Enhanced ethanol fennentation is one of the current issues in fennentation technology; this also is a main aim that goes through this thesis. In this thesis, S. cerevisiae KAY446 was used for ethanol fennentation under very high gravity (VHG) conditions. This strain showed an ability to fennent under these conditions since an average ethanol concentration of 117.5 giL was detennined for continuous fennentation. In tenns of deeper biological aspects, a proteomic analysis of S. cerevisiae was also perfonned to gain an understanding of this micro-organism in responding to stress conditions due to high glucose concentrations in the broths. Therefore, the iTRAQ teclmique was used as a proteomics tool for this purpose. The reliability of this technique was analysed via three different domains of life, including S. cerevisiae (eukaryotes), S. solfatarictls (archaea) and SynecllOcystis sp. (bacteria). Based on multiple replicate analyses, an increment of peptidcs (proteins) was found, as well 'as a tcchnical replicate to confinn the reliability of this method. The main functions of the protcomic analyses in this thesis were to discover three crucial objectives: firstly, to Investigate the cellular response of S. cerevisiae to osmotic stress generated by VHG conditions, as well as to immobilization of cells; secondly, to examine the response of this micro-organism to VHG conditions with an amino acids supplementation; and finally, to investigate the main triggers for the fluctuation of fennentation parameters observed during continuous fennentation process.

No description available.

No description available.

Genomic libraries of S. lavendulae and S. subrutilus were constructed in S. lividans using the technique of shotgun cloning. S. lividans is a genetically well characterised recipient for heterologous DNA, but plasmid deletions or the entry of plasmids with small inserts occurred during transformation of the DNA into S. lividans This was not due to a straightforward restriction-modif ication system as this possibility was checkea using the KC301 phage. Several gene libraries were produced, using high and low copy number plasmids, and the resultant transformants screened. The nature of deoxynojirimycin (DNJ) and its lack of microbial activity prevented use of a bioassay. However, the inhibition of cc-glucosidase by DNJ was exploited by development of a quantitative assay system f or DNJ and nojirimycin (NOJ). The assay could not only detect DNJ and NOJ-producing clones, but could also assess the titre of DNJ and NOJ in culture broths. The assay was used to demonstrate differential production of DNJ and NOJ by selected StreRtOIFYces strains of cluster 61 (the S-lavendulae species group). The assay also confirmed the effectiveness of using microtitre plates as an effective screening procedure. The microtitre screening programme generated further data and statistical treatment of the results delimited the number of isolates for further examination. No DNJ-producing colony was detected and examination of the size of the DNA inserts showed almost all were too small to contain the DNJ gene cluster. Additionally, blocked DNJ production mutants were characterised by the feeding of NOJ, one of the mutants successfully converted NOJ to DNJ.

