A lithotrophic microbial fuel cell operated with pseudomonads‐dominated iron‐oxidizing bacteria enriched at the anode

In this study, we attempted to enrich neutrophilic iron bacteria in a microbial fuel cell (MFC)‐type reactor in order to develop a lithotrophic MFC system that can utilize ferrous iron as an inorganic electron donor and operate at neutral pHs. Electrical currents were steadily generated at an average level of 0.6 mA (or 0.024 mA cm^–2 of membrane area) in reactors initially inoculated with microbial sources and operated with 20 mM Fe²⁺ as the sole electron donor and 10 ohm external resistance; whereas in an uninoculated reactor (the control), the average current level only reached 0.2 mA (or 0.008 mA cm^–2 of membrane area). In an inoculated MFC, the generation of electrical currents was correlated with increases in cell density of bacteria in the anode suspension and coupled with the oxidation of ferrous iron. Cultivation‐based and denaturing gradient gel electrophoresis analyses both show the dominance of some Pseudomonas species in the anode communities of the MFCs. Fluorescent in‐situ hybridization results revealed significant increases of neutrophilic iron‐oxidizing bacteria in the anode community of an inoculated MFC. The results, altogether, prove the successful development of a lithotrophic MFC system with iron bacteria enriched at its anode and suggest a chemolithotrophic anode reaction involving some Pseudomonas species as key players in such a system. The system potentially offers unique applications, such as accelerated bioremediation or on‐site biodetection of iron and/or manganese in water samples.     

Inhibitory effect of iron‐oxidizing bacteria on ferrous‐promoted chalcopyrite leaching

It is generally accepted that iron‐oxidizing bacteria, Thiobacillus ferrooxidans, enhance chalcopyrite leaching. However, this article details a case of the bacteria suppressing chalcopyrite leaching. Bacterial leaching experiments were performed with sulfuric acid solutions containing 0 or 0.04 mol/dm³ ferrous sulfate. Without ferrous sulfate, the bacteria enhance copper extraction and oxidation of ferrous ions released from chalcopyrite. However, the bacteria suppressed chalcopyrite leaching when ferrous sulfate was added. This is mainly due to the bacterial consumption of ferrous ions which act as a promoter for chalcopyrite oxidation with dissolved oxygen. Coprecipitation of copper ions with jarosite formed by the bacterial ferrous oxidation also causes the bacterial suppression of copper extraction. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 478–483, 1999.     

Micro‐ and nano‐scale mineralogical characterization of Fe(II)‐oxidizing bacterial stalks

Neutrophilic, microaerobic Fe(II)‐oxidizing bacteria (FeOB) from marine and freshwater environments are known to generate twisted ribbon‐like organo‐mineral stalks. These structures, which are extracellularly precipitated, are susceptible to chemical influences in the environment once synthesized. In this paper, we characterize the minerals associated with freshwater FeOB stalks in order to evaluate key organo‐mineral mechanisms involved in biomineral formation. Micro‐Raman spectroscopy and Field Emission Scanning Electron Microscopy revealed that FeOB isolated from drinking water wells in Sweden produced stalks with ferrihydrite, lepidocrocite and goethite as main mineral components. Based on our observations made by micro‐Raman Spectroscopy, field emission scanning electron microscopy and scanning transmission electron microscope combined with electron energy‐loss spectroscopy, we propose a model that describes the crystal‐growth mechanism, the Fe‐oxidation state, and the mineralogical state of the stalks, as well as the biogenic contribution to these features. Our study suggests that the main crystal‐growth mechanism in stalks includes nanoparticle aggregation and dissolution/re‐precipitation reactions, which are dominant near the organic exopolymeric material produced by the microorganism and in the peripheral region of the stalk, respectively.     

