Dry trehalose was then added and completely dissolved. Finally, 4- vinylbenzyl chloride was added dropwise. The reaction was stirred for 22 hours at 22 °C. The reaction mixture was then precipitated into a rapidly stirring solution of methylene chloride and hexanes . The solids were collected by vacuum filtration through a sintered glass funnel equipped with a filter flask.Solvents were further removed in vacuo over 10 hours and then solids were broken up with a spatula to increase surface area and make drying more efficient. The solids were dried for an additional 24 hours before being used for gelation without further purification. To synthesize the gels, the crude trehalose monomer and cross-linker mixture was completely dissolved in Milli Q water . To this, tetramethylethylenediamine was added. This mixture and a 10 mg mL-1 stock solution of ammonium persulfate in Milli Q water were separately degassed for 30 minutes by sparging with argon. Under an inert atmosphere of argon gas, the APS solution was added to the crude styrenyl-trehalose solids and TEMED for a final ratio of 1 g crude material for every 1 mL Milli Q water, 5 µL TEMED, and 250 µL APS solution . The solution was gently shaken for 12 hours to form a gel. The crude gel was washed with a Soxhlet extractor for three days with deionized water to removed unreacted monomers, crosslinkers, and other impurities,fabrica de macetas plasticas providing a clear gel. The yields of gelations were based on comparing the moles of limiting reagent, 4-vinylbenzyl chloride, to the moles of final product.
The final product molecular weight was calculated based on the molecular weights of each of the individual components of the crude mixture, and the distribution of these products was determined by LCMS . Finally, the gel was lyophilized and then ground to produce a fine, white powder. The overall yield of the two-step synthesis was 87.5%, providing 155.9 mg of gel. The synthesis was increased 100-fold and carried out as outlined above with the following exceptions: the monomer/cross-linker reaction was stirred for 46 hours instead of 22 hours as a longer reaction time was required to have sufficient styrene-functionalization on trehalose, the reaction was then precipitated in 100 mL aliquots into DCM and hexanes at an approximate rate of 150 mL per minute while the suspension stirred at 800 rpm, and the final crude gel was washed for 7 days with deionized water in a Soxhlet extractor. The reaction gave an overall yield of 75.6%, providing 81.02 g of monomer/cross linker and 13.8 g of gel. Gelation was confirmed by examining physical properties of the gels, e.g. storage and loss modulus as well as swelling ratio. Storage and loss modulus were measured by trimming hydrogels to 8-mm diameters to match the top parallel plate geometry and with an applied constant strain of 1% and angular frequency range of 0.1 to 10 rad/s at 22 °C. Swelling ratio was determined by swelling completely dried hydrogels in Milli Q water over 72 hours and calculating the mass ratio between the swollen gels and their initial dry weights. All physical properties are displayed as the average and standard deviations of three independent hydrogel measurements.Phytase activity was measured by modifying a previously reported method.Phytase stock solution was added to trehalose gels and prepared as described above. Heated and control hydrogels were removed by centrifugation after addition of sodium acetate buffer and incubation.
Supernatant was added to 1 mL of 0.2 M sodium citrate buffer, pH 5.5. Aliquots were transferred to Lobind Eppendorf tubes. To all sample tubes, 10 µL of 1% phytic acid was added. The reactions were then incubated at 37 °C for 15 minutes before quenching with 15% trichloroacetic acid and then diluted ten-fold with Milli Q water . Aliquots were transferred to a 96-well plate and then diluted with a 1:3:1 solution of 2.5% ammonium molybdate , 10% sulfuric acid , and 10% ascorbic acid . The plate was covered with parafilm and then incubated at 50 °C in a water bath for 15 minutes, cooled at 4 °C for 15 minutes, and absorbance measurements were taken at 820 nm. Phytase activity was defined as the quantity of enzyme that catalyzes the liberation of 1.0 µmol of inorganic phosphate from 1 % phytic acid per minute at 37 °C and pH 5.5. Assay was run in triplicate. Note that generally this assay is difficult to reproduce due to the fast reaction between phytase and phytic acid. We advise that the assay be done as quickly as possible, using a multi-pipetter.Stock solution of b-glucanase was added to trehalose gels and prepared as described above. Heated and control hydrogels were centrifuged after addition of sodium acetate buffer and incubation. Supernatant was pre-warmed along with azoBarley glucan substrate provided in Megazyme assay kit at 30 °C for five minutes. Due to the viscous nature of the glucan substrate, it was transferred using a positive displacement pipet. Aliquots of the supernatant was added to azo-Barley glucan substrate and then mixed vigorously before incubating at 30 °C for 10 minutes. Precipitation solution was made by dissolving sodium acetate and zinc acetate in distilled water . The pH was then adjusted to 5.0 with concentrated hydrochloric acid, and the volume was adjusted to 200.0 mL. Finally, 2-methoxyethanol was added. An aliquot of this precipitation solution was added to each sample, and the contents were mixed vigorously, incubated at ambient conditions for five minutes, and then mixed vigorously again.
