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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

A novel semi-structured kinetic model of methanotroph-photoautotroph cocultures for biogas conversion

Through metabolic coupling of methane oxidation and oxygenic photosynthesis, methanotroph-photoautotroph (M-P) cocultures offer a highly promising technology platform for biogas conversion. However, there has not been any quantitative modeling of the coculture growth kinetics. This is mainly due to the inherent difficulty associated with real time characterization of the M-P cocultures and the complex interactions such as the cross-feeding mechanism within the coculture. To address this challenge, we recently developed a novel experimental-computational (E-C) protocol to accurately characterize the M-P coculture in real-time, and validated its accuracy through cell counting. Enabled by the E-C protocol, this work presents the very first kinetic model for M-P cocultures. By explicitly modeling the exchange of in situ produced O 2 /CO 2 within the M-P coculture and coupling the individual biomass growth with mass transfer between the gas and liquid phases, the semi-structured kinetic model accurately predicts the growth dynamics of the M-P coculture under a wide range of growth conditions. The proposed model is validated by a series of wet-lab experiments using Methylomicrobium buryatense 5GB1 - Arthrospira platensis as the model coculture. Although it has been speculated that there may exist other emergent metabolic interactions within the M-P coculture, in addition to the exchange of in situ produced O 2 /CO 2 , there has not been any experimental validation prior to this study. By integrating designed experiments with the semi-structured kinetic model, this study is the first to confirm the existence of the additional emergent metabolic exchanges within the coculture. Furthermore, this study further quantifies the effect of these unknown metabolic interactions on the growth of both species in the model coculture, supporting further research to identify these exchanged metabolites for metabolic engineering.

42 ENGINEERING↗
DOE OSTI2022

The potential of caproate (hexanoate) production using Clostridium kluyveri syntrophic cocultures with Clostridium acetobutylicum or Clostridium saccharolyticum

Caproate (hexanoate) and other medium-chain fatty acids are valuable platform chemicals produced by processes utilizing petroleum or plant oil. Clostridium kluyveri , growing on short chain alcohols (notably ethanol) and carboxylic acids (such as acetate) is noted for its ability to perform chain elongation to produce 4- to 8-carbon carboxylates. C. kluyveri has been studied in monoculture and coculture conditions, which lead to relatively modest carboxylate titers after long fermentation times. To assess the biosynthetic potential of C. kluyveri for caproate production from sugars through coculture fermentations, in the absence of monoculture data in the literature suitable for our coculture experiments, we first explored C. kluyveri monocultures. Some monocultures achieved caproate titers of 150 to over 200 mM in 40–50 h with a production rate of 7.9 mM/h. Based on that data, we then explored two novel, syntrophic coculture partners for producing caproate from sugars: Clostridium acetobutylicum and Clostridium saccharolyticum . Neither species has been cocultured with C. kluyveri before, and both demonstrate promising results. Our experiments of C. kluyveri monocultures and C. kluyveri — C. saccharolyticum cocultures demonstrate exceptionally high caproate titers (145–200 mM), fast production rates (3.25–8.1 mM/h), and short fermentation times (18–45 h). These results represent the most caproate produced by a C. kluyveri coculture in the shortest known fermentation time. We also explored the possibility of heterologous cell fusion between the coculture pairs similar to the results seen previously in our group with C. acetobutylicum and Clostridium ljungdahlii. Fusion events were observed only in the C. acetobutylicum — C. kluyveri coculture pair, and we offer an explanation for the lack of fusion between C. saccharolyticum and C. kluyveri . This work supports the promise of coculture biotechnology for sustainable production of caproate and other platform chemicals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Transcriptomic Network Analysis of Cyanobacterial-Methylotroph Interactions in Coculture and Axenic Conditions

