Scholarly Work - Chemical & Biological Engineering
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Item Environment Constrains Fitness Advantages of Division of Labor in Microbial Consortia Engineered for Metabolite Push or Pull Interactions(American Society for Microbiology, 2022-08) Beck, Ashely E.; Pintar, Kathryn; Schepens, Diana; Schrammeck, Ashely; Johnson, Timothy; Bleem, Alissa; Du, Martina; Harcombe, William R.; Bernstein, Hans C.; Heys, Jeffrey J.; Gedeon, Tomas; Carlson, Ross P.Fitness benefits from division of labor are well documented in microbial consortia, but the dependency of the benefits on environmental context is poorly understood. Two synthetic Escherichia coli consortia were built to test the relationships between exchanged organic acid, local environment, and opportunity costs of different metabolic strategies. Opportunity costs quantify benefits not realized due to selecting one phenotype over another. The consortia catabolized glucose and exchanged either acetic or lactic acid to create producer-consumer food webs. The organic acids had different inhibitory properties and different opportunity costs associated with their positions in central metabolism. The exchanged metabolites modulated different consortial dynamics. The acetic acid-exchanging (AAE) consortium had a “push” interaction motif where acetic acid was secreted faster by the producer than the consumer imported it, while the lactic acid-exchanging (LAE) consortium had a “pull” interaction motif where the consumer imported lactic acid at a comparable rate to its production. The LAE consortium outperformed wild-type (WT) batch cultures under the environmental context of weakly buffered conditions, achieving a 55% increase in biomass titer, a 51% increase in biomass per proton yield, an 86% increase in substrate conversion, and the complete elimination of by-product accumulation all relative to the WT. However, the LAE consortium had the trade-off of a 42% lower specific growth rate. The AAE consortium did not outperform the WT in any considered performance metric. Performance advantages of the LAE consortium were sensitive to environment; increasing the medium buffering capacity negated the performance advantages compared to WT.Item Artificial consortium demonstrates emergent properties of enhanced cellulosic-sugar degradation and biofuel synthesis(2020-12) Park, Heejoon; Patel, Ayushi; Hunt, Kristopher A.; Henson, Michael A.; Carlson, Ross P.Planktonic cultures, of a rationally designed consortium, demonstrated emergent properties that exceeded the sums of monoculture properties, including a >200% increase in cellobiose catabolism, a >100% increase in glycerol catabolism, a >800% increase in ethanol production, and a >120% increase in biomass productivity. The consortium was designed to have a primary and secondary-resource specialist that used crossfeeding with a positive feedback mechanism, division of labor, and nutrient and energy transfer via necromass catabolism. The primary resource specialist was Clostridium phytofermentans (a.k.a. Lachnoclostridium phytofermentans), a cellulolytic, obligate anaerobe. The secondary-resource specialist was Escherichia coli, a versatile, facultative anaerobe, which can ferment glycerol and byproducts of cellobiose catabolism. The consortium also demonstrated emergent properties of enhanced biomass accumulation when grown as biofilms, which created high cell density communities with gradients of species along the vertical axis. Consortium biofilms were robust to oxic perturbations with E. coli consuming O2, creating an anoxic environment for C. phytofermentans. Anoxic/oxic cycling further enhanced biomass productivity of the biofilm consortium, increasing biomass accumulation ~250% over the sum of the monoculture biofilms. Consortium emergent properties were credited to several synergistic mechanisms. E. coli consumed inhibitory byproducts from cellobiose catabolism, driving higher C. phytofermentans growth and higher cellulolytic enzyme production, which in turn provided more substrate for E. coli. E. coli necromass enhanced C. phytofermentans growth while C. phytofermentans necromass aided E. coli growth via the release of peptides and amino acids, respectively. In aggregate, temporal cycling of necromass constituents increased flux of cellulose-derived resources through the consortium. The study establishes a consortia-based, bioprocessing strategy built on naturally occurring interactions for improved conversion of cellulose-derived sugars into bioproducts.Item Multiscale analysis of autotroph-heterotroph interactions in a high-temperature microbial community(2018-09) Hunt, Kristopher A.; Jennings, Ryan deM.; Inskeep, William P.; Carlson, Ross P.Interactions among microbial community members can lead to emergent properties, such as enhanced productivity, stability, and robustness. Iron-oxide mats in acidic (pH 