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INTRODUCTION: In dryland systems, biological soil crusts (biocrusts) can occupy large areas of plant interspaces, where they fix carbon following rain. Although distinct biocrust types contain different dominant photoautotrophs, few studies to date have documented carbon exchange over time from various biocrust types. This is especially true for gypsum soils. Our objective was to assess the carbon exchange of biocrust types established at the worlds largest gypsum dune field at White Sands National Park. METHODS: We sampled five different biocrust types from a sand sheet location in three different years and seasons (summer 2020, fall 2021, and winter 2022) for carbon exchange measurements in controlled lab conditions. Biocrusts were rehydrated to full saturation and light incubated for 30 min, 2, 6, 12, 24, and 36 h. Samples were then subject to a 12-point light regime with a LI-6400XT photosynthesis system to determine carbon exchange. RESULTS: Biocrust carbon exchange values differed by biocrust type, by incubation time since wetting, and by date of field sampling. Lichens and mosses had higher gross and net carbon fixation rates than dark and light cyanobacterial crusts. High respiration rates were found after 0.5 h and 2 h incubation times as communities recovered from desiccation, leveling off after 6 h incubation. Net carbon fixation of all types increased with longer incubation time, primarily as a result of decreasing respiration, which suggests rapid recovery of biocrust photosynthesis across types. However, net carbon fixation rates varied from year to year, likely as a product of time since the last rain event and environmental conditions preceding collection, with moss crusts being most sensitive to environmental stress at our study sites. DISCUSSION: Given the complexity of patterns discovered in our study, it is especially important to consider a multitude of factors when comparing biocrust carbon exchange rates across studies. Understanding the dynam

Microbial interactions impact the functioning of both natural and engineered systems, yet our ability to directly monitor these highly dynamic and spatially resolved interactions in living cells is very limited. Here, we developed a synergistic approach coupling single-cell Raman microspectroscopy with 15N2 and 13CO2 stable isotope probing in a microfluidic culture system (RMCS-SIP) for live tracking of the occurrence, rate, and physiological shift of metabolic interactions in active microbial assemblages. Quantitative and robust Raman biomarkers specific for N2 and CO2 fixation in both model and bloom-forming diazotrophic cyanobacteria were established and cross-validated. By designing a prototype microfluidic chip allowing simultaneous microbial cultivation and single-cell Raman acquisition, we achieved temporal tracking of both intercellular (between heterocyst and vegetative cells of cyanobacteria) and interspecies N and C metabolite exchange (from diazotroph to heterotroph). Moreover, single-cell N and C fixation and bidirectional transfer rate in living cells were quantified via SIP-induced characteristic Raman shifts. Remarkably, RMCS captured physiological responses of metabolically active cells to nutrient stimuli through comprehensive metabolic profiling, providing multimodal information on the evolution of microbial interactions and functions under fluctuating conditions. This noninvasive RMCS-SIP is an advantageous approach for live-cell imaging and represents an important advancement in the single-cell microbiology field. This platform can be extended for real-time tracking of a wide range of microbial interactions with single-cell resolution and advances the understanding and manipulation of microbial interactions for societal benefit.

The unicellular picocyanobacterium Prochlorococcus is the most abundant photoautotroph and contributes substantially to global CO2 fixation. In the vast euphotic zones of the open ocean, Prochlorococcus converts CO2 into organic compounds and supports diverse organisms, forming an intricate network of interactions that regulate the magnitude of carbon cycling and storage in the ocean. An understanding of the biological interactions with Prochlorococcus is critical for accurately estimating the contributions of Prochlorococcus and interacting organisms to the marine carbon cycle. This review synthesizes the primary production contributed by Prochlorococcus in the global ocean. We outline recent progress on the interactions of Prochlorococcus with heterotrophic bacteria, phages, and grazers that multifacetedly determine Prochlorococcus carbon production and fate. We discuss that climate change might affect the biological interactions with Prochlorococcus and thus the marine carbon cycle. © 2023 Elsevier Ltd

We report the mixotrophic growth of Escherichia coli based on recombinant 2-oxoglutarate:ferredoxin oxidoreductase (OGOR) to assimilate CO2 using malate as an auxiliary carbon source and hydrogen as an energy source. We employ a long-term (~184 days) two-stage adaptive evolution to convert heterotrophic E. coli into mixotrophic E. coli. In the first stage of evolution with serine, diauxic growth emerges as a prominent feature. At the end of the second stage of evolution with malate, the strain exhibits mixotrophy with CO2 as an essential substrate for growth. We expect this work will open new possibilities in the utilization of OGOR for microbial CO2 assimilation and future hydrogen-based electro-microbial conversion.

Carbon fixation is the process by which CO2 is converted from a gas into biomass. The Calvin-Benson-Bassham cycle (CBB) is the dominant carbon-consuming pathway on Earth, driving >99.5% of the ∼120 billion tons of carbon that are converted to sugar by plants, algae, and cyanobacteria. The carboxylase enzyme in the CBB, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fixes one CO2 molecule per turn of the cycle into bioavailable sugars. Despite being critical to the assimilation of carbon, rubisco's kinetic rate is not very fast, limiting flux through the pathway. This bottleneck presents a paradox: Why has rubisco not evolved to be a better catalyst? Many hypothesize that the catalytic mechanism of rubisco is subject to one or more trade-offs and that rubisco variants have been optimized for their native physiological environment. Here, we review the evolution and biochemistry of rubisco through the lens of structure and mechanism in order to understand what trade-offs limit its improvement. We also review the many attempts to improve rubisco itself and thereby promote plant growth.

Syngas fermentation processes with acetogens represent a promising process for the reduction of CO₂ emissions alongside bulk chemical production. However, to fully realize this potential the thermodynamic limits of acetogens need to be considered when designing a fermentation process. An adjustable supply of H₂ as electron donor plays a key role in autotrophic product formation. In this study an anaerobic laboratory scale continuously stirred tank reactor was equipped with an All-in-One electrode allowing for in-situ H₂ generation via electrolysis. Furthermore, this system was coupled to online lactate measurements to control the co-culture of a recombinant lactate-producing Acetobacterium woodii strain and a lactate-consuming Clostridium drakei strain to produce caproate. When C. drakei was grown in batch cultivations with lactate as substrate, 1.6 g·L⁻¹ caproate were produced. Furthermore, lactate production of the A. woodii mutant strain could manually be stopped and reinitiated by controlling the electrolysis. Applying this automated process control, lactate production of the A. woodii mutant strain could be halted to achieve a steady lactate concentration. In a co-culture experiment with the A. woodii mutant strain and the C. drakei strain, the automated process control was able to dynamically react to changing lactate concentrations and adjust H₂ formation respectively. This study confirms the potential of C. drakei as medium chain fatty acid producer in a lactate-mediated, autotrophic co-cultivation with an engineered A. woodii strain. Moreover, the monitoring and control strategy presented in this study reinforces the case for autotrophically produced lactate as a transfer metabolite in defined co-cultivations for value-added chemical production.

The Central Arctic Ocean is a repository of rich and diverse biota, whose major portion is one of the most important drivers of global biogeochemical cycles, including carbon cycling. In this study, the functional potential of the microbiota to fix carbon with alternative carbon fixation pathways were investigated along with their chemolithotrophic characteristics. Samples from two expeditions MOSAiC & SAS-Oden (2019-2021) resulted in metagenomic data consisting of about 1200 mOTUs (metagenomic Operational Taxonomic Units). Kofamscan based annotation explained by KEGG pathways database was analysed to explore prevalence of the alternative Carbon Fixation pathways across different taxa. From the six carbon fixation pathways, three were consolidated for their presence (rTCA, DC-HP, HP-HP). In order to explain the other metabolic processes that these organisms employ to survive, a functional annotation tool for metabolic pathways was used. that Reductive Tricarboxylic acid cycle pathway was found to be most present and observed in 5 out of 6 mOTUs selected from filtering the dataset. The taxa include bacterial phyla Proteobacteria, Actinobacteria, Chloroflexota and Marinisomatota and archaeal phyla Thermoplasmota. However, for the other pathways and less studied organisms less resolution were observed across the dataset for the presence of other pathways. These CFPs found were also supported by oxidation of inorganic compounds with high redox potentials. This study provides a glimpse of the metabolic potential of the Central Arctic Ocean microbiota, shines light on the importance of understanding and unravelling the intricacies of this rich and diverse environment.

The Central Arctic Ocean is a repository of rich and diverse biota, whose major portion is one of the most important drivers of global biogeochemical cycles, including carbon cycling. In this study, the functional potential of the microbiota to fix carbon with alternative carbon fixation pathways were investigated along with their chemolithotrophic characteristics. Samples from two expeditions MOSAiC & SAS-Oden (2019-2021) resulted in metagenomic data consisting of about 1200 mOTUs (metagenomic Operational Taxonomic Units). Kofamscan based annotation explained by KEGG pathways database was analysed to explore prevalence of the alternative Carbon Fixation pathways across different taxa. From the six carbon fixation pathways, three were consolidated for their presence (rTCA, DC-HP, HP-HP). In order to explain the other metabolic processes that these organisms employ to survive, a functional annotation tool for metabolic pathways was used. that Reductive Tricarboxylic acid cycle pathway was found to be most present and observed in 5 out of 6 mOTUs selected from filtering the dataset. The taxa include bacterial phyla Proteobacteria, Actinobacteria, Chloroflexota and Marinisomatota and archaeal phyla Thermoplasmota. However, for the other pathways and less studied organisms less resolution were observed across the dataset for the presence of other pathways. These CFPs found were also supported by oxidation of inorganic compounds with high redox potentials. This study provides a glimpse of the metabolic potential of the Central Arctic Ocean microbiota, shines light on the importance of understanding and unravelling the intricacies of this rich and diverse environment.

