Susan T.L. Harrison

LIPID PRODUCTIVITY AS A KEY CHARACTERISTIC FOR CHOOSING ALGAL SPECIES FOR BIODIESEL PRODUCTION

Microalgae are a promising alternative source of lipid for biodiesel production. One of the most important decisions is the choice of species to use. High lipid productivity is a key desirable characteristic of a species for biodiesel production. This paper reviews information available in the literature on microalgal growth rates, lipid content and lipid productivities for 55 species of microalgae, including 17 Chlorophyta, 11 Bacillariophyta and five Cyanobacteria as well as other taxa. The data available in the literature are far from complete and rigorous comparison across experiments carried out under different conditions is not possible. However, the collated information provides a framework for decision-making and a starting point for further investigation of species selection. Shortcomings in the current dataset are highlighted. The importance of lipid productivity as a selection parameter over lipid content and growth rate individually is demonstrated.

BIOLOGICAL OXIDATION OF FERROUS SULPHATE BY THIOBACILLUS FERROOXIDANS : A REVIEW ON THE KINETIC ASPECTS

Abstract Biological oxidation of ferrous sulphate by Thiobacillus ferrooxidans has proved to be a significant step in the bioleaching of sulphide minerals and treatment of acid mine drainage. The same bioreaction also has beneficial applications in the desulphurization of coal and removal of hydrogen sulphide from gaseous effluents. Owing to the numerous potential industrial applications, the process of biocatalytic oxidation of ferrous iron has been studied extensively over the years. In the present article different aspects of this biological reaction from both a microbiological and engineering point of view are discussed and an overview of the current knowledge with respect to T. ferrooxidans and the process it catalyses is provided.

Bacterial cell disruption: A key unit operation in the recovery of intracellular products

The need for microbial cell disruption has hindered the large scale production of commercial biotechnological products of intracellular derivation. The intracellular nature of many recombinant products and the potential use of the bacterial storage product, PHB as a commodity thermoplastic have renewed interest in the improvement of this unit operation. This paper provides a review of processes of a mechanical, physical, chemical or biological nature used for cell disruption on both the laboratory and large scale. Applicability of the techniques to large scale operation is discussed. Modification of existing processes is suggested for the reduction of energy requirements and improved process economics. The requirements for the liberation of granular intracellular products such as inclusion bodies and virus-like yeast particles are distinguished from those for the liberation of soluble products, mainly proteinaceous in nature. The integrated nature of the process with both upstream and downstream processes is addressed. Finally, the recent approach of selective liberation of soluble products of interest is reviewed.

Selection of Direct Transesterification as the Preferred Method for Assay of Fatty Acid Content of Microalgae

Assays for total lipid content in microalgae are usually based on the Folch or the Bligh and Dyer methods of solvent extraction followed by quantification either gravimetrically or by chromatography. Direct transesterification (DT) is a method of converting saponifiable lipids in situ directly to fatty acid methyl esters which can be quantified by gas chromatography (GC). This eliminates the extraction step and results in a rapid, one-step procedure applicable to small samples. This study compared the effectiveness of DT in quantifying the total fatty acid content in three species of microalgae to extraction using the Folch, the Bligh and Dyer and the Smedes and Askland methods, followed by transesterification and GC. The use of two catalysts in sequence, as well as the effect of reaction water content on the efficiency of DT were investigated. The Folch method was the most effective of the extraction methods tested, but comparison with DT illustrated that all extraction methods were incomplete. Higher levels of fatty acid in the cells were obtained with DT in comparison with the extraction-transesterification methods. A combination of acidic and basic transesterification catalysts was more effective than each individually when the sample contained water. The two-catalyst reaction was insensitive to water up to 10% of total reaction volume. DT proved a convenient and more accurate method than the extraction techniques for quantifying total fatty acid content in microalgae.

Interference by pigment in the estimation of microalgal biomass concentration by optical density

Optical density is used as a convenient indirect measurement of biomass concentration in microbial cell suspensions. Absorbance of light by a suspension can be related directly to cell density using a suitable standard curve. However, inaccuracies can be introduced when the pigment content of the cells changes. Under the culture conditions used, pigment content of the microalga Chlorella vulgaris varied between 0.5 and 5.5% of dry weight with age and culture conditions. This led to significant errors in biomass quantification over the course of a growth cycle, due to the change in absorbance. Using a standard curve generated at a single time point in the growth cycle to calculate dry weight (dw) from optical density led to average relative errors across the growth cycle, relative to actual dw, of between 9 and 18% at 680 nm and 5 and 13% at 750 nm. When a standard curve generated under low pigment conditions was used to estimate biomass under normal pigment conditions, average relative errors in biomass estimation relative to actual dw across the growth cycle were 52% at 680 nm and 25% at 750 nm. Similar results were found with Scenedesmus, Spirulina and Nannochloropsis. Suggested strategies to minimise error include selection of a wavelength that minimises absorbance by the pigment, e.g. 750 nm where chlorophyll is the dominant pigment, and generation of a standard curve towards the middle, or across the entire, growth cycle.

