Enhanced
saccharification of lignocellulosic biomass in Myb61a knock-down mutants and by
pretreatment with ammonium hydroxide
JILLIAN
C. VAUGHT
Department of Plant Biology, The
University of Oklahoma, Norman, OK
30
April 2015
Abstract
Lignocellulosic
material is highly abundant in agricultural waste. Plant cell walls are dense
networks of lignin, highly cross-linked with microfibrils of sugar
polysaccharides. Sugar found bound in these cell walls could be used to produce
a sustainable amount of biofuel and reduce the carbon footprint. Unfortunately,
the strong network of sugar polysaccharides makes cell walls resistant to
degradation. The deconstruction of lignocellulose to its component sugars is
energy intensive. However, a combination of new genetic targets for cell wall
mutations and methods of biomass pretreatment are promising for creating a more
effective protocol for sugar extraction. Previous data from a wheat straw
deconstruction indicates that biomass pretreated with 16.8% NH4OH is
3-fold more digestible. The result of my deconstruction experiment supports
these findings in rice straw, showing up to a 9-fold increase in glucose sugar
release, or saccharification, when pretreated with 15% NH4OH. In
addition to pretreatment methods, targeting the cell wall regulating gene,
Myb61a, to produce a knock-down mutant results in biomass with up to 3-fold
increase in saccharification. A combination of basic pretreatment (pH 11-12)
and genetic modifications to rice could be key to producing a source of
digestible biomass for biofuels.
Introduction
Plant
cell walls contain a strong network of sugar chains bound in lignocellulose.
Sugar released from the cell walls of plants can be converted into biofuels by
means of yeast or other microbes that undergo fermentation (1). Currently, the
industrial burning of fossil fuels explains the buildup of atmospheric carbon
dioxide, creating an immense carbon footprint (2). Using plant biomass to
harvest sugar for biofuels has the potential to be a sustainable source of energy.
With a global total of 23% of agricultural waste in rice straw alone, it is a
promising candidate for harvestable biomass (3). Unfortunately, current methods
of extracting sugar from plant cell walls are expensive and energy intensive. Barriers
to inefficient sugar extraction include both understanding the mechanisms by
which plants build up cell walls and determining pretreatment methods, which
enhance the digestibility of cell walls by cellulase enzymes.
The
composition of plant cell walls varies among and within plant species. Rice and
other monocots exhibit distinct cell wall characteristics including remarkably
developed secondary cell walls. The main components of secondary cell walls are
cellulose, hemicellulose, and lignin (4). Collectively, these components are
referred to as lignocellulose. Cellulose is composed of linear chains of
glucose and can make up nearly 50% of the cell wall. Lignin is deposited in
plant cells during the maturation of the plant to maintain structural support
for the growing tissues. Both cellulose and lignin are dense and chemically
cross-linked within the cell walls (1). This dense network of lignin and
cellulose contributes to the recalcitrance, or the resistance to digestibility,
of secondary cell walls. Highly recalcitrant cell walls are difficult to
deconstruct and therefore the amount sugar release from harvested biomass is
consistently low.
One
approach to decreasing the recalcitrance of cell walls is identifying genes involved
in cell wall synthesis. One family of MYB proteins has been found to bind to
structural genes and other transcription factors involved in cell wall
synthesis (4). Of these MYB proteins, MYB61 in Arabidopsis has been found to
regulate the closure of the stomatal aperture (5). Additional data finds MYB61
in Arabidopsis to be involved in the deposition of mucilage (6). In unpublished
data of a co-expression network, Myb61a is found to be co-expressed with four
cell wall biosynthesis enzymes: COMT1, 4CL3, AT5, and AT4 (Zhao and Bartley,
unpublished). Data from this co-expression
network suggests that Myb61a in rice plants is a transcriptional activator
involved in the regulation of cell wall synthesis. Preliminary evidence leads
to the hypothesis that if Myb61a is knocked-down in rice plants, then the
biomass will be more digestible than wild-type rice straw when deconstructed
under the standard conditions described in the Materials and Methods section.