Acid‐tolerant microaerophilic Fe(II)‐oxidizing bacteria promote Fe(III)‐accumulation in a fen

The ecological importance of Fe(II)‐oxidizing bacteria (FeOB) at circumneutral pH is often masked in the presence of O₂ where rapid chemical oxidation of Fe(II) predominates. This study addresses the abundance, diversity and activity of microaerophilic FeOB in an acidic fen (pH ～5) located in northern Bavaria, Germany. Mean O₂ penetration depth reached 16 cm where the highest dissolved Fe(II) concentrations (up to 140 µM) were present in soil water. Acid‐tolerant FeOB cultivated in gradient tubes were most abundant (10⁶ cells g^?1 peat) at the 10–20 cm depth interval. A stable enrichment culture was active at up to 29% O₂ saturation and Fe(III) accumulated 1.6 times faster than in abiotic controls. An acid‐tolerant, microaerophilic isolate (strain CL21) was obtained which was closely related to the neutrophilic, lithoautotrophic FeOB Sideroxydans lithotrophicus strain LD‐1. CL21 oxidized Fe(II) between pH 4 and 6.0, and produced nanoscale‐goethites with a clearly lower mean coherence length (7 nm) perpendicular to the (110) plane than those formed abiotically (10 nm). Our results suggest that an acid‐tolerant population of FeOB is thriving at redox interfaces formed by diffusion‐limited O₂ transport in acidic peatlands. Furthermore, this well‐adapted population is successfully competing with chemical oxidation and thereby playing an important role in the microbial iron cycle.     

On the biogenicity of Fe‐oxyhydroxide filaments in silicified low‐temperature hydrothermal deposits: Implications for the identification of Fe‐oxidizing bacteria in the rock record

Microaerophilic Fe(II)‐oxidizing bacteria produce biomineralized twisted and branched stalks, which are promising biosignatures of microbial Fe oxidation in ancient jaspers and iron formations. Extracellular Fe stalks retain their morphological characteristics under experimentally elevated temperatures, but the extent to which natural post‐depositional processes affect fossil integrity remains to be resolved. We examined siliceous Fe deposits from laminated mounds and chimney structures from an extinct part of the Jan Mayen Vent Fields on the Arctic Mid‐Ocean Ridge. Our aims were to determine how early seafloor diagenesis affects morphological and chemical signatures of Fe‐oxyhydroxide biomineralization and how extracellular stalks differ from abiogenic features. Optical and scanning electron microscopy in combination with focused ion beam‐transmission electron microscopy (FIB‐TEM) was used to study the filamentous textures and cross sections of individual stalks. Our results revealed directional, dendritic, and radial arrangements of biogenic twisted stalks and randomly organized networks of hollow tubes. Stalks were encrusted by concentric Fe‐oxyhydroxide laminae and silica casings. Element maps produced by energy dispersive X‐ray spectroscopy (EDS) in TEM showed variations in the content of Si, P, and S within filaments, demonstrating that successive hydrothermal fluid pulses mediate early diagenetic alteration and modify the chemical composition and surface features of stalks through Fe‐oxyhydroxide mineralization. The carbon content of the stalks was generally indistinguishable from background levels, suggesting that organic compounds were either scarce initially or lost due to percolating hydrothermal fluids. Dendrites and thicker abiotic filaments from a nearby chimney were composed of nanometer‐sized microcrystalline iron particles and silica and showed Fe growth bands indicative of inorganic precipitation. Our study suggests that the identification of fossil stalks and sheaths of Fe‐oxidizing bacteria in hydrothermal paleoenvironments may not rely on the detection of organic carbon and demonstrates that abiogenic filaments differ from stalks and sheaths of Fe‐oxidizing bacteria with respect to width distribution, ultrastructure, and textural context.     

