Substrate selectivity and catalytic activation of the type III CRISPR ancillary nuclease Can2.

Type III CRISPR-Cas systems provide adaptive immunity against foreign mobile genetic elements through RNA-guided interference. Sequence-specific recognition of RNA targets by the type III effector complex triggers the generation of cyclic oligoadenylate (cOA) second messengers that activate ancillary effector proteins, thus reinforcing the host immune response. The ancillary nuclease Can2 is activated by cyclic tetra-AMP (cA4); however, the mechanisms underlying cA4-mediated activation and substrate selectivity remain elusive. Here we report crystal structures of Thermoanaerobacter brockii Can2 (TbrCan2) in substrate- and product-bound complexes. We show that TbrCan2 is a single strand-selective DNase and RNase that binds substrates via a conserved SxTTS active site motif, and reveal molecular interactions underpinning its sequence preference for CA dinucleotides. Furthermore, we identify a molecular interaction relay linking the cA4 binding site and the nuclease catalytic site to enable divalent metal cation coordination and catalytic activation. These findings provide key insights into the molecular mechanisms of Can2 nucleases in type III CRISPR-Cas immunity and may guide their technological development for nucleic acid detection applications.

Characterization of ferredoxins from the thermophilic, acetogenic bacterium Thermoanaerobacter kivui.

A major electron carrier involved in energy and carbon metabolism in the acetogenic model organism Thermoanaerobacter kivui is ferredoxin, an iron-sulfur-containing, electron-transferring protein. Here, we show that the genome of T. kivui encodes four putative ferredoxin-like proteins (TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530). All four genes were cloned, a His-tag encoding sequence was added and the proteins were produced from a plasmid in T. kivui. The purified proteins had an absorption peak at 430 nm typical for ferredoxins. The determined iron-sulfur content is consistent with the presence of two predicted [4Fe4S] clusters in TKV_c09620 and TKV_c19530 or one predicted [4Fe4S] cluster in TKV_c16450 and TKV_c10420 respectively. The reduction potential (Em ) for TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530 was determined to be -386 ± 4 mV, -386 ± 2 mV, -559 ± 10 mV and -557 ± 3 mV, respectively. TKV_c09620 and TKV_c16450 served as electron carriers for different oxidoreductases from T. kivui. Deletion of the ferredoxin genes led to only a slight reduction of growth on pyruvate or autotrophically on H2 + CO2 . Transcriptional analysis revealed that TKV_c09620 was upregulated in a ΔTKV_c16450 mutant and vice versa TKV_c16450 in a ΔTKV_c09620 mutant, indicating that TKV_c09620 and TKV_c16450 can replace each other. In sum, our data are consistent with the hypothesis that TKV_c09620 and TKV_c16450 are ferredoxins involved in autotrophic and heterotrophic metabolism of T. kivui.

Crystallographic snapshots of ternary complexes of thermophilic secondary alcohol dehydrogenase from Thermoanaerobacter pseudoethanolicus reveal the dynamics of ligand exchange and the proton relay network.

Three-dimensional structures of I86A and C295A mutant secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter pseudoethanolicus were determined by x-ray crystallography. The tetrameric structure of C295A-SADH soaked with NADP+ and dimethyl sulfoxide (DMSO) was determined to 1.85 Å with an Rfree of 0.225. DMSO is bound to the tetrahedral zinc in each subunit, with ligands from SG of Cys-37, NE2 of His-59, and OD2 of Asp-150. The nicotinamide ring of NADP is hydrogen-bonded to the N of Ala-295 and the O of Val-265 and Gly-293. The O of DMSO is connected to a network of hydrogen bonds with OG of Ser-39, the 3'-OH of NADP, and ND1 of His-42. The structure of I86A-SADH soaked with 2-pentanol and NADP+ contains (R)-2-pentanol bound in each subunit, ligated to the tetrahedral zinc, and connected to the proton relay network. The structure of I86A-SADH soaked with 3-methylcyclohexanol and NADP+ has alcohol bound in three subunits. Two of the sites have the alcohol ligated to the zinc in an axial position, with OE2 of Glu-60 in the other axial position of a trigonal bipyramidal complex. One site has 3-methylcyclohexanol bound noncovalently, with the zinc in an inverted tetrahedral geometry with Glu-60. The fourth site also has the zinc in a trigonal bipyramidal complex with axial Glu-60 and water ligands. These structures demonstrate that ligand exchange of SADH involves pentacoordinate and inverted zinc complexes with Glu-60. Furthermore, we see a network of hydrogen bonds connecting the substrate oxygen to the external solvent that is likely to play a role in the mechanism of SADH.

