Carbon-limited Environments Research Articles

Taxonomic and genomic attributes of oligotrophic soil bacteria.

Not all bacteria are fast growers. In soil as in other environments, bacteria exist along a continuum-from copiotrophs that can grow rapidly under resource-rich conditions to oligotrophs that are adapted to life in the "slow lane." However, the field of microbiology is built almost exclusively on the study of copiotrophs due, in part, to the ease of studying them in vitro. To begin understanding the attributes of soil oligotrophs, we analyzed three independent datasets that represent contrasts in organic carbon availability. These datasets included 185 samples collected from soil profiles across the USA, 950 paired bulk soil and rhizosphere samples collected across Europe, and soils from a microcosm experiment where carbon availability was manipulated directly. Using a combination of marker gene sequencing and targeted genomic analyses, we identified specific oligotrophic taxa that were consistently more abundant in carbon-limited environments (subsurface, bulk, unamended soils) compared to the corresponding carbon-rich environment (surface, rhizosphere, glucose-amended soils), including members of the Dormibacterota and Chloroflexi phyla. In general, putative soil oligotrophs had smaller genomes, slower maximum potential growth rates, and were under-represented in culture collections. The genomes of oligotrophs were more likely to be enriched in pathways that allow oligotrophs to metabolize a range of energy sources and store carbon, while genes associated with energy-intensive functions like chemotaxis and motility were under-represented. However, few genomic attributes were shared, highlighting that oligotrophs likely use a range of different metabolic strategies and regulatory pathways to thrive in resource-limited soils.

Itaconic acid and dimethyl itaconate exert antibacterial activity in carbon-enriched environments through the TCA cycle

Itaconic acid (IA), a metabolite generated by the tricarboxylic acid (TCA) cycle in eukaryotic immune cells, and its derivative dimethyl itaconate (DI) exert antibacterial functions in intracellular environments. Previous studies suggested that IA and DI only inhibit bacterial growth in carbon-limited environments; however, whether IA and DI maintain antibacterial activity in carbon-enriched environments remains unknown. Here, IA and DI inhibited the bacteria with minimum inhibitory concentrations of 24.02 mM and 39.52 mM, respectively, in a carbon-enriched environment. The reduced bacterial pathogenicity was reflected in cell membrane integrity, motility, biofilm formation, AI-2/luxS, and virulence. Mechanistically, succinate dehydrogenase (SDH) activity and fumaric acid levels decreased in the IA and DI treatments, while isocitrate lyase (ICL) activity was upregulated. Inhibited TCA circulation was also observed through untargeted metabolomics. In addition, energy-related aspartate metabolism and lysine degradation were suppressed. In summary, these results indicated that IA and DI reduced bacterial pathogenicity while exerting antibacterial functions by inhibiting TCA circulation. This study enriches knowledge on the inhibition of bacteria by IA and DI in a carbon-mixed environment, suggesting an alternative method for treating bacterial infections by immune metabolites.

Saprotrophic Fungus Induces Microscale Mineral Weathering to Source Potassium in a Carbon-Limited Environment

Plants rely on potassium for many critical biological processes, but most soils are potassium limited. Moving potassium from the inaccessible, mineral-bound pool to a more bioavailable form is crucial for sustainably increasing local potassium concentrations for plant growth and health. Here, we use a synthetic soil habitat (mineral doped micromodels) to study and directly visualize how the saprotrophic fungus, Fusarium sp. DS 682, weathers K-rich soil minerals. After 30 days of fungal growth, both montmorillonite and illite (secondary clays) had formed as surface coatings on primary K-feldspar, biotite, and kaolinite grains. The distribution of montmorillonite differed depending on the proximity to a carbon source, where montmorillonite was found to be associated with K-feldspar closer to the carbon (C) source, which the fungus was inoculated on, but associated with biotite at greater distances from the C source. The distribution of secondary clays is likely due to a change in the type of fungal exuded organic acids; from citric to tartaric acid dominated production with increasing distance from the C source. Thus, the main control on the ability of Fusarium sp. DS 682 to weather K-feldspar is proximity to a C source to produce citric acid via the TCA cycle.