Hydrogen is seen as a potential fuel for the future; its choice is driven by the increasing awareness of the necessity for clean fuel. Together with the simultaneous development of “green technologies” and sustainable development, a current goal is to convert waste to energy or to create energy from a renewable resource. Biological processing [of renewables] or bioremediation of waste to create hydrogen as a product fulfils this goal and, as such, is widely researched. In this work, an already established process, using a hydrogenase up‐regulated strain ‐ was characterised and the important process parameters were established. This bacterial strain has the potential for industrial‐scale hydrogen production from, for example, waste sugars. Previous work, repeated here, showed that hydrogen could be generated by E. coli HD701 using a two‐phase process (growth in shake flasks, followed by hydrogen production within a bioreactor). Ideally a commercial process would need to be in a single vessel (bioreactor), which therefore resulted in this investigation of the scale‐up of twophase fermentations to 5 L stirred tank bioreactors. Within the initial two‐phase process, shake flask growth in 2 L shake flasks (employing a 50% working volume) achieved a dry cell weight of 1.33 +- 0.1 mg mL‐1 which then, when transferred to a 5 L bioreactor (containing 2 L of culture and 2 L of hydrogen production substrate), achieved a maximum hydrogen production rate of (200 mL h‐1) 150 mL g(dcw)‐1 h‐1. The first step in scale‐up was to simply transfer the process to a bioreactor and see the effect it had on hydrogen production. This approach did not yield any hydrogen and therefore consequent experimentation sought to see if the hydrogen production was growth phase dependant. However all phases of growth evolved no hydrogen upon the addition of substrate. The next approach was to take the conclusion drawn from a literature survey that showed a need for microaerobiosis or anaerobiosis during growth (for mixed acid fermentation to occur) along with a high formate concentration necessary for the transcription of the FHL complex (the hydrogen gas evolving enzyme). For this reason the KLa from the initial shake flask growth (calculated from literature correlations) was applied to the bioreactor. Experiments used to simulate the shake flask mass transfer coefficient (kLa) in a bioreactor did not generate hydrogen; the physical system within the shake flask used for growth in the initial process allows for this to occur, but the consequent process change to a bioreactor did not. This inability to produce hydrogen was concluded to be due to the lack of microaerobiosis/anaerobiosis required for mixed acid fermentation (the metabolic precursor to hydrogen production). The criterion of KLa was inappropriate for scale up in this case due to the physical differences between the shake flask and the bioreactor, as the oxygen transfer within the shake flask is not limited to transfer between the liquid and gas phase (the effect of transfer across the shake flask closure must be considered). This fact led to the novel use of gas blending for dissolved oxygen tension control. Gas blending was used in a bioreactor to track the changes observed during growth in the shake flask. This created a process that mirrored the shake flask in both growth and hydrogen production. The outcome was a dry cell weight of 1.34 +- 0.02 mg mL‐1 and a maximum hydrogen production rate of 200 mL h‐1 i.e. 150 mL g(dcw)‐1 h‐1, exhibiting almost identical process results to the two‐stage process. This characterisation reinforced the necessity for microaerobiosis during growth to allow subsequent post‐growth hydrogen production. Microaerobiosis in the latter stages of growth allows mixed acid fermentation to occur, which was found to be essential for hydrogen production. Process intensification took place by increasing cell density. This was achieved by increasing the medium concentration, then by changing the medium (two differing fed batch media were chosen; each medium used was experimentally linked with multiple feeds) and finally by utilising the novel technique of combining gas blending with fed batch cultivation to ensure microaerobiosis during growth. This, along with the use of a low (\(\mu\)=0.05 h‐1) growth rate for feed calculation, led to an eight‐fold increase in cell density. The low growth rate was employed to reduce inhibitory acetate formation while the multiple feeds were used to investigate nitrate depletion. The maximum increase in cell density led to a hydrogen evolution rate of 1800 mL h‐1, thus producing hydrogen that could be converted into energy at a rate eleven‐fold greater than the rate at which it consumed energy for agitation.

This work discusses optimisation of the cryopreservation of Bacillus licheniformis cell banks, used as inoculum for α-amylase producing 5 L batch fermentations. The effect of the presence of various cryopreservants including glycerol, Tween 80 and dimethyl sulphoxide on final fermentation performance measured by biomass and α-amylase concentration was investigated using optical density, dry cell weight, colony forming units, and multi-parameter flow cytometry. The application of multi-parameter flow cytometry using the fluorophores DiBac\(_4\)(3) and PI allowed real time viability measurements of individual microbial cells to be monitored before and after cryopreservation and during the fermentation process; viability here being defined as a cell having an intact and fully polarised cytoplasmic membrane. It was found that the concentration and type of cryopreservant used had a significant effect on microbial cell physiology and population heterogeneity during resuscitation recovery immediately after thawing. Cell banks prepared with Tween 80 were fastest to recover after freezing in comparison to cell banks prepared with dimethyl sulphoxide which showed the slowest growth rates. Interestingly cells preserved in glycerol recovered at a similar rate to cells frozen without cryopreservant. Despite different responses to the freezing process when each cell bank was used as inoculum for 5 L batch fermentations very little difference was noticed in overall process performance with respect to α-amylase production, growth rate and final biomass concentration.