2–4), high-temperature (> 65 °C) springs of Yellowstone National Park contain relatively simple microbial communities and are well-characterized geochemically. Consequently, these communities are excellent model systems for studying the metabolic activity of individual populations and key microbial interactions. The primary goals of the current study were to integrate data collected in situ with in silico calculations across process-scales encompassing enzymatic activity, cellular metabolism, community interactions, and ecosystem biogeochemistry, as well as to predict and quantify the functional limits of autotroph-heterotroph interactions. Metagenomic and transcriptomic data were used to reconstruct carbon and energy metabolisms of an important autotroph (Metallosphaera yellowstonensis) and heterotroph (Geoarchaeum sp. OSPB) from the studied Fe(III)-oxide mat communities. Standard and hybrid elementary flux mode and flux balance analyses of metabolic models predicted cellular- and community-level metabolic acclimations to simulated environmental stresses, respectively. In situ geochemical analyses, including oxygen depth-profiles, Fe(III)-oxide deposition rates, stable carbon isotopes and mat biomass concentrations, were combined with cellular models to explore autotroph-heterotroph interactions important to community structure-function. Integration of metabolic modeling with in situ measurements, including the relative population abundance of autotrophs to heterotrophs, demonstrated that Fe(III)-oxide mat communities operate at their maximum total community growth rate (i.e. sum of autotroph and heterotroph growth rates), as opposed to net community growth rate (i.e. total community growth rate subtracting autotroph consumed by heterotroph), as predicted from the maximum power principle. Integration of multiscale data with ecological theory provides a basis for predicting autotroph-heterotroph interactions and community-level cellular organization.Item Competitive resource allocation to metabolic pathways contributes to overflow metabolisms and emergent properties in cross-feeding microbial consortia(2018-04) Carlson, Ross P.; Beck, Ashley E.; Phalak, Poonam; Fields, Matthew W.; Gedeon, Tomas; Hanley, Luke; Harcombe, W. R.; Henson, Michael A.; Heys, Jeffrey J.Resource scarcity is a common stress in nature and has a major impact on microbial physiology. This review highlights microbial acclimations to resource scarcity, focusing on resource investment strategies for chemoheterotrophs from the molecular level to the pathway level. Competitive resource allocation strategies often lead to a phenotype known as overflow metabolism; the resulting overflow byproducts can stabilize cooperative interactions in microbial communities and can lead to cross-feeding consortia. These consortia can exhibit emergent properties such as enhanced resource usage and biomass productivity. The literature distilled here draws parallels between in silico and laboratory studies and ties them together with ecological theories to better understand microbial stress responses and mutualistic consortia functioning.Item Effects of Spatial Localization on Microbial Consortia Growth(2017-01) Venters, Michael; Carlson, Ross P.; Gedeon, Tomas; Heys, Jeffrey J.Microbial consortia are commonly observed in natural and synthetic systems, and these consortia frequently result in higher biomass production relative to monocultures. The focus here is on the impact of initial spatial localization and substrate diffusivity on the growth of a model microbial consortium consisting of a producer strain that consumes glucose and produces acetate and a scavenger strain that consumes the acetate. The mathematical model is based on an individual cell model where growth is described by Monod kinetics, and substrate transport is described by a continuum-based, non-equilibrium reaction-diffusion model where convective transport is negligible (e.g., in a biofilm). The first set of results focus on a single producer cell at the center of the domain and surrounded by an initial population of scavenger cells. The impact of the initial population density and substrate diffusivity is examined. A transition is observed from the highest initial density resulting in the greatest cell growth to cell growth being independent of initial density. A high initial density minimizes diffusive transport time and is typically expected to result in the highest growth, but this expected behavior is not predicted in environments with lower diffusivity or larger length scales. When the producer cells are placed on the bottom of the domain with the scavenger cells above in a layered biofilm arrangement, a similar critical transition is observed. For the highest diffusivity values examined, a thin, dense initial scavenger layer is optimal for cell growth. However, for smaller diffusivity values, a thicker, less dense initial scavenger