Substrate-limited Conditions Research Articles

Biological Mn(II) oxidation under organic substrate-limited conditions and its application in mine drainage remediation

Manganese (Mn)-oxidizing bacteria are considered promising tools for remediation of Mn-containing mine drainage. However, whether such microorganisms function in an environment rich in metals and poor in organic substrate remains largely unexplored. In repeated batch experiments, we found that suspensions of mine drainage sediment showed heat-labile, azide-sensitive, and Mn(II) concentration-dependent removal of dissolved Mn(II), consistent with the contribution of biological activity to the Mn(II) oxidation. In limestone-packed bioreactors, most Mn(II) and zinc (Zn) (II) ions added at 20 or 60 mg L−1 and 6 mg L−1, respectively, were removed at hydraulic retention times ranging from 0.5 days to 3.0 days. The Mn oxide deposits in the reactors showed network structures of nanosheets, which structurally incorporated most parts of Mn and Zn to form a Zn-bearing Mn(IV) oxide mineral resembling woodruffite. The Mn deposit suspensions incubated with 13C-labeled bicarbonate assimilated the inorganic carbon and this process enhanced the Mn(II) oxidation. The preferential growth of oligotrophs or methylotrophs might provide organic carbon within Mn(II)-oxidizing microbial communities. This study first demonstrates the important role of inorganic carbon assimilation in heterotrophic Mn(II)-oxidizing community maintained under metal-rich, organic substrate-limited conditions and reinforces the advantage of use of biological Mn(II) oxidation for mine drainage remediation.

Revisit the E2 Domain of Amyloid Precursor Protein: Ferroxidase, Superoxide and Peroxynitrite Scavenging Activities.

Amyloid precursor protein (APP) is the biological precursor of β-amyloids, a known histopathological hallmark associated with Alzheimer's disease (AD). The function of APP is of great interest yet remains elusive. One of the extracellular domains of APP, the E2 domain, has been proposed to possess ferroxidase activity and affect neuronal iron homeostasis. However, contradicting evidence has been reported, and its precise role remains inconclusive. Here, we studied the Cu-binding site of the E2 domain using extended X-ray absorption fine structure (EXAFS), UV-vis, and electron paramagnetic resonance (EPR) and discovered that a new labile water ligand coordinates to the Cu(II) cofactor in addition to the four known histidines. We explored the proposed ferroxidase activity of the Cu(II)-E2 domain through reactions with ferrous iron and observed single-turnover ferrous oxidation activity with a rate up to 1.0 × 102 M-1 s-1. Cu(I)-E2 reacted with molecular oxygen at a rate of only 5.3 M-1 s-1, which would restrict any potential multiturnover ferroxidase activity to this slow rate and prevents observation of activity under multiturnover conditions. The positive electrostatic potential surface of the protein indicates possible reactivity with negatively charged small substrates such as superoxide radicals (O2•-) and peroxynitrite (ONOO-) that are major contributors to the oxidative stress prevalent in the extracellular environment. Our assays showed that Cu(I)-E2 can remove O2•- at a rate of 1.6 × 105 M-1 s-1, which is slower than the rates of native SODs. However, the reaction between Cu(I)-E2 and ONOO- achieved a rate of 1.1 × 105 M-1 s-1, comparable to native ONOO- scavenger peroxiredoxins (105-107 M-1 s-1). Therefore, the E2 domain of APP can serve as an enzymatic site that may function as a ferroxidase under substrate-limiting conditions, a supplemental O2•- scavenger, and an ONOO- remover in the vicinity of the cellular iron efflux channel and protect neuron cells from reactive oxygen species (ROS) and reactive nitrogen species (RNS) damage.

Batch Experiments Demonstrating a Two-Stage Bacterial Process Coupling Methanotrophic and Heterotrophic Bacteria for 1-Alkene Production From Methane.