Electron carriers involved in autotrophic and heterotrophic acetogenesis in the thermophilic bacterium Thermoanaerobacter kivui.

Thermoanaerobacter kivui is an acetogenic model organism that reduces CO2 with electrons derived from H2 or CO, or from organic substrates in the Wood-Ljugdahl pathway (WLP). For the calculation of ATP yields, it is necessary to know the electron carriers involved in coupling of the oxidative and reductive parts of metabolism. Analyses of key catabolic oxidoreductases in cell-free extract (CFE) or with purified enzymes revealed the physiological electron carriers involved. The glyceraldehyde-3-phosphate dehydrogenase (GA3P-DH) assayed in CFE was NAD+-specific, NADP+ was used with less than 4% and ferredoxin (Fd) was not used. The methylene-THF dehydrogenase was NADP+-specific, NAD+ or Fd were not used. A Nfn-type transhydrogenase that catalyzes reduced Fd-dependent reduction of NADP+ with NADH as electron donor was also identified in CFE. The electron carriers used by the potential electron-bifurcating hydrogenase (HydABC) could not be unambiguously determined in CFE for technical reasons. Therefore, the enzyme was produced homologously in T. kivui and purified by affinity chromatography. HydABC contained 33.9 ± 4.5 mol Fe/mol of protein and FMN; it reduced NADP+ but not NAD+. The methylene-THF reductase (MetFV) was also produced homologously in T. kivui and purified by affinity chromatography. MetFV contained 7.2 ± 0.4 mol Fe/mol of protein and FMN; the complex did neither use NADPH nor NADH as reductant but only reduced Fd. In sum, these analysis allowed us to propose a scheme for entire electron flow and bioenergetics in T. kivui.

The pyruvate:ferredoxin oxidoreductase of the thermophilic acetogen, Thermoanaerobacter kivui.

Pyruvate:ferredoxin oxidoreductase (PFOR) is a key enzyme in bacterial anaerobic metabolism. Since a low-potential ferredoxin (Fd2- ) is used as electron carrier, PFOR allows for hydrogen evolution during heterotrophic growth as well as pyruvate synthesis during lithoautotrophic growth. The thermophilic acetogenic model bacterium Thermoanaerobacter kivui can use both modes of lifestyle, but the nature of the PFOR in this organism was previously unestablished. Here, we have isolated PFOR to apparent homogeneity from cells grown on glucose. Peptide mass fingerprinting revealed that it is encoded by pfor1. PFOR uses pyruvate as an electron donor and methylene blue (1.8 U·mg-1 ) and ferredoxin (Fd; 27.2 U·mg-1 ) as electron acceptors, and the reaction is dependent on thiamine pyrophosphate, pyruvate, coenzyme A, and Fd. The pH and temperature optima were 7.5 and 66 °C, respectively. We detected 13.6 mol of iron·mol of protein-1 , consistent with the presence of three predicted [4Fe-4S] clusters. The ability to provide reduced Fd makes PFOR an interesting auxiliary enzyme for enzyme assays. To simplify and speed up the purification procedure, we established a protocol for homologous protein production in T. kivui. Therefore, pfor1 was cloned and expressed in T. kivui and the encoded protein containing a genetically engineered His-tag was purified in only two steps to apparent homogeneity. The homologously produced PFOR1 had the same properties as the enzyme from T. kivui. The enzyme can be used as auxiliary enzyme in enzymatic assays that require reduced Fd as electron donor, such as electron-bifurcating enzymes, to keep a constant level of reduced Fd.

Cross-feeding and wheat straw extractives enhance growth of Clostridium thermocellum-containing co-cultures for consolidated bioprocessing.

Co-cultures consisting of three thermophilic and lignocellulolytic bacteria, namely Clostridium thermocellum, C. stercorarium, and Thermoanaerobacter thermohydrosulfuricus, degrade lignocellulosic material in a synergistic manner. When cultured in a defined minimal medium two of the members appeared to be auxotrophic and unable to grow, but the growth of all species was observed in all co-culture combinations, indicating cross-feeding of unidentified growth factors between the members. Growth factors also appeared to be present in water-soluble extractives obtained from wheat straw, allowing for the growth of the auxotrophic monocultures in the defined minimal medium. Cell enumeration during growth on wheat straw in this medium revealed different growth profiles of the members that varied between the co-cultures. End-product profiles also varied substantially between the cultures, with significantly higher ethanol production in all co-cultures compared to the mono-cultures. Understanding interactions between co-culture members, and the additional nutrients provided by lignocellulosic substrates, will aid us in consolidated bioprocessing design.

Synthesis of zinc-gallate phosphors by biomineralization and their emission properties.