Microalgae are attractive for the development of environmentally sustainable processes with the option for carbon fixation, given the photosynthetic capacity of these microorganisms. Nitrogen and phosphorus are key nutrients for microalgal growth as well as carbon fixation and product formations. These are also important nutrients in agriculture, the sustainable supply of which is a challenge. They are often lost in waste streams but can be effectively recovered using microalgae and made available for agriculture. In this investigation, we systematically examined the nitrate and phosphate uptake response of two halotolerant strains of Chlorella vulgaris exposed to four carbon and three nitrogen and phosphorus supply regimes to develop an understanding of the relationship between uptake and supply in two microalgae strains from a similar habitat. Markedly differential response to changes in carbon and nutrient supply regimes were noted between the supply regimes and the strains. Changes in carbon supply was found to significantly influence nitrogen and phosphorus uptake in the two strains. Kinetics of nutrient depletion and carbon fixation were observed to be dynamic and non-linear for both the strains, under high carbon and nutrient supply regimes. This investigation provides for the first time a clear evidence base for the role of supply regimes in the management of nutrient uptake in microalgae, including the relationship between the two, and the significance of carbon supply in nitrogen and phosphorus uptake by microalgae. This will enable development of sustainable processes and predictable strategies involving microalgae towards net-zero solutions, as well as nitrogen and phosphorus recovery from waste streams

Cyanobacteria are considered promising hosts for product synthesis directly from CO2 via photosynthetic carbon assimilation. The introduction of heterologous carbon sinks in terms of product synthesis has been reported to induce the so-called “carbon sink effect,” described as the release of unused photosynthetic capacity by the introduction of additional carbon. This effect is thought to arise from a limitation of carbon metabolism that represents a bottleneck in carbon and electron flow, thus enforcing a downregulation of photosynthetic efficiency. It is not known so far how the cellular source/sink balance under different growth conditions influences the extent of the carbon sink effect and in turn product formation from CO2, constituting a heterologous carbon sink. We compared the Synechocystis sp. strain PCC 6803 wild type (WT) with an engineered lactate-producing strain (SAA023) in defined metabolic states. Unexpectedly, high-light conditions combined with carbon limitation enabled additional carbon assimilation for lactate production without affecting biomass formation. Thus, a strong carbon sink effect only was observed under carbon and thus sink limitation, but not under high-sink conditions. We show that the carbon sink effect was accompanied by an increased rate of alternative electron flow (AEF). Thus, AEF plays a crucial role in the equilibration of source/sink imbalances, presumably via ATP/NADPH balancing. This study emphasizes that the evaluation of the biotechnological potential of cyanobacteria profits from cultivation approaches enabling the establishment of defined metabolic states and respective quantitative analytics. Factors stimulating photosynthesis and carbon fixation are discussed.

Acetobacterium woodii is known to produce mainly acetate from CO₂ and H₂, but the production of higher value chemicals is desired for the bioeconomy. Using chain-elongating bacteria, synthetic co-cultures have the potential to produce longer-chained products such as caproic acid. In this study, we present first results for a successful autotrophic co-cultivation of A. woodii mutants and a Clostridium drakei wild-type strain in a stirred-tank bioreactor for the production of caproic acid from CO₂ and H₂ via the intermediate lactic acid. For autotrophic lactate production, a recombinant A. woodii strain with a deleted Lct-dehydrogenase complex, which is encoded by the lctBCD genes, and an inserted D-lactate dehydrogenase (LdhD) originating from Leuconostoc mesenteroides, was used. Hydrogen for the process was supplied using an All-in-One electrode for in situ water electrolysis. Lactate concentrations as high as 0.5 g L–1 were achieved with the AiO-electrode, whereas 8.1 g L–1 lactate were produced with direct H₂ sparging in a stirred-tank bioreactor. Hydrogen limitation was identified in the AiO process. However, with cathode surface area enlargement or numbering-up of the electrode and on-demand hydrogen generation, this process has great potential for a true carbon-negative production of value chemicals from CO₂.

In this work, the simultaneous reduction of CO2 and NO to urea under mild conditions is achieved using nanoporous pp-HAp cubes as catalyst. In addition, a direct study of the effects of temperature, pressure, and time on the reaction is performed., En este trabajo, la reducción simultánea de CO2 y NO a urea en condiciones bajas se logra utilizando cubos nanoporosos de hidroxiapatita polarizada como catalizador. Además, se realiza un estudio directo de los efectos de la temperatura, la presión y el tiempo en la reacción., En aquest treball, s'aconsegueix la reducció simultània de CO2 i NO a urea en condicions baixes utilitzant cubs nanoporosos d'hidroxiapatita polaritzada com a catalitzador. A més, es realitza un estudi directe dels efectes de la temperatura, la pressió i el temps en la reacció.

Tropical successions are environments that cover large areas and are of great importance due to their ecological functions and extension. In Ecuador, studies on the carbon storage of these cosystems are scarce and most of the equations are developed for humid lowland areas. The aim of this research was to generate allometric models for estimation of aerial biomass of secondary montane forests in northwestern Ecuador. A forest inventory was carried out in three secondary forests of 18, 30, and 50 years old approximately. Allometric models were developed using diameter at breast height (DBH) and total height (Ht) as independent variables, and the organic matter and accumulated aerial carbon were estimated. The DAP2Ht combinatorial ariable was the best predictor variable according to the adjustment and alidation criteria. The model equations showed an R2 greater than 95 %, consequently the variables studied are reliable. It was determined that forests store 36.56, 70.36, and 156.27 Mg.C.ha-1 respectively, where the stem is the component with the highest storage with a range between 65.76 and 73.44%. Both branches and leaves represent between 20.98 - 25, 50% and 5.58 - 8.74% of the total aerial carbon respectively. These models could be applied effectively in ecosystems with similar environmental conditions., Las sucesiones tropicales son ambientes de gran importancia por sus funciones ecológicas y extensión. En el Ecuador, los estudios sobre el almacenamiento de carbono de estos ecosistemas son escasos y la mayoría de ecuaciones desarrolladas se oncentran en zonas húmedas de tierras bajas. El objetivo de la presente investigación fue generar modelos alométricos para la estimación de biomasa aérea de bosques secundarios montanos del noroccidente de Ecuador. Se realizó un inventario forestal en tres áreas de sucesión de 18, 30 y 50 años aproximadamente. Se construyeron modelos alométricos utilizando el diámetro a la altura del pecho (DAP) y la altura total (Ht) como variables independientes y se estimó la materia viva y el carbono aéreo acumulado. La variable combinatoria DAP2Ht fue la mejor predictora según los criterios de ajuste y validación. Las ecuaciones desarrolladas mostraron un R2 mayor al 95 %, por lo que son confiables para la estimación de las variables estudiadas. Se determinó que los bosques almacenan 36,56, 70,36 y 156,27 Mg.C.ha-1 respectivamente, siendo el fuste el componente de mayor almacenaje en un intervalo de 65,76 - 73,44 %, mientras que ramas y hojas representan un 20,98 - 25,50 % y 5,58 - 8,74 % del carbono aéreo total respectivamente. Estos modelos podrían aplicarse de manera efectiva en ecosistemas en condiciones ambientales similares.

Polarized hydroxyapatite (HAp) scaffolds with customized architecture at the nanoscale have been presented as a green alternative to conventional catalysts used for carbon and dinitrogen fixation. HAp printable inks with controlled nanoporosity and rheological properties have been successfully achieved by incorporating Pluronic hydrogel. Nanoporous scaffolds with good mechanical properties, as demonstrated by means of the nanoindentation technique, have been obtained by a sintering treatment and the posterior thermally induced polarization process. Their catalytic activity has been evaluated by considering three different key reactions (all in the presence of liquid water): (1) the synthesis of amino acids from gas mixtures of N2, CO2, and CH4; (2) the production of ethanol from gas mixtures of CO2 and CH4; and (3) the synthesis of ammonia from N2 gas. Comparison of the yields obtained by using nanoporous and nonporous (conventional) polarized HAp catalysts shows that both the nanoporosity and water absorption capacity of the former represent a drawback when the catalytic reaction requires auxiliary coating layers, as for example for the production of amino acids. This is because the surface nanopores achieved by incorporating Pluronic hydrogel are completely hindered by such auxiliary coating layers. On the contrary, the catalytic activity improves drastically for reactions in which the HAp-based scaffolds with enhanced nanoporosity are used as catalysts. More specifically, the carbon fixation from CO2 and CH4 to yield ethanol improves by more than 3000% when compared with nonporous HAp catalyst. Similarly, the synthesis of ammonia by dinitrogen fixation increases by more than 2000%. Therefore, HAp catalysts based on nanoporous scaffolds exhibit an extraordinary potential for scalability and industrial utilization for many chemical reactions, enabling a feasible green chemistry alternative to catalysts based on heavy metals., Peer Reviewed, Postprint (author's final draft)

Carbon fixation by chemoautotrophic microorganisms in the dark ocean has a major impact on global carbon cycling and ecological relationships in the ocean's interior. At present, six pathways of autotrophic carbon fixation have been found: the Calvin cycle, the reductive Acetyl-CoA or Wood-Ljungdahl pathway (rAcCoA), the reductive tricarboxylic acid cycle (rTCA), the 3-hydroxypropionate bicycle (3HP), the 3-hydroxypropionate/4hydroxybutyrate cycle (3HP/4HB), and the dicarboxylate/4-hydroxybutyrate cycle (DC/4HB). Although our knowledge about carbon fixation pathways in the ocean has increased significantly, carbon fixation pathways in the cold seeps are still unknown. In this study, we collected sediment samples from two cold seeps and one trough in the south China sea (SCS), and investigated with metagenomic and metagenome assembled genomes (MAGs). We found that six autotrophic carbon fixation pathways present in the cold seeps and trough with rTCA cycle was the most common pathway, whose genes were particularly high in the cold seeps and increased with sediment depths; the rAcCoA cycle mainly occurred in the cold seep regions, and the abundance of module genes increased with sediment depths. We also elucidated members of chemoautotrophic microorganisms involved in these six carbon-fixation pathways. The rAcCoA, rTCA and DC/4-HB cycles required significantly less energy probably play an important role in the deep-sea environments, especially in the cold seeps. This study provided metabolic insights into the carbon fixation pathways in the cold seeps, and laid the foundation for future detailed study on processes and rates of carbon fixation in the deep-sea ecosystems.

Tropical successions are environments that cover large areas and are of great importance due to their ecological functions and extension. In Ecuador, studies on the carbon storage of these cosystems are scarce and most of the equations are developed for humid lowland areas. The aim of this research was to generate allometric models for estimation of aerial biomass of secondary montane forests in northwestern Ecuador. A forest inventory was carried out in three secondary forests of 18, 30, and 50 years old approximately. Allometric models were developed using diameter at breast height (DBH) and total height (Ht) as independent variables, and the organic matter and accumulated aerial carbon were estimated. The DAP2Ht combinatorial ariable was the best predictor variable according to the adjustment and alidation criteria. The model equations showed an R2 greater than 95 %, consequently the variables studied are reliable. It was determined that forests store 36.56, 70.36, and 156.27 Mg.C.ha-1 respectively, where the stem is the component with the highest storage with a range between 65.76 and 73.44%. Both branches and leaves represent between 20.98 - 25, 50% and 5.58 - 8.74% of the total aerial carbon respectively. These models could be applied effectively in ecosystems with similar environmental conditions., Las sucesiones tropicales son ambientes de gran importancia por sus funciones ecológicas y extensión. En el Ecuador, los estudios sobre el almacenamiento de carbono de estos ecosistemas son escasos y la mayoría de ecuaciones desarrolladas se oncentran en zonas húmedas de tierras bajas. El objetivo de la presente investigación fue generar modelos alométricos para la estimación de biomasa aérea de bosques secundarios montanos del noroccidente de Ecuador. Se realizó un inventario forestal en tres áreas de sucesión de 18, 30 y 50 años aproximadamente. Se construyeron modelos alométricos utilizando el diámetro a la altura del pecho (DAP) y la altura total (Ht) como variables independientes y se estimó la materia viva y el carbono aéreo acumulado. La variable combinatoria DAP2Ht fue la mejor predictora según los criterios de ajuste y validación. Las ecuaciones desarrolladas mostraron un R2 mayor al 95 %, por lo que son confiables para la estimación de las variables estudiadas. Se determinó que los bosques almacenan 36,56, 70,36 y 156,27 Mg.C.ha-1 respectivamente, siendo el fuste el componente de mayor almacenaje en un intervalo de 65,76 - 73,44 %, mientras que ramas y hojas representan un 20,98 - 25,50 % y 5,58 - 8,74 % del carbono aéreo total respectivamente. Estos modelos podrían aplicarse de manera efectiva en ecosistemas en condiciones ambientales similares.