Methane (CH4) is a sustainable carbon feedstock for value-added chemical production in aerobic CH4-oxidizing bacteria (methanotrophs). Under substrate-limited (e.g., oxygen and nitrogen) conditions, CH4 oxidation results in the production of various short-chain organic acids and platform chemicals. These CH4-derived products could be broadened by utilizing them as feedstocks for heterotrophic bacteria. As a proof of concept, a two-stage system for CH4 abatement and 1-alkene production was developed in this study. Type I and Type II methanotrophs, Methylobacter tundripaludum SV96 and Methylocystis rosea SV97, respectively, were investigated in batch tests under different CH4 and air supplementation schemes. CH4 oxidation under either microaerobic or aerobic conditions induced the production of formate, acetate, succinate, and malate in M. tundripaludum SV96, accounting for 4.8–7.0% of consumed carbon from CH4 (C-CH4), while M. rosea SV97 produced the same compounds except for malate, and with lower efficiency than M. tundripaludum SV96, accounting for 0.7–1.8% of consumed C-CH4. For the first time, this study demonstrated the use of organic acid-rich spent media of methanotrophs cultivating engineered Acinetobacter baylyi ADP1 ‘tesA-undA cells for 1-alkene production. The highest yield of 1-undecene was obtained from the spent medium of M. tundripaludum SV96 at 68.9 ± 11.6 μmol mol Csubstrate–1. However, further large-scale studies on fermenters and their optimization are required to increase the production yields of organic acids in methanotrophs.

Are K-strategists yield-strategists in disguise? An example from autotrophic nitrogen removal.

The behavior of heterotrophic bacteria growing in systems with low or no external supply of chemical oxygen demand (COD) has become more relevant within the wastewater context. Growth strategies help to clarify how bacteria behave and adapt to different environmental conditions. In the case of substrate limited conditions, research has been mainly focused on the k-strategy, whereas another important strategy: the yield strategy has not been explored intensely. Some authors have, however, demonstrated the implications of bacteria pursuing the yield strategy when living in structured environments and facing low-substrate concentrations. This study uses a one-dimensional biofilm model to study the influence of the affinity constant, the maximum growth rate, and the growth yield on the heterotrophic formation of dinitrogen gas (N2 ) in a completely autotrophic partial nitritation anammoxsystem. The effect of these parameters on the composition and the diversity of the heterotrophic community is also evaluated. In a first scenario, heterotrophic bacteria are allowed to grow only on the COD produced by biomass decay. In a second step, the competition with a second group of heterotrophs using external COD as electron donor is assessed. For both evaluated scenarios, the results suggest that the yield plays a crucial role in the heterotrophic biomass and dinitrogen gas formation. Moreover, in the case of the community diversity the yield seems to be the decisive parameter. Finally, we conceptually compared the K and the yield strategy and give some insight to the possibility of both either being closely related or even being the same strategy.

Methoxyl stable isotopic constraints on the origins and limits of coal-bed methane.

Microbial coal-bed methane is an important economic resource and source of a potent greenhouse gas, but controls on its formation are poorly understood. To test whether the microbial degradability of coal limits microbial methane, we monitored methoxyl group demethylation—a reaction that feeds methanogenesis—in a global sample suite ranging in maturity from wood to bituminous coal. Carbon isotopic compositions of residual methoxyl groups were inconsistent with a thermal reaction, instead implying a substrate-limited biologic process. This suggests that deep biosphere communities participated in transforming plant matter to coal on geologic time scales and that methoxyl abundance influences coal-bed methane yield. Carbon isotopic enrichments resulting from microbial methylotrophy also explain an enigmatic offset in the carbon-13 content of microbial methane from coals and conventional hydrocarbon deposits.

Iron and sulfur speciation and cycling in the sediments of marine systems located in arid environments: the northern Red Sea

The high fluxes of iron minerals associated with aeolian dry deposition may result in an anomalously high reactive iron content and the rapid reoxidation of hydrogen sulfide in sediments. This will prevent the formation of pyrite and result in a ‘cryptic’ sulfur cycle. We studied the cycling of iron and sulfur in deep water (>800 m water depth) sediments of the Red Sea and its northern extension, the Gulf of Aqaba. We found that the reactive iron content in the surface sediments of the Gulf of Aqaba and the Red Sea is high, whereas the amount of sulfur-bound iron is very low and decreases with the water depth. The presence of trace amounts of pyrite and zero-valent sulfur, as well as the isotopic compositions of sulfate and pyrite, which are consistent with sulfate reduction under substrate-limiting conditions, suggest that cryptic sulfur cycling is likely to be a result of the rapid reoxidation of hydrogen sulfide rather than the suppression of microbial sulfate reduction. The low amount of reactive iron and high organic carbon content in the sediments of the Shaban Deep, which are overlain by hypersaline hydrothermal brines, result in a non-cryptic sulfur cycle characterized by the preservation of pyrite in the sediments. Supplementary material: Data for sulfur speciation, iron speciation and sulfur isotope composition are available at https://doi.org/10.6084/m9.figshare.c.5508155 Thematic collection: This article is part of the Sulfur in the Earth system collection available at: https://www.lyellcollection.org/cc/sulfur-in-the-earth-system

Substrate availability drives mixed culture fermentation of glucose to lactate at steady state.

