Engineering Synthetic Microbial Communities for Biomethane Production

Complex microbial communities underlie natural processes such as global chemical cycles and digestion in higher animals, and are routinely exploited for industrial scale synthesis, waste treatment and fermentation. Our basic understanding of the structures, stabilities and functions of such communities is limited, leading to the declaration of their study as the next frontier in microbial ecology, microbiology, and synthetic biology. Focusing on biomethane producing microbial communities (BMCs), we will undertake a two-tiered approach of optimising natural communities and designing synthetic communities with a focus on achieving robust, high-yield biomethane production. Within this biotechnological framework, our proposal will address several fundamental scientific questions on the link between the structure and function of microbial communities.
To ensure success in this challenging project, we assembled the strongest possible interdisciplinary research team that combines significant practical and scientific expertise in microbial ecology and evolution, systems modelling, molecular microbiology, bioengineering, genomics, and synthetic biology.
We are confident that this team will deliver and that this project will result in significant impact in the scientific and industrial domains. Through our work, described in detail below, we will; significantly improve the current understanding of the structure-function relation in microbial communities, provide the scientific community with a systematic, temporal genomics and transcriptomics dataset on complex microbial communities, develop novel computational tools for microbial community (re)design, and experimentally build synthetic BMCs that will act as model ecosystems in different research fields. These scientific developments, in turn, will accumulate in the development of more sustainable bioenergy solutions for the UK economy by optimising the communities underlying biomethane production. This will help to drive the efficiency of biomethane as an alternative fuel source.

We will employ both top-down (directed evolution) and bottom-up (synthetic biology) engineering of biomethane producing microbial communities (BMCs) with improved functionality. These two approaches are connected via the resulting BMCs, which will be further analysed in mid-scale reactors with the aim to impact biotechnological application of microbial communities.

Directed evolution of BMCs. We will combine our expertise in experimental evolution with applied expertise in biomethane production to use group selection on naturally derived BMCs to improve their biomethane productivity. Using the expertise and the infrastructure at TGAC, we will employ next generation sequencing to determine how communities change in response to selection, and whether significant evolutionary change has occurred in the transcriptomes of focal species. Our core experimental evolution setup will use 60 mini reactors to set up independent batch cultures, where biomethane production can be measured in real-time by automated monitoring of gas volume.

Rational engineering of synthetic BMCs. We will combine our expertise in kinetic modelling and flux balance analysis (FBA) with molecular biology to rationally design and experimentally implement synthetic BMCs. The starting point for both FBA and experimental work will be an existing co-culture that is capable of converting lactate into methane. The engineered communities and their temporal behaviour will be analysed using genomics and transcriptomics approaches.

Testing and scaling up of (re)engineered BMCs. We will test the performance and stability of evolved and synthetic BMCs under industrially realistic conditions in mid-scale reactors using our expertise and lab infrastructure in process engineering. For this task, we will use both anaerobic membrane reactors (AnMBRs), which allow for the maintenance of BMCs in the reactor without "washout" and more commonly used continuously stirred tank

In line with national and international policy, this research aims to produce a step change in the efficient production of biomethane, a key renewable energy source. This, in turn, will impact on government and industrial end users, who have clearly articulated their requirements for improvements in yield and reliability of biomethane production. At the scientific level, the relations between structure, composition and function in microbial communities is at the heart of several unresolved questions in the fields of microbial ecology and evolution, microbiology and synthetic biology.

1. Academic Communities
Impact on Existing Communities. This research will benefit systems microbiologists by generating a more complete understanding of the interactions found in complex microbial communities and synthetic biologists by developing improved tools and approaches for the manipulation of microbial communities. These tools will be applicable to biomethane production but will also be of interest for the production of biofuels or bio-products by accommodating bacteria into a stable productive community. In addition, our proposed research will provide the scientific community with an unprecedented data set on the composition and structure of complex microbial communities and provide novel computational tools for their study.

Educational Impact. Today's scientific challenges require bringing together scientists from diverse fields and educating younger scientists in a genuinely cross-disciplinary fashion. Being a truly integrative project that amalgamates theory and experiment towards achieving a better understanding of complex microbial communities, the proposed research will provide an ideal setting for the development of the participating staff and PhD students, and will excite a new generation of scientists.

2. Industrial Communities
The innovative nature of this project and the economic and regulatory drivers related to biomethane production have already created strong interest from industry. We have engaged end users in the development of this proposal, primarily through an industrial liaison workshop held in Exeter in December 2011. Most attendees of this workshop, as well as several other industrial companies are now members of our advisory board (AB); major users of biomethane production, SME technology development companies, and a regional industry network. There is strong interest in the potential for future commercial exploitation of the proposed basic research, and we will actively seek to pursue opportunities for commercial industrial collaborations during and post-project.

3. Policy and Society
Impact on Policy. The close link between government priorities on renewable energy and greenhouse gas emission, and biomethane production through anaerobic digestion is explicitly recognised in the DECC Strategy and Action Plan, 2011. The Government has set targets to recycle 50% of household waste by 2020, reduce greenhouse gas emissions to 34% below 1990 levels by 2020 and by 80% by 2050, and achieve greater energy security. The related regulations and innovation stimulation packages developed by the Government, heavily influences the anaerobic digestion bioindustry. Recognising this link, we have already sought advice on engagement with DECC, and following this advice, we will provide them with research briefing papers as results are made available.

Social Impact. The proposed research is extremely timely and of significant social relevance since it addresses an important aspect of a "daily" challenge, namely eco-friendly and sustainable energy production. We will capitalise on this and use the project as a way to engage with the public and funding bodies and offer collaborative opportunities to think in innovative and informed ways about systems biology, synthetic biology and microbial biotechnology.