Direct Conversion of Wet Microalgae and Oleaginous Yeast to Biodiesel Using Ionic Liquids

This thesis examined the development of an oleaginous yeast or microalgae based biorefinery process. First, improvements were made to the Nile Red assay, a high-throughput method for monitoring lipid accumulation in oleaginous organisms resulting in a significantly more reproducible and accurate assay. This assay was then used to optimize lipid production during heterotrophic cultivation of microalgae on glucose using response surface methodology resulting in microalgae with high lipid content (37.6% wt.). In order to improve the renewability of heterotrophic lipid production, both oleaginous yeast and microalgae were cultivated using pyrolytic sugars, produced via fast pyrolysis of pinewood waste. The effects of inhibitors on glucose consumption and lipid accumulation as well as the quality of the produced fatty acid methyl esters (FAME) were examined. Upon the establishment of cultivation processes for these two organisms, the overall objective of this work shifted towards the development of a fractionation process for producing and recovering multiple end products. Over 20 ionic liquids were screened for the ability to disruption microalgal cell structure. This was the first report of non-imidazolium ionic liquids assessed for algal bioprocessing applications and the first examining room temperature ionic liquids. The leading candidate ionic liquid was further studied for its ability to disrupt fresh microalgal cultures which were dewatered containing up to 82% water content. This was the first in depth report of the effect of process parameters on the use of ionic liquds for algae disruption. The resulting lipid extraction process was minimized to a simple 1.5 h process conducted at ambient temperature with wet algae. This was further extended to include a catalyst in order to directly convert intracellular lipid to biodiesel from whole yeast biomass. The effects of the reagent ratios, reaction temperatures, and reaction time were studied in depth using response surface methodology. Recovery of the ionic liquid and catalyst for reuse was quantified. Finally, the carbohydrates and protein fractions were recovered after the ionic liquid lipid extraction process using microalgae and it was demonstrated that the resulting sugars primarily in the form of starch could be directly fermented to biobutanol, bioethanol, and acetone using a traditional ABE fermentation process

ABE fermentation from microalgae-derived carbohydrates after lipid extraction

Although lignocellulosic biomass is an abundant substrate, major challenges in their pretreatment and digestion remain a serious limitation for large scale second generation biofuel production. First generation feedstocks which are primarily starchy substrates like corn have much more favourable economics as they are simple to mash. However, concerns related to the use of water and land for the production of biofuel crops have generated a need for alternative biomass sources for renewable biofuel production from a non-food crop. One biomass which satisfies all of these concerns is microalgae. Many strains are capable of growing in waste water, can accumulate carbon intracellularly as starch, are not eaten as food in any significant amount, and also have the added bonus of fixing CO2 during photosynthetic growth. However, production of microalgae biomass has its own particular challenges such as the high cost of dewatering and drying microalgal biomass. In order to demonstrate a possible biofuel production strategy using microalgal biomass, Chlorella vulgaris was cultivated at the pilot scale (100 L) and harvested using centrifugation. Lipids were extracted for biodiesel production using either a water compatible ionic liquid based process or using traditional (non water compatible) solvent based process. The residual biomass containing proteins and carbohydrates was recovered from both processes and designated either ionic liquid extracted algae (ILEA) or hexane extracted algae (HEA). To convert these micro-algal carbohydrates into solvents (ABE), HEA and ILEA was either acid hydrolysed into glucose before fermentation or directly fermented as it is. The highest butanol titers (8.05 g/L) was obtained with the fermentation of acid hydrolysates of HEA, which however required detoxification to support solvent production while ILEA did not. Interestingly, both ILEA and HEA can be fermented directly without any additional steps and resulted in a butanol titer of 4.99 and 6.63 g/L, respectively, which significantly simplified the LEA to butanol process. Further study has shown that butanol titers close to the toxicity limits are possible with higher substrate loadings, however a a fed-batch approach is required in order to mitigate increased culture viscosity issues during direct fermentations. These results indicate that lipid extracted microalgae are a readily consumed substrate for biofuel production. Please click Additional Files below to see the full abstract

Simultaneous clarification and purification of recombinant penicillin G acylase using tangential flow filtration anion-exchange membrane chromatography

Downstream purification often represents the most cost-intensive step in the manufacturing of recombinant proteins. Conventional purification processes are lengthy, technically complicated, product specific and time-consuming. To address this issue, herein we develop a one step purification system that due to the nature of the non-selective secretion system and the versatility of ion-exchange membrane chromatography can be widely applied to the production of many recombinant proteins. This was achieved through the integration of the intrinsically coupled upstream, midstream and downstream processes, a connection that is rarely exploited. A bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography medium while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange medium, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level of 487 U/L/OD600, with more than 90% was localized in the extracellular medium. Both, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. Furthermore, formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. iv At harvest, the whole non-clarified culture broth was applied directly to a tangential flow filtration anion-exchange membrane chromatography system. One-step purification of recombinant PAC was achieved based on the dual nature of membrane chromatography (i.e. microfiltration-sized pores and anion-exchange chemistry). Due to their size, cells remained in the retentate while the extracellular medium penetrated the membrane. Most contaminate proteins were captured by the anion-exchange membrane, whereas the purified PAC was collected in the filtrate. The batch time for both cultivation and purification was less than 24 h and recombinant PAC with high purity (19 U/mg), process yield (74%), and productivity (41 mg/L) was obtained

