Influence of cohabiting bacteria on carbohydrate accumulations in Chlorella vulgaris CCAP 211/21A

Microalgae are considered to be an ideal developmental platform for capture of CO2 from the atmosphere to produce biomass with potential to be of value as feedstock for conversion to biofuels and bioproducts with low negative impact on the environment. Carbohydrate is an energy storage component in algae cells which can be converted to several bioproducts, including biofuels. Therefore, increasing the content and productivity of carbohydrates from microalgae becomes a significantly impactful component to consider in developing algae as a sustainable feedstock. Co-culture is one approach that has been used to increase lipids and carbohydrates. The primary hypothesis we wished to study is if we can use cohabiting bacteria associated with algae as a biotic stressor to increase carbohydrate accumulation in microalgae cells, and the specific questions we addressed are: (1) whether the concentration of bacteria introduced and the specific time point of introduction of bacteria has a role to play in increasing carbohydrate accumulations, and (2) is there a difference in influence between single and multiple bacterial types? In this project, Chlorella vulgaris CCAP 211/21A, a halotolerant microalga that has been shown to accumulate high carbohydrates was investigated. Cultivation of non-axenic C. vulgaris under nutrient replete and deplete conditions was studied aiming to increase carbohydrate accumulation. Nutrient limitations resulted in a carbohydrate yield of 47% DCW, which is 74% higher than that found in replete medium. Three cohabiting bacterial species were isolated from all tested conditions. These were identified by 16S rRNA sequence to belong to Halomonas 2sp. and Muricauda sp. Distribution of the bacterial population was influenced by nutrient depletion/repletion in algae cultures, Halomonas sp. WSR2 was the dominant isolate under all tested conditions. All three isolates were studied in isolation to characterise the optimal conditions for bacterial growth. Different media that contain different nutrient concentrations were tested; f/2+R2A was found to be a suitable medium to grow all isolates. Moreover, the bacterial isolates were cultivated in a range of pH and temperatures. It was found that both Halomonas sp. grew optimally at pH 7.5 and 30°C whilst the optimal conditions for growth of Muricauda sp. was at pH 8.5 and a temperature of 25°C. The dominant isolate, Halomonas sp. WSR2, was cultivated with axenic C. vulgaris in co-cultures in different ratios and different inoculation time during algal cultivation. Two inoculum concentrations (1 CFU/ml and 104 CFU/ml) of Halomonas sp. WSR2 were introduced separately into the algae culture during the start of cultivation. This resulted in doubling of algal maximum specific growth rate and a 99% increase in fold change of carbohydrate yield, for a bacterial concentration of 104 CFU/ml, compared to control axenic cultures. Introducing the same concentration of bacteria on day 2 of algae cultivation (beginning of stationary phase) resulted in a 82% increase in fold change of carbohydrate yield. However, 175% increase in maximum carbohydrate productivity could be achieved with the addition of 1 CFU/ml. In addition, mixed bacterial species (Halomonas sp. WSR2: Halomonas sp. WS1: Muricauda sp. WSR) have also been cultivated with algae in two different ratios at the start of cultivation that both showed a doubling of growth rate of the algae. We conclude that introducing single type of bacteria on the lag phase (day 0) or at the beginning of stationary phase (day 2) in high concentration (104 CFU/ml) resulted in high carbohydrate yield whilst adding small concentration (1 CFU/ml) on day 2 achieved high carbohydrate productivity compared to control. These findings could increase chances for using co-culture of C. vulgaris with Halomonas sp. on a large scale for biofuels and bioproducts production