Mixed-culture fermentation (MCF) enables carbon recycling from complex organic waste streams into valuable feedstock chemicals. Using complex microbial consortia, MCF systems can be tuned to produce a range of biochemicals to meet market demand. However, the metabolic mechanisms and community interactions which drive biochemical production changes under differing conditions are currently poorly understood. These mechanisms are critical to useful MCF production models. Furthermore, predictable product transitions are currently limited to pH-driven changes between butyrate and ethanol, and chain-elongation (fed by lactate, acetate, and ethanol) to butyrate, valerate, and hexanoate. Lactate, a high-value biopolymer feedstock chemical, has been observed in transition states, but sustained production has not been described. In this study, steady state lactate production was achieved by increasing the organic loading rate of a butyrate-producing system from limiting to nonlimiting conditions at pH 5.5. Crucially, butyrate production resumed upon return to substrate-limited conditions. 16S ribosomal DNA community profiling combined with metaproteomics demonstrated that the butyrate-producing lineage Megasphaera redirected carbon flow through the methylglyoxal bypass when substrate was nonlimiting, which altered the community structure and metabolic expression toward lactate production. This metabolic mechanism can be included in future MCF models to describe the changes in product generation in substrate nonlimiting conditions.

Total Cellular ATP Production Changes With Primary Substrate in MCF7 Breast Cancer Cells.

Cancer growth is predicted to require substantial rates of substrate catabolism and ATP turnover to drive unrestricted biosynthesis and cell growth. While substrate limitation can dramatically alter cell behavior, the effects of substrate limitation on total cellular ATP production rate is poorly understood. Here, we show that MCF7 breast cancer cells, given different combinations of the common cell culture substrates glucose, glutamine, and pyruvate, display ATP production rates 1.6-fold higher than when cells are limited to each individual substrate. This increase occurred mainly through faster oxidative ATP production, with little to no increase in glycolytic ATP production. In comparison, non-transformed C2C12 myoblast cells show no change in ATP production rate when substrates are limited. In MCF7 cells, glutamine allows unexpected access to oxidative capacity that pyruvate, also a strictly oxidized substrate, does not. Pyruvate, when added with other exogenous substrates, increases substrate-driven oxidative ATP production, by increasing both ATP supply and demand. Overall, we find that MCF7 cells are highly flexible with respect to maintaining total cellular ATP production under different substrate-limited conditions, over an acute (within minutes) timeframe that is unlikely to result from more protracted (hours or more) transcription-driven changes to metabolic enzyme expression. The near-identical ATP production rates maintained by MCF7 and C2C12 cells given single substrates reveal a potential difficulty in using substrate limitation to selectively starve cancer cells of ATP. In contrast, the higher ATP production rate conferred by mixed substrates in MCF7 cells remains a potentially exploitable difference.

Micro–level evaluation of organic compounds transformation in anaerobic digestion under feast and famine conditions assisted by iron–based materials – Revealing the true mechanism of AD enhancement

Conductive materials have been applied to assist syntrophic metabolism in anaerobic digestion. However, their role in the transformation of organic compounds, particularly recalcitrant compounds, has not been revealed. In this study, iron-based materials – magnetite nanoparticles and Fe2+– were employed to explore their effects on the transformation of different organic matters in anaerobic system. Prompted methane production rates and quantity in iron–based materials groups were found due to the improved solubilization of organic particles, enhanced degradation of recalcitrant compounds, and maintained microbial activity under substrate–limited conditions. Specifically, the proportion of the reducing functional groups (C–C/H or CC) and O/C ratio were always significantly lower in iron–based materials supplemented groups (Fe groups) compared to Control group, despite hydrolysis was greatly enhanced in Fe groups. The greater dehydrogenation oxidation was confirmed in the presence of iron–based materials. The remaining humic-like substances (HS), a typical type of recalcitrant compound, was about 2.5 times higher in Control group (221.2 ± 5.3 mg/L-C) compared to Fe groups after 30 days degradation. By tracking the aromaticity of HS and individual compounds at molecular level, this study reveals that iron–based materials were more effective in stimulating the degradation of aliphatic moieties than the aromatic moieties of recalcitrant compounds. When readily biodegradable substrates were limited, Fe groups continued methane generation by using recalcitrant compounds (e.g. thiethylperazine and fluvoxamino acid) as carbon source, and the microbial activity was maintained according to higher relative abundance of protonated nitrogen and continuous methanogenesis activity at starvation phase.