Microbial Survival in an Extreme Martian Analog Ecosystem: Poás Volcano, Costa Rica

Past acid-sulfate hydrothermal systems on Mars have promise in their ability to have hosted life for billions of years. One method for analyzing these systems is to study analog environments on Earth. To assess the astrobiological potential of Martian acid-sulfate hydrothermal systems, the crater lake of the active Poás Volcano, Laguna Caliente, was sampled in 2013 and 2017. Laguna Caliente presents an extremely dynamic terrestrial environment with near-ambient to boiling temperatures, pH fluctuations from −0.87 to 1.5, a wide range of chemistries and redox potential, and frequent phreatic-to-phreatomagmatic eruptions. Samples of lake fluid, sulfur clumps, and lake bottom sediment underwent 16S rRNA gene sequencing and metagenomic “shotgun” sequencing, which revealed this lake hosts an extremely low biodiversity of microorganisms dominated by Acidiphilium spp. Shotgun metagenomics of the samples suggests this community has numerous genetic adaptations that confer survival, including functional pathways to reduce the effects of toxic metals and numerous metabolic pathways utilizing a variety of simple and complex sugar molecules. The identification of these various metabolic pathways suggests adaptations related to carbon limited environments, fulfillment of high energy requirements, and survival in a hostile volcanic setting. The perseverance of life in Laguna Caliente indicates life on Mars could have thrived in analogous environments, stressing the need for the search for life in relict Martian acid-sulfate hydrothermal systems.

Partnering of anammox and denitrifying bacteria benefits anammox's recovery from starvation and complete nitrogen removal

The cooperative metabolic activity of anammox and denitrifying bacteria could speed up anammox's recovery and reduce nitrate generated from the anammox reaction. In this study, a laboratory-scale model system containing a defined anammox culture AMX and a simultaneous nitrification and denitrification (SND) bacterium - Thauera sp. strain SND5 was established and investigated. Several lines of evidence revealed that strain SND5 consumed soluble microbial products (SMPs) generated by culture AMX (as high as 1.5 mg/L), stimulating anammox activity after long-term starvation. At low C/N ratios with an optimal C/N of 1, SND5 completely consumed organic carbon first at anoxic condition, storing carbon intracellularly as poly-β-hydroxybutyrate (PHB) (as high as 0.6 mg/L biomass), thereby creating a favorable environment for the growth of anammox bacteria. The anammox reaction and nitrate reduction supported by PHB catabolism could then proceed simultaneously, resulting in enhanced nitrogen removal. Cooperative interactions between anammox and denitrifying bacteria involving SMPs consumption and PHB synthesis may play a significant role in nitrogen cycling at nitrite- and carbon-limited environments.

Bacterial community dynamics and functional profiling of soils from conventional and organic cropping systems

Soil microbiomes play an integral role in agricultural production systems. Understanding of the complex microbial community structure and responses to conventional compared to organic cropping systems is crucial for sustainable production and ecosystems health. This study investigated soil microbial community structure responses based on a four year long field experiment. Bacterial communities characterizing conventional and organic cropping systems were evaluated using Illumina MiSeq high-throughput sequencing targeting the V4-V5 variable region of the 16S rRNA gene. Soil bacterial community structure showed a cropping system dependant distribution, with nitrogen cycling taxa (Bacillus, Niastella, Kribbella, and Beijerinckia) dominant in conventional cropping systems, while carbon cycling taxa (Dokdonella, Caulobacter, Mathylibium, Pedobacter, Cellulomonas and Chthoniobacter and Sorangium) were abundant in organic cropping systems. Functional prediction of the bacterial biomes showed conventional cropping systems to harbour a community adapted to carbon-limited environments, with organic cropping systems dominated by those involved in the degradation of complex organic compounds. These findings suggest the existence of niche specific communities and functional specialization between cropping systems with potential use in soil management through selective promotion of organisms beneficial to soil health.