Finally, the samples were centrifuged at 6,000 rpm for 10 minutes, supernatant was added to a 96-well plate, and the absorbance was read at 590 nm. b-Glucanase activity was defined as the quantity of enzyme that catalyzes the liberation of 1.0 µmol of glucose reducing sugar equivalent from azo-Barley glucan substrate per minute at 37 °C and pH 4.6. Experiments were repeated in triplicate.Note that all LB media used throughout these studies contained 50 µg/mL kanamycin to prevent other strains of bacteria from growing. A colony of kanamycin-resistant strain of BL21 E. Coli bacteria was grown in 50 mL LB media in a 250 mL sterilized Erlenmeyer flask at 37 °C and 200 rpm. At an OD600 of 0.426, the bacteria was diluted in 50 mL LB media and incubated at 37 °C and 200 rpm for an additional 1.5 hours. The bacteria was diluted 1:1 in LB media containing P3, free trehalose or no excipient . The samples were frozen and lyophilized for 24 hours. Following lyophilization stress, 200 µL of LB media was added to each condition. Aliquots of 150 µL were added to 3 mL of fresh LB media in culture tubes and incubated at 37 °C and 200 rpm. Cell growth was monitored by measuring the absorbance at 600 nm.As drought frequency, severity, and duration are exacerbated by climate change,improving the efficiency of water resources is crucial for a sustainable future. Drought affects agriculture globally and poorly affects food security, water availability, and rural livelihoods. In the developing world alone, drought caused $29 billion agriculture revenue loss between 2005 and 2015.Drought cannot be avoided, but mitigation practices can negate its deleterious effects. In particular, drought reduces crop productivity due to high temperatures and limited water,but on-farm water and soil management have proven successful in abating these issues. Despite this, many inefficient practices, such as flood irrigation, are still widely applied.Technologies that prevent agricultural water wastage must be developed and implemented to improve the health of crops subjected to drought. Hydrogels are hydrophilic polymeric materials capable of absorbing and releasing water many times their weight.In soil, swollen hydrogels act as water reservoirs by slowly releasing captured water through a diffusion-driven mechanism that arises from humidity variation between the internal environment of the material and the soil surrounding it. Hydrogels have been mixed into soil to prevent water irrigation loss caused by drainage and evaporation. They also offer a potential scaffold for controlled release of nutrients,and provide better oxygenation to plant roots by increasing soil porosity. By improving the water holding capacity of soil and water available to plant roots, hydrogels have demonstrated the ability to increase plant survival rate, water use efficiency, and growth.While superabsorbent polyacrylate gels have demonstrated success as soil conditioners,precio de macetas de plastico it is hypothesized that anionic moieties within hydrogels create electrostatic repulsions with negative charges on the surface of soil particles.The anion-anion repulsive forces can reduce adsorption of the hydrogel to soil and therefore allow the polymer to be leached by water over time. The development of alternative hydrophilic gels for soil conditioning could help overcome these issues and potentially demonstrate other advantages. The Maynard lab has designed a scalable, two-step synthesis of a trehalose-based hydrogel for the thermal stabilization of enzymes.The synthesis yield was greatly improved from 17 % to 88 %, scaled 100-fold while retaining a high yield at 76 %, and was optimized to eliminate the use of halogenated and toxic solvents .
This multi-gram, green synthesis makes the gel more practical for agricultural applications where materials need to be cost-efficient and scalable.20 Moreover, trehalose has been shown to stabilize desiccant-intolerant soil bacteria necessary for plant growth.As such, trehalose hydrogels have great potential for water management as well as stabilization and delivery of plant nutrients while being beneficial to soil. Here, two hydrogels, a commercially available poly-based gel, Terra-sorb , and a trehalose hydrogel, synthesized by our lab as described in Chapter 2, were separately applied as soil amendments for tomato plants, Solanum lycopersicum, subjected to drought conditions. Performance of the gels was evaluated by monitoring tomato plant health through chlorophyll content, water potential, stomatal conductance, and relative growth rate measurements. We hypothesized that presence of the trehalose hydrogel would boost tomato plants’ physiological function after extended droughts . We also hypothesized that since the trehalosehydrogels were less hydrophilic than the Terra-sorb hydrogels, they would likely not be as efficacious as the positive control.Due to climate change, water availability has become more sporadic with cycles of drought and rewatering, which ultimately stresses plants.We therefore tested the ability for the hydrogels to retain their swelling ratio through repeated drying and wetting cycles. After purification and lyophilization, the trehalose hydrogels were swollen to their maximum capacity in 72 hours in deionized water. This drying-swelling cycle was repeated where the dry weight was taken after lyophilization and swollen weight was taken after swelling the gel in deionized water. The swelling ratio was calculated for each cycle by dividing the difference between the gels’ swollen weight and dry weight by the dry weight . Over the course of ten drying-swelling cycles , the hydrogels swelling ratio decreased from 16.3 ± 2.9 to 14.9 ± 1.1 . This minimal loss in swelling ratio during these cycles is an indicator that the gel could be subjected to multiple drought cycles without compromising its swelling abilities. Next, we evaluated how the water holding capacity of a sandy loam soil is affected by Terra-sorb and the trehalose hydrogel. We applied Terra-sorb at the manufacture’s recommended concentration, 0.4 wt %, and trehalose hydrogel at 0.4 wt % and 0.8 wt % . We saturated the soil then allowed it to desaturate over eight days while monitoring water loss by weight. All of the amendments improved the water holding capacity of the soil over the entirety of the experiment. Consistently, soil amended with Terra-sorb gels had the highest WHC, followed by soil with trehalose hydrogel at 0.8 wt % and then 0.4 wt %. We then rehydrated the soils to evaluate the gels’ capacities to work through multiple drying cycles. The conditioners maintained their previous trends and most of their WHC percentages. While WHC is an important factor for soil health, the water held by hydrogels is not necessarily available to crops.As such, it is vital to monitor plant growth in soil with the hydrogel amendments.Previous reports have demonstrated that hydrogel soil conditioners are not always effective in improving plant health and growth, and, in fact, are sometimes detrimental, depending on the soil type, plant species, and experimental conditions.So, before testing trehalose hydrogels directly, we ensured that tomato plants and our simulated drought conditions could benefit from soil conditioners by using commercially available hydrogel, Terra-sorb at 0.4 wt %, that has previously demonstrated delayed moisture loss for Quercus ruba seedlings subjected to short-term desiccation stress.