ACBD6 protein controls acyl chain availability and specificity of the N-myristoylation modification of proteins

Members of the human acyl-CoA binding domain-containing (ACBD) family regulate processes as diverse as viral replication, stem-cell self-renewal, organelle organization, and protein acylation. These functions are defined by nonconserved motifs present downstream of the ACBD. The human ankyrin-repeat-containing ACBD6 protein supports the reaction catalyzed by the human and PlasmodiumN-myristoyltransferase (NMT) enzymes. Likewise, the newly identified Plasmodium ACBD6 homologue regulates the activity of the NMT enzymes. The relatively low abundance of myristoyl-CoA in the cell limits myristoylation. Binding of myristoyl-CoA to NMT is competed by more abundant acyl-CoA species such as palmitoyl-CoA. ACBD6 also protects the Plasmodium NMT enzyme from lauryl-CoA and forces the utilization of the myristoyl-CoA substrate. The phosphorylation of two serine residues of the acyl-CoA binding domain of human ACBD6 improves ligand binding capacity, prevents competition by unbound acyl-CoAs, and further enhances the activity of NMT. Thus, ACBD6 proteins promote N-myristoylation in mammalian cells and in one of their intracellular parasites under unfavorable substrate-limiting conditions.

Role of feed water biodegradable substrate concentration on biofouling: Biofilm characteristics, membrane performance and cleanability

Biofouling severely impacts operational performance of membrane systems increasing the cost of water production. Understanding the effect of critical parameters of feed water such as biodegradable substrate concentration on the developed biofilm characteristics enables development of more effective biofouling control strategies.In this study, the effect of substrate concentration on the biofilm characteristics was examined using membrane fouling simulators (MFSs). A feed channel pressure drop (PD) increase of 200 mbar was used as a benchmark to study the developed biofilm. The amount and characteristics of the formed biofilm were analysed in relation to membrane performance indicators: feed channel pressure drop and permeate flux. The effect of the characteristics of the biofilm developed at three substrate concentrations on the removal efficiency of the different biofilms was evaluated applying acid/base cleaning.Results showed that a higher feed water substrate concentration caused a higher biomass amount, a faster PD increase, but a lower permeate flux decline. The permeate flux decline was affected by the spatial location and the physical characteristics of the biofilm rather than the total amount of biofilm. The slower growing biofilm developed at the lowest substrate concentration was harder to remove by NaOH/HCl cleanings than the biofilm developed at the higher substrate concentrations.Effective biofilm removal is essential to prevent a fast biofilm regrowth after cleaning. While substrate limitation is a generally accepted biofouling control strategy delaying biofouling, development of advanced cleaning methods to remove biofilms formed under substrate limited conditions is of paramount importance.

Leucine, Palmitate, or Leucine/Palmitate Cotreatment Enhances Myotube Lipid Content and Oxidative Preference

Branched-chain amino acids (BCAA) such as leucine stimulate favorable metabolic processes involved in lean tissue preservation and skeletal muscle metabolism. However, higher levels of circulating BCAA correlate with severity of metabolic disease (including diabetes/insulin resistance), and may result from dysregulated BCAA catabolism. Past observations have demonstrated potential interaction between BCAA and dietary fat; however, much of this relationship remains underexplored. This study investigated the effect of leucine both with and without palmitate on oxidative and glycolytic metabolism, as well as indicators of BCAA catabolism using cultured skeletal muscle cells. Specifically, C2C12 myotubes were treated with or without varying concentrations of leucine both with and without palmitate for 24 h. Leucine treatment significantly elevated mRNA expression of metabolic regulators including peroxisome proliferator-activated receptor-gamma coactivator 1-alpha versus leucine with concurrent palmitate treatment. Interestingly, leucine-only, palmitate-only, and leucine with palmitate all significantly increased cellular lipid content, which translated into significantly increased oxidative capacity under substrate-limited conditions. However, upon the addition of excess substrate and carnitine, discrepancies in peak metabolic capacities between various treatments were no longer observed, suggesting leucine, palmitate, or the combination thereof causes a shift in metabolic preference from glycolytic to oxidative. These data also suggest leucine's effect on mitochondrial metabolism may result in part from increased lipid stores in addition to other previously documented pathways.

