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- Doctoral Thesis (6)
This study investigated the potential of fungal biomass as industrial catalyst through developing an alternative catalyst for biodiesel production using fungal biomasses for whole-cell catalysis. The research explored various fungal isolates and identified the most potent isolates. The transesterification catalytic capability of the selected isolates was improved through random genetic mutagenesis and optimization of the cultivation conditions of the chosen mutants.
Aspergillus flavus and Rhizopus stolonifer two fungal species belong to two different phyla (Ascomycota and Zygomycota) revealed the capability of their biomasses to catalyze the transesterification reaction efficiently up to 95.5 %, in relatively short reaction time (24 h.). The biomass produced was also capable of catalyzing the transesterification reaction using different acyl-acceptors. A. flavus and R. stolonifer biomasses proved the probability of using biomasses cocktails to catalyze transesterification reaction. In addition, the fungal biomass investigated exhibited considerable ability to catalyze the hydrolysis of triglycerides, which means an opportunity of producing multifunctional catalytic fungal biomass. A. flavus showed a potential to be implemented in biorefinery processes that include wastewater treatment by microalgae as part of a circular economy approach, where A. flavus biomass produced in a biorefinery approach was effectively capable of catalyzing the modification of lipids.
No doubt that the whole world facing energy crisis problems, therefore, three different biofuel generations have been developed to overcome fossil fuel depletion. As first generation biofuels have been shown to be unsustainable and insufficient to meet the increasing energy demands, due to the competition for arable lands and second generation biofuels have potential negative effects on carbon balances and biodiversity, Algal-based “third generation” biofuels, typically based on microalgae can overcome the drawbacks of the two others biofuel generations. To make the biofuel (biodiesel and biogas) production process from microalgae sustainable, it is essential that further cost reductions are applied to all steps and to make use of all the process waste and convert it to energy positive outcomes. Three scientific papers have been developed in the framework of my PhD thesis contributing process sustainability.
In the first paper (published) “Isolation and Screening of Non-toxic Thermo-tolerant Microalgae Strains Cultivated in Different Growth Media for Further Outdoor Applications”, we focused on robust strains selection which is the major bottleneck towards the process sustainability. Fresh water samples from Nile River / Egypt were investigated to select the most robust strains which have high growth rate and high lipid content at 40°C. Afterword the promising strains were cultivated in different growth media for further outdoor cultivation. Seven unicellular strains were able to grow fast with high lipid accumulation at 40°C; two from them can grow in synthetic human urine (SHU) as a synthetic wastewater. Lipid quantification has been done using Nile red stain method. Toxicity experiment carried out using Artemia salina shrimps to avoid using any toxic strains in outdoor cultivation. In addition, the promising strains were identified at molecular level by 18S and 16S rDNA sequencing.
The incredible increase in world population, which could reach nine billion by 2050, and the rapid progress of globalization in recent decades have put pressure on the food and energy sectors. The resources currently available for energy production are insufficient to meet future demand. These facts are pushing governments and scientific organizations all over the world to search for alternative renewable energy sources.
Microalgae present an ideal, resurgent resource for the production of biofuels, especially biodiesel and biogas, because their lipid productivity is greater than that of other terrestrial food crops. However, from a biotechnological point of view, the use of microalgae requires further investigation and development to be economically viable, particularly in regard to cost and biomass production.
The most important step in the use of microalgae for biofuel production is strain selection. The optimal strain must be able to withstand outdoor conditions and survive seasonality. Four related manuscripts were prepared during my Ph.D. project. Two of them have been published, one has been submitted for publication, and the fourth is ready for submission. Together, they focus on new strategies for strain selection, lipid production increase, lipid vesicles detection and imaging, total cost reduction strategies, biodiesel production from promising strains, and biogas production from the remaining microalgal residues.
From a practical perspective, only a few microalgal species have been investigated for pharmaceutical and industrial applications. Throughout my Ph.D. project, I have identified microalgal strains able to grow at high temperature and under light stress, as a step toward the development of sustainable microalgal fuels. The four manuscripts that have resulted from my project are described below.