If Myb61a is down-regulated then it will not be able to transcriptionally
activate other cell wall genes involved in the biosynthesis of the cell wall,
leaving the walls of mutant rice plants less cross-linked and less dense
compared to wild-type cell walls. Data from the deconstruction of mutant rice
straw in this experiment finds that Myb61a knock-down mutants have a 1.2-fold
increase in digestibility compared to the wild-type.
In
addition to genetically modifying rice plants, modifications to the
deconstruction process itself can facilitate increased glucose yield. The
process to converting biomass to biofuel can be divided into three main steps.
First, biomass is produced and harvested from a plant source such as rice.
Next, the destruction of biomass includes pulverization of materials and the
breakdown of lignocellulose into its component sugars such as glucose. The breakdown
of lignocellulose is catalyzed by expensive enzymes that are specific to the
components of lignocellulose being targeted for sugar release. Examples of
these enzymes include cellulase, beta-glucosidase, and xylanase. Lastly, the
sugar released from lignocellulose by enzymes is fed to microbes that undergo
fermentation to produce biofuels such as ethanol. Experimental evidence
suggests that modifications to the deconstruction process can enhance sugar
release from biomass. One crucial modification to the protocol is pretreating
the pulverized biomass with a harsh acid or base before the biomass is
subjected to cell wall enzymes.
The
chemical pretreatment of the pulverized biomass has the potential to open up
the structure of lignocellulose fibers, making them accessible to enzymes (7).
Pretreatment chemicals must not only modify the matrix of the lignocellulose
complex, but also be affordable. Previous experiments used 16.8% NH4OH
to pretreat wheat straw. The results of this experiment show a 3-fold increase
in saccharification, or sugar yield, by pretreated biomass (8). Based on data
from this experiment, I hypothesized that pretreating rice straw with 15% NH4OH
will increase the glucose yield compared to biomass under standard deconstruction
with 0.1M Citrate Buffer. If rice straw is subjected to 15% NH4OH
(ammonium hydroxide), the strong base will decrease the cross-linking of lignin
fibers and decrease cellulose crystallinity, allowing enzymes to access the
glucose polysaccharides. In this experiment, ammonium hydroxide pretreatment
increased glucose yields 1.9-fold in pretreated wild-type rice straw compared
to rice straw deconstructed under standard conditions with 0.1M Citrate Buffer.
Materials
and Methods
Preparation of Biomass
Rice
straw from Myb61a-1 knock-down mutants and negative segregant (wild-type) was
dried at approximately 45° C. Straw was chopped
and ground to a uniform size to produced milled rice straw.
Pretreatment of
Milled Rice Straw
To prepare the biomass for enzymatic digestion, samples were
pre-treated with either a strong base or a weak acid. Milled rice straw samples
between approximately 5-7 mg were weighed and assigned to screw cap tubes. The
tubes were organized based on pre-treatment and biomass genotype (Table
S1). Four technical replicates of both mutant and wild-type biomass
were pre-treated with 15% ammonium hydroxide (NH4OH) or citrate
buffer. Biomass was gently shaken in 500 ul of 100mM Citrate Buffer or NH4OH
for 5 minutes. Samples were then incubated at 99° C for one hour. After one hour, samples were removed and cooled.
Neutralization
of Pre-treated Rice Straw
According to the Novoenzyme Biomass Kit protocol, samples must be
neutralized to a pH of 4.5-6.5 for the cellulase complex and beta-glucosidase enzymes
to perform. Samples pre-treated with NH4OH were neutralized by
adding 2-5 ul of 4M NaCl until the pH neutralized to approximately a pH of
5.00.
Saccharification
To digest the lignocellulosic material into glucose, two enzymes
supplied by Novoenzyme Biomass Kit are added. Stock solutions of NS50013
(cellulase) at a 5-fold dilution and NS50010 (B-glucosidase) at a 100-fold
dilution were mixed at a 1:1 ration. Four ul of stock enzyme solution were added
to all neutralized samples.
