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Achieving microbial compliance levels in biosolids storage is complicated by the unpredictable increase of Escherichia coli (E. coli), which serves as an important indicator for pathogen presence risk. Meeting required microbial specifications validates sludge treatment processes and ensures that a safe product is applied to agricultural land. Controlled indicator monitoring provides confidence for farmers, retailers and the food industry, safeguarding the sludge-to-land application route. Following mechanical dewatering biosolids products are stored before microbial compliance testing permits agricultural application. During storage, concentrations of E. coli bacteria can become elevated and prevent the product from meeting the conventional or enhanced levels of treatment outlined in The Safe Sludge Matrix guidelines. Literature research identified innate characteristics of sludge and ambient environmental parameters of storage which are factors likely to influence E. coli behaviour in stored biosolids. The research hypothesis tested whether E. coli growth and death in dewatered sewage sludge can be controlled by the modification of physical-chemical factors in the cake storage environment. Parameters including nutrient availability, temperature, moisture content and atmospheric influences were investigated through a series of laboratory-scale experiments. Controlled dewatering and the assessment of modified storage environments using traditional microbial plating and novel flow cytometry analysis have been performed. At an operational scale, pilot trials and up-scaled monitoring of the sludge storage environment have been conducted enabling verification of laboratory results. Understanding the dynamics of cell health within the sludge matrix in relation to nutrient availability has provided a valuable understanding of the mechanisms that may be affecting bacterial growth post-dewatering. The importance of elevated storage temperatures on E. coli death rates and results showing the benefits of a controlled atmosphere storage environment provide important considerations for utilities.

Microalgae are a diverse group of oxygenic photosynthetic microorganisms which show great promise as a source of biofuel. However, significant challenges still remain before microalgae can be considered a viable source of biofuel. The main current challenges are nutrient sourcing and recycling as well as downstream processing. The algal cell wall and especially the presence of an algaenan cell wall in some Chlorophyte algae could be an important variable in determining downstream processing costs but not much comparative research has been done to elucidate this. The first part of the present study focuses on the recently isolated alga Pseudochoricystis ellipsoidea (Trebouxiophyceae) and its improvement and assessment for biofuel production. Random mutagenesis and FACS screening protocols were developed for the isolation of pigment and cell wall mutants but despite considerable efforts no suitable mutants could be identified in the first half of this project. Two 500 L raceway ponds as well as an algal growth room and bubble column bioreactors were set up to facilitate algal research at the University of Bath and assess the performance of P. ellipsoidea in realistic culture conditions. P. ellipsoidea showed a maximum growth of 1.53 divisions day-1 in semi-open raceway ponds, resistance to contamination and a 30% lipid content, making it particularly suitable for raceway pond cultures. In the second part of this project six species of Chlorophyte (“green”) algae, three of which produced algaenan, were compared for suitability to growth in anaerobic digestate and municipal wastewater as well as cell wall strength, permeability and suitability to hydrothermal liquefaction. We found that anaerobic digestate was a good medium for the growth of all species independently of autoclaving and that non-autoclaved wastewater was a very challenging medium. Algaenan production did not affect cell disruption by ultrasonication but growth stage and cell wall thickness did. Lipid extraction kinetics by chloroform/methanol were greatly affected by algaenan, meaning that this material is relatively impermeable to organic solvents. Cell wall thickness, cell volume and lipid content also had an effect on lipid extraction kinetics but this was only measurable after 180 minutes of extraction. 8 Hydrothermal liquefaction showed high solid and low oil yields, very low sulphur (≤0.1 %) as well as a 1.1 % -1.8 % nitrogen content which is significantly lower than most algal HTL studies to date. This suggests that stationary stage algae are more difficult to process but give a cleaner biocrude and reduce the loss of nitrogen through incorporation in the oil. Significant opportunities for optimisation still exist in the HTL process.