A previous study demonstrated the potential for Cyanobacterial-Methylotroph cocultures to facilitate biogas processing as well as to be used in other biotechnological applications. To advance this technology, we investigated potential interactions between Cyanobacterium stanieri HL-69 (HL69) and Methylotuvimicrobium alkaliphilum 20Z (20Z) by inferring and analyzing gene co-expression networks under co-culture and axenic conditions. Five different co-expression networks were examined. These networks were inferred using gene expression profiles for 20Z axenic condition, HL-69 axenic, HL-69, 20Z coculture, HL-69 coculture, and cross-species HL-69-20Z coculture. Through the analysis of node (gene) betweenness and node normalized degree values in all five network cases, we compared adjustments in gene expression between growth conditions (axenic vs co-culture) as well as identify biological functions relevant to interspecies interactions. This analysis was done to distinguish between gene interactions within an organism and gene interactions between two organisms. Moreover, for all five cases we investigated two different network cutoff levels of 3,000 and 10,000. By shedding light on inter- and intra- species interactions, we hope to gain a better understanding of how these two organisms interact. This research will allow the investigation of further biotechnological applications of coculture systems and optimization of such applications for biotechnological purposes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Matlab implementation of a novel semi-structured kinetic model for methanotroph-photoautotroph cocultures

This paper presents the MatLab implementation details of a novel semi-structured kinetic model for methanotroph-photoautotroph cocultures. This includes the parameterization of the modeling equations, and the initialization of the simulation based on experimental conditions. More importantly, it provides details on how the differential equations governing mass balances in both gas and liquid phases are integrated together to simulate the system dynamics over time. The semi-structured kinetic model for methanotroph-photoautotroph coculture is validated using a wide range of experimental conditions. The model: Accurate predicts both the coculture growth in liquid phase and the gas composition changes in head space over time; Explicitly models the exchange of in situ produced O 2 and CO 2 within the coculture; Considers the self-shading effect on the growth of photoautotroph.

42 ENGINEERING↗
DOE OSTI2022

Photosynthetic biohybrid coculture for tandem and tunable CO 2 and N 2 fixation

Solar-driven bioelectrosynthesis represents a promising approach for converting abundant resources into value-added chemicals with renewable energy. Microorganisms powered by electrochemical reducing equivalents assimilate CO 2 , H 2 O, and N 2 building blocks. However, products from autotrophic whole-cell biocatalysts are limited. Furthermore, biocatalysts tasked with N 2 reduction are constrained by simultaneous energy-intensive autotrophy. To overcome these challenges, we designed a biohybrid coculture for tandem and tunable CO 2 and N 2 fixation to value-added products, allowing the different species to distribute bioconversion steps and reduce the individual metabolic burden. This consortium involves acetogen Sporomusa ovata, which reduces CO 2 to acetate, and diazotrophic Rhodopseudomonas palustris, which uses the acetate both to fuel N 2 fixation and for the generation of a biopolyester. We demonstrate that the coculture platform provides a robust ecosystem for continuous CO 2 and N 2 fixation, and its outputs are directed by substrate gas composition. Moreover, we show the ability to support the coculture on a high–surface area silicon nanowire cathodic platform. The biohybrid coculture achieved peak faradaic efficiencies of 100, 19.1, and 6.3% for acetate, nitrogen in biomass, and ammonia, respectively, while maintaining product tunability. Finally, we established full solar to chemical conversion driven by a photovoltaic device, resulting in solar to chemical efficiencies of 1.78, 0.51, and 0.08% for acetate, nitrogenous biomass, and ammonia, correspondingly. Ultimately, our work demonstrates the ability to employ and electrochemically manipulate bacterial communities on demand to expand the suite of CO 2 and N 2 bioelectrosynthesis products.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

DNA transfer between two different species mediated by heterologous cell fusion in Clostridium coculture

Investigations of natural multispecies microbiomes and synthetic microbial cocultures are attracting renewed interest for their potential application in biotechnology, ecology, and medical fields. Previously, we have shown the syntrophic coculture of C. acetobutylicum and C. ljungdahlii undergoes heterologous cell-to-cell fusion, which facilitates the exchange of cytoplasmic protein and RNA between the two organisms. We now show that heterologous cell fusion between the two Clostridium organisms can facilitate the exchange of DNA. By applying selective pressures to this coculture system, we isolated clones of wild-type C. acetobutylicum which acquired the erythromycin resistance (erm) gene from the C. ljungdahlii strain carrying a plasmid with the erm gene. Single-molecule real-time sequencing revealed that the erm gene was integrated into the genome in a mosaic fashion. Our data also support the persistence of hybrid C. acetobutylicum / C. ljungdahlii cells displaying hybrid DNA-methylation patterns.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1985

