1. 1.Introduction GLYPHOSATE Glyphosate (N-(phosphonomethyl)glycine) is the most commonly used conventional herbicide active ingredients according to 2007 statistics In 2011, about 180 million acres of land were treated with Roundup, an herbicide with glyphosate as its major active ingredient. Glyphosate constrains weeds’ ability to synthesize amino acids by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme localized in the chloroplasts responsible for catalyzing biosynthesis of essential amino acids. The inhibition of enzyme produces growth inhibitors and prevents the production of growth promoters. The chemical is degraded by microbes, as it reacts with glyoxylate to yield aminomethylphosphonic acid (AMPA) and glyoxylic acid. These two chemicals further degrade into carbon dioxide (CO2) and phosphate, as shown in the figure. HYPHAE - 80% of all plants are involved in mycorrhizal association - Tubes absorbing and transporting nutrients from the environment - Indication of environmental change due to sensitivity to environmental stimuli with apical growth and localization of chitin synthesis CLIMATE CHANGE Carbon dioxide are at an average of 399.50 parts per million, the highest rate in 650,000 years. The predicted rate of climate increase over the next century is at least 20 times faster than that of the previous years. As much as 50-70% of CO2 in the atmosphere are emitted by fungi. Image courtesy of NASA 2. Methods Dry soil samples from moderately vegetated areas were collected on July 14 th, 2014 in the San Joaquin Marsh Reserve All procedures were conducted at the Treseder Lab in University of California, Irvine Carbon dioxide emission was measured using a CO 2 analyzer The project was partitioned into three parts: Set 1, Set 2, and Set 3 Set 1 was an experimental set to assess the parameters and finalize procedures of the experiment Set 2 was initiated on August 28 th with five replicates in both control and treatment Set 3 was initiated on September17th with ten replicates in both control and treatment Set 2 and 3 shared the same protocol for measuring carbon dioxide emission of fungi samples Hyphal extraction microscopy calculations were conducted on Set 2 to compare hyphal length CO 2 emissions and hyphal lengths were compared between the control and treatment groups using analysis of variance test (ANOVA) at α = 0.05 level. CARBON DIOXIDE EMISSION 20 grams of soil were inserted into a clear vial for both control and treatment groups Control groups were treated with 10 mL of deionized water Treatment groups were treated with 10 mL of 100% RoundUp® herbicide solution At designated time points, each of the samples were sealed with lids that allow gas collection After the samples accumulated CO 2 for 30 minutes, emission was measured and recorded HYPHAL EXTRACTION MICROSCOPY Approximately 0.400 grams of soil from each of the replicates in Set 2 were measured and inserted into a 50 mL solution of sodium hexametaphosphate (NaPO 3 ) 6 10 mL of the slurry was added into a 90mL (NaPO 3 ) 6 solution to dilute the solution further 5 mL of the solution was pipetted with a syringe into a filtering apparatus The sample was stained with acid fuchsin The filter was mounted onto a glass slide and placed in the drying oven overnight Filter was examined under the light microscope under 40x objective to count hyphae Hyphal length was recorded 3.2 Results HYPOTHESIS: Glyphosate concentrate will increase carbon dioxide (CO 2 ) emission of the fungi samples and decrease their biomass, measured by fungal hyphal length, as the chemical reacts with glyoxylate to emit CO 2 and hinder growth promoters. Abstract The purpose of the experiment is to assess the effect of glyphosate from the herbicide RoundUp® on the CO 2 emission and biomass of fungal samples. Glyphosate, or N-(phosphonomethyl) glycine, is one of the most widely used ingredients for herbicide, and presumed to have no adverse effect on the ecosystem with its ability to target specific weeds. The chemical is degraded in the soil to aminomethylphosphonic acid (AMPA), which then reacts with glyoxylate to release carbon dioxide. With the rising concern of global warming and increasing carbon dioxide levels, the effect of glyphosate in widely applied herbicide on fungal carbon dioxide emissions and biomass was assessed to evaluate the potential role of herbicide and fungus on global warming. To measure the CO 2 emission levels of the treatment and control groups, the samples were sealed off to accumulate gas at ten designated time points. After the accumulation of gas from each sample, the gas was analyzed using a CO 2 analyzer to determine the CO 2 levels for the sample. To assess the difference in biomass, hyphal extraction microscopy calculations were done with the treatment and control groups and compared. The CO 2 emission levels for the treatment group were consistently higher than