Effect of decanoic acid and 10-hydroxydecanoic acid on the biotransformation of methyl decanoate to sebacic acid

Biotransformation of fatty acid methyl esters to dicarboxylic acids has attracted much attention in recent years; however, reports of sebacic acid production using such biotransformation remain few. The toxicity of decanoic acid is the main challenge for this process. Decane induction has been reported to be essential to activate the enzymes involved in the α,ω-oxidation pathway before initiating the biotransformation of methyl decanoate to sebacic acid. However, we observed the accumulation of intermediates (decanoic acid and 10-hydroxydecanoic acid) during the induction period. In this study, we examined the effects of these intermediates on the biotransformation process. The presence of decanoic acid, even at a low concentration (0.2 g/L), inhibited the transformation of 10-hydroxydecanoic acid to sebacic acid. Moreover, about 24–32% reduction in the decanoic acid oxidation was observed in the presence of 0.5–1.5 g/L 10-hydroxydecanoic acid. To eliminate these inhibitory effects, we applied substrate-limiting conditions during the decane induction process, which eliminated the accumulation of decanoic acid. Although the productivity of sebacic acid (34.5 ± 1.10 g/L) was improved, by 28% over that achieved using the previously methods, after 54 h, the accumulation of 10-hydroxydecanoic acid was still detected. The accumulation of 10-hydroxydecanoic acid even under the decane limiting conditions could be an evidence that oxidation of 10-hydroxydecanoic acid could be the rate-limiting step in this process. The improvement of this reaction should be an important objective for further development of the production of sebacic acid using biotransformation.

Extended anaerobic conditions in the biological wastewater treatment: Higher reduction of toxicity compared to target organic micropollutants

Extended anaerobic conditions during biological wastewater treatment may enhance the biodegradation of micropollutants. To explore this, we combined iron-reducing or substrate-limited anaerobic conditions and aerobic pilot-scale reactors directly at a wastewater treatment plant. To investigate the detoxification by these processes, we applied two in vitro bioassays for baseline toxicity (Microtox) and reactive toxicity (AREc32) as well as in vivo bioassays with aquatic model species in two laboratory experiments (Desmodesmus subspicatus, Daphnia magna) and two on-site, flow-through experiments (Potamopyrgus antipodarum, Lumbriculus variegatus). Moreover, we analyzed 31 commonly occurring micropollutants and 10 metabolites.The baseline toxicity of raw wastewater was effectively removed in full-scale and reactor scale activated sludge treatment (>85%), while the oxidative stress response was only partially removed (>61%). A combination of an anaerobic pre-treatment under iron reducing conditions and an aerobic nitrification significantly further reduced the residual in vitro toxicities by 46–60% and outperformed the second combination consisting of an aerobic pre-treatment and an anaerobic post-treatment under substrate-limiting conditions (27–43%). Exposure to effluents of the activated sludge treatment did not induce adverse in vivo effects in aquatic invertebrates. Accordingly, no further improvement in water quality could be observed. Compared to that, the removal of persistent micropollutants was increased. However, this observation was restricted to a limited number of compounds and the removal of the sum concentration of all target micropollutants was relative low (14–17%).In conclusion, combinations of strictly anaerobic and aerobic processes significantly enhanced the removal of specific and non-specific in vitro toxicities. Thus, an optimization of biological wastewater treatment can lead to a substantially improved detoxification. These otherwise hidden capacities of a treatment technology can only be uncovered by a complementary biological analysis.

Oxidation of alkane and alkene moieties with biologically inspired nonheme iron catalysts and hydrogen peroxide: from free radicals to stereoselective transformations.