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Smith, A. R., Mueller, R., Fisk, M. R., & Colwell, F. S. Ancient metabolisms of a thermophilic subseafloor bacterium. Frontiers in Microbiology, 12, (2021): 764631, https://doi.org/10.3389/fmicb.2021.764631., The ancient origins of metabolism may be rooted deep in oceanic crust, and these early metabolisms may have persisted in the habitable thermal anoxic aquifer where conditions remain similar to those when they first appeared. The Wood–Ljungdahl pathway for acetogenesis is a key early biosynthetic pathway with the potential to influence ocean chemistry and productivity, but its contemporary role in oceanic crust is not well established. Here, we describe the genome of a novel acetogen from a thermal suboceanic aquifer olivine biofilm in the basaltic crust of the Juan de Fuca Ridge (JdFR) whose genome suggests it may utilize an ancient chemosynthetic lifestyle. This organism encodes the genes for the complete canonical Wood–Ljungdahl pathway, but is potentially unable to use sulfate and certain organic carbon sources such as lipids and carbohydrates to supplement its energy requirements, unlike other known acetogens. Instead, this organism may use peptides and amino acids for energy or as organic carbon sources. Additionally, genes involved in surface adhesion, the import of metallic cations found in Fe-bearing minerals, and use of molecular hydrogen, a product of serpentinization reactions between water and olivine, are prevalent within the genome. These adaptations are likely a reflection of local environmental micro-niches, where cells are adapted to life in biofilms using ancient chemosynthetic metabolisms dependent on H2 and iron minerals. Since this organism is phylogenetically distinct from a related acetogenic group of Clostridiales, we propose it as a new species, Candidatus Acetocimmeria pyornia., Metagenome sequencing was made possible by the Deep Carbon Observatory Census of Deep Life supported by the Alfred P. Sloan Foundation and was performed at the Marine Biological Laboratory (Woods Hole, MA, United States). This work was funded by NASA grant NNX08AO22G and a graduate fellowship from the NSF Center for Dark Energy Biosphere Investigations. The flow cells were funded under J0972A from the U.S. Science Support Program of Joint Oceanographic Institutions.

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Smith, A. R., Mueller, R., Fisk, M. R., & Colwell, F. S. Ancient metabolisms of a thermophilic subseafloor bacterium. Frontiers in Microbiology, 12, (2021): 764631, https://doi.org/10.3389/fmicb.2021.764631., The ancient origins of metabolism may be rooted deep in oceanic crust, and these early metabolisms may have persisted in the habitable thermal anoxic aquifer where conditions remain similar to those when they first appeared. The Wood–Ljungdahl pathway for acetogenesis is a key early biosynthetic pathway with the potential to influence ocean chemistry and productivity, but its contemporary role in oceanic crust is not well established. Here, we describe the genome of a novel acetogen from a thermal suboceanic aquifer olivine biofilm in the basaltic crust of the Juan de Fuca Ridge (JdFR) whose genome suggests it may utilize an ancient chemosynthetic lifestyle. This organism encodes the genes for the complete canonical Wood–Ljungdahl pathway, but is potentially unable to use sulfate and certain organic carbon sources such as lipids and carbohydrates to supplement its energy requirements, unlike other known acetogens. Instead, this organism may use peptides and amino acids for energy or as organic carbon sources. Additionally, genes involved in surface adhesion, the import of metallic cations found in Fe-bearing minerals, and use of molecular hydrogen, a product of serpentinization reactions between water and olivine, are prevalent within the genome. These adaptations are likely a reflection of local environmental micro-niches, where cells are adapted to life in biofilms using ancient chemosynthetic metabolisms dependent on H2 and iron minerals. Since this organism is phylogenetically distinct from a related acetogenic group of Clostridiales, we propose it as a new species, Candidatus Acetocimmeria pyornia., Metagenome sequencing was made possible by the Deep Carbon Observatory Census of Deep Life supported by the Alfred P. Sloan Foundation and was performed at the Marine Biological Laboratory (Woods Hole, MA, United States). This work was funded by NASA grant NNX08AO22G and a graduate fellowship from the NSF Center for Dark Energy Biosphere Investigations. The flow cells were funded under J0972A from the U.S. Science Support Program of Joint Oceanographic Institutions.

Tropical successions are environments that cover large areas and are of great importance due to their ecological functions and extension. In Ecuador, studies on the carbon storage of these cosystems are scarce and most of the equations are developed for humid lowland areas. The aim of this research was to generate allometric models for estimation of aerial biomass of secondary montane forests in northwestern Ecuador. A forest inventory was carried out in three secondary forests of 18, 30, and 50 years old approximately. Allometric models were developed using diameter at breast height (DBH) and total height (Ht) as independent variables, and the organic matter and accumulated aerial carbon were estimated. The DAP2Ht combinatorial ariable was the best predictor variable according to the adjustment and alidation criteria. The model equations showed an R2 greater than 95 %, consequently the variables studied are reliable. It was determined that forests store 36.56, 70.36, and 156.27 Mg.C.ha-1 respectively, where the stem is the component with the highest storage with a range between 65.76 and 73.44%. Both branches and leaves represent between 20.98 - 25, 50% and 5.58 - 8.74% of the total aerial carbon respectively. These models could be applied effectively in ecosystems with similar environmental conditions., Las sucesiones tropicales son ambientes de gran importancia por sus funciones ecológicas y extensión. En el Ecuador, los estudios sobre el almacenamiento de carbono de estos ecosistemas son escasos y la mayoría de ecuaciones desarrolladas se oncentran en zonas húmedas de tierras bajas. El objetivo de la presente investigación fue generar modelos alométricos para la estimación de biomasa aérea de bosques secundarios montanos del noroccidente de Ecuador. Se realizó un inventario forestal en tres áreas de sucesión de 18, 30 y 50 años aproximadamente. Se construyeron modelos alométricos utilizando el diámetro a la altura del pecho (DAP) y la altura total (Ht) como variables independientes y se estimó la materia viva y el carbono aéreo acumulado. La variable combinatoria DAP2Ht fue la mejor predictora según los criterios de ajuste y validación. Las ecuaciones desarrolladas mostraron un R2 mayor al 95 %, por lo que son confiables para la estimación de las variables estudiadas. Se determinó que los bosques almacenan 36,56, 70,36 y 156,27 Mg.C.ha-1 respectivamente, siendo el fuste el componente de mayor almacenaje en un intervalo de 65,76 - 73,44 %, mientras que ramas y hojas representan un 20,98 - 25,50 % y 5,58 - 8,74 % del carbono aéreo total respectivamente. Estos modelos podrían aplicarse de manera efectiva en ecosistemas en condiciones ambientales similares.

Cyanobacteria are one of the most promising microorganisms to produce biofuels and renewable chemicals due to their oxygenic autotrophic growth properties. However, to rely on photosynthesis, which is one of the main reasons for slow growth, low carbon assimlation rate and low production, is a bottleneck. To address this challenge, optimizing the Calvin-Benson-Bassham (CBB) cycle is one of the strategies since it is the main carbon fixation pathway. In a previous study, we showed that overexpression of either aldolase (FBA), transketolase (TK), or fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), enzymes responsible for RuBP regeneration and vital for controlling the CBB carbon flux, led to higher production rates and titers in ethanol producing strains of Synechocystis PCC 6803. In the present study, we investigated the combined effects of the above enzymes on ethanol production in Synechocystis PCC 6803. The ethanol production of the strains overexpressing two CBB enzymes (FBA + TK, FBP/SBPase + FBA or FBP/SBPase + TK) was higher than the respective control strains, overexpressing either FBA or TK. The co-overexpression of FBA and TK led to more than 9 times higher ethanol production compared to the overexpression of FBA. Compared to TK the respective increase is 4 times more ethanol production. Overexpression of FBP/SBPase in combination with FBA showed 2.5 times higher ethanol production compared to FBA. Finally, co-overexpression of FBP/SBPase and TK reached about twice the production of ethanol compared to overexpression of only TK. This study clearly demonstrates that overexpression of two selected CBB enzymes leads to significantly increased ethanol production compared to overexpression of a single CBB enzyme.

Cyanobacteria are one of the most promising microorganisms to produce biofuels and renewable chemicals due to their oxygenic autotrophic growth properties. However, to rely on photosynthesis, which is one of the main reasons for slow growth, low carbon assimlation rate and low production, is a bottleneck. To address this challenge, optimizing the Calvin-Benson-Bassham (CBB) cycle is one of the strategies since it is the main carbon fixation pathway. In a previous study, we showed that overexpression of either aldolase (FBA), transketolase (TK), or fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), enzymes responsible for RuBP regeneration and vital for controlling the CBB carbon flux, led to higher production rates and titers in ethanol producing strains of Synechocystis PCC 6803. In the present study, we investigated the combined effects of the above enzymes on ethanol production in Synechocystis PCC 6803. The ethanol production of the strains overexpressing two CBB enzymes (FBA + TK, FBP/SBPase + FBA or FBP/SBPase + TK) was higher than the respective control strains, overexpressing either FBA or TK. The co-overexpression of FBA and TK led to more than 9 times higher ethanol production compared to the overexpression of FBA. Compared to TK the respective increase is 4 times more ethanol production. Overexpression of FBP/SBPase in combination with FBA showed 2.5 times higher ethanol production compared to FBA. Finally, co-overexpression of FBP/SBPase and TK reached about twice the production of ethanol compared to overexpression of only TK. This study clearly demonstrates that overexpression of two selected CBB enzymes leads to significantly increased ethanol production compared to overexpression of a single CBB enzyme.