The increasing cost of fossil fuels, combined with concerns about their impact on our environment has led to a renewed interest in hydrogen as a clean, sustainable, alternative energy vector. Using sewage biosolids as the substrate for fermentative hydrogen production offers several advantages over the use of other biomass sources. It is available at little or no cost and is abundant, being produced wherever there are human settlements, with 1.3 million tonnes (dry solids) per year currently being produced in the U.K alone. This research demonstrated the feasibility of hydrogen production from sewage biosolids via anaerobic fermentation. To do this a number of issues specifically relating to the nature of sewage biosolids had to be addressed. Firstly, the solids content and rheology made automatic feeding difficult. The feedstock also contained high levels of indigenous microorganisms and a high ratio of insoluble to soluble carbohydrate. To address these challenges, a novel reactor design using wide bore tubing and computer controlled pumping equipment was successfully used to construct a working continuously fed bio-reactor. A combination of heat treatment at 70°C for one hour and pre-treatment with a commercially available food processing enzyme mixture was found to be the most efficient method of inactivating competing microorganisms and improving substrate quality. Hydrogen was successfully produced via batch fermentation of primary sewage biosolids which had undergone heat treatment and enzymatic digestion. When fermentation took place at pH 5.5 a peak hydrogen production rate of 3.75 cm3 min"1 was observed. At this pH the hydrogen yield was 0.37 mol H2 mol~ : carbohydrate, equivalent to 18.14 L H2 kg"1 dry solids. Fermentative hydrogen production from sewage biosolids was also demonstrated in a five litre, continuously fed bio-reactor for the first time. A comparison of different hydraulic retention times showed that hydrogen production was most stable at a HRT of 24 hours. A hydrogen producing fermenter was successfully linked to a methanogenic bio-reactor in a two stage digestion process.

Two staining protocols were formulated that enabled the detection of cellular stress at the single-cell level for Bacillus cereus. Both DiOC6(3) and RedoxSensor Green™ can be employed to detect perturbations in the energetic status of the cell at concentrations of 0.30 \mug.mL-1 and 3.0 \muM respectively. These methods can be employed for sensitive analysis of bacteria of both industrial and clinical interest. Flow cytometry was used throughout this work in order to assess the quality of recombinant Escherichia coli populations present within an agitated bioreactor. It was demonstrated in shake-flask culture that the cells could be grown to moderate cell densities (OD600nm 7 25) whilst producing measurable levels of antibody fragment. Despite being described in a patent which claims invention of a 100 % effective repression system (Hodgson et al., 2006), there was extensive evidence of promoter leakiness. Fab production was usually synonymous with cellular breakdown, however, a strategy based on simultaneous feeding and induction, before the exhaustion of the primary carbon source, yielded the highest concentration of Fab, 105 mg.L-1, with more than 50 mg.L-1 successfully targeted to the extracellular environment. Unlike all the previous cultures, this attainment also preceded the breakdown in the cellular structure.

The purpose of this research was to contribute to the development of H\(_2\) production technologies from biomass. The study integrated thermochemical processes to achieve biomass hydrolysis with biological methods to then obtain H2 by the fermentation of these hydrolysates using E. coli. Different strains of E. coli were tested under controlled conditions in 3 L scale fermentations with the aim to find the most useful strain for the fermentation process in terms of H\(_2\) produced and the subsequent hydrogen production potential of the organic acid co-products in a downstream photofermentation. Among the strains tested FTD89, FTD67 and RL009 gave the best results, however ethanol was successfully abolished by strain RL009 making this strain more suitable for long term fermentations. Model polysaccharide compounds such as starch and cellulose, and representative food and lignocellulosic wastes were hydrolysed in hot compressed water in the presence of CO\(_2\) under pressure and various temperatures to produce hydrolysates with high sugar content suitable for fermentation for H\(_2\) production. Optimum hydrolysis conditions for maximum sugar yields for each compound were determined. Fermentation of the obtained hydrolysates yielded acceptable amounts of H\(_2\) after their ‘detoxification’ with activated carbon (AC), comparable to H\(_2\) yielded by the glucose controls in all cases. The maximum yield of glucose after HCW treatment was obtained from starch at 200 °C yielding 548 g C.kg C initial starch\(^{-1}\); maximum glucose yield from cellulose was 225 g C.Kg C initial cellulose\(^{-1}\) obtained from cellulose hydrolysis at 250 °C, and the glucose yield from food waste was 45.5 g.g food waste\(^{-1}\). The main degradation product (DP) from these hydrolysates was 5 Hydroxymethylfurfural (5-HMF), whereas the main DP obtained from the lignocellulosic wastes was Furfural. Both were successfully removed by AC treatment. The best hydrolysate obtained from wastes was evaluated for H\(_2\) production at 3 L scale. Despite obtaining low H\(_2\) yields improvements would be possible and are discussed. Fermentations for H\(_2\) production at pilot plant scale were also trialled, indicating key areas for future development for successful scale up.