Toward in silico CMC: An industrial collaborative approach to model-based process development.

The Third Modeling Workshop focusing on bioprocess modeling was held in Kenilworth, NJ in May 2019. A summary of these Workshop proceedings is captured in this manuscript. Modeling is an active area of research within the biotechnology community, and there is a critical need to assess the current state and opportunities for continued investment to realize the full potential of models, including resource and time savings. Beyond individual presentations and topics of novel interest, a substantial portion of the Workshop was devoted toward group discussions of current states and future directions in modeling fields. All scales of modeling, from biophysical models at the molecular level and up through large scale facility and plant modeling, were considered in these discussions and are summarized in the manuscript. Model life cycle management from model development to implementation and sustainment are also considered for different stages of clinical development and commercial production. The manuscript provides a comprehensive overview of bioprocess modeling while suggesting an ideal future state with standardized approaches aligned across the industry.

Biodiversity-function relationships in methanogenic communities.

Methanogenic communities play a crucial role in carbon cycling and biotechnology (anaerobic digestion), but our understanding of how their diversity, or composition in general, determines the rate of methane production is very limited. Studies to date have been correlational because of the difficulty in cultivating their constituent species in pure culture. Here, we investigate the causal link between methanogenesis and diversity in laboratory anaerobic digesters by experimentally manipulating the diversity of cultures by dilution and subsequent equilibration of biomass. This process necessarily leads to the loss of the rarer species from communities. We find a positive relationship between methane production and the number of taxa, with little evidence of functional saturation, suggesting that rare species play an important role in methane-producing communities. No correlations were found between the initial composition and methane production across natural communities, but a positive relationship between species richness and methane production emerged following ecological selection imposed by the laboratory conditions. Our data suggest methanogenic communities show little functional redundancy, and hence, any loss of diversity-both natural and resulting from changes in propagation conditions during anaerobic digestion-is likely to reduce methane production.

A Single Community Dominates Structure and Function of a Mixture of Multiple Methanogenic Communities.

The ecology of microbes frequently involves the mixing of entire communities (community coalescence), for example, flooding events, host excretion, and soil tillage [1, 2], yet the consequences of this process for community structure and function are poorly understood [3-7]. Recent theory suggests that a community, due to coevolution between constituent species, may act as a partially cohesive unit [8-11], resulting in one community dominating after community coalescence. This dominant community is predicted to be the one that uses resources most efficiently when grown in isolation [11]. We experimentally tested these predictions using methanogenic communities, for which efficient resource use, quantified by methane production, requires coevolved cross-feeding interactions between species [12]. After propagation in laboratory-scale anaerobic digesters, community composition (determined from 16S rRNA sequencing) and methane production of mixtures of communities closely resembled that of the single most productive community grown in isolation. Analysis of each community's contribution toward the final mixture suggests that certain combinations of taxa within a community might be co-selected as a result of coevolved interactions. As a corollary of these findings, we also show that methane production increased with the number of inoculated communities. These findings are relevant to the understanding of the ecological dynamics of natural microbial communities, as well as demonstrating a simple method of predictably enhancing microbial community function in biotechnology, health, and agriculture [13].

Metabolic modelling in a dynamic evolutionary framework predicts adaptive diversification of bacteria in a long-term evolution experiment.

BACKGROUND: Predicting adaptive trajectories is a major goal of evolutionary biology and useful for practical applications. Systems biology has enabled the development of genome-scale metabolic models. However, analysing these models via flux balance analysis (FBA) cannot predict many evolutionary outcomes including adaptive diversification, whereby an ancestral lineage diverges to fill multiple niches. Here we combine in silico evolution with FBA and apply this modelling framework, evoFBA, to a long-term evolution experiment with Escherichia coli. RESULTS: Simulations predicted the adaptive diversification that occurred in one experimental population and generated hypotheses about the mechanisms that promoted coexistence of the diverged lineages. We experimentally tested and, on balance, verified these mechanisms, showing that diversification involved niche construction and character displacement through differential nutrient uptake and altered metabolic regulation. CONCLUSION: The evoFBA framework represents a promising new way to model biochemical evolution, one that can generate testable predictions about evolutionary and ecosystem-level outcomes.

A stable genetic polymorphism underpinning microbial syntrophy.