Cyanobacteria are one of the most promising microorganisms to produce biofuels and renewable chemicals due to their oxygenic autotrophic growth properties. However, to rely on photosynthesis, which is one of the main reasons for slow growth, low carbon assimlation rate and low production, is a bottleneck. To address this challenge, optimizing the Calvin-Benson-Bassham (CBB) cycle is one of the strategies since it is the main carbon fixation pathway. In a previous study, we showed that overexpression of either aldolase (FBA), transketolase (TK), or fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), enzymes responsible for RuBP regeneration and vital for controlling the CBB carbon flux, led to higher production rates and titers in ethanol producing strains of Synechocystis PCC 6803. In the present study, we investigated the combined effects of the above enzymes on ethanol production in Synechocystis PCC 6803. The ethanol production of the strains overexpressing two CBB enzymes (FBA + TK, FBP/SBPase + FBA or FBP/SBPase + TK) was higher than the respective control strains, overexpressing either FBA or TK. The co-overexpression of FBA and TK led to more than 9 times higher ethanol production compared to the overexpression of FBA. Compared to TK the respective increase is 4 times more ethanol production. Overexpression of FBP/SBPase in combination with FBA showed 2.5 times higher ethanol production compared to FBA. Finally, co-overexpression of FBP/SBPase and TK reached about twice the production of ethanol compared to overexpression of only TK. This study clearly demonstrates that overexpression of two selected CBB enzymes leads to significantly increased ethanol production compared to overexpression of a single CBB enzyme.

Cyanobacteria are one of the most promising microorganisms to produce biofuels and renewable chemicals due to their oxygenic autotrophic growth properties. However, to rely on photosynthesis, which is one of the main reasons for slow growth, low carbon assimlation rate and low production, is a bottleneck. To address this challenge, optimizing the Calvin-Benson-Bassham (CBB) cycle is one of the strategies since it is the main carbon fixation pathway. In a previous study, we showed that overexpression of either aldolase (FBA), transketolase (TK), or fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), enzymes responsible for RuBP regeneration and vital for controlling the CBB carbon flux, led to higher production rates and titers in ethanol producing strains of Synechocystis PCC 6803. In the present study, we investigated the combined effects of the above enzymes on ethanol production in Synechocystis PCC 6803. The ethanol production of the strains overexpressing two CBB enzymes (FBA + TK, FBP/SBPase + FBA or FBP/SBPase + TK) was higher than the respective control strains, overexpressing either FBA or TK. The co-overexpression of FBA and TK led to more than 9 times higher ethanol production compared to the overexpression of FBA. Compared to TK the respective increase is 4 times more ethanol production. Overexpression of FBP/SBPase in combination with FBA showed 2.5 times higher ethanol production compared to FBA. Finally, co-overexpression of FBP/SBPase and TK reached about twice the production of ethanol compared to overexpression of only TK. This study clearly demonstrates that overexpression of two selected CBB enzymes leads to significantly increased ethanol production compared to overexpression of a single CBB enzyme.

Cyanobacteria are one of the most promising microorganisms to produce biofuels and renewable chemicals due to their oxygenic autotrophic growth properties. However, to rely on photosynthesis, which is one of the main reasons for slow growth, low carbon assimlation rate and low production, is a bottleneck. To address this challenge, optimizing the Calvin-Benson-Bassham (CBB) cycle is one of the strategies since it is the main carbon fixation pathway. In a previous study, we showed that overexpression of either aldolase (FBA), transketolase (TK), or fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), enzymes responsible for RuBP regeneration and vital for controlling the CBB carbon flux, led to higher production rates and titers in ethanol producing strains of Synechocystis PCC 6803. In the present study, we investigated the combined effects of the above enzymes on ethanol production in Synechocystis PCC 6803. The ethanol production of the strains overexpressing two CBB enzymes (FBA + TK, FBP/SBPase + FBA or FBP/SBPase + TK) was higher than the respective control strains, overexpressing either FBA or TK. The co-overexpression of FBA and TK led to more than 9 times higher ethanol production compared to the overexpression of FBA. Compared to TK the respective increase is 4 times more ethanol production. Overexpression of FBP/SBPase in combination with FBA showed 2.5 times higher ethanol production compared to FBA. Finally, co-overexpression of FBP/SBPase and TK reached about twice the production of ethanol compared to overexpression of only TK. This study clearly demonstrates that overexpression of two selected CBB enzymes leads to significantly increased ethanol production compared to overexpression of a single CBB enzyme.

Fully understanding the environmental implications of engineered nanomaterials is crucial for their safe and sustainable use. Cyanobacteria, as the pioneers of the planet earth, play important roles in global carbon and nitrogen cycling. Here, we evaluated the biological effects of molybdenum disulfide (MoS2) nanosheets on a N2-fixation cyanobacteria (Nostoc sphaeroides) by monitoring growth and metabolome changes. MoS2 nanosheets did not exert overt toxicity to Nostoc at the tested doses (0.1 and 1 mg/L). On the contrary, the intrinsic enzyme-like activities and semiconducting properties of MoS2 nanosheets promoted the metabolic processes of Nostoc, including enhancing CO2-fixation-related Calvin cycle metabolic pathway. Meanwhile, MoS2 boosted the production of a range of biochemicals, including sugars, fatty acids, amino acids, and other valuable end products. The altered carbon metabolism subsequently drove proportional changes in nitrogen metabolism in Nostoc. These intracellular metabolic changes could potentially alter global C and N cycles. The findings of this study shed light on the nature and underlying mechanisms of bio-nanoparticle interactions, and offer the prospect of utilization bio-nanomaterials for efficient CO2 sequestration and sustainable biochemical production.

CO2 capture and utilization has been a hot topic for decades. Photoautotrophic microalgae production is a carbon-friendly industry that converts CO2 to biomass with value-added proteins and bio-oils. This study aims at facilitating the microalgal fixation of CO2 via two different methods. Firstly, the thesis investigates a liquid-liquid membrane contactor to deliver CO2 captured from large point sources by a chemical solvent to the microalgae culture. The CO2 mass transfer coefficients of two membrane modules with varied membrane areas were studied. The solvent flow was controlled by pH feedback. The microalgae productivity and CO2 utilization efficiency with different membrane modules and CO2 supply conditions were compared. Three methods to immobilize carbonic anhydrase (CA), as a CO2 capturing enzyme are then investigated. These methods are layer-by-layer electrostatic adsorption, hollow fiber entrapment, and tannic acid-mediated assembly. The methods are compared based on enzyme activity, cyclic stability, and their enhancement to microalgae production. A fourth immobilization method combining CA-glutaraldehyde cross-linking and buoyant calcium alginate beads encapsulation is developed and used to demonstrate the use of interfacial CA catalyzed atmospheric CO2 fixation by microalgae. The CA beads were characterized with microscopic, protein bonding and activity assays. The biomass productivity, dissolved carbon concentration stability, culture water loss and enzyme cyclic performance were monitored, and the cost was calculated to assess the potential of applying immobilized CA in the microalgae industry. It was found that CA-encapsulated buoyant alginate beads provided a recyclable, stable and effective additive to the microalgal pond. Finally, a study is conducted on inhibitive effects of the microalgae medium on CA activity by the Wilbur-Anderson assay. A substitute culture medium was suggested based on the assay results and the performance of CA in the new me

Carbon dioxide (CO2) emissions from human activities are driving climate change, but the pending crisis could be mitigated by a circular carbon economy where released CO2 is recycled into commodity chemicals. Autotrophic microbes can make a contribution by producing chemicals, such as biofuels, from CO2 and renewable energy. The primary natural CO2 fixation pathway is the Calvin cycle, in which the enzyme Rubisco carboxylates ribulose-1,5-bisphosphate. The present investigation used computational systems biology methods to map adaptations and constraints in autotrophic microbial metabolism based on the Calvin cycle. First, the metabolic network of the Calvin cycle-capable photoautotrophic cyanobacterium Synechocystis was contrasted with that of heterotrophic E. coli. Intracellular metabolite concentration ranges differed, leading to different capacity to provide thermodynamic driving forces to chemical production pathways. Second, the Calvin cycle in Synechocystis was modeled kinetically, showing that certain enzyme saturation and metabolite levels, for example high ribulose-1,5-bisphosphate concentration, were detrimental to stability. Control over reaction rates was distributed, but making certain enzymes faster, for example fructose-1,6-bisphosphatase, could increase overall carbon fixation rate. Third, Synechocystis was starved of CO2 and ribosome profiling was used to track the effect on translation. Stress response and CO2 uptake were upregulated, but constant Rubisco expression and ribosome pausing in 5' untranslated regions indicated readiness for reappearance of CO2. Finally, microbial genomes with and without the Calvin cycle were contrasted, revealing metabolic, energetic, and regulatory adaptations that describe the properties of a functional autotroph. These findings provide a background for future study and engineering of autotrophs for direct conversion of CO2 into commodity chemicals., Utsläpp av koldioxid (CO2) från mänskliga aktiviteter driver klimatförändringarna, men den stundande krisen skulle kunna mildras av en cirkulär kolekonomi där CO2 som släpps ut återvinns till råvarukemikalier. Autotrofa mikrober kan bidra genom att producera kemikalier, såsom biobränslen, från CO2 och förnybar energi. Den primära naturliga syntesvägen för CO2-fixering är calvincykeln, i vilken enzymet Rubisco karboxylerar ribulos-1,5-bisfosfat. Undersökningen som ligger till grund för denna avhandling använde systembiologiska beräkningsmetoder för att kartlägga anpassningar och begränsningar i autotrof mikrobiell metabolism baserad på calvincykeln. För det första kontrasterades det metaboliska nätverket hos den calvincykelkapabla fotoautotrofa cyanobakterien Synechocystis med det hos heterotrofen E. coli. De intracellulära metabolitkoncentrationerna var olika, vilket ledde till olika kapacitet att bistå med termodynamisk drivkraft till kemiska syntesvägar. För det andra modellerades calvincykeln i Synechocystis kinetiskt, vilket visade att vissa enzymsatureringsnivåer och metabolitkoncentrationer, bland annat hög ribulos-1,5-bisfosfatkoncentration, motverkade stabiliteten. Kontroll över reaktionshastigheter var distribuerad, men ökning av hastigheten hos vissa enzymer, till exempel fruktos-1,6-bisfosfatas, skulle kunna öka den generalla kolfixeringshastigheten. För det tredje svältes Synechocystis på CO2 och ribosomprofilering användes för att följa effekten på translationen. Stressrespons och CO2-upptag uppreglerades, men konstant uttryck av Rubisco och pausning av ribosomer i de icketranslaterade 5'-regionerna indikerade beredskap för ett återuppträdande av CO2. Slutligen jämfördes mikrobiella genom med och utan calvincykeln, vilket avslöjade metaboliska, energetiska, och regulatoriska anpassningar som beskriver egenskaperna hos en funktionell autotrof. Dessa upptäckter ger en bakgrund för framtida studier och ingenjörsmässig design av autotrofer för direkt omvand, QC 2021-04-07

