Microbial ecology for engineering biology

Engineering biology has become synonymous with synthetic biology where genes are snapped together, with Lego-like ease, to go beyond the natural metabolic diversity of microorganisms and deliver novel biotechnologies. The molecular microbiology toolkits of synthetic biology are awe-inspiring. However, for the biotechnologies to be effective and financially viable then the artificial organisms or consortia must persist and continue to function as intended. Any modification of the genetic code will affect an organism's fitness. Thus, it is desirable to know the fate of organisms a priori when exposed to the vagaries of real-world engineered or natural environments. In most engineering disciplines, ease of construction is a secondary facet of a design. First and foremost, the engineer must anticipate failure and use mathematical models to build a product that is robust and resilient. This principal needs to be adopted for all engineered biological systems, synthetic or otherwise, if engineering biology is to realise its full potential and to sit alongside more established engineering disciplines in delivering solutions in a timely and cost-effective manner. This paper demonstrates the vulnerability of synthetic organisms and the potential for even the most parsimonious of population biology models to yield insights on designing resilient systems. 

A natural question to ask in applied microbial ecology is to what extent our understanding of the ecology of large, multicellular organisms can be transferred to microorganisms. To answer it, it might help to lean on the emerging consensus that there is an explanatory separation between ecological processes involving just two or a few species on one hand, and processes involving large numbers of species and large spatial and temporal scales on the other. The latter, it appears, can be understood without a detailed understanding of the former. The underlying great unifier is a phenomenon called ecological structural instability. It denotes a high sensitivity of local ecological communities to press perturbations or species invasions that arises as species richness reaches a certain critical value from below. This high sensitivity easily leads to species extinctions, which is why in nature species richness tends to saturate shortly below the critical value. This value and other macrocological phenomena, which Dr Rossberg will discuss, are controlled essentially only by the mean, variance, and correlation of the distribution of interaction strengths between species. Details don’t matter. In what forms this leads to parallelism between microbial and traditional macroecology is an exciting question we should address. 

In environmental biotechnology, microbial transformations of organic molecules are often carried out by a microbial community as opposed to one single type of microbe. Within these communities, microorganisms are forced to interact with each other. In the most general case, microbes compete for space, a resource needed by all community members. More specific interactions may involve competition for shared substrates or a mutual requirement for each other's metabolic products. Understanding the network of interactions between the members of an existing community may enable us to introduce and maintain in an augmented community a thermodynamically possible but currently unrealised ecosystem function. This reasoning was successfully used to engineer a granular, phototrophic and methane-consuming community, able to convert dissolved methane into biomass without externally supplied oxygen. Despite competition for oxygen with traditional heterotrophic respiration, the initially introduced methanotrophic activity was maintained over prolonged times in a continuously operated reactor system. Microbial community analysis revealed that a more complex foodweb established than anticipated. Methane oxidation likely involved subsequent steps of partial oxidations by different bacteria, and possibly even intergranule dependencies. Theoretical considerations are a necessary starting point but need to be sufficiently backed up by experiments to account for unexpected behaviour.

Microbial communities in open microbial systems such as activated sludge are subject to a continual influx of microbes in the influent stream. The microbes entering the system have a range of fates – they may die within the system, leaving behind organic matter, they may pass through the system with little impact, or they may exhibit a positive growth rate and become a permanent member of the community. At a high enough rate, this continual immigration of organisms can have a disproportionate impact on the structure of a local community, which, in ecological terms, is referred to as the mass effects archetype of community assembly. In this talk, Dr Fowler will discuss the prevalence of mass effects within biological treatment systems and present estimates for migration rates within different systems. Dr Fowler will then consider the potential consequences of this archetype on the structure, function and stability of microbial communities.

Microbial communities (‘microbiomes') have been employed to benefit society for thousands of years. However, the vast majority of the microbial world’s transformative capabilities have yet to be unlocked and harnessed for engineering applications. A key reason for this is the lack of tools available to quantitatively probe and experimentally discover the structure and in situ activity (ie fluxes) of biochemical networks operating in poorly characterised and uncultivated microorganisms. While understanding and control of microbiome metabolic flux is the ultimate goal, Dr Lawson and his team have started with unraveling metabolism and fluxes in anaerobic microbes and microbial communities driving anaerobic ammonium oxidisation (anammox) and anaerobic fermentation. Together, these microbiomes are responsible for controlling nitrogen and carbon cycling on a global-scale and for performing sustainable wastewater treatment and resource recovery through complex metabolic interactions. Dr Lawson will show how isotopic tracers combined with quantitative metabolomic analysis were used to probe and illuminate the biochemical pathways operating in anammox bacteria. He will also discuss how the integration of metagenomics, 13C-metaproteomics, and metabolic modeling were used to determine metabolic interactions between anammox, nitrifying, and denitrifying bacteria in complex microbiomes. These analyses have resulted in several key discoveries on the enigmatic metabolism of anammox bacteria and their metabolic interactions with other poorly characterised nitrogen cycle bacteria. He will conclude with discussion on preliminary efforts to assemble and engineer synthetic consortia of anaerobic fungi and bacteria to recover valuable products from renewable biomass, while unmasking basic principles for microbiome engineering.