Syntrophies are metabolic cooperations, whereby two organisms co-metabolize a substrate in an interdependent manner. Many of the observed natural syntrophic interactions are mandatory in the absence of strong electron acceptors, such that one species in the syntrophy has to assume the role of electron sink for the other. While this presents an ecological setting for syntrophy to be beneficial, the potential genetic drivers of syntrophy remain unknown to date. Here, we show that the syntrophic sulfate-reducing species Desulfovibrio vulgaris displays a stable genetic polymorphism, where only a specific genotype is able to engage in syntrophy with the hydrogenotrophic methanogen Methanococcus maripaludis. This 'syntrophic' genotype is characterized by two genetic alterations, one of which is an in-frame deletion in the gene encoding for the ion-translocating subunit cooK of the membrane-bound COO hydrogenase. We show that this genotype presents a specific physiology, in which reshaping of energy conservation in the lactate oxidation pathway enables it to produce sufficient intermediate hydrogen for sustained M. maripaludis growth and thus, syntrophy. To our knowledge, these findings provide for the first time a genetic basis for syntrophy in nature and bring us closer to the rational engineering of syntrophy in synthetic microbial communities.

Microbial diversity arising from thermodynamic constraints.

The microbial world displays an immense taxonomic diversity. This diversity is manifested also in a multitude of metabolic pathways that can utilise different substrates and produce different products. Here, we propose that these observations directly link to thermodynamic constraints that inherently arise from the metabolic basis of microbial growth. We show that thermodynamic constraints can enable coexistence of microbes that utilise the same substrate but produce different end products. We find that this thermodynamics-driven emergence of diversity is most relevant for metabolic conversions with low free energy as seen for example under anaerobic conditions, where population dynamics is governed by thermodynamic effects rather than kinetic factors such as substrate uptake rates. These findings provide a general understanding of the microbial diversity based on the first principles of thermodynamics. As such they provide a thermodynamics-based framework for explaining the observed microbial diversity in different natural and synthetic environments.

Challenges in microbial ecology: building predictive understanding of community function and dynamics.

The importance of microbial communities (MCs) cannot be overstated. MCs underpin the biogeochemical cycles of the earth's soil, oceans and the atmosphere, and perform ecosystem functions that impact plants, animals and humans. Yet our ability to predict and manage the function of these highly complex, dynamically changing communities is limited. Building predictive models that link MC composition to function is a key emerging challenge in microbial ecology. Here, we argue that addressing this challenge requires close coordination of experimental data collection and method development with mathematical model building. We discuss specific examples where model-experiment integration has already resulted in important insights into MC function and structure. We also highlight key research questions that still demand better integration of experiments and models. We argue that such integration is needed to achieve significant progress in our understanding of MC dynamics and function, and we make specific practical suggestions as to how this could be achieved.

Widespread Distribution and Expression of Gamma A (UMB), an Uncultured, Diazotrophic, γ-Proteobacterial nifH Phylotype.

Marine dinitrogen (N2) fixation studies have focused nearly exclusively on cyanobacterial diazotrophs; however γ-proteobacteria are an abundant component of the marine community and have been largely overlooked until recently. Here we present a phylogenetic analysis of all nifH γ-proteobacterial sequences available in public databases and qPCR data of a γ-proteobacterial phylotype, Gamma A (UMB), obtained during several research cruises. Our analysis revealed a complex diversity of diazotrophic γ-proteobacteria. One phylotype in particular, Gamma A, was described in several traditional and quantitative PCR studies. Though several γ-proteobacterial nifH sequences have been described as laboratory contaminants, Gamma A is part of a large cluster of sequences isolated from marine environments and distantly related to the clade of contaminants. Using a TaqMan probe and primer set, Gamma A nifH DNA abundance and expression were analyzed in nearly 1000 samples collected during 15 cruises to the Atlantic and Pacific Oceans. The data showed that Gamma A is an active, cosmopolitan diazotroph found throughout oxygenated, oligotrophic waters reaching maximum abundances of 8 x 104 nifH DNA copies l-1 and 5 x 105 nifH transcript copies l-1. Gamma A nifH transcript abundances were on average 3 fold higher than nifH DNA abundances. The widespread distribution and activity of Gamma A indicate that it has potential to be a globally important N2 fixing organism.

Facets of diazotrophy in the oxygen minimum zone waters off Peru.