those of the control group, and ANOVA test comparing the two groups were significant at α = 0.05 level at five time points out of ten. There was a significant difference between hyphal lengths, with the average of the treatment group being approximately 139.75 m/g of dry soil and the average of the control group being approximately 346.78 m/g. The discrepancies of the gas emission levels may be due to the direct effect of glyphosate on the respiration rates of the microbial community or the degradation of the chemical into AMPA, releasing gas as a byproduct. The differences in hyphal length may be due to the disturbance of the symbiotic relations between the fungi and different soil components. By observing the correlation between the application of herbicide and fungal carbon dioxide emission and biomass, this experimentation model was successful in indicating a relation between chemical manipulation of the environment and increased carbon dioxide levels and hindered biological activities. Table II: Set 2 Control v. Treatment Average CO2 emission (ug CO2-C/gram dry soil/per hour) 0 hr.4 hrs.28 hr.30 hr.168 hr. 360 hr. 432 hr. 480 hr. 528 hr. 600 hr. 648 hr. Control0.1440.2630.133-0.0150.0900.2590.2040.1890.1680.0990.138 Treatment 0.8290.4470.4740.2400.1270.3700.2960.3860.4790.2990.365 Table III: Set 3 Control v. Treatment Average CO2 emission (ug CO2-C/gram dry soil/per hour) 0 hr.24 hr. 48 hrs. 120 hr. 168 hr. 192hr 288 hr. 312 hr. 360 hr. 504 hr. 528 hr. Control0.1120.0930.1440.1150.1520.0370.1320.1430.1750.2770.205 Treatment 0.2900.1290.1820.2060.3200.1680.4060.4890.6950.8210.599 3.1 Results Fig. 1. Standardized CO 2 emission levels for set 2 Fig. 2. Standardized CO 2 emission levels for set 3 Fig. 5. Comparison of hyphal lengths for set 2 Highlighted values in Tables I and II denote significance at α = 0.05 level. Time points where the average difference between the control and the treatment was significant at α=0.05 level were denoted in the following tables with a highlight Percent deviations were relatively high The average values for Set 2 CO 2 emission ranged from 0.132 to 0.277(ug CO2-C/gram dry soil/per hour) according to different time points The average values for Set 3 CO 2 emission ranged from 0.129 at 24 hours to 0.821(ug CO2-C/gram dry soil/per hour for different time points There was a large discrepancy between the control and treatment groups when the average hyphal lengths were examined * * KEY: *: Denotes significant difference at α= 0.05 level 4. Analysis CO2 emission levels for treatment samples were consistently higher than control samples, which may be due to the glyphosate degrading to AMPA and glyoxylate, producing carbon dioxide. Hyphal lengths for the treatment group (139.75) were significantly less than those of the control group (346.78), possibly due to the changes in symbiotic relationships of the soil and fungi, along with altered growth factors At least 200% increase in carbon dioxide emission levels with treatment Approximately 60% decrease in hyphal length with treatment More significance at the 5% level was shown towards the end of the experiment, possibly suggesting that the glyphosate concentrate do not directly and/or immediately affect the biological structure and systems of the fungal community 5. Conclusion The data supported the hypothesis CO 2 emission values for the control group were less than those for the treatment ANOVA indicated significant differences towards the end of the experiment, suggesting that the effect of glyphosate on the respiration rates of the microbial community was not immediate Biomass as shown by fungal hyphal length was smaller in the treatment group, possibly denoting decreased bioactivity and symbiosis Decreased hyphae of fungi denote less absorption and transportation of nutrients by the organisms Manipulation of herbicide treatment may help reduce the rate of climate change, as carbon dioxide emission is predicted to be reduced With fungi responsible for as much as 50-70% of the CO2 in the atmosphere, the 200% increase in emission levels with glyphosate treatment contributes significantly to human’s carbon footprint. Acknowledgements Dr. Yev Marusenko for advice and direction throughout the project Dr. Kathleen Treseder for providing this opportunity. Mr. Antrim and Ms. Cooper for their support Family members References Company, Monsanto. Backgrounder: Glyphosate and Environmental Fate Studies (n.d.): n. pag. Web. "Glyphosate." Technical Fact Sheet. N.p., n.d. Web. 18 Jan. 2015.. Lane, Matthew. The Effect of Glyphosate on Soil Microbial Communities. Thesis. Ohio State University, 2011. N.p.: n.p., n.d. Web. 11 Jan. 2015.. "Mycology - Structure and Function - Hyphal Structure." Mycology. University of Sydney, n.d. Web. 19 Jan. 2015.. Table I: Set 2 Control v. Treatment Average Hyphal Extraction Microscopy (m/g dry weight soil) Control346.78 Treatment139.75**