Time Points
To determine glucose release over time, a 30 ul aliquot was
removed from each spun down test tube and added to a thermocycle strip tube.
Thermocycle strips were maintained in a freezer. Samples were incubated at 50° C. Time points were taken at 0, 1, 6,
22, 25.5, and 46 hours in the first experiment and at 0, 1, 6, and 19.5 hours
in the follow-up experiment.
DNS Assay
To quantify the amount of sugar released over time from each time
point, 90 ul of DNS reagent was added to each thermocycle strip well. Aliquots
of dilutions for the standard curve were also added to the strip. Strips were
covered to prevent evaporation and incubated at 95° C for 6 minutes in the thermocycler machine. Samples were then
transferred to a microtiter plate. Absorbance of each sample was read at 540 nm
by a Biotek plate reader.
Standard Curve
The standard curve was prepared by diluting 0.1M glucose with
citrate buffer to known concentrations. Thirty ul of each dilution was added to
the thermocyler plate and absorbance was determined. Microsoft Excel was used
to plot absorbance vs. concentration. A linear range line was fit to the data
and the trendline equation as well as R2 values were determined. The
standard curve line was used to calculate the amount of sugar released in mg at
each absorbance value (Figure S2).
Statistical
Analysis
Sugar release in mg/mL was calculated at each absorbance using the
linear trendline generated by the standard curve. Sugar release from each
sample was divided by the amount of biomass in mg originally added to each test
tube. Sugar release in mg/mg was averaged across each of the four technical
replicates and graphed in a scatter plot in Excel. Standard deviation for each
sample was calculated across the technical replicate at each time point and
added to the graph using errors bars. A two-tailed student’s t-tests was used
to calculate the significance of the final time points from each treatment
sample. A t-test p-value of 0.05 or less was considered significant.
Results
Part I: Myb61a
knock-down mutant rice straw shows enhanced saccharification.
Myb61a
knock-down mutant rice straw harvested from a single greenhouse rice mutant
plant shows enhanced glucose release compared to wild-type, negative segregant
rice plants.
Biomass from the rice straw of a mutant and wild-type plants was dried and
ground to uniform size. A standard deconstruction method and DNS assay was used
to measure glucose release over a period of 46 hours. Milled rice straw from a
mutant and wild-type plants was incubated in 0.1M Citrate Buffer for one hour.
Cellulase (CTec2) and beta-glucosidase (NS50010) enzymes were then allowed to
begin digesting the biomass material. Glucose release in mg per mg of biomass
were measured with DNS assay over six time points (Figure 1). As expected, glucose release from the Myb61a mutant rice
straw displayed greater glucose release than the pool of wild-type rice straw.
Figure 1. Myb61a
knock-down mutant (MT) rice straw pretreated with Citrate Buffer (CT) shows
increased glucose release compared to wild-type (WT). Ground biomass from
Myb61a mutants and negative segregants was pretreated with 0.1M Citrate Buffer.
Cellulase complex (CTec2) and B-glucosidase (NS50010) enzymes were added to the
pre-treated biomass. Myb61a mutant biomass was taken from a single rice plant
and wild-type biomass was pooled from all negative segregant rice plants. DNS
assay measured glucose release in mg over a period of 46 hours. Mutant rice
straw shows a 3-fold increase in glucose yield compared to wild-type with a
p-value of 0.0013. A positive control of Kitake wild-type rice straw confirms
glucose release from deconstructed rice straw. A linear equation from the
glucose standard curve was used to determine glucose in mg at each absorbance.
Error bars were calculated using standard deviation. Some error bars are within
the symbol of the time point. Asterisks (**) indicate significant data.
Results from the deconstruction of a Myb61a knock-down mutant and
wild-type rice straw show the mutant biomass has increased sugar release at the
final 46-hour-time-point when compared to wild-type rice straw. Each time point
represents the average glucose release in mg/mg of biomass over four technical
replicates deconstructed from both mutant and wild-type. As indicated by
asterisks, a student’s t-test compares mutant (Mut) glucose release to the
final glucose release of the wild-type (WT) to give a p-value of 0.0013. The
p-value of this t-test is considered significant because it is less than 0.05.