Denitrification of Mature Landfill Leachate with High Nitrite in Simulated Landfill Columns Packed with Solid Digestate from Organic Fraction of Municipal Solid Waste

PURPOSE: The management of solid digestate (SD) from organic fraction of municipal solid waste is essential component of sustainable biogas plants. Beyond land use, this study aims to evaluate the application of digestate in simulated landfill columns to enhance nitrogen conversion of partially nitrified landfill leachate (PNL). METHODS: Two identical simulated landfill reactors filled with SD were setup and PNL was fed through the columns. The PNL characteristics were 1004 mg N–NO₂⁻/L, 428 mg N–NO₃⁻/L, and 434 mg N–NH₄⁺/L. RESULTS: After 109 days of operation, the average N–NO₂⁻and N–NO₃⁻ removal efficiencies were 92.6% and 85.8%, respectively. The high concentrations of nitrite did not significantly inhibit methane production during denitrification in the digestate-added landfill columns as the average specific methane production yield was 20.1 normal liter (NL) CH₄/kg VS. CONCLUSIONS: These results suggest that the novelty of applying SD in a landfill can achieve denitrification in a carbon limited environment observed in mature landfill leachate and stabilization of the digestate.

The Influence of Polyploidy on the Evolution of Yeast Grown in a Sub-Optimal Carbon Source.

Polyploidization events have occurred during the evolution of many fungi, plant, and animal species and are thought to contribute to speciation and tumorigenesis, however little is known about how ploidy level contributes to adaptation at the molecular level. Here we integrate whole genome sequencing, RNA expression analysis, and relative fitness of ∼100 evolved clones at three ploidy levels. Independent haploid, diploid, and tetraploid populations were grown in a low carbon environment for 250 generations. We demonstrate that the key adaptive mutation in the evolved clones is predicted by a gene expression signature of just five genes. All of the adaptive mutations identified encompass a narrow set of genes, however the tetraploid clones gain a broader spectrum of adaptive mutations than haploid or diploid clones. While many of the adaptive mutations occur in genes that encode proteins with known roles in glucose sensing and transport, we discover mutations in genes with no canonical role in carbon utilization (IPT1 and MOT3), as well as identify novel dominant mutations in glucose signal transducers thought to only accumulate recessive mutations in carbon limited environments (MTH1 and RGT1). We conclude that polyploid cells explore more genotypic and phenotypic space than lower ploidy cells. Our study provides strong evidence for the beneficial role of polyploidization events that occur during the evolution of many species and during tumorigenesis.

Evolution of organismal stoichiometry in a long-term experiment with Escherichia coli.

Organismal stoichiometry refers to the relative proportion of chemical elements in the biomass of organisms, and it can have important effects on ecological interactions from population to ecosystem scales. Although stoichiometry has been studied extensively from an ecological perspective, much less is known about the rates and directions of evolutionary changes in elemental composition. We measured carbon, nitrogen and phosphorus content of 12 Escherichia coli populations that evolved under controlled carbon-limited, serial-transfer conditions for 50 000 generations. The bacteria evolved higher relative nitrogen and phosphorus content, consistent with selection for increased use of the more abundant elements. Total carbon assimilated also increased, indicating more efficient use of the limiting element. We also measured stoichiometry in one population repeatedly through time. Stoichiometry changed more rapidly in early generations than later on, similar to the trajectory seen for competitive fitness. Altogether, our study shows that stoichiometry evolved over long time periods, and that it did so in a predictable direction, given the carbon-limited environment.

Stoichiometric modelling of assimilatory and dissimilatory biomass utilisation in a microbial community

Assimilatory and dissimilatory utilisation of autotroph biomass by heterotrophs is a fundamental mechanism for the transfer of nutrients and energy across trophic levels. Metagenome data from a tractable, thermoacidophilic microbial community in Yellowstone National Park was used to build an in silico model to study heterotrophic utilisation of autotroph biomass using elementary flux mode analysis and flux balance analysis. Assimilatory and dissimilatory biomass utilisation was investigated using 29 forms of biomass-derived dissolved organic carbon (DOC) including individual monomer pools, individual macromolecular pools and aggregate biomass. The simulations identified ecologically competitive strategies for utilizing DOC under conditions of varying electron donor, electron acceptor or enzyme limitation. The simulated growth environment affected which form of DOC was the most competitive use of nutrients; for instance, oxygen limitation favoured utilisation of less reduced and fermentable DOC while carbon-limited environments favoured more reduced DOC. Additionally, metabolism was studied considering two encompassing metabolic strategies: simultaneous versus sequential use of DOC. Results of this study bound the transfer of nutrients and energy through microbial food webs, providing a quantitative foundation relevant to most microbial ecosystems.

