0.98). When all data were
microbial growth and decomposition
considered, there was no significant correlation between bacteria, fungi or laccase gene copy dynamics.
number with BTD mass loss. However, within the drought treatment alone, the correlation • Blackwood, C. B., M. P. Waldrop, D. R. Zak, R.
between ITS gene copy number and BTD mass loss was significant, as well as laccase gene L. Sinsabaugh. 2007. Molecular analysis of
copy number to mass loss and laccase gene copy number to ITS gene copy number. After fungal communities and laccase genes in
eliminating one outlying datapoint, however, these results became insignificant except for the decomposing litter reveals differences among
Before Outlier Was After Outlier Was
ITS gene copy number to mass loss comparison. The outlier differed from other BTDs in that it forest types but no impact of nitrogen
Removed Removed
deposition. Environmental Microbiology
Laccase Gene Copy # to Mass Loss Laccase Gene Copy # to Mass Loss

Three classes of lignin-degrading enzymes include was considerably softer and lighter in color.
9:1306-1316
14 12

laccase, manganese-dependent peroxidases, and 12

y = 8E-08x + 5.4255
10
• D’Souza, T. M., K. Boominathan, C. A. Reddy.
R2 = 0.3427

lignin peroxidases (3). Laccase, especially, is 10 8

1168 Typic

Mass Loss (%)

considered necessary for efficient lignin breakdown 8
6
1996. Isolation of laccase gene-specific
(3;2). 6
4 y = -6E-07x + 5.6947 al sequences from white rot and brown rot fungi
Climate change may either increase or decrease the
4
2
R2 = 0.0604

Sampl by PCR. Applied and Environmental

rate of decomposition by a variety of mechanisms,
2
0 Microbiology 62:3739-3744.
including by reducing soil moisture. The purpose of
0
0.00E+00 2.00E+07 4.00E+07 6.00E+07
# of Gene C opies/gDW
8.00E+07 1.00E+08
0.00E+0 5.00E+0 1.00E+0 1.50E+0 2.00E+0 2.50E+0 3.00E+0 3.50E+0 4.00E+0
0 5 6 6 6 6
# of Gene Copies/gDW
6 6 6 e • Fierer, N., J. A. Jackson, R. Vilgalys, R. B.
this study is to examine the effect of a simulated Jackson. 2005. Assessment of soil microbial
drought in boreal forest soil on the abundance of Laccase Gene Copy # to ITS Gene Copy # Laccase Gene Copy # to ITS Gene Copy #
community structure by use of taxon-specific
lignin-degrading microbes as well as to explore the 3E+11 y = 3105.8x + 1E+10
R 2 = 0.9668
50000000000
45000000000 quantitative PCR assays. Applied and
correlation between the presence of these microbes

# of Gene Copies/gDW (ITS)

3E+11
Environmental Microbiology 71:4117-4120.
40000000000
# of Gene Copies/gDW (ITS)

35000000000
and the decomposition rate. According to Schimel 2E+11
30000000000
• Lee, C.C., D. W. S. Wong, G. H. Robertson.
et al. (2007), drought may cause resource limitation
25000000000 y = 2639.3x + 1E+10
2E+11
20000000000 R2 = 0.034
2001. Cloning and characterization of two
for microbes, thus slowing down biogeochemical 1E+11 15000000000
10000000000 Mass Loss (%) of Buried BTDs
cellulase genes from Lentinula edodes. FEMS
processes, including decomposition. Drought forces 5E+10 5000000000

METHODS
microbes to gather solutes, which are energetically 0
0
0.00E+ 5.00E+ 1.00E+ 1.50E+ 2.00E+ 2.50E+ 3.00E+ 3.50E+ 4.00E+
35
Microbiology Letters 205:355-360.
expensive, in obtained
order to reduce
0.00E+00 2.00E+07 4.00E+07 6.00E+07 8.00E+07 1.00E+08 00 05 06 06 06 06 06 06 06 30
• Luis, P., G. Walther, H. Kellner, F. Martin, F.
Samples were from anthe risk of study
ongoing # of Gene Copies/gDW (Laccase) # of Gene Copies/gDW (Laccase)

Buscot. 2004. Diversity of laccase genes from

25

dehydration andloss
death (9). Bacteria use amino

Mass Loss (%)

measuring mass of buried birch tongue 20
basidiomycetes in a forest soil. Soil Biology &
ITS Gene Copy # to Mass Loss ITS Gene Copy # to Mass Loss
compounds(BTDs)
depressors as their
as primary solutes
an indicator while fungi usein
of decomposition 3E+11 50000000000
15

polyols.versus
Bacteria are thought to be less out
drought- Biochemistry 36:1025-1036.
control a simulated drought/rain treatment
45000000000 10
2.5E+11

tolerant • Ohtoko, K., M. Ohkuma, S. Moriya, T. Inoue, R.