It is important emphasize that mutant rice straw was only taken from a single
mutant rice plant, while wild-type rice straw was pooled from all wild-type
plants. To confirm that the deconstruction process is releasing glucose, Kitake
wild-type biomass was used as a positive control. A negative control (data
excluded from Figure 1) consisted of only Novoenzymes, CTec2 and NS50010 in
0.1M Citrate Buffer. DNS assay did not indicate any glucose release from the
negative control over the 46-hour time period. Absorbance readings from the
negative control were considered background and were subtracted from the
absorbance readings of mutant and wild-type biomass wells. The low, significant
p-value of increased mutant glucose release provides confidence in the
hypothesis that Myb61a mutant biomass is more digestible to cell wall enzymes.
The
deconstruction of Myb61a knock-down mutants and wild-type rice straw was
repeated with a pool of biomass from the Myb61a mutant plants and wild-type
rice straw. The
results obtained from the initial experiment testing saccharification of mutant
and wild-type straw needed to be confirmed using pooled Myb61a mutant plant
material to confirm the significance of increased glucose release during the
digestion of mutant plants. The standard deconstruction process and DNS assay
was replicated with four technical replicates of each mutant and wild-type
deconstruction. In this experiment, only four time points were taken over a
period of 19.5 hours. Kitake wild-type rice straw was repeated as a positive
control and showed constant sugar release. The negative control (excluded from
the Figure) included a well of citrate buffer and enzymes and was measured with
DNS assay over the four time points. The results in Figure 2 confirm the trend
of increased glucose release by mutant rice straw but do not confirm that
Myb61a knock-down mutants show a significant p-value from glucose released at
the final time point compared to wild-type rice straw (Figure 2).
Figure 2. Biomass pooled
from Myb61a knock-down mutants (MT) pretreated with Citrate Buffer (CT) shows
increased glucose release compared to wild-type (WT). Ground biomass from
Myb61a mutants and negative segregants was pretreated with 0.1M Citrate Buffer.
Cellulase complex (CTec2) and B-glucosidase (NS50010) enzymes were added to the
pre-treated biomass. Myb61a mutant biomass and wild-type biomass was pooled
from all available rice plants. DNS assay measured glucose release in mg over a
period of 19.5 hours. Mutant rice straw shows a 1.2-fold increase in glucose
yield compared to wild-type with a p-value of 0.4326. Glucose yield from both
mutant and wild-type are increased in this experiment compared to the endpoint
yield in the previous deconstruction. A positive control of Kitake wild-type
rice straw confirms glucose release from deconstructed rice straw. A linear
equation from the glucose standard curve was used to determine glucose in mg at
each absorbance. Error bars were calculated using standard deviation. Some
error bars are within the symbol of the time point.
Data from Figure 2 confirms the trend of increased glucose release
from Myb61a mutant rice straw. However, a student’s t-test gives a p-value of
0.4326 indicating that although mutant rice straw has a greater final glucose
release than the wild-type, it cannot be explained by the Myb61a knock-down
mutation.
Part II:
Pretreatment of rice straw with 15% NH4OH shows enhanced
saccharification compared to standard pretreatment with 0.1M Citrate Buffer.
Wild-type rice
straw pretreated with 15% NH4OH shows significant increase in
glucose release compared to rice straw pretreated under standard conditions
with 0.1M Citrate Buffer. Wild-type rice straw was pooled across all available rice plants.
Uniformly ground straw was divided into four technical replicates for both the
pretreatment of ammonium hydroxide (NH4OH) and Citrate Buffer. 15%
NH4OH was added to four biomass samples and 0.1M Citrate Buffer was
added to the remaining four biomass samples. All samples were incubated for one
hour. After this time, NH4OH (pH 11.63) pretreated samples were
neutralized. Cell wall enzymes, CTec2 (Cellulase) and NS50010
(beta-glucosidase) were added to all samples. Time points were taken over 46
hours and glucose release was measured by DNS assay (Figure 3). As hypothesized, NH4OH pretreated samples
showed a significant amount of glucose release at the final time point compared
to samples undergoing only standard deconstruction.