Nitrogen fixation, the biological reduction of dinitrogen gas (N2) to ammonium (NH4(+)), is quantitatively the most important external source of new nitrogen (N) to the open ocean. Classically, the ecological niche of oceanic N2 fixers (diazotrophs) is ascribed to tropical oligotrophic surface waters, often depleted in fixed N, with a diazotrophic community dominated by cyanobacteria. Although this applies for large areas of the ocean, biogeochemical models and phylogenetic studies suggest that the oceanic diazotrophic niche may be much broader than previously considered, resulting in major implications for the global N-budget. Here, we report on the composition, distribution and abundance of nifH, the functional gene marker for N2 fixation. Our results show the presence of eight clades of diazotrophs in the oxygen minimum zone (OMZ) off Peru. Although proteobacterial clades dominated overall, two clusters affiliated to spirochaeta and archaea were identified. N2 fixation was detected within OMZ waters and was stimulated by the addition of organic carbon sources supporting the view that non-phototrophic diazotrophs were actively fixing dinitrogen. The observed co-occurrence of key functional genes for N2 fixation, nitrification, anammox and denitrification suggests that a close spatial coupling of N-input and N-loss processes exists in the OMZ off Peru. The wide distribution of diazotrophs throughout the water column adds to the emerging view that the habitat of marine diazotrophs can be extended to low oxygen/high nitrate areas. Furthermore, our statistical analysis suggests that NO2(-) and PO4(3-) are the major factors affecting diazotrophic distribution throughout the OMZ. In view of the predicted increase in ocean deoxygenation resulting from global warming, our findings indicate that the importance of OMZs as niches for N2 fixation may increase in the future.

Synthetic microbial communities.

While natural microbial communities are composed of a mix of microbes with often unknown functions, the construction of synthetic microbial communities allows for the generation of defined systems with reduced complexity. Used in a top-down approach, synthetic communities serve as model systems to ask questions about the performance and stability of microbial communities. In a second, bottom-up approach, synthetic microbial communities are used to study which conditions are necessary to generate interaction patterns like symbiosis or competition, and how higher order community structure can emerge from these. Besides their obvious value as model systems to understand the structure, function and evolution of microbial communities as complex dynamical systems, synthetic communities can also open up new avenues for biotechnological applications.

Giant hydrogen sulfide plume in the oxygen minimum zone off Peru supports chemolithoautotrophy.

In Eastern Boundary Upwelling Systems nutrient-rich waters are transported to the ocean surface, fuelling high photoautotrophic primary production. Subsequent heterotrophic decomposition of the produced biomass increases the oxygen-depletion at intermediate water depths, which can result in the formation of oxygen minimum zones (OMZ). OMZs can sporadically accumulate hydrogen sulfide (H2S), which is toxic to most multicellular organisms and has been implicated in massive fish kills. During a cruise to the OMZ off Peru in January 2009 we found a sulfidic plume in continental shelf waters, covering an area >5500 km(2), which contained ∼2.2×10(4) tons of H2S. This was the first time that H2S was measured in the Peruvian OMZ and with ∼440 km(3) the largest plume ever reported for oceanic waters. We assessed the phylogenetic and functional diversity of the inhabiting microbial community by high-throughput sequencing of DNA and RNA, while its metabolic activity was determined with rate measurements of carbon fixation and nitrogen transformation processes. The waters were dominated by several distinct γ-, δ- and ε-proteobacterial taxa associated with either sulfur oxidation or sulfate reduction. Our results suggest that these chemolithoautotrophic bacteria utilized several oxidants (oxygen, nitrate, nitrite, nitric oxide and nitrous oxide) to detoxify the sulfidic waters well below the oxic surface. The chemolithoautotrophic activity at our sampling site led to high rates of dark carbon fixation. Assuming that these chemolithoautotrophic rates were maintained throughout the sulfidic waters, they could be representing as much as ∼30% of the photoautotrophic carbon fixation. Postulated changes such as eutrophication and global warming, which lead to an expansion and intensification of OMZs, might also increase the frequency of sulfidic waters. We suggest that the chemolithoautotrophically fixed carbon may be involved in a negative feedback loop that could fuel further sulfate reduction and potentially stabilize the sulfidic OMZ waters.

Doubling of marine dinitrogen-fixation rates based on direct measurements.

