Utilisation of novel marine yeast and seawater-based media for the production of bioethanol

Bioethanol can be considered as one of the best replacements for petrol because of its positive impact on environment and many other advantages. Currently, bioethanol accounts for around 2% of the global road fuels and this is projected to increase to around 10% within the next few decades. Bioethanol is a very high water consuming product, with an average global water footprint of 2855 L H2O/L ethanol. A growing worldwide demand for bioethanol has raised concerns over the use of freshwater resources. This PhD project aimed to establish a marine fermentation strategy for bioethanol production where seawater replaced freshwater for the preparation of fermentation media in conjunction with use of marine yeast as a biological catalyst, and potentially utilising a marine biomass (i.e. seaweed) as a carbon source substrate. Yeasts that are present in marine environments have evolved to survive hostile environments. Therefore, yeast isolated from marine environments could have potentially interesting characteristics for industrial applications. Current methods for marine yeast isolation suffer several limitations as they usually encourage the growth of filamentous fungi and produce low number of yeast isolates. A new method was developed in this study, which included: a 3-cycle enrichment step followed by an isolation step and a confirmation step. By applying this method on 14 marine samples (collected in the UK, Egypt and the USA), a large number of marine yeast isolates was obtained without any bacterial or filamentous fungal contamination. Amongst these isolates, 116 marine yeast isolates were evaluated for their capacity for utilising monomeric fermentable sugars (glucose, xylose, mannitol and galactose) using a seawaterbased media, this assessment of sugar utilisation was performed in a phenotypic microarray assay. Following determination of sugar utilisation, 21 isolates that representing the best sugar utilisers were further characterised using YT-plates (BioLog) and identified by DNA sequencing using ITS and D1/D2 primers. The identified isolates belonged to 8 species: Saccharomyces cerevisiae (5 strains), Candida tropicalis (4 strains), Candida viswanathii (4 strains), Wickerhamomyces anomalus (3 strains), Candida glabrata (2 strain), Pichia kudriavzevii (1 strain), Issatchenkia orientalis (1 strain) and Candida albicans (1 strain). Out of the 21 identified yeasts, 9 strains representing different species were screened for ethanol production using YPD media containing 6% (w/v) glucose and prepared by freshwater (ROW) and seawater (SW). Results revealed that 3 marine S. cerevisiae strains (S65, S71, and S118) had the best fermentation rates when using SW media. These yeasts were therefore taken forwarded for investigation into their growth performance under high concentrations of glucose and seawater salts (the components of synthetic seawater). Results determined that these marine strains were significantly more tolerant when compared with a reference terrestrial S. cerevisiae strain. Fermentation experiments using YPD media containing 6% glucose were prepared using synthetic seawater (SSW), 2x SSW and different sodium chloride (NaCl) concentrations (3, 6 and 9%) and results confirmed that the marine strain S65 was a highly halotolerant and osmotolerant yeast with high fermentative capacity. In a batch fermentation using 15 L bioreactors, strain S65 produced 73 g/L ethanol from 165 g/L of glucose within 20 h of fermentation, with ethanol productivity of approximately 4 g/L/h. In a batch fermentation, using sugarcane molasses (about 14% sugar) prepared in SW, strain S65 produced 52.23 g/L of ethanol after 48 h. According to literature, determination of sugars in samples which contain chloride salts was inaccurate when applying an existing HPLC method because chloride ions and sugars (especially glucose and sucrose) elute at a similar retention time. In this study seawater - which contains high concentration of NaCl (about 2.8%) - was used for preparing the fermentation media and therefore, developing a new method for sugar determination was necessary. Subsequently, an accurate and reliable HPLC method for the simultaneous quantification of chloride salts, sugars, organic acids and alcohols was developed. The method was validated for the accurate quantification of NaCl and successfully applied on fermentation samples as well as variety of food samples from retail market. The results obtained in this study highlighted the potential for using marine yeasts and the suitability of seawater-based media for the production of bioethanol. They also provide a new strategy for increasing the efficiency of bioethanol production at the industrial level with positive impact on food and freshwater scarcity issues.