The selective oxidation of hydrocarbons is a challenging reaction for synthetic chemists, but common in nature. Iron oxygenases activate the O-O bond of dioxygen to perform oxidation of alkane and alkenes moieties with outstanding levels of regio-, chemo- and stereoselectivity. Along a bioinspired approach, iron coordination complexes which mimic structural and reactivity aspects of the active sites of nonheme iron oxygenases have been explored as oxidation catalysts. This review describes the evolution of this research field, from the early attempts to reproduce the basic reactivity of nonheme iron oxygenases to the development of effective iron oxidation catalysts. The work covers exclusively nonheme iron complexes which rely on H2O2 or O2 as terminal oxidants. First, it delineates the key steps and the essential catalyst design principles required to activate the peroxide bond at nonheme iron centers without (or at least minimizing) the release of free-diffusing radicals. It follows with a critical description of the mechanistic pathways which govern the reaction between iron complexes and H2O2 to generate the oxidizing species. Eventually, the work presents a state-of-the-art report on the use of these catalysts in aliphatic C-H oxidation, olefin epoxidation and alkene syn-dihydroxylation, under substrate-limiting conditions. A special focus is given on the main strategies elaborated to tune catalyst activity and selectivity by modification of its structure. The work is concluded by a concise discussion on the essential progresses of these oxidation catalysts together with the challenges that remain still to be tackled.

Advancing Biological Wastewater Treatment: Extended Anaerobic Conditions Enhance the Removal of Endocrine and Dioxin-like Activities

Conventional activated sludge treatment of wastewater does not completely remove micropollutants. Here, extending anaerobic conditions may enhance biodegradation. To explore this, we combined iron-reducing or substrate-limiting and aerobic pilot-scale reactors directly at a wastewater treatment plant. To assess the removal of endocrine disrupting chemicals (EDCs) as group of micropollutants that adversely affects wildlife, we applied a bioanalytical approach. We used in vitro bioassays covering seven receptor-mediated mechanisms of action, including (anti)androgenicity, (anti)estrogenicity, retinoid-like, and dioxin-like activity. Untreated wastewater induced antiandrogenic, estrogenic, antiestrogenic, and retinoid-like activity. Full-scale as well as reactor-scale activated sludge treatment effectively removes the observed effects. Nevertheless, high antiandrogenic and minor dioxin-like and estrogenic effects persisted in the treated effluent that may still be environmentally relevant. The anaerobic post-treatment under substrate-limiting conditions resulted in an additional removal of endocrine activities by 17-40%. The anaerobic pre-treatment under iron-reducing conditions significantly enhanced the removal of the residual effects by 40-75%. In conclusion, this study demonstrates that a further optimization of biological wastewater treatment is possible. Here, implementing iron-reducing anaerobic conditions preceding aerobic treatment appears promising to improve the removal of receptor-mediated toxicity.

Technical Note: A generic law-of-the-minimum flux limiter for simulating substrate limitation in biogeochemical models

Abstract. We present a generic flux limiter to account for mass limitations from an arbitrary number of substrates in a biogeochemical reaction network. The flux limiter is based on the observation that substrate (e.g., nitrogen, phosphorus) limitation in biogeochemical models can be represented as to ensure mass conservative and non-negative numerical solutions to the governing ordinary differential equations. Application of the flux limiter includes two steps: (1) formulation of the biogeochemical processes with a matrix of stoichiometric coefficients and (2) application of Liebig's law of the minimum using the dynamic stoichiometric relationship of the reactants. This approach contrasts with the ad hoc down-regulation approaches that are implemented in many existing models (such as CLM4.5 and the ACME (Accelerated Climate Modeling for Energy) Land Model (ALM)) of carbon and nutrient interactions, which are error prone when adding new processes, even for experienced modelers. Through an example implementation with a CENTURY-like decomposition model that includes carbon, nitrogen, and phosphorus, we show that our approach (1) produced almost identical results to that from the ad hoc down-regulation approaches under non-limiting nutrient conditions, (2) properly resolved the negative solutions under substrate-limited conditions where the simple clipping approach failed, (3) successfully avoided the potential conceptual ambiguities that are implied by those ad hoc down-regulation approaches. We expect our approach will make future biogeochemical models easier to improve and more robust.

Spatial Interaction of Archaeal Ammonia-Oxidizers and Nitrite-Oxidizing Bacteria in an Unfertilized Grassland Soil.