Under diazotrophic conditions, the model filamentous, heterocystforming cyanobacterium Anabaena sp. strain PCC 7120 develops a metabolic strategy based on the physical separation of the processes of oxygenic photosynthesis, in vegetative cells, and N2 fixation, in heterocysts. This strategy requires the exchange of carbon and nitrogen metabolites and their distribution along the filaments, which takes place through molecular diffusion via septal junctions involving FraCD proteins. Here, Anabaena was incubated in a time course (up to 20 h) with [13C]bicarbonate and 15N2 and analyzed by secondary ion mass spectrometry imaging (SIMS) (large-geometry SIMS [LG-SIMS] and NanoSIMS) to quantify C and N assimilation and distribution in the filaments. The 13C/12C and 15N/14N ratios measured in wild-type filaments showed a general increase with time. The enrichment was relatively homogeneous in vegetative cells along individual filaments, while it was reduced in heterocysts. Heterocysts, however, accumulated recently fixed N at their poles, in which the cyanophycin plug [multi-L-arginyl-poly(L-aspartic acid)] is located. In contrast to the rather homogeneous label found along stretches of vegetative cells, 13C/12C and 15N/14N ratios were significantly different between filaments both at the same and different time points, showing high variability in metabolic states. A fraC fraD mutant did not fix N2, and the 13C/12C ratio was homogeneous along the filament, including the heterocyst in contrast to the wild type. Our results show the consumption of reduced C in the heterocysts associated with the fixation and export of fixed N and present an unpredicted heterogeneity of cellular metabolic activity in different filaments of an Anabaena culture under controlled conditions. IMPORTANCE Filamentous, heterocyst-forming cyanobacteria represent a paradigm of multicellularity in the prokaryotic world. Physiological studies at the cellular level in model organisms are crucial to understa

Chloroflexus aggregans is a metabolically versatile, thermophilic, anoxygenic phototrophic member of the phylum Chloroflexota (formerly Chloroflexi), which can grow photoheterotrophically, photoautotrophically, chemoheterotrophically, and chemoautotrophically. In hot spring-associated microbial mats, C. aggregans co-exists with oxygenic cyanobacteria under dynamic micro-environmental conditions. To elucidate the predominant growth modes of C. aggregans, relative transcription levels of energy metabolism-and CO2 fixation-related genes were studied in Nakabusa Hot Springs microbial mats over a diel cycle and correlated with microscale in situ measurements of O2 and light. Metatranscriptomic analyses indicated two periods with different modes of energy metabolism of C. aggregans: (1) phototrophy around midday and (2) chemotrophy in the early morning hours. During midday, C. aggregans mainly employed photoheterotrophy when the microbial mats were hyperoxic (400–800 µmol L−1 O2 ). In the early morning hours, relative transcription peaks of genes encoding uptake hydrogenase, key enzymes for carbon fixation, respiratory complexes as well as enzymes for TCA cycle and acetate uptake suggest an aerobic chemomixotrophic lifestyle. This is the first in situ study of the versatile energy metabolism of C. aggregans based on gene transcription patterns. The results provide novel insights into the metabolic flexibility of these filamentous anoxygenic phototrophs that thrive under dynamic environmental conditions.

Under diazotrophic conditions, the model filamentous, heterocystforming cyanobacterium Anabaena sp. strain PCC 7120 develops a metabolic strategy based on the physical separation of the processes of oxygenic photosynthesis, in vegetative cells, and N-2 fixation, in heterocysts. This strategy requires the exchange of carbon and nitrogen metabolites and their distribution along the filaments, which takes place through molecular diffusion via septal junctions involving FraCD proteins. Here, Anabaena was incubated in a time course (up to 20 h) with [C-13]bicarbonate and N-15(2) and analyzed by secondary ion mass spectrometry imaging (SIMS) (large-geometry SIMS [LG- SIMS] and NanoSIMS) to quantify C and N assimilation and distribution in the filaments. The C-13/C-12 and N-15/N-14 ratios measured in wild-type filaments showed a general increase with time. The enrichment was relatively homogeneous in vegetative cells along individual filaments, while it was reduced in heterocysts. Heterocysts, however, accumulated recently fixed N at their poles, in which the cyanophycin plug [multi-L-arginyl-poly(L-aspartic acid)] is located. In contrast to the rather homogeneous label found along stretches of vegetative cells, C-13/C-12 and N-15/N-14 ratios were significantly different between filaments both at the same and different time points, showing high variability in metabolic states. A fraC fraD mutant did not fix N-2, and the C-13/C-12 ratio was homogeneous along the filament, including the heterocyst in contrast to the wild type. Our results show the consumption of reduced C in the heterocysts associated with the fixation and export of fixed N and present an unpredicted heterogeneity of cellular metabolic activity in different filaments of an Anabaena culture under controlled conditions. IMPORTANCE Filamentous, heterocyst- forming cyanobacteria represent a paradigm of multicellularity in the prokaryotic world. Physiological studies at the cellular level in model organisms are c

The increased human dependence on fossil-based fuels, industrial chemicals, and structural materials has caused a drastic increase in atmospheric CO2 levels during the past 50 years. Consequences of this have been observed in the disruption of terrestrial and aquatic ecosystems. Bioengineering of photosynthetic organisms capable of converting CO2 into biomass and value-added organic molecules provide a promising solution to the problem. However, faster CO2 fixation rates are needed in order to meet industrial scale production capacities. Previously published studies have mainly focused on the overexpression of Calvin cycle enzymes or engineering of ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) whereas fewer have looked into the engineering of enzymes downstream of RuBisCO. In this thesis, a malachite green-based colorimetric assay was optimized and used in characterizing the kinetic properties of wild type bifunctional cyanobacterial fructose/sedoheptulose bisphosphatase (cy-F/SBPase) and variants of the enzyme with respect to the hydrolysis of one of its substrates (fructose 1,6-bisphosphate). The assay was validated by successfully assaying a set of previously kinetically characterized cy-F/SBPase variants as well as the wild type. Subsequently, a group of rationally de novo designed mutants of the enzyme was kinetically characterized. This way, a crude understanding of new aspects of the protein structure-function relationship was established, which could potentially guide future protein engineering efforts on cy-F/SBPase. The developed malachite green assay could also complement such research efforts as a result of its multiplexed format which is desirable when assaying larger protein libraries., De årliga koldioxidutsläppen har under de senaste 50 åren ökat med nästan 200% som påföljd av människans användning av fossila bränslen och råvaror. De negativa konsekvenserna av denna ökning syns idag världen över i land- och havsbaserade ekosystem, samt inom den biologiska mångfalden. Forskningens rön visar lovande lösningar genom bioteknisk ingenjörskonst av fotosyntetiska organismer med förmågan att ta upp och konvertera koldioxid till biomassa och förädlade kemikalier. Trots att dessa resultat ser lovande ut behövs mer forskning för att skala upp denna process i en industriell miljö. I detta examensarbete undersöks ett metaboliskt enzym som är involverat i att påskynda fotosyntesen. Målet var huvudsakligen att optimera och testa en färgbaserad detektionsmetod som kan mäta den enzymatiska aktiviteten. Därefter mättes den enzymatiska aktiviteten av en rad nyskapade varianter av enzymet för att undersöka hur individuella fragment inom enzymet påverkar den katalytiska förmågan. Studiens resultat lyckades erhålla en grundläggande förståelse för hur vissa fragment påverkar enzymets katalysförmåga, som i sin tur skulle kunna tillämpas i framtida experiment inom enzymoptimering.

Knowledge of the genetic basis for autotrophic metabolism is valuable since it relates to both the emergence of life and to the metabolic engineering challenge of incorporating CO2 as a potential substrate for biorefining. The most common CO2 fixation pathway is the Calvin cycle, which utilizes Rubisco and phosphoribulokinase enzymes. We searched thousands of microbial genomes and found that 6.0% contained the Calvin cycle. We then contrasted the genomes of Calvin cycle-positive, non-cyanobacterial microbes and their closest relatives by enrichment analysis, ancestral character estimation, and random forest machine learning, to explore genetic adaptations associated with acquisition of the Calvin cycle. The Calvin cycle overlaps with the pentose phosphate pathway and glycolysis, and we could confirm positive associations with fructose-1,6-bisphosphatase, aldolase, and transketolase, constituting a conserved operon, as well as ribulose-phosphate 3-epimerase, ribose-5-phosphate isomerase, and phosphoglycerate kinase. Additionally, carbohydrate storage enzymes, carboxysome proteins (that raise CO2 concentration around Rubisco), and Rubisco activases CbbQ and CbbX accompanied the Calvin cycle. Photorespiration did not appear to be adapted specifically for the Calvin cycle in the non-cyanobacterial microbes under study. Our results suggest that chemoautotrophy in Calvin cycle-positive organisms was commonly enabled by hydrogenase, and less commonly ammonia monooxygenase (nitrification). The enrichment of specific DNA-binding domains indicated Calvin-cycle associated genetic regulation. Metabolic regulatory adaptations were illustrated by negative correlation to AraC and the enzyme arabinose-5-phosphate isomerase, which suggests a downregulation of the metabolite arabinose-5-phosphate, which may interfere with the Calvin cycle through enzyme inhibition and substrate competition. Certain domains of unknown function that were found to be important in the analysis may indica, QC 20210406