layer provides maximal growth. The overall conclusion is that high density clustering of members of a food chain is optimal under most common transport conditions, but under some slow transport conditions, high density clustering may not be optimal for microbial growth.Item Quantitative NMR metabolite profiling of methicillin-resistant and methicillin-susceptible Staphylococcus aureus discriminates between biofilm and planktonic phenotypes(2013-06) Ammons, Mary Cloud B.; Tripet, Brian P.; Carlson, Ross P.; Kirker, Kelly R.; Gross, M. A.; Stanisich, Jessica J.; Copie, ValerieWound bioburden in the form of colonizing biofilms is a major contributor to nonhealing wounds. Staphylococcus aureus is a Gram-positive, facultative anaerobe commonly found in chronic wounds; however, much remains unknown about the basic physiology of this opportunistic pathogen, especially with regard to the biofilm phenotype. Transcriptomic and proteomic analysis of S. aureus biofilms have suggested that S. aureus biofilms exhibit an altered metabolic state relative to the planktonic phenotype. Herein, comparisons of extracellular and intracellular metabolite profiles detected by 1H NMR were conducted for methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) S. aureus strains grown as biofilm and planktonic cultures. Principal component analysis distinguished the biofilm phenotype from the planktonic phenotype, and factor loadings analysis identified metabolites that contributed to the statistical separation of the biofilm from the planktonic phenotype, suggesting that key features distinguishing biofilm from planktonic growth include selective amino acid uptake, lipid catabolism, butanediol fermentation, and a shift in metabolism from energy production to assembly of cell-wall components and matrix deposition. These metabolite profiles provide a basis for the development of metabolite biomarkers that distinguish between biofilm and planktonic phenotypes in S. aureus and have the potential for improved diagnostic and therapeutic use in chronic wounds.Item Physiological, elemental composition, and proteomic analyses of Escherichia coli ammonium-limited chemostat growth with comparison to iron- and glucose-limited chemostat growth.(2015-08) Folsom, James Patrick; Carlson, Ross P.Escherichia coli physiological, biomass elemental composition and proteome acclimations to ammonium-limited chemostat growth were measured at four levels of nutrient scarcity controlled via chemostat dilution rate. These data were compared with published iron- and glucose-limited growth data collected from the same strain and at the same dilution rates to quantify general and nutrient-specific responses. Severe nutrient scarcity resulted in an overflow metabolism with differing organic byproduct profiles based on limiting nutrient and dilution rate. Ammonium-limited cultures secreted up to 35 % of the metabolized glucose carbon as organic byproducts with acetate representing the largest fraction; in comparison, iron-limited cultures secreted up to 70 % of the metabolized glucose carbon as lactate, and glucose-limited cultures secreted up to 4 % of the metabolized glucose carbon as formate. Biomass elemental composition differed with nutrient limitation; biomass from ammonium-limited cultures had a lower nitrogen content than biomass from either iron- or glucose-limited cultures. Proteomic analysis of central metabolism enzymes revealed that ammonium- and iron-limited cultures had a lower abundance of key tricarboxylic acid (TCA) cycle enzymes and higher abundance of key glycolysis enzymes compared with glucose-limited cultures. The overall results are largely consistent with cellular economics concepts, including metabolic tradeoff theory where the limiting nutrient is invested into essential pathways such as glycolysis instead of higher ATP-yielding, but non-essential, pathways such as the TCA cycle. The data provide a detailed insight into ecologically competitive metabolic strategies selected by evolution, templates for controlling metabolism for bioprocesses and a comprehensive dataset for validating in silico representations of metabolism.Item Modelling metabolism of the diatom Phaeodactylum tricornutum(2015-11) Singh, Dipali; Carlson, Ross P.; Fell, David; Poolman, MarkMarine diatoms have potential as a biotechnological production platform, especially for lipid-derived products, including biofuels. Here we introduce some features of diatom metabolism, particularly with respect to photosynthesis, photorespiration and lipid synthesis and their differences relative to other photosynthetic eukaryotes. Since structural metabolic modelling of other photosynthetic organisms has been shown to be capable of representing their metabolic capabilities realistically, we briefly review the main approaches to this type of modelling. We then propose that genome-scale modelling of the diatom Phaeodactylum