Tissue‐resident macrophages actively suppress IL‐1beta release via a reactive prostanoid/IL‐10 pathway

The alarm cytokine interleukin‐1β (IL‐1β) is a potent activator of the inflammatory cascade following pathogen recognition. IL‐1β production typically requires two signals: first, priming by recognition of pathogen‐associated molecular patterns leads to the production of immature pro‐IL‐1β; subsequently, inflammasome activation by a secondary signal allows cleavage and maturation of IL‐1β from its pro‐form. However, despite the important role of IL‐1β in controlling local and systemic inflammation, its overall regulation is still not fully understood. Here we demonstrate that peritoneal tissue‐resident macrophages use an active inhibitory pathway, to suppress IL‐1β processing, which can otherwise occur in the absence of a second signal. Programming by the transcription factor Gata6 controls the expression of prostacyclin synthase, which is required for prostacyclin production after lipopolysaccharide stimulation and optimal induction of IL‐10. In the absence of secondary signal, IL‐10 potently inhibits IL‐1β processing, providing a previously unrecognized control of IL‐1β in tissue‐resident macrophages

Immune adjuvant effect of V. cholerae O1 derived Proteoliposome coadministered by intranasal route with Vi polysaccharide from Salmonella Typhi

Proteoliposome derived from Vibrio cholerae O1 (PLc) is a nanoscaled structure obtained by detergent extraction process. Intranasal (i.n) administration of PLc was immunogenic at mucosal and systemic level vs. V. cholerae; however the adjuvant potential of this structure for non-cholera antigens has not been proven yet. The aim of this work was to evaluate the effect of coadministering PLc with the Vi polysaccharide antigen (Poli Vi) of S. Typhi by i.n route. The results showed that Poli Vi coadministered with PLc (PLc+Poli Vi) induce higher IgA response in saliva (p0.05) to that induced in a group of mice immunised by parenteral route with the Cuban anti-typhoid vaccine vax-TyVi®, although this vaccine did not induce mucosal response. In conclusion, this work demonstrates that PLc can be used as mucosal adjuvant to potentiate the immune response against a polysaccharide antigen like Poli Vi

Combining energy efficiency measure approaches and occupancy patterns in building modelling in the UK residential context

The UK faces a significant retrofit challenge, especially with its housing stock of old, hard-to-treat solid walled dwellings. In this work, we investigate the delivery of heated thermal comfort with a lower energy demand through four types of energy efficiency interventions: passive system, conversion device, method of service control, and level of service demanded. These are compared for three distinct household occupancy patterns, corresponding to a working family, a working couple and a daytime-present couple. Energy efficiency measures are considered singly and in combination, to study whether multiple lower cost measures can achieve comparable savings to higher cost individual measures. Scenarios are simulated using engineering building modelling software TRNSYS with data taken from literature. Upgraded insulation of wall and roof resulted in highest savings in all occupancy scenarios, but comparable savings were calculated for reduced internal temperature and partial spatial heating in scenarios in which the house is not at maximum capacity. Zonal heating control is expected to achieve greatest savings for the working couple who had a flexible occupancy pattern. The results from this modelling work show the extent to which energy consumption depends on the appropriate matching between energy efficiency measures and occupant type

Evidence that breast cancer risk at the 2q35 locus is mediated through IGFBP5 regulation.

GWAS have identified a breast cancer susceptibility locus on 2q35. Here we report the fine mapping of this locus using data from 101,943 subjects from 50 case-control studies. We genotype 276 SNPs using the 'iCOGS' genotyping array and impute genotypes for a further 1,284 using 1000 Genomes Project data. All but two, strongly correlated SNPs (rs4442975 G/T and rs6721996 G/A) are excluded as candidate causal variants at odds against >100:1. The best functional candidate, rs4442975, is associated with oestrogen receptor positive (ER+) disease with an odds ratio (OR) in Europeans of 0.85 (95% confidence interval=0.84-0.87; P=1.7 × 10(-43)) per t-allele. This SNP flanks a transcriptional enhancer that physically interacts with the promoter of IGFBP5 (encoding insulin-like growth factor-binding protein 5) and displays allele-specific gene expression, FOXA1 binding and chromatin looping. Evidence suggests that the g-allele confers increased breast cancer susceptibility through relative downregulation of IGFBP5, a gene with known roles in breast cell biology

Genome-wide association analysis of more than 120,000 individuals identifies 15 new susceptibility loci for breast cancer.

