Using flow cytometry and genomics to characterise and optimise microalgal-bacterial consortia cultivated on Wastewater to produce biomass for Biofuel

Currently producing biofuel from microalgae is commercially prohibited. This is partly due to the high economic costs associated with the nitrogen and phosphorus nutrients required to sustain photosynthetic production. These inorganic nutrients can be found in abundance in industrial wastewater. There are also issues with the requirement of water for cultivating microalgae. Marine microalgae need to be situated near to the coast for utilisation of seawater and freshwater microalgae systems are dependent on large and continuous freshwater supplies, diverting supply away from arable farming etc. Therefore, producing biofuel from microalgae cultivated onwastewater has clear environmental and economic advantages.

In any large scale microalgal cultivation system, and particularly when using wastewater, a consortia will be present consisting of a single or several species of algae together with a complement of inherent bacteria; these associated bacteria have been shown to boost lipid production. There are many challenges associated with understanding such a complex and dynamic system. For example, interactions in the system will include metabolic changes occurring both within individual species and between the species. Ultimately the quantity and quality of the biomass suitable for biofuel will be related directly with the growth and the composition of the consortia which will dependent on interactions within it. Currently we have a poor understanding on the composition, development, function and interactions occurring with microalgae consortia.

This project will bring the three centres to develop new understanding on developing microalga-bacterial consortia cultivated on industrial water to produce biomass for biofuel. Bharathidasan University (BDU) has expertise on molecular techniques and cultivation of microalgae for bioenergy products. Phycospectrum Environmental Research Centre (PERC) has expertise on robust algal consortia and working with industry on wastewater treatment. Plymouth Marine Laboratory (PML) has expertise on the biology and chemistry to understand microbial community structure and on microbial dynamics. BDU and PERC will focus on optimising strains under industrially relevant conditions and results will be brought together with those from PML who will focus on understanding the microbial dynamics in controlled synthetic wastewater experiments.

The project will undertake community composition analysis to obtain understanding on how microalgal consortia change and function at both the cellular and molecular level. We will test the effect that both bacteria and addtional organic carbon have on influencing the growth and composition of the algal biomass as a biofuel feedstock. We will assess both the lipid and carbohydrate content of the algae for potential in biodiesel and bioethanol respectively. Put simplistically we will measure 'who is there? (community/taxonomic analysis), 'how do they compare? (comparative analysis)' and 'what are they doing? (functional analysis)' under the different conditions to optimise the amount and type of biomass suitable for conversion into biofuel.

To do this we will use the novel and powerful combination of flow cytometry tools to separate both algal and bacterial populations and genomic tools to characterise the communities. Whilst these tools have recently been applied to study marine microbial ecosystems, they have not been applied to any great extent to understanding wastewater microalgal-bacterial consortia. Knowledge gained will lead to potential to optimise consortia to improve growth rates and the amounts of lipids and/or carbohydrates. This could be achieved through controlling or adding bacteria, the addition of a waste carbon source, or through manipulation of metabolic pathways. The research will contribute to creating solutions to producing biofuel from microalgae grown on wastewater with consideration to both the environment and the economy.

Producing biofuel from microalgal consortia grown using wastewater has environmental and economic advantages. Currently we have a poor understanding on the composition, development, function and interactions occurring with microalgal consortia. The project brings three partners, Bharathidasan University(BDU, India), the Phycospectrum Environmental Research Centre (PERC) and Plymouth Marine Laboratory (PML,UK) together to develop new understanding on developing microalga-bacterial consortia to produce biomass for biofuel. BDU and PERC will focus on optimising strains and scale-up with industrial wastewaters and the UK partner will focus on understanding the microbial dynamics under controlled synthetic wastewater conditions

We will determine how the structure, diversity, abundance and function of taxonomic groups relate to microalgal growth and biofuel precursor production. A prevalent wastewater consortium consisting of Scenedesmus, Chlorella and Phormidium will be qualitatively and quantitatively examined over periods of semi-continuous culture in industrial and synthetic wastewaters (PML, BDU). Cellular and molecular characterisation will be achieved using flow cytometry techniques (PML) combined with genomic analysis of community populations (PML,BDU). Growth and photosynthetic efficiency of cultures will be measured together with dissolved organic carbon, particular carbon and nitrogen, nutrients, lipids and carbohydrates. Comparative experiments will be undertaken with and without bacteria, and with an additional carbon source. This will be followed by a more detailed metatranscriptome study of metabolic function (PML). Knowledge gained will have potential to manipulate consortia to improve growth rates and biofuel precursor production through control and or supplementation of bacteria, through the addition of a waste organic carbon source, or through manipulation of metabolic pathways.

The research has the potential to impact on a wide range of industries increasingly looking to reduce the cost of the environmental waste they produce and its environmental impact and produce additional products (biofuels) in a sustainable fashion. For example, in India PERC have already worked with several industries on algal-wastewater treatment projects including the alginate industry, leather processing chemicals industry, detergent industry, electroplating industry, confectionery industry, textile dyeing industries, oil drilling effluent treatment plant and a copper smelting industry and have signed recent MOUs with a cement and oil refining industries. Clearly, through considered engagement throughout the work programme,there is significant potential for similar UK industries to benefit from the expertise built up by PERC over the last 20 years. Moreover, if the biofuel products are subject to subsidies or indeed can be utilised by the industries themselves to reduce energy requirements then this may result in significant savings. Further, it is envisaged that the development of said technologies will create new opportunities for employment within the UK. For example, Aragreen are a company based in Gloucestershire that are inthe early stages of developing the use of algae from wastewater, with a current focus on developing products such as antioxidants, pigments and proteins for human and animal consumption. There is enormous scope to make a big impact in this area. There is also potential in the use of algae cultivated on sewage wastewater although that is not the focus of the current project.

From an academic impact perspective, genomic approaches and sequence information are beginning to be applied to understanding the composition and function of marine microbes and pipelines are being developed to interpret the large quantities of data generated from metagenome projects and in particular in linking metagenome with metatranscriptome information. We will apply such tools to enable new insight into understanding the structure and function of the algal consortia. The use of the genomic toolbox together with flow cytometric approaches enables a unique and novel understanding on the quantitative and qualitative nature of both the bacteria and algal consortia communities. This will impact more widely on the developing algal bioenergy community of researchers and also on researchers focussed on natural aquatic microbe dynamics in terms microbial food webs and the carbon cycle linked to climate change.

Highlights from the project will be promoted to the public through web and press releases and through engagement in school curriculum activities.