Figure 3. Wild-type (WT)
rice straw pretreated with Ammonium Hydroxide (NH4OH) shows increased glucose
release compared to pretreatment with Citrate Buffer (Cit). Ground biomass from wild-type
rice straw was pretreated with either 0.1M Citrate Buffer or 15% NH4OH.
Cellulase complex (CTec2) and B-glucosidase (NS50010) enzymes were added to the
pre-treated biomass. Wild-type biomass was pooled from all negative segregant (WT)
rice plants. DNS assay measured glucose release in mg over a period of 46
hours. NH4OH pretreated rice straw shows a 9-fold increase in
glucose yield compared to Citrate Buffer pretreated with a p-value of 0.0045. A
positive control of Kitake wild-type rice straw confirms glucose release from
deconstructed rice straw. A linear equation from the glucose standard curve was
used to determine glucose in mg at each absorbance. Error bars were calculated
using standard deviation. Some error bars are within the symbol of the time
point. Asterisks (**) indicate significant data.
Data from the deconstruction of rice straw with a strong basic
pretreatment of NH4OH proves to show significant glucose release as
hypothesized. A positive control of Kitake wild-type plant confirms that
glucose release increased over a period of incubation due to the digestion of
the biomass material by the enzymes. The negative control consisted of only
enzymes and citrate buffer and did not show any glucose release which confirmed
that the enzymes and citrate buffer showed no signs of contamination.
Absorbance from the negative control was subtracted from absorbance of the
technical replicates. A standard curve was used to determine mg of glucose
release at each absorbance and glucose release was averaged across all
technical replicates of each pretreatment condition. A student’s t-test was
preformed on the end points of the two pretreatment conditions (NH4OH and Cit)
to give a p-value of 0.0045 for the glucose release of the NH4OH
pretreated biomass compared to the Citrate Buffer only treated biomass. A
p-value less than 0.05 was considered significant. Rice straw pretreated with
NH4OH has a 9-fold increase in glucose release compared to the
standard deconstructed rice straw. Data from the t-test confirms our hypothesis
that a strong basic pretreatment with ammonium hydroxide will alter the
structure of the biomass leaving it more digestible to the cell wall enzymes.
The
pretreatment of wild-type rice straw with NH4OH compared to 0.1M
Citrate Buffer standard deconstruction was repeated to confirm the significant
increase in glucose release from ammonium hydroxide pretreated biomass. The deconstruction of
wild-type rice straw was repeated with four technical replicates of both NH4OH
and Citrate Buffer pretreatment conditions. The initial pretreatment experiment
was replicated with the same protocol except during this replication, time
points for DNS assay were taken over a period of 19.5 hours (Figure 4). The results from the
replicated experiment confirm the significance of the initial experiment.
Figure 4. Wild-type (WT)
rice straw pretreated with Ammonium Hydroxide (NH4OH) shows increased glucose
release compared to pretreatment with Citrate Buffer (Cit). Ground biomass from
Myb61a mutants and negative segregants were pretreated with 0.1M Citrate Buffer.
Cellulase complex (CTec2) and B-glucosidase (NS50010) enzymes were added to the
pre-treated biomass. Myb61a mutant biomass and wild-type biomass was pooled
from all negative segregant rice plants. DNS assay measured glucose release in
mg over a period of 19.5 hours. NH4OH pretreated rice straw shows a 1.9-fold
increase in glucose yield compared to wild-type with a p-value of 0.0042.
Glucose yield from both treatments are increased in this experiment compared to
the endpoint yield in the previous pretreatment experiment shown in Figure 3. A
positive control of Kitake wild-type rice straw confirms glucose release from
deconstructed rice straw. A linear equation from the glucose standard curve was
used to determine glucose in mg at each absorbance. Error bars were calculated
using standard deviation. Some error bars are within the symbol of the time
point. Asterisks (**) indicate significant data.