Genome-wide association studies (GWAS) and large-scale replication studies have identified common variants in 79 loci associated with breast cancer, explaining ∼14% of the familial risk of the disease. To identify new susceptibility loci, we performed a meta-analysis of 11 GWAS, comprising 15,748 breast cancer cases and 18,084 controls together with 46,785 cases and 42,892 controls from 41 studies genotyped on a 211,155-marker custom array (iCOGS). Analyses were restricted to women of European ancestry. We generated genotypes for more than 11 million SNPs by imputation using the 1000 Genomes Project reference panel, and we identified 15 new loci associated with breast cancer at P < 5 × 10(-8). Combining association analysis with ChIP-seq chromatin binding data in mammary cell lines and ChIA-PET chromatin interaction data from ENCODE, we identified likely target genes in two regions: SETBP1 at 18q12.3 and RNF115 and PDZK1 at 1q21.1. One association appears to be driven by an amino acid substitution encoded in EXO1.BCAC is funded by Cancer Research UK (C1287/A10118, C1287/A12014) and by the European Community's Seventh Framework Programme under grant agreement 223175 (HEALTH-F2-2009-223175) (COGS). Meetings of the BCAC have been funded by the European Union COST programme (BM0606). Genotyping on the iCOGS array was funded by the European Union (HEALTH-F2-2009-223175), Cancer Research UK (C1287/A10710, C8197/A16565), the Canadian Institutes of Health Research (CIHR) for the CIHR Team in Familial Risks of Breast Cancer program and the Ministry of Economic Development, Innovation and Export Trade of Quebec, grant PSR-SIIRI-701. Combination of the GWAS data was supported in part by the US National Institutes of Health (NIH) Cancer Post-Cancer GWAS initiative, grant 1 U19 CA148065-01 (DRIVE, part of the GAME-ON initiative). For a full description of funding and acknowledgments, see the Supplementary Note.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ng.324

Germline HOXB13 mutations p.G84E and p.R217C do not confer an increased breast cancer risk

In breast cancer, high levels of homeobox protein Hox-B13 (HOXB13) have been associated with disease progression of ER-positive breast cancer patients and resistance to tamoxifen treatment. Since HOXB13 p.G84E is a prostate cancer risk allele, we evaluated the association between HOXB13 germline mutations and breast cancer risk in a previous study consisting of 3,270 familial non-BRCA1/2 breast cancer cases and 2,327 controls from the Netherlands. Although both recurrent HOXB13 mutations p.G84E and p.R217C were not associated with breast cancer risk, the risk estimation for p.R217C was not very precise. To provide more conclusive evidence regarding the role of HOXB13 in breast cancer susceptibility, we here evaluated the association between HOXB13 mutations and increased breast cancer risk within 81 studies of the international Breast Cancer Association Consortium containing 68,521 invasive breast cancer patients and 54,865 controls. Both HOXB13 p.G84E and p.R217C did not associate with the development of breast cancer in European women, neither in the overall analysis (OR = 1.035, 95% CI = 0.859-1.246, P = 0.718 and OR = 0.798, 95% CI = 0.482-1.322, P = 0.381 respectively), nor in specific high-risk subgroups or breast cancer subtypes. Thus, although involved in breast cancer progression, HOXB13 is not a material breast cancer susceptibility gene.Peer reviewe

Fine-Scale Mapping of the 4q24 Locus Identifies Two Independent Loci Associated with Breast Cancer Risk

Background: A recent association study identified a common variant (rs9790517) at 4q24 to be associated with breast cancer risk. Independent association signals and potential functional variants in this locus have not been explored. Methods: We conducted a fine-mapping analysis in 55,540 breast cancer cases and 51,168 controls from the Breast Cancer Association Consortium. Results: Conditional analyses identified two independent association signals among women of European ancestry, represented by rs9790517 [conditional P = 2.51 × 10−4; OR, 1.04; 95% confidence interval (CI), 1.02–1.07] and rs77928427 (P = 1.86 × 10−4; OR, 1.04; 95% CI, 1.02–1.07). Functional annotation using data from the Encyclopedia of DNA Elements (ENCODE) project revealed two putative functional variants, rs62331150 and rs73838678 in linkage disequilibrium (LD) with rs9790517 (r2 ≥ 0.90) residing in the active promoter or enhancer, respectively, of the nearest gene, TET2. Both variants are located in DNase I hypersensitivity and transcription factor–binding sites. Using data from both The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), we showed that rs62331150 was associated with level of expression of TET2 in breast normal and tumor tissue. Conclusion: Our study identified two independent association signals at 4q24 in relation to breast cancer risk and suggested that observed association in this locus may be mediated through the regulation of TET2. Impact: Fine-mapping study with large sample size warranted for identification of independent loci for breast cancer risk