In the first manuscript, entitled “Isolation and characterization of thermo-tolerant Egyptian marine microalgae as proposed candidates for biodiesel prod
Caulobacter crescentus is well-known for its unique dimorphic life style. It is used as a model organism to study cell division and differentiation. A range of interesting features constitute its unique nature. It is normally found in dilute organic environments and was believed to be lacking any genes coding for porin like proteins. We found channel forming activity in the enriched cell wall extracts of the organism. The protein responsible for the porin like activity was found to be a member of the OmpW family. The protein formed small cation selective channels in artificial lipid bilayers. In order to confirm that the studied protein is responsible for the channel forming activity, an ompW knockout strain of C. crescentus was developed. Enriched outer membrane extracts from the mutant strain did not show channel forming activity.
We also identified and characterized a homologue of hVDAC-1 in Legionella pneumophila. L. pneumophila has genes coding for a range of eukaryotic like protein. We were especially interested in the gene lpg1974 which codes for Lpg1974, a protein which had reasonable similarity to hVDAC-1. The protein was found to produce large anion selective channels in artificial lipid bilayers. We also developed a homology structure for the protein which had remarkable similarity to hVDAC-1. Here we further studied the properties of the protein, by expressing the protein without its predicted N-terminal signal peptide (Lpg1974Δ1-21). There are a series of diverse reports about the importance of the N-terminal sequence. The truncated protein formed large anion selective channels. The voltage sensitivity of the protein was not affected by the signal peptide deletion.
In my thesis two main projects of my PhD is explained the in chapters 2 and 3 following a pertinent introduction for understanding the projects better in Chapter 1.
In chapter 2, the effect of charge repulsion on homo repeat hexapeptides will be explained. The peptides investigated are homo repeats of either acidic (Glu,Asp) or a basic (Arg, His and Lys) amino acid and labeled at two ends for measuring their end-to-end distance and flexibility by methods explained in great detail in chapters 1 and 2. As a function of pH, negative or positive charges appear on the peptide side chains. The repulsion occurs mainly between the N-terminus and the side chains in basic peptides. Astonishingly, no effective negative charge repulsion among the acidic side chains or the C-terminus with the side chains was observed. This can be explained by the difference in binding affinities of water towards carboxylates versus positively charged groups, which results in screening of the negative charges. Charge repulsion between negative charges was recovered when I changed the solvent from water to a 92.5% methanol/water.
In the second project –that was explained in Chapter 3− an in vivo host‒dye displacement was carried out: In this project V79 and CHO cells were loaded with a host−dye complex called p-sulfonatocalix[4]arene (CX4) / lucigenin (LCG). The fluorescence of LCG is quenched inside the CX4. The cationic analytes acetylcholine, choline and protamine were sent into the cells. The dye displaced with analyte and made a fluorescence turn-on signal. The invented method can be used for assaying the analyte in the cells. My results also show that CX4 facilitate the membrane passage of LCG. Therefore, CX4 can be considered as a carrier.
The catalytic domain of the Dnmt3a DNA-(cytosine C5)-methyltransferase (Dnmt3a-C) is active in isolated form but like full-length Dnmt3a it shows only weak DNA methylation activity. To improve this activity by directed evolution, we set up a selection system in which Dnmt3a-C methylate its own expression plasmid in E. coli and protect it from cleavage with methylation specific restriction enzymes. However, despite screening about 400 clones which were selected in 3 rounds from a library of 60000 clones, we were not able to isolate a variant with improved activity, most likely because of a background of uncleaved plasmids and plasmids which have lost the restriction site. We also showed that the catalytic domain of mouse Dnmt3a DNA methyltransferase is able to transfer the methyl group from S-adenosyl-L-methionine (AdoMet) to a cysteine residue in its catalytic centre. This reaction is reversible and slow. The yield of auto-methylation is increased by addition of Dnmt3L, which functions as a stimulator of Dnmt3a AdoMet complexes. In the presence of CpG containing double stranded DNA, the transfer of the methyl group from AdoMet to the flipped target base was preferred and auto-methylation was not detected. This reaction might constitute a regulatory mechanism which could inactivate unused Dnmt3a in the cell.