Data from the replicated deconstruction of rice straw with a
strong basic pretreatment of NH4OH confirms the significance of the
initial results from Figure 3. As in the initial experiment, a positive control
of Kitake wild-type plant confirms the glucose release from rice straw and the
negative control (not shown in Figure 4) did not show any glucose release which
confirmed that the enzymes and citrate buffer showed no signs of glucose or
biomass contamination. Absorbance from the negative control was subtracted from
the absorbance of the technical replicates to adjust for background noise. A
standard curve was used to determine mg of glucose release at each absorbance
and glucose release was averaged across all technical replicates of each
pretreatment condition. A student’s t-test was preformed on the end points of
the two pretreatment conditions (NH4OH and Cit) to give a p-value of 0.0042 for
the glucose release of the NH4OH pretreated biomass compared to the
Citrate Buffer only treated biomass. A p-value less than 0.05 was considered
significant. Rice straw pretreated with NH4OH had a 1.9-fold
increase in glucose release compared to the standard deconstructed rice straw.
Although the replicated experiment shows less of a difference between the two
pretreatments, the t-test confirms our hypothesis that a NH4OH
pretreatment can be used to increase saccharification.
Part III:
Myb61a mutant rice straw pretreated with 15% NH4OH shows increased
saccharification compared to mutant rice straw deconstructed under standard
conditions with 0.1M Citrate Buffer.
In addition to measuring the digestibility Myb61a mutant rice
straw under standard deconstruction, the combination of mutant rice straw
pretreated with 15% NH4OH was measured to confirm that mutant rice
would display even more increased saccharification when pretreated under highly
basic conditions. Ground Myb61a mutant biomass was incubated for one hour in
15% NH4OH and then neutralized to a pH of around 5.00. Cell wall
enzymes (CTec2 and beta-glucosidase) were then added to the biomass slurry. DNS
assay was used to measure glucose release over a period of 46 hours (Figure 5). The results of the
deconstruction of Myb61a with NH4OH pretreatment and under standard
conditions as well as the deconstruction of wild-type rice straw with NH4OH
and under standard conditions are displayed in Figure 5.
Figure 5. Myb61a
knock-down mutant (MT) rice straw pretreated with Ammonium Hydroxide (NH4OH)
shows significant glucose release compared to Citrate Buffer pretreated mutant
rice straw. Ground
biomass from Myb61a mutants and negative segregants (WT) was pretreated with
0.1M Citrate Buffer or 15% NH4OH. Cellulase complex (CTec2) and
B-glucosidase (NS50010) enzymes were added to the pre-treated biomass. DNS
assay measured glucose release in mg over a period of 46 hours. NH4OH
pretreated mutant rice straw shows a 4.7-fold increase in glucose yield
compared to citrate pretreated mutants with a p-value of 0.0485. A positive
control of Kitake wild-type rice straw confirms glucose release from
deconstructed rice straw. All combinations of wild-type and mutant
pretreatments are graphed for the purpose of comparison. A linear equation from
the glucose standard curve was used to determine glucose in mg at each
absorbance. Error bars were calculated using standard deviation. Some error
bars are within the symbol of the time point. Asterisks (**) indicate
significant data.
Additional data from the deconstruction of Myb61a mutant rice
straw with 15% NH4OH pretreatment confirms that an ammonium
hydroxide pretreatment improves the saccharification of both wild-type and
mutant rice straw. The positive control and negative control were consistent in
the deconstruction of Myb61a mutant straw in 15% NH4OH with the
previous controls. When compared to Citrate Buffer treated mutant rice straw,
the pretreatment of mutant rice straw with NH4OH increased glucose yield by
4.7-fold with a p-value of 0.0485. A p-value of less than 0.05 confirms that
this basic pretreatment significantly improves the deconstruction of Myb61a
mutant plants. This deconstruction provides data to support the advantages of
combining successful pretreatments with digestible mutant plants to even
further improve saccharification.