Carbon dioxide (CO2) emissions from human activities are driving climate change, but the pending crisis could be mitigated by a circular carbon economy where released CO2 is recycled into commodity chemicals. Autotrophic microbes can make a contribution by producing chemicals, such as biofuels, from CO2 and renewable energy. The primary natural CO2 fixation pathway is the Calvin cycle, in which the enzyme Rubisco carboxylates ribulose-1,5-bisphosphate. The present investigation used computational systems biology methods to map adaptations and constraints in autotrophic microbial metabolism based on the Calvin cycle. First, the metabolic network of the Calvin cycle-capable photoautotrophic cyanobacterium Synechocystis was contrasted with that of heterotrophic E. coli. Intracellular metabolite concentration ranges differed, leading to different capacity to provide thermodynamic driving forces to chemical production pathways. Second, the Calvin cycle in Synechocystis was modeled kinetically, showing that certain enzyme saturation and metabolite levels, for example high ribulose-1,5-bisphosphate concentration, were detrimental to stability. Control over reaction rates was distributed, but making certain enzymes faster, for example fructose-1,6-bisphosphatase, could increase overall carbon fixation rate. Third, Synechocystis was starved of CO2 and ribosome profiling was used to track the effect on translation. Stress response and CO2 uptake were upregulated, but constant Rubisco expression and ribosome pausing in 5' untranslated regions indicated readiness for reappearance of CO2. Finally, microbial genomes with and without the Calvin cycle were contrasted, revealing metabolic, energetic, and regulatory adaptations that describe the properties of a functional autotroph. These findings provide a background for future study and engineering of autotrophs for direct conversion of CO2 into commodity chemicals., Utsläpp av koldioxid (CO2) från mänskliga aktiviteter driver klimatförändringarna, men den stundande krisen skulle kunna mildras av en cirkulär kolekonomi där CO2 som släpps ut återvinns till råvarukemikalier. Autotrofa mikrober kan bidra genom att producera kemikalier, såsom biobränslen, från CO2 och förnybar energi. Den primära naturliga syntesvägen för CO2-fixering är calvincykeln, i vilken enzymet Rubisco karboxylerar ribulos-1,5-bisfosfat. Undersökningen som ligger till grund för denna avhandling använde systembiologiska beräkningsmetoder för att kartlägga anpassningar och begränsningar i autotrof mikrobiell metabolism baserad på calvincykeln. För det första kontrasterades det metaboliska nätverket hos den calvincykelkapabla fotoautotrofa cyanobakterien Synechocystis med det hos heterotrofen E. coli. De intracellulära metabolitkoncentrationerna var olika, vilket ledde till olika kapacitet att bistå med termodynamisk drivkraft till kemiska syntesvägar. För det andra modellerades calvincykeln i Synechocystis kinetiskt, vilket visade att vissa enzymsatureringsnivåer och metabolitkoncentrationer, bland annat hög ribulos-1,5-bisfosfatkoncentration, motverkade stabiliteten. Kontroll över reaktionshastigheter var distribuerad, men ökning av hastigheten hos vissa enzymer, till exempel fruktos-1,6-bisfosfatas, skulle kunna öka den generalla kolfixeringshastigheten. För det tredje svältes Synechocystis på CO2 och ribosomprofilering användes för att följa effekten på translationen. Stressrespons och CO2-upptag uppreglerades, men konstant uttryck av Rubisco och pausning av ribosomer i de icketranslaterade 5'-regionerna indikerade beredskap för ett återuppträdande av CO2. Slutligen jämfördes mikrobiella genom med och utan calvincykeln, vilket avslöjade metaboliska, energetiska, och regulatoriska anpassningar som beskriver egenskaperna hos en funktionell autotrof. Dessa upptäckter ger en bakgrund för framtida studier och ingenjörsmässig design av autotrofer för direkt omvand, QC 2021-04-07

Chloroflexus aggregans is a metabolically versatile, thermophilic, anoxygenic phototrophic member of the phylum Chloroflexota (formerly Chloroflexi), which can grow photoheterotrophically, photoautotrophically, chemoheterotrophically, and chemoautotrophically. In hot spring-associated microbial mats, C. aggregans co-exists with oxygenic cyanobacteria under dynamic micro-environmental conditions. To elucidate the predominant growth modes of C. aggregans, relative transcription levels of energy metabolism-and CO2 fixation-related genes were studied in Nakabusa Hot Springs microbial mats over a diel cycle and correlated with microscale in situ measurements of O2 and light. Metatranscriptomic analyses indicated two periods with different modes of energy metabolism of C. aggregans: (1) phototrophy around midday and (2) chemotrophy in the early morning hours. During midday, C. aggregans mainly employed photoheterotrophy when the microbial mats were hyperoxic (400–800 µmol L−1 O2 ). In the early morning hours, relative transcription peaks of genes encoding uptake hydrogenase, key enzymes for carbon fixation, respiratory complexes as well as enzymes for TCA cycle and acetate uptake suggest an aerobic chemomixotrophic lifestyle. This is the first in situ study of the versatile energy metabolism of C. aggregans based on gene transcription patterns. The results provide novel insights into the metabolic flexibility of these filamentous anoxygenic phototrophs that thrive under dynamic environmental conditions.

Carbon dioxide (CO2) emissions from human activities are driving climate change, but the pending crisis could be mitigated by a circular carbon economy where released CO2 is recycled into commodity chemicals. Autotrophic microbes can make a contribution by producing chemicals, such as biofuels, from CO2 and renewable energy. The primary natural CO2 fixation pathway is the Calvin cycle, in which the enzyme Rubisco carboxylates ribulose-1,5-bisphosphate. The present investigation used computational systems biology methods to map adaptations and constraints in autotrophic microbial metabolism based on the Calvin cycle. First, the metabolic network of the Calvin cycle-capable photoautotrophic cyanobacterium Synechocystis was contrasted with that of heterotrophic E. coli. Intracellular metabolite concentration ranges differed, leading to different capacity to provide thermodynamic driving forces to chemical production pathways. Second, the Calvin cycle in Synechocystis was modeled kinetically, showing that certain enzyme saturation and metabolite levels, for example high ribulose-1,5-bisphosphate concentration, were detrimental to stability. Control over reaction rates was distributed, but making certain enzymes faster, for example fructose-1,6-bisphosphatase, could increase overall carbon fixation rate. Third, Synechocystis was starved of CO2 and ribosome profiling was used to track the effect on translation. Stress response and CO2 uptake were upregulated, but constant Rubisco expression and ribosome pausing in 5' untranslated regions indicated readiness for reappearance of CO2. Finally, microbial genomes with and without the Calvin cycle were contrasted, revealing metabolic, energetic, and regulatory adaptations that describe the properties of a functional autotroph. These findings provide a background for future study and engineering of autotrophs for direct conversion of CO2 into commodity chemicals., Utsläpp av koldioxid (CO2) från mänskliga aktiviteter driver klimatförändringarna, men den stundande krisen skulle kunna mildras av en cirkulär kolekonomi där CO2 som släpps ut återvinns till råvarukemikalier. Autotrofa mikrober kan bidra genom att producera kemikalier, såsom biobränslen, från CO2 och förnybar energi. Den primära naturliga syntesvägen för CO2-fixering är calvincykeln, i vilken enzymet Rubisco karboxylerar ribulos-1,5-bisfosfat. Undersökningen som ligger till grund för denna avhandling använde systembiologiska beräkningsmetoder för att kartlägga anpassningar och begränsningar i autotrof mikrobiell metabolism baserad på calvincykeln. För det första kontrasterades det metaboliska nätverket hos den calvincykelkapabla fotoautotrofa cyanobakterien Synechocystis med det hos heterotrofen E. coli. De intracellulära metabolitkoncentrationerna var olika, vilket ledde till olika kapacitet att bistå med termodynamisk drivkraft till kemiska syntesvägar. För det andra modellerades calvincykeln i Synechocystis kinetiskt, vilket visade att vissa enzymsatureringsnivåer och metabolitkoncentrationer, bland annat hög ribulos-1,5-bisfosfatkoncentration, motverkade stabiliteten. Kontroll över reaktionshastigheter var distribuerad, men ökning av hastigheten hos vissa enzymer, till exempel fruktos-1,6-bisfosfatas, skulle kunna öka den generalla kolfixeringshastigheten. För det tredje svältes Synechocystis på CO2 och ribosomprofilering användes för att följa effekten på translationen. Stressrespons och CO2-upptag uppreglerades, men konstant uttryck av Rubisco och pausning av ribosomer i de icketranslaterade 5'-regionerna indikerade beredskap för ett återuppträdande av CO2. Slutligen jämfördes mikrobiella genom med och utan calvincykeln, vilket avslöjade metaboliska, energetiska, och regulatoriska anpassningar som beskriver egenskaperna hos en funktionell autotrof. Dessa upptäckter ger en bakgrund för framtida studier och ingenjörsmässig design av autotrofer för direkt omvand, QC 2021-04-07

Knowledge of the genetic basis for autotrophic metabolism is valuable since it relates to both the emergence of life and to the metabolic engineering challenge of incorporating CO2 as a potential substrate for biorefining. The most common CO2 fixation pathway is the Calvin cycle, which utilizes Rubisco and phosphoribulokinase enzymes. We searched thousands of microbial genomes and found that 6.0% contained the Calvin cycle. We then contrasted the genomes of Calvin cycle-positive, non-cyanobacterial microbes and their closest relatives by enrichment analysis, ancestral character estimation, and random forest machine learning, to explore genetic adaptations associated with acquisition of the Calvin cycle. The Calvin cycle overlaps with the pentose phosphate pathway and glycolysis, and we could confirm positive associations with fructose-1,6-bisphosphatase, aldolase, and transketolase, constituting a conserved operon, as well as ribulose-phosphate 3-epimerase, ribose-5-phosphate isomerase, and phosphoglycerate kinase. Additionally, carbohydrate storage enzymes, carboxysome proteins (that raise CO2 concentration around Rubisco), and Rubisco activases CbbQ and CbbX accompanied the Calvin cycle. Photorespiration did not appear to be adapted specifically for the Calvin cycle in the non-cyanobacterial microbes under study. Our results suggest that chemoautotrophy in Calvin cycle-positive organisms was commonly enabled by hydrogenase, and less commonly ammonia monooxygenase (nitrification). The enrichment of specific DNA-binding domains indicated Calvin-cycle associated genetic regulation. Metabolic regulatory adaptations were illustrated by negative correlation to AraC and the enzyme arabinose-5-phosphate isomerase, which suggests a downregulation of the metabolite arabinose-5-phosphate, which may interfere with the Calvin cycle through enzyme inhibition and substrate competition. Certain domains of unknown function that were found to be important in the analysis may indica, QC 20210406

Knowledge of the genetic basis for autotrophic metabolism is valuable since it relates to both the emergence of life and to the metabolic engineering challenge of incorporating CO2 as a potential substrate for biorefining. The most common CO2 fixation pathway is the Calvin cycle, which utilizes Rubisco and phosphoribulokinase enzymes. We searched thousands of microbial genomes and found that 6.0% contained the Calvin cycle. We then contrasted the genomes of Calvin cycle-positive, non-cyanobacterial microbes and their closest relatives by enrichment analysis, ancestral character estimation, and random forest machine learning, to explore genetic adaptations associated with acquisition of the Calvin cycle. The Calvin cycle overlaps with the pentose phosphate pathway and glycolysis, and we could confirm positive associations with fructose-1,6-bisphosphatase, aldolase, and transketolase, constituting a conserved operon, as well as ribulose-phosphate 3-epimerase, ribose-5-phosphate isomerase, and phosphoglycerate kinase. Additionally, carbohydrate storage enzymes, carboxysome proteins (that raise CO2 concentration around Rubisco), and Rubisco activases CbbQ and CbbX accompanied the Calvin cycle. Photorespiration did not appear to be adapted specifically for the Calvin cycle in the non-cyanobacterial microbes under study. Our results suggest that chemoautotrophy in Calvin cycle-positive organisms was commonly enabled by hydrogenase, and less commonly ammonia monooxygenase (nitrification). The enrichment of specific DNA-binding domains indicated Calvin-cycle associated genetic regulation. Metabolic regulatory adaptations were illustrated by negative correlation to AraC and the enzyme arabinose-5-phosphate isomerase, which suggests a downregulation of the metabolite arabinose-5-phosphate, which may interfere with the Calvin cycle through enzyme inhibition and substrate competition. Certain domains of unknown function that were found to be important in the analysis may indica, QC 20210406