Barite encrustation of benthic sulfur‐oxidizing bacteria at a marine cold seep

Crusts and chimneys composed of authigenic barite are found at methane seeps and hydrothermal vents that expel fluids rich in barium. Microbial processes have not previously been associated with barite precipitation in marine cold seep settings. Here, we report on the precipitation of barite on filaments of sulfide‐oxidizing bacteria at a brine seep in the Gulf of Mexico. Barite‐mineralized bacterial filaments in the interiors of authigenic barite crusts resemble filamentous sulfide‐oxidizing bacteria of the genus Beggiatoa. Clone library and iTag amplicon sequencing of the 16S rRNA gene show that the barite crusts that host these filaments also preserve DNA of Candidatus Maribeggiatoa, as well as sulfate‐reducing bacteria. Isotopic analyses show that the sulfur and oxygen isotope compositions of barite have lower δ³⁴S and δ¹⁸O values than many other marine barite crusts, which is consistent with barite precipitation in an environment in which sulfide oxidation was occurring. Laboratory experiments employing isolates of sulfide‐oxidizing bacteria from Gulf of Mexico seep sediments showed that under low sulfate conditions, such as those encountered in brine fluids, sulfate generated by sulfide‐oxidizing bacteria fosters rapid barite precipitation localized on cell biomass, leading to the encrustation of bacteria in a manner reminiscent of our observations of barite‐mineralized Beggiatoa in the Gulf of Mexico. The precipitation of barite directly on filaments of sulfide‐oxidizing bacteria, and not on other benthic substrates, suggests that sulfide oxidation plays a role in barite formation at certain marine brine seeps where sulfide is oxidized to sulfate in contact with barium‐rich fluids, either prior to, or during, the mixing of those fluids with sulfate‐containing seawater in the vicinity of the sediment/water interface. As with many other geochemical interfaces that foster mineral precipitation, both biological and abiological processes likely contribute to the precipitation of barite at marine brine seeps such as the one studied here.     

Oxidation of Fe(II)‐EDTA by nitrite and by two nitrate‐reducing Fe(II)‐oxidizing Acidovorax strains

The enzymatic oxidation of Fe(II) by nitrate‐reducing bacteria was first suggested about two decades ago. It has since been found that most strains are mixotrophic and need an additional organic co‐substrate for complete and prolonged Fe(II) oxidation. Research during the last few years has tried to determine to what extent the observed Fe(II) oxidation is driven enzymatically, or abiotically by nitrite produced during heterotrophic denitrification. A recent study reported that nitrite was not able to oxidize Fe(II)‐EDTA abiotically, but the addition of the mixotrophic nitrate‐reducing Fe(II)‐oxidizer, Acidovorax sp. strain 2AN, led to Fe(II) oxidation (Chakraborty & Picardal, 2013). This, along with other results of that study, was used to argue that Fe(II) oxidation in strain 2AN was enzymatically catalyzed. However, the absence of abiotic Fe(II)‐EDTA oxidation by nitrite reported in that study contrasts with previously published data. We have repeated the abiotic and biotic experiments and observed rapid abiotic oxidation of Fe(II)‐EDTA by nitrite, resulting in the formation of Fe(III)‐EDTA and the green Fe(II)‐EDTA‐NO complex. Additionally, we found that cultivating the Acidovorax strains BoFeN1 and 2AN with 10 mm nitrate, 5 mm acetate, and approximately 10 mm Fe(II)‐EDTA resulted only in incomplete Fe(II)‐EDTA oxidation of 47–71%. Cultures of strain BoFeN1 turned green (due to the presence of Fe(II)‐EDTA‐NO) and the green color persisted over the course of the experiments, whereas strain 2AN was able to further oxidize the Fe(II)‐EDTA‐NO complex. Our work shows that the two used Acidovorax strains behave very differently in their ability to deal with toxic effects of Fe‐EDTA species and the further reduction of the Fe(II)‐EDTA‐NO nitrosyl complex. Although the enzymatic oxidation of Fe(II) cannot be ruled out, this study underlines the importance of nitrite in nitrate‐reducing Fe(II)‐ and Fe(II)‐EDTA‐oxidizing cultures and demonstrates that Fe(II)‐EDTA cannot be used to demonstrate unequivocally the enzymatic oxidation of Fe(II) by mixotrophic Fe(II)‐oxidizers.     