Production of butanol by fermentation in the presence of cocultures of clostridium

Sugars are converted to a mixture of solvents including butanol by a fermentation process employing a coculture of microorganisms of the Clostridium genus, one of said microorganisms favoring the production of butyric acid and the other of which converts the butyric acid so produced to butanol. The use of a coculture substantially increases the yield of butanol over that obtained using a culture employing only one microorganism.

Bergstrom, S. L.↗
NASA NTRS1994

Three-Dimensional Coculture Of Human Small-Intestine Cells

Complex three-dimensional masses of normal human epithelial and mesenchymal small-intestine cells cocultured in process involving specially designed bioreactors. Useful as tissued models for studies of growth, regulatory, and differentiation processes in normal intestinal tissues; diseases of small intestine; and interactions between cells of small intestine and viruses causing disease both in small intestine and elsewhere in body. Process used to produce other tissue models, leading to advances in understanding of growth and differentiation in developing organisms, of renewal of tissue, and of treatment of myriad of clinical conditions. Prior articles describing design and use of rotating-wall culture vessels include "Growing And Assembling Cells Into Tissues" (MSC-21559), "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662), and "In Vitro, Matrix-Free Formation Of Solid Tumor Spheroids" (MSC-21843).

Wolf, David↗
NASA NTRS1995

Growing Three-Dimensional Cocultures Of Cells

Laboratory process provides environmental conditions favoring simultaneous growth of cocultures of mammalian cells of more than one type. Cultures become three-dimensional tissuelike assemblies serving as organoid models of differentiation of cells. Process used, for example, to study growth of human colon cancers, starting from mixtures of normal colonic fibroblasts and partially differentiated colon adenocarcinoma cells.

Wolf, David A.↗
DOE OSTI2023

Construction and Modeling of a Coculture Microplate for Real-Time Measurement of Microbial Interactions

The dynamic structures of microbial communities emerge from the complex network of interactions between their constituent microorganisms. Quantitative measurements of these interactions are important for understanding and engineering ecosystem structure. Here, we present the development and application of the BioMe plate, a redesigned microplate device in which pairs of wells are separated by porous membranes. BioMe facilitates the measurement of dynamic microbial interactions and integrates easily with standard laboratory equipment. We first applied BioMe to recapitulate recently characterized, natural symbiotic interactions between bacteria isolated from the Drosophila melanogaster gut microbiome. Specifically, the BioMe plate allowed us to observe the benefit provided by two Lactobacillus strains to an Acetobacter strain. We next explored the use of BioMe to gain quantitative insight into the engineered obligate syntrophic interaction between a pair of Escherichia coli amino acid auxotrophs. We integrated experimental observations with a mechanistic computational model to quantify key parameters associated with this syntrophic interaction, including metabolite secretion and diffusion rates. This model also allowed us to explain the slow growth observed for auxotrophs growing in adjacent wells by demonstrating that, under the relevant range of parameters, local exchange between auxotrophs is essential for efficient growth. The BioMe plate provides a scalable and flexible approach for the study of dynamic microbial interactions.

3D printed device↗
NASA NTRS1985

Coculture Production of Butanol by Clostridium Bacteria

Production of butanol by anaerobic fermentation of sugars enhanced by use of two Clostridium species, one of which feeds on metabolic product of other. Renewed interest in fermentation process for making butanol stimulated by potential use of butanol as surfactant in enhanced oil recovery. Butanol also used as fuel or as chemical feedstock and currently produced synthetically from petroleum.