Knowledge of the genetic basis for autotrophic metabolism is valuable since it relates to both the emergence of life and to the metabolic engineering challenge of incorporating CO2 as a potential substrate for biorefining. The most common CO2 fixation pathway is the Calvin cycle, which utilizes Rubisco and phosphoribulokinase enzymes. We searched thousands of microbial genomes and found that 6.0% contained the Calvin cycle. We then contrasted the genomes of Calvin cycle-positive, non-cyanobacterial microbes and their closest relatives by enrichment analysis, ancestral character estimation, and random forest machine learning, to explore genetic adaptations associated with acquisition of the Calvin cycle. The Calvin cycle overlaps with the pentose phosphate pathway and glycolysis, and we could confirm positive associations with fructose-1,6-bisphosphatase, aldolase, and transketolase, constituting a conserved operon, as well as ribulose-phosphate 3-epimerase, ribose-5-phosphate isomerase, and phosphoglycerate kinase. Additionally, carbohydrate storage enzymes, carboxysome proteins (that raise CO2 concentration around Rubisco), and Rubisco activases CbbQ and CbbX accompanied the Calvin cycle. Photorespiration did not appear to be adapted specifically for the Calvin cycle in the non-cyanobacterial microbes under study. Our results suggest that chemoautotrophy in Calvin cycle-positive organisms was commonly enabled by hydrogenase, and less commonly ammonia monooxygenase (nitrification). The enrichment of specific DNA-binding domains indicated Calvin-cycle associated genetic regulation. Metabolic regulatory adaptations were illustrated by negative correlation to AraC and the enzyme arabinose-5-phosphate isomerase, which suggests a downregulation of the metabolite arabinose-5-phosphate, which may interfere with the Calvin cycle through enzyme inhibition and substrate competition. Certain domains of unknown function that were found to be important in the analysis may indica, QC 20210406

The major bottleneck in commercializing biofuels and other commodities produced by microalgae is the high cost associated with phototrophic cultivation. Improving microalgal productivities could be a solution to this problem. Synthetic biology methods have recently been used to engineer the downstream production pathways in several microalgal strains. However, engineering upstream photosynthetic and carbon fixation metabolism to enhance growth, productivity, and yield has barely been explored in microalgae. We describe strategies to improve the generation of reducing power from light, as well as to improve the assimilation of CO2 by either the native Calvin cycle or synthetic alternatives. Overall, we are optimistic that recent technological advances will prompt long-awaited breakthroughs in microalgal research.

Tropical successions are environments that cover large areas and are of great importance due to their ecological functions and extension. In Ecuador, studies on the carbon storage of these ecosystems are scarce and most of the equations are developed for humid lowland areas. The aim of this research was to generate allometric models for estimation of aerial biomass of secondary montane forests in northwestern Ecuador. A forest inventory was carried out in three secondary forests of 18, 30, and 50 years old approximately. Allometric models were developed using diameter at breast height (DBH) and total height (Ht) as independent variables, and the organic matter and accumulated aerial carbon were estimated. The DAP2Ht combinatorial variable was the best predictor variable according to the adjustment and validation criteria. The model equations showed an R2 greater than 95 %, consequently the variables studied are reliable. It was determined that forests store 36.56, 70.36, and 156.27 Mg.C.ha-1 respectively, where the stem is the component with the highest storage with a range between 65.76 and 73.44%. Both branches and leaves represent between 20.98 - 25, 50% and 5.58 - 8.74% of the total aerial carbon respectively. These models could be applied effectively in ecosystems with similar environmental conditions., Las sucesiones tropicales son ambientes de gran importancia por sus funciones ecológicas y extensión. En el Ecuador, los estudios sobre el almacenamiento de carbono de estos ecosistemas son escasos y la mayoría de ecuaciones desarrolladas se concentran en zonas húmedas de tierras bajas. El objetivo de la presente investigación fue generar modelos alométricos para la estimación de biomasa aérea de bosques secundarios montanos del noroccidente de Ecuador. Se realizó un inventario forestal en tres áreas de sucesión de 18, 30 y 50 años aproximadamente. Se construyeron modelos alométricos utilizando el diámetro a la altura del pecho (DAP) y la altura total (Ht) como variables independientes y se estimó la materia viva y el carbono aéreo acumulado. La variable combinatoria DAP2Ht fue la mejor predictora según los criterios de ajuste y validación. Las ecuaciones desarrolladas mostraron un R2 mayor al 95 %, por lo que son confiables para la estimación de las variables estudiadas. Se determinó que los bosques almacenan 36,56, 70,36 y 156,27 Mg.C.ha-1 respectivamente, siendo el fuste el componente de mayor almacenaje en un intervalo de 65,76 - 73,44 %, mientras que ramas y hojas representan un 20,98 - 25,50 % y 5,58 - 8,74 % del carbono aéreo total respectivamente. Estos modelos podrían aplicarse de manera efectiva en ecosistemas en condiciones ambientales similares.

Carbon dioxide (CO2) emissions from human activities are driving climate change, but the pending crisis could be mitigated by a circular carbon economy where released CO2 is recycled into commodity chemicals. Autotrophic microbes can make a contribution by producing chemicals, such as biofuels, from CO2 and renewable energy. The primary natural CO2 fixation pathway is the Calvin cycle, in which the enzyme Rubisco carboxylates ribulose-1,5-bisphosphate. The present investigation used computational systems biology methods to map adaptations and constraints in autotrophic microbial metabolism based on the Calvin cycle. First, the metabolic network of the Calvin cycle-capable photoautotrophic cyanobacterium Synechocystis was contrasted with that of heterotrophic E. coli. Intracellular metabolite concentration ranges differed, leading to different capacity to provide thermodynamic driving forces to chemical production pathways. Second, the Calvin cycle in Synechocystis was modeled kinetically, showing that certain enzyme saturation and metabolite levels, for example high ribulose-1,5-bisphosphate concentration, were detrimental to stability. Control over reaction rates was distributed, but making certain enzymes faster, for example fructose-1,6-bisphosphatase, could increase overall carbon fixation rate. Third, Synechocystis was starved of CO2 and ribosome profiling was used to track the effect on translation. Stress response and CO2 uptake were upregulated, but constant Rubisco expression and ribosome pausing in 5' untranslated regions indicated readiness for reappearance of CO2. Finally, microbial genomes with and without the Calvin cycle were contrasted, revealing metabolic, energetic, and regulatory adaptations that describe the properties of a functional autotroph. These findings provide a background for future study and engineering of autotrophs for direct conversion of CO2 into commodity chemicals., Utsläpp av koldioxid (CO2) från mänskliga aktiviteter driver klimatförändringarna, men den stundande krisen skulle kunna mildras av en cirkulär kolekonomi där CO2 som släpps ut återvinns till råvarukemikalier. Autotrofa mikrober kan bidra genom att producera kemikalier, såsom biobränslen, från CO2 och förnybar energi. Den primära naturliga syntesvägen för CO2-fixering är calvincykeln, i vilken enzymet Rubisco karboxylerar ribulos-1,5-bisfosfat. Undersökningen som ligger till grund för denna avhandling använde systembiologiska beräkningsmetoder för att kartlägga anpassningar och begränsningar i autotrof mikrobiell metabolism baserad på calvincykeln. För det första kontrasterades det metaboliska nätverket hos den calvincykelkapabla fotoautotrofa cyanobakterien Synechocystis med det hos heterotrofen E. coli. De intracellulära metabolitkoncentrationerna var olika, vilket ledde till olika kapacitet att bistå med termodynamisk drivkraft till kemiska syntesvägar. För det andra modellerades calvincykeln i Synechocystis kinetiskt, vilket visade att vissa enzymsatureringsnivåer och metabolitkoncentrationer, bland annat hög ribulos-1,5-bisfosfatkoncentration, motverkade stabiliteten. Kontroll över reaktionshastigheter var distribuerad, men ökning av hastigheten hos vissa enzymer, till exempel fruktos-1,6-bisfosfatas, skulle kunna öka den generalla kolfixeringshastigheten. För det tredje svältes Synechocystis på CO2 och ribosomprofilering användes för att följa effekten på translationen. Stressrespons och CO2-upptag uppreglerades, men konstant uttryck av Rubisco och pausning av ribosomer i de icketranslaterade 5'-regionerna indikerade beredskap för ett återuppträdande av CO2. Slutligen jämfördes mikrobiella genom med och utan calvincykeln, vilket avslöjade metaboliska, energetiska, och regulatoriska anpassningar som beskriver egenskaperna hos en funktionell autotrof. Dessa upptäckter ger en bakgrund för framtida studier och ingenjörsmässig design av autotrofer för direkt omvand, QC 2021-04-07

Knowledge of the genetic basis for autotrophic metabolism is valuable since it relates to both the emergence of life and to the metabolic engineering challenge of incorporating CO2 as a potential substrate for biorefining. The most common CO2 fixation pathway is the Calvin cycle, which utilizes Rubisco and phosphoribulokinase enzymes. We searched thousands of microbial genomes and found that 6.0% contained the Calvin cycle. We then contrasted the genomes of Calvin cycle-positive, non-cyanobacterial microbes and their closest relatives by enrichment analysis, ancestral character estimation, and random forest machine learning, to explore genetic adaptations associated with acquisition of the Calvin cycle. The Calvin cycle overlaps with the pentose phosphate pathway and glycolysis, and we could confirm positive associations with fructose-1,6-bisphosphatase, aldolase, and transketolase, constituting a conserved operon, as well as ribulose-phosphate 3-epimerase, ribose-5-phosphate isomerase, and phosphoglycerate kinase. Additionally, carbohydrate storage enzymes, carboxysome proteins (that raise CO2 concentration around Rubisco), and Rubisco activases CbbQ and CbbX accompanied the Calvin cycle. Photorespiration did not appear to be adapted specifically for the Calvin cycle in the non-cyanobacterial microbes under study. Our results suggest that chemoautotrophy in Calvin cycle-positive organisms was commonly enabled by hydrogenase, and less commonly ammonia monooxygenase (nitrification). The enrichment of specific DNA-binding domains indicated Calvin-cycle associated genetic regulation. Metabolic regulatory adaptations were illustrated by negative correlation to AraC and the enzyme arabinose-5-phosphate isomerase, which suggests a downregulation of the metabolite arabinose-5-phosphate, which may interfere with the Calvin cycle through enzyme inhibition and substrate competition. Certain domains of unknown function that were found to be important in the analysis may indica, QC 20210406