Evaluating four mathematical models for nitrous oxide production by autotrophic ammonia‐oxidizing bacteria

There is increasing evidence showing that ammonia‐oxidizing bacteria (AOB) are major contributors to N₂O emissions from wastewater treatment plants (WWTPs). Although the fundamental metabolic pathways for N₂O production by AOB are now coming to light, the mechanisms responsible for N₂O production by AOB in WWTP are not fully understood. Mathematical modeling provides a means for testing hypotheses related to mechanisms and triggers for N₂O emissions in WWTP, and can then also become a tool to support the development of mitigation strategies. This study examined the ability of four mathematical model structures to describe two distinct mechanisms of N₂O production by AOB. The production mechanisms evaluated are (1) N₂O as the final product of nitrifier denitrification with NO as the terminal electron acceptor and (2) N₂O as a byproduct of incomplete oxidation of hydroxylamine (NH₂OH) to NO. The four models were compared based on their ability to predict N₂O dynamics observed in three mixed culture studies. Short‐term batch experimental data were employed to examine model assumptions related to the effects of (1) NH concentration variations, (2) dissolved oxygen (DO) variations, (3) NO accumulations and (4) NH₂OH as an externally provided substrate. The modeling results demonstrate that all these models can generally describe the NH, NO, and NO data. However, none of these models were able to reproduce all measured N₂O data. The results suggest that both the denitrification and NH₂OH pathways may be involved in N₂O production and could be kinetically linked by a competition for intracellular reducing equivalents. A unified model capturing both mechanisms and their potential interactions needs to be developed with consideration of physiological complexity. Biotechnol. Bioeng. 2013; 110: 153–163. © 2012 Wiley Periodicals, Inc.     

Weak phylogenetic signal in physiological traits of methane‐oxidizing bacteria

The presence of phylogenetic signal is assumed to be ubiquitous. However, for microorganisms, this may not be true given that they display high physiological flexibility and have fast regeneration. This may result in fundamentally different patterns of resemblance, that is, in variable strength of phylogenetic signal. However, in microbiological inferences, trait similarities and therewith microbial interactions with its environment are mostly assumed to follow evolutionary relatedness. Here, we tested whether indeed a straightforward relationship between relatedness and physiological traits exists for aerobic methane‐oxidizing bacteria (MOB). We generated a comprehensive data set that included 30 MOB strains with quantitative physiological trait information. Phylogenetic trees were built from the 16S rRNA gene, a common phylogenetic marker, and the pmoA gene which encodes a subunit of the key enzyme involved in the first step of methane oxidation. We used a Blomberg's K from comparative biology to quantify the strength of phylogenetic signal of physiological traits. Phylogenetic signal was strongest for physiological traits associated with optimal growth pH and temperature indicating that adaptations to habitat are very strongly conserved in MOB. However, those physiological traits that are associated with kinetics of methane oxidation had only weak phylogenetic signals and were more pronounced with the pmoA than with the 16S rRNA gene phylogeny. In conclusion, our results give evidence that approaches based solely on taxonomical information will not yield further advancement on microbial eco‐evolutionary interactions with its environment. This is a novel insight on the connection between function and phylogeny within microbes and adds new understanding on the evolution of physiological traits across microbes, plants and animals.     

The potential of methane‐oxidizing bacteria for applications in environmental biotechnology

Methanotrophic bacteria possess a unique set of enzymes enabling them to oxidize, degrade and transform organic molecules and synthesize new compounds. Therefore, they have great potential in environmental biotechnology. The application of these unique properties was demonstrated in three case studies: (i) Methane escaping from leaky gas pipes may lead to massive mortality of trees in urban areas. Lack of oxygen within the soil surrounding tree roots caused by methanotrophic activity was identified as one of the reasons for this phenomenon. The similarity between metabolic reactions performed by the key enzymes of methanotrophs (methane monooxygenase) and ammonium oxidizers (ammonium monooxygenase) might offer a solution to this problem by applying commercially available nitrification and urease inhibitors. (ii) Methanotrophs are able to co‐metabolically degrade contaminants such as low‐molecular‐weight‐chlorinated hydrocarbons in soil and water in the presence of methane. Batch and continuous trichloroethylene degradation experiments in laboratory‐scale reactors using Methylocystis sp. GB 14 were performed, partly with cells entrapped in a polymer matrix. (iii) Using a short, two‐stage pilot‐scale process, the intracellular polymer accumulation of poly‐β‐hydroxybutyrate (PHB) in methanotrophs reached a maximum of 52%. Interestingly, an ultra‐high‐molecular‐weight PHB of 3.1 MDa was accumulated under potassium deficiency. Under strictly controlled conditions (temperature, pH and methane supply) this process can be nonsterile because of the establishment of a stable microbial community (dominant species Methylocystis sp. GB 25 ≥86% by biomass). The possibility to substitute methane with biogas from renewable sources facilitates the development of a methane‐based PHB production process that yields a high‐quality biopolymer at competitive costs.     