Biological dinitrogen fixation provides the largest input of nitrogen to the oceans, therefore exerting important control on the ocean's nitrogen inventory and primary productivity. Nitrogen-isotope data from ocean sediments suggest that the marine-nitrogen inventory has been balanced for the past 3,000 years (ref. 4). Producing a balanced marine-nitrogen budget based on direct measurements has proved difficult, however, with nitrogen loss exceeding the gain from dinitrogen fixation by approximately 200 Tg N yr−1 (refs 5, 6). Here we present data from the Atlantic Ocean and show that the most widely used method of measuring oceanic N2-fixation rates underestimates the contribution of N2-fixing microorganisms (diazotrophs) relative to a newly developed method. Using molecular techniques to quantify the abundance of specific clades of diazotrophs in parallel with rates of 15N2 incorporation into particulate organic matter, we suggest that the difference between N2-fixation rates measured with the established method and those measured with the new method can be related to the composition of the diazotrophic community. Our data show that in areas dominated by Trichodesmium, the established method underestimates N2-fixation rates by an average of 62%. We also find that the newly developed method yields N2-fixation rates more than six times higher than those from the established method when unicellular, symbiotic cyanobacteria and γ-proteobacteria dominate the diazotrophic community. On the basis of average areal rates measured over the Atlantic Ocean, we calculated basin-wide N2-fixation rates of 14 ± 1 Tg N yr−1 and 24 ±1 Tg N yr−1 for the established and new methods, respectively. If our findings can be extrapolated to other ocean basins, this suggests that the global marine N2-fixation rate derived from direct measurements may increase from 103 ± 8 Tg N yr−1 to 177 ± 8 Tg N yr−1, and that the contribution of N2 fixers other than Trichodesmium is much more significant than was previously thought.

Direct and Indirect Costs of Dinitrogen Fixation in Crocosphaera watsonii WH8501 and Possible Implications for the Nitrogen Cycle.

The recent detection of heterotrophic nitrogen (N(2)) fixation in deep waters of the southern Californian and Peruvian OMZ questions our current understanding of marine N(2) fixation as a process confined to oligotrophic surface waters of the oceans. In experiments with Crocosphaera watsonii WH8501, a marine unicellular diazotrophic (N(2) fixing) cyanobacterium, we demonstrated that the presence of high nitrate concentrations (up to 800 µM) had no inhibitory effect on growth and N(2) fixation over a period of 2 weeks. In contrast, the environmental oxygen concentration significantly influenced rates of N(2) fixation and respiration, as well as carbon and nitrogen cellular content of C. watsonii over a 24-h period. Cells grown under lowered oxygen atmosphere (5%) had a higher nitrogenase activity and respired less carbon during the dark cycle than under normal oxygen atmosphere (20%). Respiratory oxygen drawdown during the dark period could be fully explained (104%) by energetic needs due to basal metabolism and N(2) fixation at low oxygen, while at normal oxygen these two processes could only account for 40% of the measured respiration rate. Our results revealed that under normal oxygen concentration most of the energetic costs during N(2) fixation (∼60%) are not derived from the process of N(2) fixation per se but rather from the indirect costs incurred for the removal of intracellular oxygen or by the reversal of oxidative damage (e.g., nitrogenase de novo synthesis). Theoretical calculations suggest a slight energetic advantage of N(2) fixation relative to assimilatory nitrate uptake, when oxygen supply is in balance with the oxygen requirement for cellular respiration (i.e., energy generation for basal metabolism and N(2) fixation). Taken together our results imply the existence of a niche for diazotrophic organisms inside oxygen minimum zones, which are predicted to further expand in the future ocean.

Methodological underestimation of oceanic nitrogen fixation rates.

The two commonly applied methods to assess dinitrogen (N(2)) fixation rates are the (15)N(2)-tracer addition and the acetylene reduction assay (ARA). Discrepancies between the two methods as well as inconsistencies between N(2) fixation rates and biomass/growth rates in culture experiments have been attributed to variable excretion of recently fixed N(2). Here we demonstrate that the (15)N(2)-tracer addition method underestimates N(2) fixation rates significantly when the (15)N(2) tracer is introduced as a gas bubble. The injected (15)N(2) gas bubble does not attain equilibrium with the surrounding water leading to a (15)N(2) concentration lower than assumed by the method used to calculate (15)N(2)-fixation rates. The resulting magnitude of underestimation varies with the incubation time, to a lesser extent on the amount of injected gas and is sensitive to the timing of the bubble injection relative to diel N(2) fixation patterns. Here, we propose and test a modified (15)N(2) tracer method based on the addition of (15)N(2)-enriched seawater that provides an instantaneous, constant enrichment and allows more accurate calculation of N(2) fixation rates for both field and laboratory studies. We hypothesise that application of N(2) fixation measurements using this modified method will significantly reduce the apparent imbalances in the oceanic fixed-nitrogen budget.