Carbon dioxide (CO2) emissions from human activities are driving climate change, but the pending crisis could be mitigated by a circular carbon economy where released CO2 is recycled into commodity chemicals. Autotrophic microbes can make a contribution by producing chemicals, such as biofuels, from CO2 and renewable energy. The primary natural CO2 fixation pathway is the Calvin cycle, in which the enzyme Rubisco carboxylates ribulose-1,5-bisphosphate. The present investigation used computational systems biology methods to map adaptations and constraints in autotrophic microbial metabolism based on the Calvin cycle. First, the metabolic network of the Calvin cycle-capable photoautotrophic cyanobacterium Synechocystis was contrasted with that of heterotrophic E. coli. Intracellular metabolite concentration ranges differed, leading to different capacity to provide thermodynamic driving forces to chemical production pathways. Second, the Calvin cycle in Synechocystis was modeled kinetically, showing that certain enzyme saturation and metabolite levels, for example high ribulose-1,5-bisphosphate concentration, were detrimental to stability. Control over reaction rates was distributed, but making certain enzymes faster, for example fructose-1,6-bisphosphatase, could increase overall carbon fixation rate. Third, Synechocystis was starved of CO2 and ribosome profiling was used to track the effect on translation. Stress response and CO2 uptake were upregulated, but constant Rubisco expression and ribosome pausing in 5' untranslated regions indicated readiness for reappearance of CO2. Finally, microbial genomes with and without the Calvin cycle were contrasted, revealing metabolic, energetic, and regulatory adaptations that describe the properties of a functional autotroph. These findings provide a background for future study and engineering of autotrophs for direct conversion of CO2 into commodity chemicals., Utsläpp av koldioxid (CO2) från mänskliga aktiviteter driver klimatförändringarna, men den stundande krisen skulle kunna mildras av en cirkulär kolekonomi där CO2 som släpps ut återvinns till råvarukemikalier. Autotrofa mikrober kan bidra genom att producera kemikalier, såsom biobränslen, från CO2 och förnybar energi. Den primära naturliga syntesvägen för CO2-fixering är calvincykeln, i vilken enzymet Rubisco karboxylerar ribulos-1,5-bisfosfat. Undersökningen som ligger till grund för denna avhandling använde systembiologiska beräkningsmetoder för att kartlägga anpassningar och begränsningar i autotrof mikrobiell metabolism baserad på calvincykeln. För det första kontrasterades det metaboliska nätverket hos den calvincykelkapabla fotoautotrofa cyanobakterien Synechocystis med det hos heterotrofen E. coli. De intracellulära metabolitkoncentrationerna var olika, vilket ledde till olika kapacitet att bistå med termodynamisk drivkraft till kemiska syntesvägar. För det andra modellerades calvincykeln i Synechocystis kinetiskt, vilket visade att vissa enzymsatureringsnivåer och metabolitkoncentrationer, bland annat hög ribulos-1,5-bisfosfatkoncentration, motverkade stabiliteten. Kontroll över reaktionshastigheter var distribuerad, men ökning av hastigheten hos vissa enzymer, till exempel fruktos-1,6-bisfosfatas, skulle kunna öka den generalla kolfixeringshastigheten. För det tredje svältes Synechocystis på CO2 och ribosomprofilering användes för att följa effekten på translationen. Stressrespons och CO2-upptag uppreglerades, men konstant uttryck av Rubisco och pausning av ribosomer i de icketranslaterade 5'-regionerna indikerade beredskap för ett återuppträdande av CO2. Slutligen jämfördes mikrobiella genom med och utan calvincykeln, vilket avslöjade metaboliska, energetiska, och regulatoriska anpassningar som beskriver egenskaperna hos en funktionell autotrof. Dessa upptäckter ger en bakgrund för framtida studier och ingenjörsmässig design av autotrofer för direkt omvand, QC 2021-04-07

BackgroundFerrovum spp. are abundant in acid mine drainage sites globally where they play an important role in biogeochemical cycling. All known taxa in this genus are Fe(II) oxidizers. Thus, co-occurring members of the genus could be competitors within the same environment. However, we found multiple, co-occurring Ferrovum spp. in Cabin Branch, an acid mine drainage site in the Daniel Boone National Forest, KY.ResultsHere we describe the distribution of Ferrovum spp. within the Cabin Branch communities and metagenome assembled genomes (MAGs) of two new Ferrovum spp. In contrast to previous studies, we recovered multiple 16S rRNA gene sequence variants suggesting the commonly used 97% cutoff may not be appropriate to differentiate Ferrovum spp. We also retrieved two nearly-complete Ferrovum spp. genomes from metagenomic data. The genomes of these taxa differ in several key ways relating to nutrient cycling, motility, and chemotaxis.ConclusionsPreviously reported Ferrovum genomes are also diverse with respect to these categories suggesting that the genus Ferrovum contains substantial metabolic diversity. This diversity likely explains how the members of this genus successfully co-occur in Cabin Branch and why Ferrovum spp. are abundant across geochemical gradients.

Vegetated coastal ecosystems along the Red Sea and Arabian Gulf coasts of Saudi Arabia thrive in an extremely arid and oligotrophic environment, with high seawater temperatures and salinity. Mangrove, seagrass and saltmarsh ecosystems have been shown to act as efficient sinks of sediment organic carbon, earning these vegetated ecosystems the moniker 'blue carbon' ecosystems. However, their role as nitrogen and phosphorus (N and P) sinks remains poorly understood. In this study, we examine the capacity of blue carbon ecosystems to trap and store nitrogen and phosphorous in their sediments in the central Red Sea and Arabian Gulf. We estimated the N and P stocks (in 0.2 m thick-sediments) and accumulation rates (for the last century based on 210Pb and for the last millennia based on 14C) in mangrove, seagrass and saltmarsh sediments from eight locations along the coast of Saudi Arabia (81 cores in total). The N and P stocks contained in the top 20 cm sediments ranged from 61 g N m-2 in Red Sea seagrass to 265 g N m-2 in the Gulf saltmarshes and from 70 g P m-2 in Red Sea seagrass meadows and mangroves to 58 g P m-2 in the Gulf saltmarshes. The short-term N and P accumulation rates ranged from 0.09 mg N cm-2 yr-1 in Red Sea seagrass to 0.38 mg N cm-2 yr-1 in Gulf mangrove, and from 0.027 mg P cm-2 yr-1 in the Gulf seagrass to 0.092 mg P cm-2 yr-1 in Red Sea mangroves. Short-term N and P accumulation rates were up to 10-fold higher than long-term accumulation rates, highlighting increasing sequestration of N and P over the past century, likely due to anthropogenic activities such as coastal development and wastewater inputs. © 2020 The Author(s). Published by IOP Publishing Ltd.

The Calvin – Benson cycle is the most widespread metabolic pathway capable of fixing CO2 in nature and a target of very high interest to metabolic engineers worldwide. In this study, 12 metabolites (ATP, AMP, NADP, NADPH, 2PG, 3PGA, FBP, RuBP, PEP, AKG, Ac-CoA and phenylalanine) were tested for protein – metabolite interactions against the proteome of Cupriavidus necator (previously Ralstonia eutropha) in the hopes of finding potential examples of allosteric regulation of the Calvin – Benson cycle. This is accomplished through the use of the LiP-SMap method, a recently developed shotgun proteomics method described by Piazza et al. capable of testing a metabolite of interest for interactions with the entire proteome of an organism at once. A functional protocol was developed and 234 protein – metabolite interactions between ATP and the proteome of C. necator are identified, 103 of which are potentially novel. Due to time constraints and setbacks in the lab, significant results were not produced for the other 11 metabolites tested. C. necator is an industrially relevant chemolithoautotroph that can be engineered to produce many valuable products and is capable of growth on CO2 and hydrogen gas. The bacteria were grown in continuous cultures after which the proteome was extracted while retaining its native state. Subsequently, the proteome was incubated with a metabolite of interest and subjected to limited, non-specific proteolysis. The resulting peptide mix was analyzed by liquid chromatography coupled tandem mass spectrometry (LC – MS/MS)., Calvin-Benson-cykeln är den mest utbredda metaboliska processen i naturen med vilken det är möjligt att fixera CO2 och en måltavla av högsta intresse för bioteknologer världen över. I den här studien testades 12 metaboliter (ATP, AMP, NADP, NADPH, 2PG, 3PGA, FBP, RuBP, PEP, AKG, Ac-CoA and phenylalanine) för interaktioner mot proteomet från Cupriavidus necator (tidigare Ralstonia eutropha) i hopp om att hitta potentiella exempel på allosterisk reglering av Calvin-Benson-cykeln. Detta uppnåddes genom användning av LiP-SMap-metoden, en nyligen utvecklad proteomikmetod beskriven av Piazza et al. kapabel av att testa en metabolit av intresse mot en organisms hela proteom simultant. Ett funktionellt protokoll utvecklades och 234 interaktioner mellan ATP och proteomet av C. necator identifierades, varav 103 potentiellt är nyupptäckta. På grund av tidsbrist och motgångar i labbet producerades inga signifikanta resultat för de resterande 11 metaboliterna som testades. C. necator är en industriellt relevant kemolitoautotrof som kan växa på CO2 och vätgas, samt manipuleras till att producera många värdefulla produkter. Bakterierna odlades i kemostater varefter proteomet extraherades i sitt naturliga tillstånd. Sedan inkuberades proteomet med en metabolit av intresse och utsattes för begränsad, icke-specifik proteolys. Den resulterande peptidblandningen analyserades via tandem masspektrometri kopplad till vätskekromatografi (LC – MS/MS).

Vegetated coastal ecosystems along the Red Sea and Arabian Gulf coasts of Saudi Arabia thrive in an extremely arid and oligotrophic environment, with high seawater temperatures and salinity. Mangrove, seagrass and saltmarsh ecosystems have been shown to act as efficient sinks of sediment organic carbon, earning these vegetated ecosystems the moniker 'blue carbon' ecosystems. However, their role as nitrogen and phosphorus (N and P) sinks remains poorly understood. In this study, we examine the capacity of blue carbon ecosystems to trap and store nitrogen and phosphorous in their sediments in the central Red Sea and Arabian Gulf. We estimated the N and P stocks (in 0.2 m thick-sediments) and accumulation rates (for the last century based on 210Pb and for the last millennia based on 14C) in mangrove, seagrass and saltmarsh sediments from eight locations along the coast of Saudi Arabia (81 cores in total). The N and P stocks contained in the top 20 cm sediments ranged from 61 g N m-2 in Red Sea seagrass to 265 g N m-2 in the Gulf saltmarshes and from 70 g P m-2 in Red Sea seagrass meadows and mangroves to 58 g P m-2 in the Gulf saltmarshes. The short-term N and P accumulation rates ranged from 0.09 mg N cm-2 yr-1 in Red Sea seagrass to 0.38 mg N cm-2 yr-1 in Gulf mangrove, and from 0.027 mg P cm-2 yr-1 in the Gulf seagrass to 0.092 mg P cm-2 yr-1 in Red Sea mangroves. Short-term N and P accumulation rates were up to 10-fold higher than long-term accumulation rates, highlighting increasing sequestration of N and P over the past century, likely due to anthropogenic activities such as coastal development and wastewater inputs. © 2020 The Author(s). Published by IOP Publishing Ltd.