Interrelated successive transformation steps of nitrification are performed by distinct microbial groups – the ammonia-oxidizers, comprising ammonia-oxidizing archaea (AOA) and bacteria (AOB), and nitrite-oxidizers such as Nitrobacter and Nitrospira, which are the dominant genera in the investigated soils. Hence, not only their presence and activity in the investigated habitat is required for nitrification, but also their temporal and spatial interactions. To demonstrate the interdependence of both groups and to address factors promoting putative niche differentiation within each group, temporal and spatial changes in nitrifying organisms were monitored in an unfertilized grassland site over an entire vegetation period at the plot scale of 10 m2. Nitrifying organisms were assessed by measuring the abundance of marker genes (amoA for AOA and AOB, nxrA for Nitrobacter, 16S rRNA gene for Nitrospira) selected for the respective sub-processes. A positive correlation between numerically dominant AOA and Nitrospira, and their co-occurrence at the same spatial scale in August and October, suggests that the nitrification process is predominantly performed by these groups and is restricted to a limited timeframe. Amongst nitrite-oxidizers, niche differentiation was evident in observed seasonally varying patterns of co-occurrence and spatial separation. While their distributions were most likely driven by substrate concentrations, oxygen availability may also have played a role under substrate-limited conditions. Phylogenetic analysis revealed temporal shifts in Nitrospira community composition with an increasing relative abundance of OTU03 assigned to sublineage V from August onward, indicating its important role in nitrite oxidation.

Growth and activity of ANME clades with different sulfate and sulfide concentrations in the presence of methane.

Extensive geochemical data showed that significant methane oxidation activity exists in marine sediments. The organisms responsible for this activity are anaerobic methane-oxidizing archaea (ANME) that occur in consortia with sulfate-reducing bacteria. A distinct zonation of different clades of ANME (ANME-1, ANME-2a/b, and ANME-2c) exists in marine sediments, which could be related to the localized concentrations of methane, sulfate, and sulfide. In order to test this hypothesis we performed long-term incubation of marine sediments under defined conditions with methane as a headspace gas: low or high sulfate (±4 and ±21 mM, respectively) in combination with low or high sulfide (±0.1 and ±4 mM, respectively) concentrations. Control incubations were also performed, with only methane, high sulfate, or high sulfide. Methane oxidation was monitored and growth of subtypes ANME-1, ANME-2a/b, and ANME-2c assessed using qPCR analysis. A preliminary archaeal community analysis was performed to gain insight into the ecological and taxonomic diversity. Almost all of the incubations with methane had methane oxidation activity, with the exception of the incubations with combined low sulfate and high sulfide concentrations. Sulfide inhibition occurred only with low sulfate concentrations, which could be due to the lower Gibbs free energy available as well as sulfide toxicity. ANME-2a/b appears to mainly grow in incubations which had high sulfate levels and methane oxidation activity, whereas ANME-1 did not show this distinction. ANME-2c only grew in incubations with only sulfate addition. These findings are consistent with previously published in situ profiling analysis of ANME subclusters in different marine sediments. Interestingly, since all ANME subtypes also grew in incubations with only methane or sulfate addition, ANME may also be able to perform anaerobic methane oxidation under substrate limited conditions or alternatively perform additional metabolic processes.

Trimethylamine and Organic Matter Additions Reverse Substrate Limitation Effects on the δ13C Values of Methane Produced in Hypersaline Microbial Mats.

Methane production has been observed in a number of hypersaline environments, and it is generally thought that this methane is produced through the use of noncompetitive substrates, such as the methylamines, dimethylsulfide and methanol. Stable isotope measurements of the produced methane have also suggested that the methanogens are operating under conditions of substrate limitation. Here, substrate limitation in gypsum-hosted endoevaporite and soft-mat hypersaline environments was investigated by the addition of trimethylamine, a noncompetitive substrate for methanogenesis, and dried microbial mat, a source of natural organic matter. The δ(13)C values of the methane produced after amendments were compared to those in unamended control vials. At all hypersaline sites investigated, the δ(13)C values of the methane produced in the amended vials were statistically lower (by 10 to 71‰) than the unamended controls, supporting the hypothesis of substrate limitation at these sites. When substrates were added to the incubation vials, the methanogens within the vials fractionated carbon isotopes to a greater degree, resulting in the production of more (13)C-depleted methane. Trimethylamine-amended samples produced lower methane δ(13)C values than the mat-amended samples. This difference in the δ(13)C values between the two types of amendments could be due to differences in isotope fractionation associated with the dominant methane production pathway (or substrate used) within the vials, with trimethylamine being the main substrate used in the trimethylamine-amended vials. It is hypothesized that increased natural organic matter in the mat-amended vials would increase fermentation rates, leading to higher H2 concentrations and increased CO2/H2 methanogenesis.