A role for programmed cell death in the microbial loop.

The microbial loop is the conventional model by which nutrients and minerals are recycled in aquatic eco-systems. Biochemical pathways in different organisms become metabolically inter-connected such that nutrients are utilized, processed, released and re-utilized by others. The result is that unrelated individuals end up impacting each others' fitness directly through their metabolic activities. This study focused on the impact of programmed cell death (PCD) on a population's growth as well as its role in the exchange of carbon between two naturally co-occurring halophilic organisms. Flow cytometric, biochemical, 14C radioisotope tracing assays, and global transcriptomic analyses show that organic algal photosynthate released by Dunalliela salina cells undergoing PCD complements the nutritional needs of other non-PCD D. salina cells. This occurs in vitro in a carbon limited environment and enhances the growth of the population. In addition, a co-occurring heterotroph Halobacterium salinarum re-mineralizes the carbon providing elemental nutrients for the mixoheterotrophic chlorophyte. The significance of this is uncertain and the archaeon can also subsist entirely on the lysate of apoptotic algae. PCD is now well established in unicellular organisms; however its ecological relevance has been difficult to decipher. In this study we found that PCD in D. salina causes the release of organic nutrients such as glycerol, which can be used by others in the population as well as a co-occurring halophilic archaeon. H. salinarum also re-mineralizes the dissolved material promoting algal growth. PCD in D. salina was the mechanism for the flow of dissolved photosynthate between unrelated organisms. Ironically, programmed death plays a central role in an organism's own population growth and in the exchange of nutrients in the microbial loop.

Estimate of the Decay Rate Constant of Hydrogen Sulfide from Drywall in a Simulated Bench-Scale Study

AbstractResearch was conducted to investigate the impact of particle size on H2S gas emissions and estimate a decay rate constant for H2S gas generation from the anaerobic decomposition of drywall. Three different particle sizes of regular drywall and one particle size of paperless drywall were evaluated. The experiment was conducted in a carbon-limited environment. The paperless drywall generated significantly less H2S gas than regular drywall at the same particle size. No significant differences were found between the rates of H2S gas generated from the different particle sizes of regular drywall investigated (p=0.44), which most likely was the result of the carbon limitation. The volume of H2S gas emitted from drywall was modeled as a first-order decay with correction factors that take into account the impacts of pH, temperature, and liquid-gas partitioning. The first-order decay model resulted in an average decay constant value of 0.14±0.01 and 0.031 year−1 for regular and paperless drywall, respecti...

A study of the flavin response by Shewanella cultures in carbon-limited environments

Mediated electron transfer has been implicated as a primary mechanism of extracellular electron transfer to insoluble electron acceptors in anaerobic cultures of the facultative anaerobe Shewanella oneidensis. In this work, planktonic and biofilm cultures of S. oneidensis exposed to carbon-limited environments trigger an electrochemical response thought to be the signature of an electrochemically active metabolite. This metabolite was detected via cyclic voltammetry for S. oneidensis MR-1 biofilms. The observed electrochemical potentials correspond to redox potentials of flavin-containing molecules. Chromatographic techniques were then used to quantify concentrations of riboflavin by the carbon-limited environmental response of planktonic S. oneidensis. Further evidence of flavin redox chemistry was associated with biofilm formation on multi-walled carbon nanotube-modified Toray paper under carbon-starved environments. By encapsulating one such electrode in silica, the encapsulated biofilm exhibits riboflavin redox activity earlier than a non-encapsulated system after media replacement. This work explores the electrochemical nature of riboflavin interaction with an electrode after secretion from S. oneidensis and in comparison to abiotic systems.

Hunger artists: yeast adapted to carbon limitation show trade-offs under carbon sufficiency.