Respiratory interactions of soil bacteria with (semi)conductive iron‐oxide minerals

Pure‐culture studies have shown that dissimilatory metal‐reducing bacteria are able to utilize iron‐oxide nanoparticles as electron conduits for reducing distant terminal acceptors; however, the ecological relevance of such energy metabolism is poorly understood. Here, soil microbial communities were grown in electrochemical cells with acetate as the electron donor and electrodes (poised at 0.2 V versus Ag/AgCl) as the electron acceptors in the presence and absence of iron‐oxide nanoparticles, and respiratory current generation and community structures were analysed. Irrespective of the iron‐oxide species (hematite, magnetite or ferrihydrite), the supplementation with iron‐oxide minerals resulted in large increases (over 30‐fold) in current, while only a moderate increase (～10‐fold) was observed in the presence of soluble ferric/ferrous irons. During the current generation, insulative ferrihydrite was transformed into semiconductive goethite. Clone‐library analyses of 16S rRNA gene fragments PCR‐amplified from the soil microbial communities revealed that iron‐oxide supplementation facilitated the occurrence of Geobacter species affiliated with subsurface clades 1 and 2. We suggest that subsurface‐clade Geobacter species preferentially thrive in soil by utilizing (semi)conductive iron oxides for their respiration.     

Manganese‐oxidizing bacteria mediate the degradation of 17α‐ethinylestradiol

Manganese (II) and manganese‐oxidizing bacteria were used as an efficient biological system for the degradation of the xenoestrogen 17α‐ethinylestradiol (EE2) at trace concentrations. Mn²⁺‐derived higher oxidation states of Mn (Mn³⁺, Mn⁴⁺) by Mn²⁺‐oxidizing bacteria mediate the oxidative cleavage of the polycyclic target compound EE2. The presence of manganese (II) was found to be essential for the degradation of EE2 by Leptothrix discophora, Pseudomonas putida MB1, P. putida MB6 and P. putida MB29. Mn²⁺‐dependent degradation of EE2 was found to be a slow process, which requires multi‐fold excess of Mn²⁺ and occurs in the late stationary phase of growth, implying a chemical process taking place. EE2‐derived degradation products were shown to no longer exhibit undesirable estrogenic activity.     

Detection of novel syntrophic acetate‐oxidizing bacteria from biogas processes by continuous acetate enrichment approaches

To enrich syntrophic acetate‐oxidizing bacteria (SAOB), duplicate chemostats were inoculated with sludge from syntrophic acetate oxidation (SAO)‐dominated systems and continuously supplied with acetate (0.4 or 7.5 g l^?1) at high‐ammonia levels. The chemostats were operated under mesophilic (37°C) or thermophilic (52°C) temperature for about six hydraulic retention times (HRT 28 days) and were sampled over time. Irrespective of temperature, a methane content of 64–69% and effluent acetate level of 0.4–1.0 g l^?1 were recorded in chemostats fed high acetate. Low methane production in the low‐acetate chemostats indicated that the substrate supply was below the threshold for methanization of acetate via SAO. Novel representatives within the family Clostridiales and genus Syntrophaceticus (class Clostridia) were identified to represent putative SAOB candidates in mesophilic and thermophilic conditions respectively. Known SAOB persisted at low relative abundance in all chemostats. The hydrogenotrophic methanogens Methanoculleus bourgensis (mesophilic) and Methanothermobacter thermautotrophicus (thermophilic) dominated archaeal communities in the high‐acetate chemostats. In line with the restricted methane production in the low‐acetate chemostats, methanogens persisted at considerably lower abundance in these chemostats. These findings strongly indicate involvement in SAO and tolerance to high ammonia levels of the species identified here, and have implications for understanding community function in stressed anaerobic processes.     

