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
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.
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.
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.
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.
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.
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).
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.
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.
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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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.