Subashchandrabose, Gobalakrishnan (2019) Generating and evaluating salinity and temperature resilient cyanobacteria for tropical outdoor cultivation in Australia. PhD thesis, James Cook University.

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Abstract

Global population levels, anticipated to increase to >9 billion by 2050, present serious worldwide challenges, such as energy-, food- and freshwater security. In addition, rising greenhouse gas (GHG) emissions lead to climatic instability, reduce the availability of freshwater and challenge agricultural productivity, which is exacerbated by decreasing arable land availability. Hence, the carbon- and freshwater-constrained economy demands industries to limit freshwater usage and carbon emissions. In this context, photosynthetic microalgae or cyanobacteria offer great promise for remediating carbon-dioxide emissions and high-nutient wastewaters, which can be coupled with renewable resource production to cater for large-volume low-value markets, such as animal feed, bio-fertiliser, and energy-production. The required scale of production for these markets, however, has to date not been realised, as outdoor cultivation presents severe challenges, including access to sufficient non-arable land in close proximity to water, nutrients (inorganic fertilisers: nitrogen and phosphate) and carbon-dioxide sources. In addition, high temperature and variable salinities are major limitations to cost-effective commercial microalgal production, as these factors are challenging to control.

Ectoine, a valuable osmolyte, is produced by extremophile microbes in response to variable salinities and high temperature stress. Ectoine synthesis is mediated by an ectABC gene cassette. Given this, my research aimed at engineering a de novo biosynthesis pathway for ectoine production into the freshwater cyanobacterium Synechococcus elongatus PCC 7942 - to examine: a) its effect on temperature and/or salinity tolerance and b) potential downstream effects of ectoine on fertilisation requirements and biochemical profiles of this cyanobacterium, as the latter affects bio-product potential. Synechococcus elongatus PCC7942 was chosen as a model cyanobacterium, as its genome is small and fully sequenced, commercial vectors for transformation are available and it is exempt from restriction of laboratory transformation experiments by the Office of Gene Technology Regulator (OGTR).

For ectABC transformation of S. elongatus PCC7942, a codon-optimised ectABC_pSyn_6 plasmid was constructed, based on the ectABC gene nucleotide sequence from the temperature- and salinity-tolerant bacterium Halomonas elongata DSM4043. ectABC-transformed, untransformed pSyn_6 vector (lacking ectABC insert) controls and wild-type (no vector, WT) S. elongatus PCC 7942 were subjected to a three temperature (35, 40, and 45°C), three salinity (0, 18, 36 ppt) factorial design experimental challenge without acclimation. Our data confirmed that ectABC-transformed S. elongatus PCC7942 had improved temperature tolerance up to 45°C and salinity tolerance up to 18 ppt at 35°C, compared to WT and pSyn_6 empty vector controls. Limited growth was observed at 36 ppt salinity in WT, pSyn-6 and ectABC transformants, irrespective of temperature. ectABC-transformant population growth rates were highest at 35°C. High pressure liquid chromatography analysis of these ectABC transformants confirmed ectoine production, albeit minimal. Further studies are necessary at the molecular level to resolve impediments associated with the low level of ectoine expression, should ectoine be chosen as a high-value co-product for the cosmetics industry.

In terms of commercial production, it is vital to assess ectABCtransformed S. elongatus PCC 7942 fertilisation requirements. Results showed that nitrogen-requirements of ectABC-transformants were higher than that of WT and pSyn_6 empty vector controls at an elevated salinity of 18 ppt, but lower at 45°C temperature stress. Phosphate uptake was lowest in ectABC-transformants at temperature and salinity stress of 45°C and 18 ppt, respectively. Fertilisation costs require serious consideration for commercial-scale cultivation of large-volume, low-value bio-products markets. Thus, the stress-induced increased nitrogen fertilisation requirements of ectABC-transformants suggest that co-location with nitrogen-rich wastewater streams would be beneficial, thereby also reducing nutrient run-off into the local river systems.

Regarding the biochemical profile of hydrocarbon-based biofuel production, ectABC transformants had increased lipid and fatty acid production under both temperature (45°C) and salinity (18 ppt) stress. Thus, this research addresses an area of importance for transitioning to a bio‐economy as a whole and for implementing environmentally and economically sustainable production of renewable biofuels, animal feed, bio-fertilisers, which are perhaps best achieved through co-production of some high-value bio-products, such as ectoine or the high-value pigment - c-phycocyanin.

To investigate this potential, a modelling approach using multi-criteria analysis and geographical information system analysis was adopted. ArcGIS was used to evaluate potential sites suitable for co‐locating microalgal and sugarcane production in the Great Barrier Reef (GBR) catchment region in Queensland, Australia – whilst taking into account climatic, land-use and economic factors that consider energy balances for each facility. Critical resource inputs such as land, water, CO₂, energy and climatic factors such as temperature and rainfall were considered when estimating the available resources at sugar mills in the Wet Tropics region, adjacent to the GBR. Our economic analysis revealed that co-locating microalgal biomass production with such an industry is economically feasible in the Wet Tropics, by achieving significant cost-reductions and improved economic performance. As such, this research produces valuable information for investors, policy makers, government and industry to make informed decisions about the location potential for microalgal production sites that focus on salinity and temperature-resilient microalgal cultivation for high-value compounds (e.g. the osmolyte ectoine) or low-value animal feed as their principal commodity, whilst reducing CO₂ emissions and nutrient runoff to the GBR, both of which attract tradeable credits which offer additional economic returns over and above the returns from the production and sales process.

Item ID:	63687
Item Type:	Thesis (PhD)
Keywords:	salinity resilient, temperature resilient, cyanobacteria, microalgae, freshwater, agriculture, sugarcane production, bioremediation, greenhouse gas emissions, Wet Tropics
Copyright Information:	Copyright © 2019 Gobalakrishnan Subashchandrabose.
Date Deposited:	03 Jul 2020 04:11
FoR Codes:	06 BIOLOGICAL SCIENCES > 0607 Plant Biology > 060701 Phycology (incl Marine Grasses) @ 50% 10 TECHNOLOGY > 1002 Environmental Biotechnology > 100203 Bioremediation @ 50%
SEO Codes:	86 MANUFACTURING > 8698 Environmentally Sustainable Manufacturing > 869802 Management of Greenhouse Gas Emissions from Manufacturing Activities @ 100%
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