Tobacco seed performance is traditionally measured as percentage germination at 14 d under controlled laboratory conditions. However, under greenhouse conditions, seed lots with equal 14-d germination may exhibit substantial differences in uniformity of early seedling growth and spiral root incidence that impact the number of usable transplants. A seedling performance index (SPI) was developed to quantitatively describe greenhouse tobacco seedling performance. The 14-d emergence, relative leaf area uniformity, and seedling leaf area determined by computer image analysis were used to calculate the index. Greenhouse tobacco seed trials demonstrated that seed with the Rickard pellet had higher emergence, but the higher spiral root incidence associated with the Rickard pellet lowered the SPI compared to the Cross Creek pellet. Primed seed lots of flue-cured cultivars (NC 72 and NC 71) had a significantly higher SPI than the nonprimed seed lots at one location but not at a second location. Seed lots sown in Premier Pro-Mix TA commercial medium had a higher spiral root incidence, which resulted in a lower SPI compared to Carolina Choice, Carolina Gold, and Sunshine LP5 commercial media. The index quantitatively determined differences in seedling performance under greenhouse conditions not reflected by standard germination tests. Significant differences in the SPI were observed among seed lots with certified 90% germination. The SPI is a simple method to describe seedling performance because the data used to calculate the SPI is obtained from one seed tray image. In contrast, frequent counting and seedling evaluations are involved with standard germination and vigor tests. / Master of Science

The maximum potential of biorefineries cannot be achieved without the valorisation of their residues. The two main residues from wheat biorefineries, bran and wet distiller's grains and solubles (WDGS), have a projected production of 7 and 3 million tonnes/year respectively by 2025 just in the European Union. These residues can be mixed to form moist animal feed. However, the product can undergo growth of contaminating fungi. This bio-deterioration causes significant losses and represents a hazard for the animals. Currently, commercial preservatives (from the petrochemical industry) are added to prevent bio-deterioration but these add to the production cost. The research reported in this thesis was focused on the utilisation of solid state bioprocessing (SSB) to prevent the bio-deterioration of the moist feed. The method is based on the inhibition of contaminating fungi by an edible fungus, R. oryzae, considered safe for animal consumption which is a lactic acid producer. This fungus is known to produce very dense mycelia causing significant oxygen transfer limitations and this, together with the reduction in pH and substrate available for contaminating fungi, was explored as a potential mechanism to bio-preserve the moist feed. SSB, has been targeted as a key technology for the treatment of agro-industrial solid residues. However, the measurement of parameters in SSB is very complicated, hampering the development of the technology. Consequently, methods for the measurement of pH and growth in SSB were also studied. The measurement of pH is normally performed in extract solutions but, although widely used, the method has not been standardised. There are many extraction variables, such as contact time, type of solids and solvent, solid:water ratio and agitation velocity, involved in the measurement. Experiments revealed that changes in the extraction conditions affect pH readings. The degree of impact depends upon the variable but results presented in this thesis clearly highlight the importance of providing a precise and comprehensive report of the extraction conditions. For the estimation of growth, a method based on digital imaging analysis (DIA) was developed in this work. DIA is a non-destructive, quick and simple method, which uses computational analysis of digital images to measure areas and colour changes on a surface. The technique provided data that allowed an objective comparison of fungal colonies on plates, making it of higher quality than simple visual evaluation. It was determined that C*Traffordgold, the model moist feed used in this research, has starch and moisture contents of 10% and 50% respectively. Bio-deterioration is caused by indigenous organisms in the bran and is observable after only 2 days of incubation at 20°C. R. oryzae was able to bio-preserve the material for at least a month. This is a very promising result since preservative treated C*Traffordgold shows considerable bio-deterioration after just 2 weeks. DIA was used for the estimation of R. oryzae growth at different temperatures, moisture contents and inoculum sizes. The fungus was able to grow at temperatures between 15° and 37°C, with optimum growth at 30°C. An increase in the moisture content resulted in faster growth of the fungus and it was determined that a minimum inoculum size of 103 spores/gC*Traffordgold is necessary to avoid bio-deterioration. The bio-preservation is a result of the reduction of substrate and oxygen available for contaminating microorganisms. Studies revealed that lactic acid does not inhibit growth of contaminating fungi even at pH 4. On the other hand, R. oryzae showed radial growth rates up to 6 times faster than those from contaminating cultures. The fungus is able to surround the competitor colony and 'enclose' it. The formation of a fungal barrier limits the oxygen available for the undesired colony, hampering its growth. It was estimated that by implementing the bio-preservation method a wheat biorefinery producing 180,000 tpa of moist feed could save circa 500,000 USD per annum. Integration of residue processing in biorefineries is necessary to make them sustainable. The application of SSB for bio-preservation could enhance the overall value of wheat biorefineries and simultaneously reduce dependency on the petrochemical industry.

Currently, the transplantation of islets of Langerhans is a viable means to maintain control of blood sugar levels and reduce the risk of hypoglycemia in defined populations with brittle type I diabetes mellitus or those requiring pancreatectomy. However, the process of islet isolation is highly variable and not all isolations result in islet numbers or quality suitable for transplantation. This thesis aimed to improve transplantation success through optimization and standardization of the isolation process and to identify pretransplant variables associated with early islet engraftment. A previously disregarded enzyme activity, tryptic-like activity (TLA), has been identified to influence pancreas digestion efficiency and islet isolation success in both the preclinical and clinical situations. For human pancreases, islet isolation success rates improved from 0% in the lowest TLA group to over 50% in the highest TLA groups without affecting islet quality. These findings should help standardize evaluation of enzymes for clinical islet isolation. A closed, automated, pump-made gradient system was compared to the open, manual method for islet separation. No differences were observed in expected gradient volumes, islet yields or total purities between the two methods. The pump-made gradient system successfully removed manual influences on density gradient production while fulfilling regulatory requirements for closed system processing. Islet quantification was evaluated with computer-assisted digital imaging analysis (DIA) and a semi-closed assessment system. By using the DIA system method, which measures islet purity and pellet volume instead of manual counting methods, variation in islet counts and purity reduced by almost half. By using a transplant outcome measurement of C-peptide adjusted by blood glucose and creatinine, we identified four pretransplant factors that affect early transplant outcome. Of the four factors, one was related to the organ transport time, one to function of the islets, and two to the transplanted tissue volume. When these four factors were put into a predictive model, it accounted for about 40% of the transplant outcome. The work contained in this thesis identifies and optimizes a number of critical elements related to islet isolation and transplantation protocols.