Discussion
A
global emphasis has been laid on decreasing the amount of carbon dioxide (CO2)
released into the atmosphere as a result of the large-scale burning of fossil
fuels. The main components of fossil fuels are methane and petroleum (8). These
chemical compounds consists of hydrocarbons that when burned release water,
energy, and carbon dioxide (9). Climate change has become a household name that
refers to the flux of carbon dioxide into the atmosphere that outstrips the
rate of carbon dioxide removal. The growth rate of CO2 emissions
increased on a global scale from 1.1% a year between 1990-1999 to greater than
3% a year between 2000-2004 (10). The Earth’s atmosphere is maintained by gases
such as CO2, water vapor, methane, ozone, nitrous oxide, and
halocarbons (9). To maintain the global carbon cycle, the rate of addition and
removal of these gases must be equal and steady. An excess of CO2
causes the Earth to retain more heat in the form of infrared rays that would
otherwise escape the atmosphere.
Currently,
98% of the energy used for transportation comes from the burning of fossil
fuels (9). International efforts are focused toward finding an alternative
source of hydrocarbons to replace fossil fuels. Plant biomass is an abundant
energy reserve of glucose sugars that can be fed to microbes to produce
biofuels. It is estimated that 98-99% of accumulated organic plant matter
decays; the energy in the sugars of this decaying matter left unutilized (10).
This
experiment makes progress in the utilization of energy stored in plant cell
walls through the genetic manipulation of Myb61a and the highly basic
pretreatment of biomass with 15% NH4OH. Not only does the
deconstruction of Myb61a knock-down mutant plants show increase
saccharification, but the improved digestibility of these mutant plants
confirms the role of Myb61a in the cell wall biosynthetic process. The results
of the Myb61a mutant deconstruction are promising for engineering a genetically
modified plant that displays increased sugar yield for the process of biofuel
production.
In
comparison to other experiments that show increased saccharification due to
genetic manipulation, my results prove to be competitive. In one experiment
from the literature, an acyl-transferase, OsAT10, involved in the acylation of
cell wall matrix polysaccharides was overexpressed in rice. The overexpression
mutant, OsAT10-D1, showed a significant increase in glucose yield of
approximately 0.035 mg of glucose per mg of biomass when compared to the
wild-type which showed approximately 0.029 mg of glucose per mg of biomass (3).
In my experiment, Myb61a knock-down mutants show approximately 0.039 mg of
glucose release per mg of biomass compared to the wild-type showing
approximately 0.013 mg of glucose release per mg of biomass (Figure 1). Data from my experiment
suggests that the Myb61a knock-down mutant contends well with other published
data for genetically modified rice plants.
In
addition to rice, genetic mutants exhibiting increased digestibility have also
been identified in maize. A phenotypic mutation known as brown-midrib (bmr) mutation can be induced in maize as
well as sorghum. This simple recessive mutation is associated with a pigment in
lignified tissues. Bmr mutants show
reduced lignin content and a 33% increase in the digestibility of cell wall
components when digested with a commercial Cellulase similar to Ctec2 (11). In
both Bmr and OsAT10-D1 mutants,
increased digestibility was found to correlate to a change in lignocellulosic
content. Evidence from my experiment confirms that significant glucose release
could in fact be related to Myb61a’s role in regulating the transcription of
cell wall components.
If
Myb61a is a transcriptional activator for cell wall synthesis, it would be
logical to infer that the knock-down mutant would display decreased
lignocellulosic content. Further experiments should be conducted to confirm
this hypothesis and the general trend of increased saccharification in Myb61a
mutant biomass.
One
important parameter to consider when examining the data of mutant versus
wild-type glucose release (Figures 1 and 2) is that in Figure 1 the Myb61a
mutant refers to biomass from a single mutant plant. In Figure 2, the
experiment was repeated using a pool of milled rice straw across all available
Myb61a mutant plants. Taking rice straw from more than one mutant plant makes
the data more credible because the mutant plants now have a greater sample
size. As expected, the comparison of a pool of mutant plants to a pool of
wild-type plant biomass resulted in an insignificant p-value. If this
experiment were to be repeated, it would be interesting to see if the general
trend of increased saccharification by mutant rice straw persisted even in a
larger sample size.