As organisms adaptively evolve to a new environment, selection results in the improvement of certain traits, bringing about an increase in fitness. Trade-offs may result from this process if function in other traits is reduced in alternative environments either by the adaptive mutations themselves or by the accumulation of neutral mutations elsewhere in the genome. Though the cost of adaptation has long been a fundamental premise in evolutionary biology, the existence of and molecular basis for trade-offs in alternative environments are not well-established. Here, we show that yeast evolved under aerobic glucose limitation show surprisingly few trade-offs when cultured in other carbon-limited environments, under either aerobic or anaerobic conditions. However, while adaptive clones consistently outperform their common ancestor under carbon limiting conditions, in some cases they perform less well than their ancestor in aerobic, carbon-rich environments, indicating that trade-offs can appear when resources are non-limiting. To more deeply understand how adaptation to one condition affects performance in others, we determined steady-state transcript abundance of adaptive clones grown under diverse conditions and performed whole-genome sequencing to identify mutations that distinguish them from one another and from their common ancestor. We identified mutations in genes involved in glucose sensing, signaling, and transport, which, when considered in the context of the expression data, help explain their adaptation to carbon poor environments. However, different sets of mutations in each independently evolved clone indicate that multiple mutational paths lead to the adaptive phenotype. We conclude that yeasts that evolve high fitness under one resource-limiting condition also become more fit under other resource-limiting conditions, but may pay a fitness cost when those same resources are abundant.

A fungal growth model fitted to carbon‐limited dynamics of Rhizoctonia solani

Here, a quasi-steady-state approximation was used to simplify a mathematical model for fungal growth in carbon-limiting systems, and this was fitted to growth dynamics of the soil-borne plant pathogen and saprotroph Rhizoctonia solani. The model identified a criterion for invasion into carbon-limited environments with two characteristics driving fungal growth, namely the carbon decomposition rate and a measure of carbon use efficiency. The dynamics of fungal spread through a population of sites with either low (0.0074 mg) or high (0.016 mg) carbon content were well described by the simplified model with faster colonization for the carbon-rich environment. Rhizoctonia solani responded to a lower carbon availability by increasing the carbon use efficiency and the carbon decomposition rate following colonization. The results are discussed in relation to fungal invasion thresholds in terms of carbon nutrition.

Comprehensive evaluation of the palatability and chemical defenses of subtidal macroalgae from the Antarctic Peninsula

The palatability of 35 non-encrusting, subtidal macroalgal species collected from the vicinity of Palmer Station, Antarctica (64° 46' S, 64° 03' W), was determined in laboratory bioassays utilizing sympatric sea stars and fish known to consume macroalgae in nature. Overall, 63% of the macroalgal species offered to sea stars and 83% of the macroalgal species offered to fish in thallus bioassays were significantly unpalatable. This included all of the ecologically dominant, overstory brown macroalgae in the region. When organic extracts of unpalatable macroalgal species were incorporated into artificial foods, 76% of the species unpalatable as thallus to sea stars were also unpalatable to them as extract, and 53% of the species unpalatable as thallus to fish were also unpalatable to them as extract. If either sea stars or fish rejected thallus of a macroalgal species, palatability of organic extracts of that species to herbivorous amphipods was determined: 63% of such algal species were unpalatable as extract to the amphipods. It was concluded that antarctic macroalgae are commonly unpalatable to sympatric consumers and that much of this unpalatability is the result of chemical defenses. As a whole, neither thallus toughness nor a variety of nutritional quality parameters appeared to be related to macroalgal palatability. We also tested the hypothesis that nitrogen-containing metabolites should be common in macroalgae from nitrogen-replete, carbon-limited environments such as the coastal waters of Antarctica. Macroalgal acid extracts targeting nitrogenous secondary metabolites were subjected to thin-layer chromatography analysis; no such compounds were detected.

Divergent evolution during an experimental adaptive radiation.

How repeatable a process is evolution? Comparative studies of multicellular eukaryotes and experimental studies with unicellular prokaryotes document the repeated evolution of adaptive phenotypes during similar adaptive radiations, suggesting that the outcome of adaptive radiation is broadly reproducible. The goal of this study was to test this hypothesis by using phenotypic traits to infer the genetic basis of adaptation to simple carbon-limited environments in an extensive adaptive radiation. We used a clone of the bacterium Pseudomonas fluorescens to found two sets of experimental lines. The first set of lines was allowed to adapt to one of 23 novel environments for 1100 generations while the second set of lines was allowed to accumulate mutations by drift for 2000 generations. All lines were then assayed in the 95 environments provided by Biolog microplates to determine the phenotypic consequences of selection and drift. Replicate selection lines propagated in a common environment evolved similar adaptive components of their phenotype but showed extensive variation in non-adaptive phenotypic traits. This variation in non-adaptive phenotypic traits primarily resulted from the ascendance of different beneficial mutations in different lines. We argue that these results reconcile experimental and comparative approaches to studying adaptation by demonstrating that the convergent phenotypic evolution that occurs during adaptive radiation may be associated with radically different sets of beneficial mutations.