Bergstrom, S. L.↗
DOE OSTI2025

Separate, separated, and together: the transcriptional program of the Clostridium acetobutylicum-Clostridium ljungdahlii syntrophy leading to interspecies cell fusion

ABSTRACT Syntrophic cocultures (hitherto assumed to be commensalistic) of Clostridium acetobutylicum and Clostridium ljungdahlii , whereby CO 2 and H 2 produced by the former feed the latter, result in interspecies cell fusion involving large-scale exchange of protein, RNA, and DNA between the two organisms. Although mammalian cell fusion is mechanistically dissected, the mechanism for such microbial-cell fusions is unknown. To start exploring this mechanism, we used RNA sequencing to identify genes differentially expressed in this coculture using two types of comparisons. One type compared coculture to the two monocultures, capturing the combined impact of interactions through soluble signals in the medium and through direct cell-to-cell interactions. The second type compared membrane-separated versus -unseparated cocultures, isolating the impact of interspecies physical contact. While we could not firmly identify specific genes that might drive cell fusion, consistent with our hypothesized model for this interspecies microbial cell fusion, we observed differential regulation of genes involved in C. ljungdahlii’s autotrophic Wood-Ljungdahl pathway metabolism and genes of the motility machinery. Unexpectedly, we also identified differential regulation of biosynthetic genes of several amino acids, and notably of arginine and histidine. We verified that they are produced by C. acetobutylicum and are metabolized by C. ljungdahlii to its growth advantage. These and other findings, and notably upregulation of C. acetobutylicum ribosomal-protein genes, paint a more complex syntrophic picture and suggest a mutualistic relationship, whereby beyond CO 2 and H 2 , C. acetobutylicum feeds C. ljungdahlii with growth-boosting amino acids, while benefiting from the H 2 utilization by C. ljungdahlii . IMPORTANCE The construction and study of synthetic microbial cocultures is a growing research area due to the untapped potential of defined multi-species industrial bioprocesses and the utility of defined cocultures for generating insight into complex, undefined, natural microbial consortia. Our previous work showed that coculturing C. acetobutylicum and C. ljungdahlii leads to a unique metabolic phenotype (production of isopropanol) and heterologous cell fusion events. Here, we used RNAseq to explore genes involved in and impacted by these fusions. First, we compared gene expression in coculture to each monoculture. Second, we utilized a transwell system to compare gene expression in mixed cocultures to cocultures with both species physically separated by a permeable membrane, isolating the impact of interspecies “touching” on the transcriptome. This study deepens our mechanistic understanding of the C. acetobutylicum-C. ljungdahlii coculture phenotype, laying the groundwork for reverse genetic studies of heterologous cell fusion in Clostridium cocultures.

Willis, Noah B. (ORCID:0009000689365955)↗
DOE OSTI2024

Probing interspecies metabolic interactions within a synthetic binary microbiome using genome-scale modeling

Metabolic interactions within a microbial community play a key role in determining the structure, function, and composition of the community. However, due to the complexity and intractability of natural microbiomes, limited knowledge is available on interspecies interactions within a community. In this work, using a binary synthetic microbiome, a methanotroph-photoautotroph (M-P) coculture, as the model system, we examined different genome-scale metabolic modeling (GEM) approaches to gain a better understanding of the metabolic interactions within the coculture, how they contribute to the enhanced growth observed in the coculture, and how they evolve over time. Using batch growth data of the model M-P coculture, we compared three GEM approaches for microbial communities. Two of the methods are existing approaches: SteadyCom, a steady state GEM, and dynamic flux balance analysis (DFBA) Lab, a dynamic GEM. We also proposed an improved dynamic GEM approach, DynamiCom, for the M-P coculture. SteadyCom can predict the metabolic interactions within the coculture but not their dynamic evolutions; DFBA Lab can predict the dynamics of the coculture but cannot identify interspecies interactions. DynamiCom was able to identify the cross-fed metabolite within the coculture, as well as predict the evolution of the interspecies interactions over time. A new dynamic GEM approach, DynamiCom, was developed for a model M-P coculture. Constrained by the predictions from a validated kinetic model, DynamiCom consistently predicted the top metabolites being exchanged in the M-P coculture, as well as the establishment of the mutualistic N-exchange between the methanotroph and cyanobacteria. The interspecies interactions and their dynamic evolution predicted by DynamiCom are supported by ample evidence in the literature on methanotroph, cyanobacteria, and other cyanobacteria-heterotroph cocultures.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Mono-mix strategy enables comparative proteomics of a cross-kingdom microbial symbiosis

