Simple, reliable and cost-effective methods for detecting pathogens are a vital part of diagnostics inside and outside the clinic, in particular in the developing world. Paper based colorimetric techniques are a promising approach for biosensors and bioassays as they can be used at the point of sampling and require little equipment. This study reports on the development of a colorimetric cellulose bioassay that can detect pathogen DNA with covalently attached single-stranded DNA probes. Chemical activation of cellulose via tosylation and oxidation was investigated. The successful activation of cellulose was characterised by Fourier transform infrared spectroscopy, scanning electron microscopy and elemental analysis. Sulfhydryl and amine functionalised oligonucleotide probes complementary to a segment of IS6110 element in Mycobacterium tuberculosis genome were covalently immobilised on the cellulose strips for recognition of target nucleic acid. The detection of biotinylated target oligonucleotides was achieved with horseradish peroxidase (HRP) linked to streptavidin that binds biotin with high affinity. HRP catalysed the oxdidation of tetramethylbenzidine by hydrogen peroxide. The successful assay was confirmed by the appearance of blue coloured spots on cellulose strips incubated with biotinylated target oligonucleotides complementary to the surface attached probe. The study also showed that tosylated cellulose is more reliable for the detection of targets. Initial experiments have shown sensitivity upto 0.1 µM and considerable specificity. High probe immobilization efficiencies (>90%) have been observed. The assay was also effectively demonstrated with mycobacterial DNA. Additionally, the development of a label free assay based on a dual-probe approach was investigated, but did not yield conclusive results. The developed assay has the potential for use as a simple test for the detection of pathogen DNA in clinical samples since it requires minimal equipment and is cost effective. In addition, it also shows the potential use of tosylated cellulose as a prospective surface for attaching other types of biomolecules in an active conformation.

Solid state fermentation (SSF) refers to the microbial fermentation, which takes place in the absence or near absence of free water, thus being close to the natural environment to which the selected microorganisms, especially fungi, are naturally adapted. The current status of SSF research globally was discussed in terms of articles publication. This was followed by discussion of the advantages of SSF and the reason for interest in SSF as a notable bioprocessing technology to be investigated and compared to submerged fermentation (SmF) for the production of various added-value products. SSF also proved to be a potential technology to treat solid waste produced from food and agricultural industry and to provide environmental benefits with solid waste treatment. A summary was made of the attempts at using modern SSF technology for future biorefineries for the production of chemicals. Many works were carried out in the Satake Centre for Grain Process Engineering (SCGPE), University of Manchester, to prove the strategy of using SSF for the production of hydrolysate rich in nutrients for sequel microbial fermentation with or without adding any commercial nutrients. The research findings presented in this thesis are based on a series of SSF experiments carried out on systems varying in complexity from simple petri dishes to our own design of bioreactor systems. They were conducted to assess a solution for biomass estimation, enzymes production, and successful mass and heat transfer. A proper technique for inoculum transfer prior to the start of the fermentation process was developed. In SSF, estimation of biomass presents difficulties as generally the fungal mycelium penetrates deep and remains attached with the solid substrate particles. Although many promising methods are available, the evaluation of microbial growth in SSF may sometimes become laborious, impractical and inaccurate. Essentially, this remains another critical issue for monitoring growth. In these studies, measurement of colour changes during SSF are presented as one of the potential techniques that can be used to describe growth, complementary to monitoring metabolic activity measurement, such as CER, OUR and heat evolution, which is directly related to growth. For the growth of Aspergillus awamori and Aspergillus oryzae on wheat bran, soybean hulls and rapeseed meal, it was confirmed that colour production was directly proportional to fungal growth. This colourimetric technique was also proved to be a feasible approach for fungal biomass estimation in SmF. This new approach is an important complementation to the existing techniques especially for basic studies. The key finding is that the colourimetric technique demonstrated and provided information of higher quality than that obtained by visual observation or spores counting. The effect of aeration arrangements on moisture content, oxygen (O2), mass and heat transfer during SSF was investigated. A. awamori and A. oryzae were cultivated on wheat bran in newly designed four tray solid state bioreactor (SSB) systems. The new tray SSB systems were: (1) single circular tray SSB, (2) multi-stacked circular tray SSB, (3) Single rectangular tray SSB and (4) multi-square tray SSB. The purpose was to study the effect, on heat and water transfer, of operating variables, fermentation on the perforated base tray and internal moist air circulation under natural and forced aeration. Temperature, O2 and carbon dioxide were measured continuously on-line. Enzyme activity, moisture content and biomass were also measured. The results suggest that the air arrangements examined have a remarkable effect on the quantity of biomass produced using measurement of spores and enzymes production. The strategy presented in these studies allowed quantitative evaluation of the effect of forced internal moist air circulation on the removal of metabolic heat. With the proposed strategy, it was possible to maintain the bed temperatures at the optimum level for growth. However, the effect on moisture content was very different for the two fungi. It was found that the ability of A. oryzae to retain moisture was much higher than that of A. awamori. This is possibly due to the higher levels of chitin in A. oryzae. Greater spores and enzymes (glucoamylase, xylanase and cellulase) production was observed for A. awamori in multi-stacked circular tray and multi-square tray SSB systems compared to the conventional petri dishes and the other two systems. A. oryzae was excellent in producing protease in the same bioreactors. A direct technique of establishing a correlation between fungal growth and CER, OUR, heat evolved was proven successful in this work. The information obtained from CER and OUR led to the estimation of respiratory quotient (RQ). RQ describes the state of the fungal population in the tray SSB and gives an indication of fungal metabolic behaviour. RQ values < 1 were obtained from 38 experiments using four tray SSB systems for the two fungi. A kinetic model based on CO2 evolution instead of biomass concentration was examined in order to simplify the required experiments for kinetic model development. A Gompertz model was used to fit the integrated CO2 data and predict the quantity of CO2 evolution in all experiments. A correlation was found between the heat evolution and CER. The performances of tray SSB systems can be improved by constructing them as multi-trays. The multi-tray systems improved the mass transfer considerably compared with single tray systems. In addition, the multi-tray systems allowed precise measurement of the gradients of CO2, enzymes, spores and fungal biomass. In addition, the air arrangements using moistened air were successful in maintaining moisture content, adequate O2 supply and control of temperature, and hence, increased the productivity of both fungi. Overall A. awamori and A. oryzae have their own ability and performance to degrade and utilise the complex compositions contained in the solid substrate and fermentation conditions may lead to possible comparisons. In addition, multi-stacked circular tray and multi-square tray SSB systems demonstrated an excellent system for further investigations of mass transfer and possibly for large scale operation, though considerable optimisation work remains to be done, for example the height/diameter ratio and total number of trays should be optimised.