tricornutum, in response to varying light intensity, could uncover the novel aspects of the metabolic potential of this organism.Item Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum(2012-06) Valenzuela, Jacob J.; Mazurie, Aurélien J.; Carlson, Ross P.; Gerlach, Robin; Cooksey, Keith E.; Bothner, Brian; Peyton, Brent M.; Fields, Matthew W.Background Phaeodactylum tricornutum is a unicellular diatom in the class Bacillariophyceae. The full genome has been sequenced (<30 Mb), and approximately 20 to 30% triacylglyceride (TAG) accumulation on a dry cell basis has been reported under different growth conditions. To elucidate P. tricornutum gene expression profiles during nutrient-deprivation and lipid-accumulation, cell cultures were grown with a nitrate to phosphate ratio of 20:1 (N:P) and whole-genome transcripts were monitored over time via RNA-sequence determination. Results The specific Nile Red (NR) fluorescence (NR fluorescence per cell) increased over time; however, the increase in NR fluorescence was initiated before external nitrate was completely exhausted. Exogenous phosphate was depleted before nitrate, and these results indicated that the depletion of exogenous phosphate might be an early trigger for lipid accumulation that is magnified upon nitrate depletion. As expected, many of the genes associated with nitrate and phosphate utilization were up-expressed. The diatom-specific cyclins cyc 7 and cyc 10 were down-expressed during the nutrient-deplete state, and cyclin B1 was up-expressed during lipid-accumulation after growth cessation. While many of the genes associated with the C3 pathway for photosynthetic carbon reduction were not significantly altered, genes involved in a putative C4 pathway for photosynthetic carbon assimilation were up-expressed as the cells depleted nitrate, phosphate, and exogenous dissolved inorganic carbon (DIC) levels. P. tricornutum has multiple, putative carbonic anhydrases, but only two were significantly up-expressed (2-fold and 4-fold) at the last time point when exogenous DIC levels had increased after the cessation of growth. Alternative pathways that could utilize HCO3- were also suggested by the gene expression profiles (e.g., putative propionyl-CoA and methylmalonyl-CoA decarboxylases). Conclusions The results indicate that P. tricornutum continued carbon dioxide reduction when population growth was arrested and different carbon-concentrating mechanisms were used dependent upon exogenous DIC levels. Based upon overall low gene expression levels for fatty acid synthesis, the results also suggest that the build-up of precursors to the acetyl-CoA carboxylases may play a more significant role in TAG synthesis rather than the actual enzyme levels of acetyl-CoA carboxylases per se. The presented insights into the types and timing of cellular responses to inorganic carbon will help maximize photoautotrophic carbon flow to lipid accumulation.Item Nutrient resupplementation arrests bio-oil accumulation in Phaeodactylum tricornutum(2013-08) Valenzuela, Jacob J.; Carlson, Ross P.; Gerlach, Robin; Cooksey, Keith E.; Peyton, Brent M.; Bothner, Brian; Fields, Matthew W.Phaeodactylum tricornutum is a marine diatom in the class Bacillariophyceae and is important ecologically and industrially with regards to ocean primary production and lipid accumulation for biofuel production, respectively. Triacylglyceride (TAG) accumulation has been reported in P. tricornutum under different nutrient stresses, and our results show that lipid accumulation can occur with nitrate or phosphate depletion. However, greater lipid accumulation was observed when both nutrients were depleted as observed using a Nile Red assay and fatty acid methyl ester (FAME) profiles. Nitrate depletion had a greater effect on lipid accumulation than phosphate depletion. Lipid accumulation in P. tricornutum was arrested upon resupplementation with the depleted nutrient. Cells depleted of nitrogen showed a distinct shift from a lipid accumulation mode to cellular growth post-resupplementation with nitrate, as observed through increased cell numbers and consumption of accumulated lipid. Phosphate depletion caused lipid accumulation that was arrested upon phosphate resupplementation. The cessation of lipid accumulation was followed by lipid consumption without an increase in cell numbers. Cells depleted in both nitrate and phosphate displayed cell growth upon the addition of both nitrate and phosphate and had the largest observed lipid consumption upon resupplementation. These results indicate that phosphate resupplementation can shut down lipid accumulation but does not cause cells to shift into cellular growth, unlike nitrate resupplementation. These data suggest that nutrient resupplementation will arrest lipid accumulation and that switching between cellular growth and lipid accumulation can be regulated upon the availability of nitrogen and phosphorus.