Cross-kingdom microbial symbioses, such as those between algae and bacteria, are key players in biogeochemical cycles. The molecular changes during initiation and establishment of symbiosis are of great interest, but quantitatively monitoring such changes can be challenging, particularly when the microorganisms differ greatly in size or are intimately associated. Here, we analyze output from label-free, data-dependent acquisition (DDA) LC-MS/MS proteomics experiments investigating the well-studied interaction between the alga Chlamydomonas reinhardtii and the heterotrophic bacterium Mesorhizobium japonicum. We found that detection of bacterial proteins decreased in coculture by 50% proteome-wide due to the abundance of algal proteins. As a result, standard differential expression analysis led to numerous false-positive reports of significantly downregulated proteins, where it was not possible to distinguish meaningful biological responses to symbiosis from artifacts of the reduced protein detection in coculture relative to monoculture. We show that data normalization alone does not eliminate the impact of altered detection on differential expression analysis of the cross-kingdom symbiosis. We assessed two additional strategies to overcome this methodological artifact inherent to DDA proteomics. In the first, we combined algal and bacterial monocultures at a relative abundance that mimicked the coculture, creating a “mono-mix” control to which the coculture could be compared. This approach enabled comparable detection of bacterial proteins in the coculture and the monoculture control. In the second strategy, we enhanced detection of lowly abundant bacterial proteins by using sample fractionation upstream of LC-MS/MS analysis. When these simple approaches were combined, they allowed for meaningful comparisons of nearly 10,000 algal proteins and over 4,000 bacterial proteins in response to symbiosis by DDA. They successfully recovered expected changes in the bacterial proteome in response to algal coculture, including upregulation of sugar-binding proteins and transporters. They also revealed novel proteomic responses to coculture that guide hypotheses about algal-bacterial interactions.

Dupuis, Sunnyjoy [University of California, Berkel↗
DOE OSTI2024

The use of a benign fast-growing cyanobacterial species to control microcystin synthesis from Microcystis aeruginosa

Introduction Microcystis aeruginosa(M. aeruginosa), one of the most abundant blue-green algae in aquatic environments, produces microcystin by causing harmful algal blooms (HABs). This study investigated the combined effects of nutrients and competition among cyanobacterial subpopulations on the synthesis of microcystin-LR. Methods Under varying nitrogen and phosphorus concentrations, cyanobacterial coculture, and the presence of algicidal DCMU, the growth was monitored by optical density analysis or microscopic counting, and the microcystin production was analyzed using high-performance liquid chromatography-UV. Furthermore, growth and toxin production were predicted using a kinetic model. Results and discussion First, coculture with the fast-growing cyanobacteriumSynechococcus elongatusUTEX 2973 (S. elongatus) reducedM. aeruginosabiomass and microcystin production at 30°C. Under high nitrogen and low phosphorus conditions,S. elongatuswas most effective, limitingM. aeruginosagrowth and toxin synthesis by up to 94.7% and 92.4%, respectively. Second, this biological strategy became less effective at 23°C, whereS. elongatusgrew more slowly. Third, the photosynthesis inhibitor DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) inhibitedM. aeruginosagrowth (at 0.1 mg/L) and microcystin production (at 0.02 mg/L). DCMU was also effective in controlling microcystin production inS. elongatus–M. aeruginosacocultures. Based on the experimental results, a multi-substrate, multi-species kinetic model was built to describe coculture growth and population interactions. Conclusion Microcystin from representative toxin-producingM. aeruginosacan be controlled by coculturing fast-growing benign cyanobacteria, which can be made even more efficient if appropriate algicide is applied. This study improved the understanding of the biological control of microcystin production under complex environmental conditions.

Microbiology↗