People with cystic fibrosis (CF) suffer from persistent, poly-microbial infections in the lung. This CF lung microbiome is a dynamical, evolving ecosystem that displays classic features of a complex system. It shows temporal changes in composition, is spatially stratified in the alveolar microenvironment and microbial interactions generate an ecological dependency structure in the community. The emergent disease dynamics are characterised by abrupt inflammatory aggravation (pulmonary exacerbations) that cause irreversible lung damage and drive patient mortality. The working hypothesis is that dysbiosis and metabolic niche reconstruction are relevant drivers of lung exacerbation. However, a strong patient-specificity of the microbiome composition as well as hyper-stable community dynamics currently limit advances in the field. Dr Widder works at the interface of complex systems, data science and microbial ecology. She will present an overview on her work about the CF lung microbiome and will discuss different theoretical angles employed for overcoming system-specific challenges. Her future vision is a rational, model-based design of interference strategies that exploit underlying ecological dependencies of the microbial community and enable controlled microbiome management. The treatment would aim to avoid microbial dysbiosis, reduce lung exacerbation and thereby prolong patient’s lives.

Functional understanding of microbial DNA is largely based on isolate genetics, where the effects of genetic manipulations on cultivable organisms are observed in isolation. Unfortunately, this provides limited insight into the workings of genes in the complex and societally relevant microbial communities that exist in nature. In order to move beyond the paradigm of manipulating microbes in confinement, Dr Rubin and collaborators have created a generalisable toolset for targeted genome editing of individual organisms within complex microbial communities. First, they have developed environmental transformation sequencing (ET-Seq) to determine in situ which microbes within a community can be edited by untargeted transposases, and with what efficiency. Second, they have repurposed RNA-guided CRISPR-Cas transposases to paste customised DNA into unique target sites within the genomes of specific microbes in a community. Third, they have applied these technologies to enrich, isolate, and track fitness of genetic mutants in soil and infant gut microbiota. The ability to make organism- and locus-specific changes within microbiomes will lead to improved understanding of microbial communities and enable meaningful modification of them.  

Process control of complex microbial communities grown in bioreactors should aim to create a niche defined by boundaries to strengthen and maintain an initially formed microbial community. Steady-state operated bioreactors are designed to reduce the effects of environmental factors on cultivated organisms, but have been shown to greatly increase the variability of community structures over long periods of time. Therefore, new ideas for reactor configurations that could lead to stabilization of community structures are needed.  Flow Cytometry as a rapid detection and assessment method was used to monitor assembly processes of microbial communities within bacterial generation times. Bioinformatics tools were used to interpret these data. Selected taxonomic sequencing of whole communities and sorted subcommunities supported the findings. The results suggest that synchronizing complex microbial communities in insular steady-state environments or keeping them in their original or desired structure can be difficult. In addition, the stability paradigm was studied in such insular environments and a fast workflow for monitoring and calculating the stability properties resistance, resilience, displacement speed, and elasticity was developed.

In this study, the researchers monitored the succession of viruses and prokaryotes in a microcosm experiment during different phases of benzene mineralisation under nitrate-reducing conditions in coarse sand. Nitrate-dependent benzene mineralisation was monitored by the addition of 13C-labelled benzene and subsequent analysis of generated ¹³CO₂. They collected solid (sand) and liquid samples at five-time points (three biological replicates per time point) for DNA extraction and shot-gun whole-genome sequencing. A total of 24 metagenomes (Illumina, 2x125 bp, 25 Mio reads) were analysed. Functional annotation of the 222 vOTUs identified 91 genes encoding 13 proteins relevant to benzene mineralisation coupled to nitrate reduction. Regarding carbon cycling, genes related to anaerobic degradation of benzoyl-CoA, and CO₂ fixation using the Wood-Ljungdahl pathway were found in our vOTUs. Similarly, genes related to nitrate reduction to nitrite, nitrite reduction to N₂, and nitrite reduction to ammonium indicate the viruses' potential contribution to the nitrogen cycle. Preliminary analysis using PHACTS suggested that 160 vOTUs (72.1%) have a lytic life cycle, indicating a potential role of viruses in carbon and nitrogen cycling. The researchers' data demonstrate the potential relevance of viruses in anaerobic benzene degradation and open new doors for the study of viruses in anaerobic ecosystems.