A kinetic study on anaerobic reduction of sulphate, Part I: Effect of sulphate concentration

The kinetics of anaerobic reduction of sulphate was studied in continuous bioreactors. The effects of initial sulphate concentration and its volumetric loading on the kinetics of reaction and activity of sulphate-reducing bacteria were investigated. The increase in initial concentration of sulphate in the range 1.0– enhanced the reaction rate from 0.007–. For a given initial sulphate concentration increasing the volumetric loading rate of sulphate led to a linear increase in volumetric reduction rate. The initial concentration of sulphate did not have a significant effect on maximum specific growth rate (μm), decay coefficient (kd) on bacterial yields (Yx/sulphate and Yx/acetate), with the values of these coefficients being bacteria/g sulphate and bacteria/g acetate, respectively. The saturation constant (Ks) was an increasing linear function of initial sulphate concentration, with the lowest and highest values being 0.027 and , respectively. Using the experimental data a kinetic model, incorporating terms for the effects of initial and residual concentrations of sulphate and biomass, was developed.

Advances in product release strategies and impact on bioprocess design

Intracellular products such as recombinant insulin, which are typically produced in microbial host cells, demand a product release step to remove them from the cell. How this is performed determines the quantity of released contaminants, the particle size distribution of cell debris and the physical properties of the resultant process stream, which all impact on the performance of the downstream operations. Thus, achieving selective release of the desired product is crucial for improving the process economics. Advances in upstream processing (the bioreactor phase) have been successful in achieving high product titres, and downstream costs now typically dominate the overall manufacturing costs. Here, we review and discuss the selective release of products as a possible means of improving the efficiency of downstream processing.

Particle size effects in bioleaching of pyrite by acidophilic thermophile Sulfolobus metallicus (BC).

Abstract The effect of mineral particle size on the bioleaching of pyrite by the acidophilic thermophile Sulfolobus metallicus was investigated in a batch bioreactor. Decreasing the particle size from a mean diameter of 202 micron (size fraction: 150–180 micron) to a mean diameter of 42.5 micron (size fraction: 25–45 micron) enhanced the bioleaching rate from 0.05 kg m−3 h−1 to 0.098 kg m−3 h−1. The particle size distribution of the mineral in this range did not influence the morphology and growth kinetics of the cells. The values of specific growth rate (μ) and yield factor (Y) were 0.018–0.025 h−1 and 0.67 × 1011–1.45 × 1011 cells (g iron)−1, respectively. Decreasing the particle size of the mineral to a mean diameter of 6.40 micron (size fraction <25 micron) adversely influenced the activity of the cells. The presence of fine particles apparently damaged the structure of the cells, resulting in their inability to oxidise pyrite.

Study of Physical and Biological Factors Involved in the Disruption of E. coli by Hydrodynamic Cavitation

Hydrodynamic cavitation results in flow restriction in a flow system causing rapid pressure fluctuations and significant fluid forces. These can be harnessed to mediate microbial cell damage. Hydrodynamic cavitation was studied for the partial disruption of E. coli and selective release of specific proteins relative to the total soluble protein. The effects of the cavitation number, the number of passes, and the specific growth rate of E. coli on the release of periplasmic and cytoplasmic proteins were studied. At the optimum cavitation number of 0.17 for this experimental configuration, 48% of the total soluble protein, 88% of acid phosphatase, and 67% of β‐galactosidase were released by hydrodynamic cavitation in comparison with the maximum release attained using multiple passes through the French Press. The higher release of the acid phosphatase over the total soluble protein suggested preferred release of periplasmic compounds. This was supported by SDS‐PAGE analysis. The absence of micronization of cell material resulting in the potential for ease of solid‐liquid separation downstream of the cell disruption operation was confirmed by TEM microscopy. E. coli cells cultivated at a higher specific growth rate (0.36 h−1) were more easily disrupted than slower grown cells (0.11 h−1). The specific activity of the enzyme of interest released by hydrodynamic cavitation, defined as the units of enzyme in solution per milligram of total soluble protein, was greater than that obtained on release by the French Press, high‐pressure homogenization, osmotic shock, and EDTA treatment. The selectivity offered indicates the potential of enzyme release by hydrodynamic cavitation to ease the purification in the subsequent downstream processing.

Plasticization of poly(hydroxybutyrate) in vivo

The influence of a variety of treatments on the mobility and crystallinity of poly(hydroxybutyrate) (PHB) in whole cells and native granules has been proved using 13C-n.m.r. spectroscopy and X-ray powder diffraction, and correlated with the known biological effects of these treatments. It was concluded that at least water is responsible for PHB plasticization in vivo, and that only native mobile PHB is susceptible to depolymerases. Another, probably hydrophobic, component appears to be involved either as plasticizer or nucleation inhibitor. Three states of the granule are identified in addition to the native, biologically-competent state: freeze-drying of whole cells leads to a partially-immobilized amorphous state which can be restored virtually to native mobility by rehydration; extended centrifugation of native granules in aqueous suspension, or treatment with hydrophobic detergents under certain conditions, leads to a crystalline state that is less susceptible to exogenous depolymerase; and heating to 95 degrees C or refrigeration has no detectable effect on mobility but leads to inactivation of the granule, presumably via damage to superficial membrane or protein.