In
addition to the effects of mutation on cell wall digestibility by the cellulase
and beta-glucosidase enzymes, biomass pretreated with ammonium hydroxide exhibits
a significant increase in saccharification. Wild-type rice straw pretreated
with NH4OH shows up to a 3–fold increase in glucose release compared
to wild-type rice straw incubated in Citrate Buffer. In a repeated experiment,
NH4OH pretreated biomass shows a 1.2-fold increase in glucose yield.
Despite the decline in glucose yield in the follow-up experiment, a student’s
t-test validates that both data sets show significant increase (p-value <0.05)
in glucose yield when pretreated with ammonium hydroxide.
The
significance of the rice straw’s increased digestibility due to 15% NH4OH
pretreatment has special implications. In a deconstruction of wheat straw, a
16.8% NH4OH pretreatment was used to enhance saccharification (11).
Results of this deconstruction also show a 3-fold increase in saccharification
of pretreated samples compared to untreated samples. Images captured by scanning
electronic microscopy (SEM) of wheat straw before and after pretreatment showed
obvious structural changes to lignocellulosic material after being pretreated
with NH4OH. Further analysis by X-ray diffraction suggests that the
crystallinity of lignocelllulose was significantly decreased by NH4OH
pretreatment. Decreasing the crystallinity of lignocellulose may open up the cell
wall structure to allow the cellulase enzymes more access to cellulosic
material. Evidence obtained by the wheat straw deconstruction suggests that
similar structural rearrangements due to the ammonium hydroxide pretreatment may
be affecting rice straw. Wheat and rice, both monocots, display the same ratio
of increased saccharification under similar pretreatment conditions, which
would lead us to presume that a pretreatment that works well for one monocot
may work just as well for another monocot.
The
data obtained from both mutant and pretreatment deconstructions was put to the
test to see if a combination of genetic mutation and ammonium hydroxide pretreatment
would yield even further glucose release. In a deconstruction of Myb61a mutant
biomass pretreated with NH4OH (Figure
5), the data indicates an outstanding 15.7-fold increase in
saccharification compared to the untreated wild-type biomass. This data is especially
important to the deconstruction of biomass because it shows that the
combination of genetic mutation and pretreatment methods can be used to more
than double the sugar yield that would otherwise be obtained by using one of
these deconstruction targets by itself. Building on Myb61a mutation and highly
basic pretreatment strategies to increase sugar yield has been validated as
time-worthy research by the data put forth by this experiment. The benefit of
increased sugar yield is magnified by the combination of unique targets outlined
in this experiments for the deconstruction of lignocellulosic biomass. It is
hopeful that one day the deconstruction of agricultural waste will yield
adequate amounts of glucose at a low energy cost.
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Supplementary
Information
Table S1. Organization of technical replicates by pre-treatment and
biomass type. Myb61a-1
mutants biomass in the first set of experiments was taken from a single
Myb61a-1 mutant rice plant. This experiment includes four technical replicates
of each pre-treatment and biomass combination. Kitake (KIT) wild-type rice
straw was used as the positive control (PC). The negative control (NC)
contained enzyme only, to adjust for background readings.
Figure S2. Standard curve of absorbance verses a known amount of
glucose dilution used to derive linear equation for determining mg of glucose
released at each absorbance. This standard curve was obtained by plotting
known glucose dilutions on the x-axis against a given absorbance determined by
the thermocycler. A linear trendline of y=0.3987x-0.0859 was obtained from the
standard curve and used to calculate mg of glucose per mL of all subsequent
thermocycler runs. An R2=0.96379 confirms that the linear trendline
obtained from the data of this graph does not display a significant amount of
error and can be used to determine glucose release.