theinBonanza
part because
Creekof theirTerm
demand for
40000000000
Gene Copy Number/gDW

within Long Ecological 5

# of Gene Copies/gDW

2E+11 35000000000

nitrogen.(LTER)
My hypothesis
Site (Fig.is3).
that thewere
simulated Usami, T. Kudo. 2000. Diverse genes of
y = 3E+09x - 2E+09
30000000000 0
Research BTDs buried in soil 1.5E+11 y = 2E+10x - 6E+10 25000000000
R2 = 0.268
Control Average Drought Average
drought
for 2 yearstreatment
and thenwould decrease
removed, washed,the weighed
abundance and 1E+11
R2 = 0.439
20000000000 Treatment cellulase homologues of glycosyl hydrolase
of lignin-degrading
stored frozen prior tomicrobes,
this studythus
(8). lowering
Total DNA the rate
was 50000000000
15000000000

10000000000 family 45 from the symbiotic protists in the

of decomposition.
extracted from subsamples of BTDs using the MoBIO 0 5000000000
hindgut of the termite Reticulitermes speratus.
0 2 4 6 8 10 12 14 0

PowerSoil DNA extraction kit with a few modifications -50000000000

Mass Loss (%)
0 2 4 6
% Mass Loss
8 10 12
Extremophiles 4:343-349.
to the manufacturer’s protocol (MoBIO, CA). Instead • Runck, S., D. Valentine, J. Yarie. Sensitivity of
of vortexing in an adapter, samples were put in a soil organic carbon dynamics to long-term
bead beater at maximum speed for 30 seconds. DNA throughfall exclusion in interior Alaska. ASA-
was eluted in 30 μL PCR grade water instead of 100 CSSA-SSSA 2006 International Meetings,
μL of solution C6. Bacterial biomass, fungal biomass DISCUSSION
Indianapolis, IN. Nov. 12-16, 2006.
and the quantity of laccase genes was determined • Schimel, J, T Balser, M Wallenstein. 2007.
using quantitative real-time PCR (qPCR). For standard There was not a significant difference in the number of laccase genes or biomass of bacteria or fungi or ACKNOWLEDGEMENTS
Microbial stress-response physiology and its
curves for qPCR, purified PCR products were used B:F ratio in the different treatments. This does not support the original hypothesis that drought would implications
I would for ecosystem
like to thank my mentor, function. Ecology
Mary Beth
after amplification using primers for bacterial 16S decrease the abundance of microbes and thus the number of gene copies. This suggests that the
rRNA (on Burkholderia xenovorans LB400), fungal ITS 88:1386-1394.
Leigh, for helping me understand my project
simulated drought has little detectable effect on the number of copies of each of the genes or the B:F • Stuardo, M., M. Vásquez, R. Vicuña, B.
(on Saccharomyces cerevesiae), and laccase genes and her input into this presentation. I would also
ratio. One reason for this may be the fact that the BTDs were washed gently prior to weighing. Although González.
(on DNA from nearby soil samples). PCR products like to thank 2004.
RobertMolecular
Burgess forapproach
helpingfor
me with
were viewed by means of gel electrophoresis to verify lignocellulose degraders are generally thought to be tightly bound to surfaces, washing would have analysis of model fungal genes encoding
lab work and his contributions. A thank you is
amplification of target genes based on fragment size. removed much of the loosely-associated biomass. Also, it is possible that both control and drought ligninolytic peroxidases in wood-decaying soil
also extended to Ian Herriott for his input into
qPCR was applied to quantify the number of copies of treatments were colonized by the same number of microbes but that they functioned at much lower rates systems. LettersI’d
in like
Applied Microbiologyall the
this presentation. to acknowledge
bacterial 16S rRNA genes, fungal ITS and laccase due to drought. Another explanation may be that although the total number of bacteria and fungi were 38:43-4
people working in the Leigh lab who helped me.
genes in each sample on the basis of BTD dry weight. similar, the community composition may have differed so that there were fewer lignocellulolytic
Special thanks to Sara Runck for sharing her
The program JMP 7 was used for all statistical organisms present in the drought treatment.
data for my project. A thank you is also
analyses (SAS). The two-sample t-test was used to It was expected that fungal biomass would correlate to mass loss of BTDs. However, in the control, the
compare the drought and control treatments for each extended to Kari and Leif of the Core Lab for
abundance of the different genes was not correlated with mass loss, while in the drought treatment, the
type of gene. The bivariate analysis was used to their patience. I would also like to thank the
abundance of the fungal ITS gene did correlated with mass loss. An outlier that was excluded from the
investigate the correlation between Fig. the3: number of RAHI program for funding.
Fig. 1: DNA extracted Fig. 2: Subsamples of a
Rainout shelters at analysis had high levels of laccase and ITS gene copy numbers, and was obtained from an unusually soft
gene
from BTD copies
subsamplesof eachBTD
gene to percentBonaza mass loss
Creek LTERof
that
and light-colored BTD sample from the drought treatment. This BTD may be worthy of further study for
simulate summer drought
BTDs. Sample size was 26 BTDs. All qPCR reactions
due to climate change
the presence of white-rot fungi, and also may be an indicator of spatial heterogeneity at the site since
were performed in duplicates for samples and
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