Magnetite as a precursor for green rust through the hydrogenotrophic activity of the iron‐reducing bacteria Shewanella putrefaciens

Magnetite (Fe^IIFe^III₂O₄) is often considered as a stable end product of the bioreduction of Fe^III minerals (e.g., ferrihydrite, lepidocrocite, hematite) or of the biological oxidation of Fe^II compounds (e.g., siderite), with green rust (GR) as a mixed Fe^II‐Fe^III hydroxide intermediate. Until now, the biotic transformation of magnetite to GR has not been evidenced. In this study, we investigated the capability of an iron‐reducing bacterium, Shewanella putrefaciens, to reduce magnetite at circumneutral pH in the presence of dihydrogen as sole inorganic electron donor. During incubation, GR and/or siderite (Fe^IICO₃) formation occurred as secondary iron minerals, resulting from the precipitation of Fe^II species produced via the bacterial reduction of Fe^III species present in magnetite. Taking into account the exact nature of the secondary iron minerals and the electron donor source is necessary to understand the exergonic character of the biotic transformation of magnetite to GR, which had been considered to date as thermodynamically unfavorable at circumneutral pH. This finding reinforces the hypothesis that GR would be the cornerstone of the microbial transformations of iron‐bearing minerals in the anoxic biogeochemical cycle of iron and opens up new possibilities for the interpretation of the evolution of Earth's history and for the understanding of biocorrosion processes in the field of applied science.     

Thermodynamic characterization of proton‐ionizable functional groups on the cell surfaces of ammonia‐oxidizing bacteria and archaea

The ammonia‐oxidizing archaeon Nitrosopumilus maritimus strain SCM1 (strain SCM1), a representative of the Thaumarchaeota archaeal phylum, can sustain high specific rates of ammonia oxidation at ammonia concentrations too low to sustain metabolism by ammonia‐oxidizing bacteria (AOB). One structural and biochemical difference between N. maritimus and AOB that might be related to the oligotrophic adaptation of strain SCM1 is the cell surface. A proteinaceous surface layer (S‐layer) comprises the outermost boundary of the strain SCM1 cell envelope, as opposed to the lipopolysaccharide coat of Gram‐negative AOB. In this work, we compared the surface reactivities of two archaea having an S‐layer (strain SCM1 and Sulfolobus acidocaldarius) with those of four representative AOB (Nitrosospira briensis, Nitrosomonas europaea, Nitrosolobus multiformis, and Nitrosococcus oceani) using potentiometric and calorimetric titrations to evaluate differences in proton‐ionizable surface sites. Strain SCM1 and S. acidocaldarius have a wider range of proton buffering (approximately pH 10–3.5) than the AOB (approximately pH 10–4), under the conditions investigated. Thermodynamic parameters describing proton‐ionizable sites (acidity constants, enthalpies, and entropies of protonation) are consistent with these archaea having proton‐ionizable amino acid side chains containing carboxyl, imidazole, thiol, hydroxyl, and amine functional groups. Phosphorous‐bearing acidic functional groups, which might also be present, could be masked by imidazole and thiol functional groups. Parameters for the AOB are consistent with surface structures containing anionic oxygen ligands (carboxyl‐ and phosphorous‐bearing acidic functional groups), thiols, and amines. In addition, our results showed that strain SCM1 has more reactive surface sites than the AOB and a high concentration of sites consistent with aspartic and/or glutamic acid. Because these alternative boundary layers mediate interaction with the local external environment, these data provide the basis for further comparisons of the thermodynamic behavior of surface reactivity toward essential nutrients.     