Seasonal Changes in Nitrogen Fractions of Coptis japonica, a Temperate Forest Evergreen Chamaephyte, and their Ecological Implications

Seasonal changes in several forms of nitrogen were investigated in Coptis japonica, an evergreen rosette hemicryptophyte in temperate deciduous forest. The concentration of total nitrogen in rhizomes and roots decreased during the period of new shoot growth from winter to spring. In the rhizomes, total soluble protein stored by early summer decreased gradually until winter, coupled with an increase in free amino acids. Nitrogen was largely stored in free amino acids in the roots, especially during summer. The total soluble protein in current-year leaves decreased from spring to summer and then increased during winter. The seasonal changes in nitrogen components were coincident with the change in light-saturated photosynthetic rates recorded in a previous study. The ratio of total soluble protein to total nitrogen decreased from spring to summer and then increased from late summer to winter in the current-year leaves. In contrast, chlorophyll content and the ratio of chlorophyll to total nitrogen were higher in summer than in other seasons. The results indicate that nitrogen was used in a manner that better utilizes the very weak light in summer and the higher light intensities in other seasons. The major component of the free amino acid pool was asparagine, in every organ throughout the season, except for the senescent leaves. Since asparagine has a high N:C ratio, we suppose that the asparagine-dominated amino acid pool is advantageous in the carbon-limited environment of the forest floor.

Selective Advantage of a Spirillum sp. in a Carbon-limited Environment. Accumulation of Poly- -hydroxybutyric Acid and Its Role in Starvation

A freshwater Spirillum sp., which apparently belongs to a niche of low nutritional status (Matin & Veldkamp, 1978), accumulated poly-beta-hydroxybutyric acid (PHB) during lactate-limited growth in continuous culture. The PHB content varied in a complex manner with the dilution rate (D), but was greatest at the lowest D value examined: about 18% (w/w) at D = 0.025 h-1. It is not known what mechanism accounted for PHB accumulation during carbon-limited growth. The resistance of cultures of Spirillum sp. to starvation after growth at various D values was compared with that of a Pseudomonas sp. which appears to belong to relatively richer environments (Matin & Veldkamp, 1978) and does not accumulate PHB. In Spirillum sp., resistance correlated directly with the PHB content of the culture subjected to starvation, whereas in Pseudomonas sp. it increased with RNA content. Further, after growth at D = 0.03 to 0.05 h-1, the Spirillum sp. was much more resistant to starvation than was the Pseudomonas sp. Since the microflora of oligotrophic environments are probably often subjected to starvation conditions, PHB accumulation by Spirillum sp. during growth in such environments may assist survival. PHB in Spirillum sp. was rapidly degraded during starvation but it had no sparing effect on RNA degradation. It is not known how PHB enhanced resistance to starvation.

The regulation of α-amylase production inBacillus licheniformis

The production ofα-amylase (α-1,4 glucan-4-glucan hydrolase; E.C. 3.2.1.1.) by a strain ofBacillus licheniformis has been studied in batch and continuous cultures. The synthesis of this enzyme was shown to be repressed by glucose or other low-molecular-weight metabolisable sugars. Consequently, amylase production in a medium which contained “liquified” starch only began after the low-molecular-weight sugars had been dissimilated. Thereafter, the dextrins in the medium were degraded by amylase produced by the bacteria to yield further quantities of metabolisable sugars. These sugars were continuously dissimilated by the growing organisms and never accumulated to concentrations where they would repress further amylase synthesis. A clear analogy could thus be drawn with bacteria growing in a carbon-limited environment in a chemostat. Therefore,α-amylase production byB. licheniformis organisms growing in 3-litre chemostats was studied. No evidence was obtained to infer that an inducer was necessary for amylase production, and it was concluded that the prime factors influencing amylase production, in this species at least, were growth rate and catabolite repression.