Screening, optimization and extraction of polyhydroxyalkanoates and peptidoglycan from Bacillus megaterium

Bioplastics are polymers (such as polyesters) produced from bacterial fermentations that are biodegradable and nonhazardous. They are produced by a wide variety of bacteria and are made only when stress conditions allow, such as when nutrient levels are low, more specifically levels of nitrogen and oxygen. These stress conditions cause certain bacteria to build up excess carbon deposits as energy reserves in the form of polyhydroxyalkanoates (PHAs). PHAs can be extracted and formed into actual plastic with the same strength of conventional, synthetic-based plastics without the need to rely on foreign petroleum. The overall goal of this project was to select for a bacteria that could grow on sugars found in the lignocellulosic biomass, and get the bacteria to produce PHAs and peptidoglycan. Once this was accomplished the goal was to extract PHAs and peptidoglycan in order to make a stronger more rigid plastic, by combing them into a co-polymer. The individual goals of this project were to: (1) Select and screen bacteria that are capable of producing PHAs by utilizing the carbon/energy sources found in lignocellulosic biomass; (2) Maximize the utilization of those sugars present in woody biomass in order to produce optimal levels of PHAs. (3) Use room temperature ionic liquids (RTILs) in order to separate the cell membrane and peptidoglycan, allowing for better extraction of PHAs and more intact peptidoglycan. B. megaterium a Gram-positive PHA-producing bacterium was selected for study in this project. It was grown on a variety of different substrates in order to maximize both its growth and production of PHAs. The optimal conditions were found to be 30°C, pH 6.0 and sugar concentration of either 30g/L glucose or xylose. After optimal growth was obtained, both RTILs and enzymatic treatments were used to break the cell wall, in order to extract the PHAs, and peptidoglycan. PHAs and peptidoglycan were successfully extracted from the cell, and will be used in the future to create a new stronger co-polymer. Peptidoglycan recovery yield was 16% of the cells’ dry weight.

UNDERSTANDING FARMERS’ PERCEPTIONS AND THE EFFECTS OF SHEA TREE Vitellaria paradoxa DISTRIBUTION IN AGROFORESTRY PARKLANDS OF THE UPPER WEST REGION, GHANA

Agroforestry parklands represent a vast majority of the agricultural landscape under subsistent-oriented farming in semi-arid West Africa. Parklands are characterized by the growth of well- maintained trees (e.g., shea) on cultivated fields as a result of both environmental and human influences. Shea (Vitellaria paradoxa) provides a cultural and economic benefit to the local people of Ghana, especially women. Periods between traditional fallow rotation systems have reduced recently due to agricultural development and a demand for higher production. As a result, shea trees, which regenerate during fallow periods, has decreased over the landscape. The aim of this study was to determine beneficial spatial distributions of V. paradoxa to maintain high yields of staple crops, and how management of V. paradoxa will differ between male and female farmers as a result of farmer based needs and use of shea. Vegetation growth and grain yield of maize (Zea mays) associated with individual trees, clumped trees, and open fields were measured. Soil moisture and light availability were also measured to determine how V. paradoxa affected resource availability of maize in either clumped or scattered distributions of V. paradoxa. As expected, light availability increased as measurement locations moved farther away from all trees. However, soil moisture was actually greater under trees in clumps than under individual trees. Maize stalk height and cob length showed no difference between clumped and single trees at each measurement location. Grain yield per plot and per cob increased as measurement locations moved farther from single trees, but was actually greater near clumped trees that in the open field subplots. Cob length and maize stalk height increased with greater light availability, but grain yield per cob or per plot showed no relationship with light, but were not affected by soil moisture. Conversely, grain yield increased with increasing soil moisture, but had no relationship with light availability. Initial farming capital is the largest constraint to female farmers; therefore the collection of shea can help provide women with added income that could meet their specific farming needs. Our data indicate that overall effects of maintaining clumped distributions of V. paradoxa provided beneficial microclimates for staple crops when compared to single trees. It is recommended that male and female farmers allow shea to grow in clumped spatial distributions rather than maintaining scattered, individual trees.

Planting and production of switchgrass (Panicum virgatum L.) as a bioenergy crop in Michigan's Upper Peninsula

Switchgrass (Panicum virgatum L.) is a perennial grass holding great promise as a biofuel resource. While Michigan’s Upper Peninsula has an appropriate land base and climatic conditions, there is little research exploring the possibilities of switchgrass production. The overall objectives of this research were to investigate switchgrass establishment in the northern edge of its distribution through: investigating the effects of competition on the germination and establishment of switchgrass through the developmental and competitive characteristics of Cave-in-Rock switchgrass and large crabgrass (Digitaria sanguinalis L.) in Michigan’s Upper Peninsula; and, determining the optimum planting depths and timing for switchgrass in Michigan’s Upper Peninsula. For the competition study, a randomized complete block design was installed June 2009 at two locations in Michigan’s Upper Peninsula. Four treatments (0, 1, 4, and 8 plants/m2) of crabgrass were planted with one switchgrass plant. There was a significant difference between switchgrass biomass produced in year one, as a function of crabgrass weed pressure. There was no significant difference between the switchgrass biomass produced in year two versus previous crabgrass weed pressure. There is a significant difference between switchgrass biomass produced in year one and two. For the depth and timing study, a completely randomized design was installed at two locations in Michigan’s Upper Peninsula on seven planting dates (three fall 2009, and four spring 2010); 25 seeds were planted 2 cm apart along 0.5 m rows at depths of: 0.6 cm, 1.3 cm, and 1.9 cm. Emergence and biomass yields were compared by planting date, and depths. A greenhouse seeding experiment was established using the same planting depths and parameters as the field study. The number of seedlings was tallied daily for 30 days. There was a significant difference in survivorship between the fall and spring planting dates, with the spring being more successful. Of the four spring planting dates, there was a significant difference between May and June in emergence and biomass yield. June planting dates had the most percent emergence and total survivorship. There is no significant difference between planting switchgrass at depths of 0.6 cm, 1.3 cm, and 1.9 cm. In conclusion, switchgrass showed no signs of a legacy effect of competition from year one, on biomass production. Overall, an antagonistic effect on switchgrass biomass yield during the establishment period has been observed as a result of increasing competing weed pressure. When planting switchgrass in Michigan’s Upper Peninsula, it should be done in the spring, within the first two weeks of June, at any depth ranging from 0.6 cm to 1.9 cm.