Evidence for iron‐mediated anaerobic methane oxidation in a crude oil‐contaminated aquifer

In a methanogenic crude oil contaminated aquifer near Bemidji, Minnesota, the decrease in dissolved CH₄ concentrations along the groundwater flow path, along with the positive shift in δ¹³C_CH4 and negative shift in δ¹³C_DIC, is indicative of microbially mediated CH₄ oxidation. Calculations of electron acceptor transport across the water table, through diffusion, recharge, and the entrapment and release of gas bubbles, suggest that these processes can account for at most 15% of the observed total reduced carbon oxidation, including CH₄. In the anaerobic plume, the characteristic Fe(III)‐reducing genus Geobacter was the most abundant of the microbial groups tested, and depletion of labile sediment iron is observed over time, confirming that reduced carbon oxidation coupled to iron reduction is an important process. Electron mass balance calculations suggest that organic carbon sources in the aquifer, BTEX and non‐volatile dissolved organic carbon, are insufficient to account for the loss in sediment Fe(III), implying that CH₄ oxidation may also be related to Fe(III) reduction. The results support a hypothesis of Fe(III)‐mediated CH₄ oxidation in the contaminated aquifer.     

Direct real‐time imaging of protein adsorption onto hydrophilic and hydrophobic surfaces

Atomic force microscopy has been used to follow in real time the adsorption from solution of two of the gliadin group of wheat seed storage proteins onto hydrophilic (mica) and hydrophobic (graphite) surfaces. The liquid cell of the microscope was used initially to acquire images of the substrate under a small quantity of pure solvent (1% acetic acid). Continuous imaging as an injection of gliadin solution entered the liquid cell enabled the adsorption process to be followed in situ from zero time. For ω‐gliadin, a monolayer was formed on the mica substrate during a period of ～2000 s, with the protein molecules oriented in parallel to the mica surface. In contrast, the ω‐gliadin had a relatively low affinity for the graphite substrate, as demonstrated by slow and weak adsorption to the surface. With γ‐gliadin, random deposition onto the mica surface was observed forming monodispersed structures, whereas on the graphite surface, monolayer islands of protein were formed with the protein molecules in a perpendicular orientation. Sequential adsorption experiments indicated strong interactions between the two proteins that, under certain circumstances, caused alterations to the surface morphologies of preadsorbed species. The results are relevant to our understanding of the interactions of proteins within the hydrated protein bodies of wheat grain and how these determine the processing properties of wheat gluten and dough. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 74–84, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Carbon isotopic heterogeneity of coenzyme F430 and membrane lipids in methane‐oxidizing archaea

Archaeal ANaerobic MEthanotrophs (ANME) facilitate the anaerobic oxidation of methane (AOM), a process that is believed to proceed via the reversal of the methanogenesis pathway. Carbon isotopic composition studies indicate that ANME are metabolically diverse and able to assimilate metabolites including methane, methanol, acetate, and dissolved inorganic carbon (DIC). Our data support the interpretation that ANME in marine sediments at methane seeps assimilate both methane and DIC, and the carbon isotopic compositions of the tetrapyrrole coenzyme F430 and the membrane lipids archaeol and hydroxy‐archaeol reflect their relative proportions of carbon from these substrates. Methane is assimilated via the methyl group of CH₃‐tetrahydromethanopterin (H₄MPT) and DIC from carboxylation reactions that incorporate free intracellular DIC. F430 was enriched in ¹³C (mean δ¹³C = ?27‰ for Hydrate Ridge and ?80‰ for the Santa Monica Basin) compared to the archaeal lipids (mean δ¹³C = ?97‰ for Hydrate Ridge and ?122‰ for the Santa Monica Basin). We propose that depending on the side of the tricarboxylic acid (TCA) cycle used to synthesize F430, its carbon was derived from 76% DIC and 24% methane via the reductive side or 57% DIC and 43% methane via the oxidative side. ANME lipids are predicted to contain 42% DIC and 58% methane, reflecting the amount of each assimilated into acetyl‐CoA. With isotope models that include variable fractionation during biosynthesis for different carbon substrates, we show the estimated amounts of DIC and methane can result in carbon isotopic compositions of ? 73‰ to ? 77‰ for F430 and ? 105‰ for archaeal lipids, values close to those for Santa Monica Basin. The F430 δ¹³C value for Hydrate Ridge was ¹³C‐enriched compared with the modeled value, suggesting there is divergence from the predicted two carbon source models.