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

Lead Research Organisation: Plymouth Marine Laboratory
Department Name: Plymouth Marine Lab


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.

Technical Summary

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.

Planned Impact

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.
Description There is increasing interest in the use of microalgae grown on wastewater to provide useful metabolites. Microalgae are often found in close association with bacteria. However the relationship between microalgae and bacteria is not always clear. This project has enabled us to better understand this close and necessary relationship. We have been able to characterize the populations of bacteria associated with the cultivation of the green microalgae, Chlorella. We have been able to determine which groups of bacteria have positive effects on the growth of Chlorella and those which have a negative effect. We have also been able to determine how nutrients in the media effects the composition, and in particular the lipid composition within the Chlorella. Two key aspects are highlighted below:

1. Bacteria enhance the growth of Chlorella vulgaris in low nutrient media: Several bacteria have been shown to affect the growth rate and quality of the algae, but it is not clear if this is specific to a particular group of bacteria or if nutrient conditions can also influence this interaction. The bacterial community associated with a freshwater Chlorella sp. isolated from open pond textile factory wastewater was characterised and a diverse group of bacteria isolated. We provide evidence that nutrient concentrations affect bacterial community composition. When grown in BG11 medium, the community was dominated by Pseudomonas sp., but when grown in the lower nutrient Chu 10 medium, the relative abundance of a Brevundimonas spp. increased. Several of the bacteria isolated were able to influence the growth of an axenic Chlorella vulgaris culture. The Pseudomonas sp. had a negative effect in all media tested whereas several isolates enhanced C. vulgaris growth, but only in Chu 10 medium. This supports the theory that bacterial stimulation of algal growth is not limited to species-specific interactions but is influenced by environmental conditions. In low nutrient conditions, Chlorella sp. may be increasingly dependent on bacteria for growth.

2: Lipid profiles in Chlorella are modulated in response to nutrient limitation: using modern polar lipid species profiling (via LC-MS/MS) we found a significant diversity of polar lipids species across all polar lipid classes in Chlorella sp. cultures, and that the polar lipid productivity, class and species composition can be modulated in response to nutrient availability.
Exploitation Route The research has led to further collaboration with the waste industry where there is an increasing necessity to reduce and reuse waste.
Sectors Agriculture, Food and Drink,Chemicals,Energy,Environment,Manufacturing, including Industrial Biotechology

Description A patent has been filed by the Bharathidisan University (India): Techno-Economic Drying of Microalgae by Frame Ooze Out Technique (FOOT). Application No. 201641042909
First Year Of Impact 2016
Sector Agriculture, Food and Drink
Impact Types Societal,Economic

Description Interrreg NWE
Amount € 5,550,000 (EUR)
Funding ID NWE520 
Organisation European Commission 
Department Interreg : European Territorial Co-operation
Sector Public
Country European Union (EU)
Start 08/2017 
End 03/2021
Description AD 
Organisation Langage-AD (UK) LLP
Country United Kingdom 
Sector Private 
PI Contribution presentation of project and findings of results
Collaborator Contribution Visit and discussion with India partners.
Impact Collaborative EU-Interreg proposal that has been successful at Step 1. Step 2 to be submitted June 2017.
Start Year 2016
Description Collaboration with India 
Organisation Bharathidasan University
Country India 
Sector Academic/University 
PI Contribution This BBSRC project is part of the SUBB intiative so we are collaborating with India both Bharathidisan University and PhycoSpectrum.
Collaborator Contribution We are having meetings to discuss results and progress.
Impact None yet.
Start Year 2013
Description Welsh Water 
Organisation Welsh Water
Country United Kingdom 
Sector Private 
PI Contribution As a result of me approaching Welsh Water to describe our work on using algae to remediate waste water to take up excess nutrients a small collaborate project (funded by Welsh Water to Swansea University and PhycoSpectrum) has been set up between India and the UK
Collaborator Contribution Phycospectrum will build and commission a small algal pilot facility at a waste water site in South wales to trial capture of waste nutrients.
Impact Plans have been drawn up to build a small pilot facility. Multidisciplinary in that engineers and biologists work together.
Start Year 2016
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact organised by BBSRC with the Objective: To provide a platform to grant holders under BBSRC-DBT Sustainable Bionenergy and Biofuels Managed programme (SuBB) and industry experts for discussion and potential collaboration for mutual benefit.

Shared knowledge on the different SuBB projects. Increased collaboration with Indian partners.
Year(s) Of Engagement Activity 2014
Description Using waste to produce value workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact A workshop, funded through the NRN-LCEE Research Development Fund, was held overlooking the Principality Stadium in Cardiff on 11th October to discuss new ways that algae could help strengthen the Anaerobic Digestion (AD) industry as part of a developing circular economy.

A team of 30 expert stakeholders including policy makers, industry practitioners and academic researchers contributed to the workshop combining expertise on agricultural nutrient management, anaerobic digestion of food and farm waste and cultivation and use of algae. Algal technologies could bring sought after solutions to help close nutrient cycling loops, addressing nutrient bottlenecks and adding value to enable further expansion of a thriving AD industry. Tim Pullen AGL Energy Ltd commented 'It is good to know that there is genuine innovation occurring in the AD industry'.

The workshop led by Swansea University (Carole Llewellyn, Associate Professor of Applied Aquatic Bioscience), Aberystwyth University (Dr Will Stiles, KE Fellow) and Bangor University (Dr Dave Styles, Lecturer in Life Cycle Assessment) demonstrated the effectiveness of bringing different communities together to create new innovative solutions to food and farm waste processing and deriving value products such as fish feed and biochemicals. Lynsey Melville, Professor of Environmental Engineering at Birmingham City University, commented 'The range of expertise in the room was invaluable and led to a really productive and interesting forum'. Many new useful contacts were made through the day and the network will continue to work together.
Year(s) Of Engagement Activity 2017
Description local industry engagement 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact meetings with three local companies to highlight our work and discuss potential collaborations
Jackie Young from UrbanAgenda SW
Southwest Water in Exeter
Tamar/Gingsters Foods
Langage Farm AD

There were clear pathways for engagement. However, as PI has relocated to Swansea University it is more likely that collaborations will be developed with wastewater companies local to Swansea.
Year(s) Of Engagement Activity 2014