Reduction of target species by microorganisms and their subsequent precipitation into sparingly soluble mineral phase nanoparticles have been referred to as microbially mediated nanomaterial synthesis. Here, we describe the microbially mediated production of nano-dimensioned spinel structured zinc-gallate (ZnGa2O4) phosphors exhibiting different emission performance with varying substituted elements. Interestingly, in the microbially mediated phosphor production described herein, there were no reducible metal- and non-metal species composing the target minerals. By varying substituted elements, zinc-gallate phosphors present typical red, green, and blue (RGB) emission. An apparent whitish emission was accomplished by blending phosphors. A promising potential for white light produced by biosynthesized mixtures of Cr-, Mn-, and Co- substituted zinc-gallates representing RGB emissions was evidenced. Microbial activity supplied a reducing driving force and provided appropriate near neutral pH and reduced Eh conditions to thermodynamically precipitate spinel structured nanomaterials from supersaturated divalent and trivalent cations. This result complemented conventional biomineralization concepts and expanded the realm of biomanufacturing nanomaterials for further applications. STATEMENT OF SIGNIFICANCE: This study substantiated that circumstances of a suitable pH/Eh derived from bacterial activity, divalent/trivalent ion supply, buffering capacity, and supersaturation could precipitate spinel structure nanoparticles. Even though live or dead cells with membrane could enhance the nuclei generation, the spinel structured phases were produced regardless of existence of live or dead cells and reducible metal or non-metal species incorporating into the produced solid phases. This finding led to production of a series of metal-substituted zinc-gallates with specific RGB emission that can result in whitish light using simple blending. We believe our findings could expand the realm of nanomaterial synthesis using low cost, highly scalable bio-nanotechnology.

Branched-chain amino acid catabolism of Thermoanaerobacter strain AK85 and the influence of culture conditions on branched-chain alcohol formation.

The bioprocessing of amino acids to branched-chain fatty acids and alcohols is described using Thermoanaerobacter strain AK85. The amino acid utilization profile was evaluated without an electron scavenger, with thiosulfate, and in a co-culture with a methanogen. There was an emphasis on the production of branched-chain alcohols and fatty acids from the branched-chain amino acids, particularly the influence of culture conditions which was investigated using isoleucine, which revealed that the concentration of thiosulfate was of great importance for the branched-chain alcohols/fatty acid ratio produced. Kinetic studies show that branched-chain amino acid fermentation is relatively slow as compared to glucose metabolism with the concentrations of the branched-chain alcohol increasing over time. To understand the flow of electrons and to investigate if the branched-chain fatty acid was being converted to branched-chain alcohol, enzyme assays and fermentation studies using 13C-labeled leucine and 3-methyl-1-butyrate were performed which indeed suggest that carboxylic acid reduction is a source of branched-chain alcohols when Thermoanaerobacter strain AK85 was cultivated with thiosulfate as an electron scavenger.

Biotransformation of organic acids to their corresponding alcohols by Thermoanaerobacter pseudoethanolicus.

Higher order alcohols, such as 1-butanol and 1-hexanol, have a large number of applications but are currently prepared from non-renewable feedstocks. Here, the ability of Thermoanaerobacter pseudoethanolicus to reduce short-chain fatty acids to their corresponding alcohols using reducing potential generated by glucose catabolism with yields between 21.0 and 61.0%. 13C-labelled acetate, 1-propionate and 1-butyrate demonstrates that exogenously added fatty acids are indeed reduced to their corresponding alcohols. This mode of producing primary alcohols from fatty acids using a thermophilic anaerobe opens the door for the production of such alcohols from low-value materials using an inexpensive source of reducing potential.

Energy conservation by a hydrogenase-dependent chemiosmotic mechanism in an ancient metabolic pathway.

The ancient reductive acetyl-CoA pathway is employed by acetogenic bacteria to form acetate from inorganic energy sources. Since the central pathway does not gain net ATP by substrate-level phosphorylation, chemolithoautotrophic growth relies on the additional formation of ATP via a chemiosmotic mechanism. Genome analyses indicated that some acetogens only have an energy-converting, ion-translocating hydrogenase (Ech) as a potential respiratory enzyme. Although the Ech-encoding genes are widely distributed in nature, the proposed function of Ech as an ion-translocating chemiosmotic coupling site has neither been demonstrated in bacteria nor has it been demonstrated that it can be the only energetic coupling sites in microorganisms that depend on a chemiosmotic mechanism for energy conservation. Here, we show that the Ech complex of the thermophilic acetogenic bacterium Thermoanaerobacter kivui is indeed a respiratory enzyme. Experiments with resting cells prepared from T. kivui cultures grown on carbon monoxide (CO) revealed CO oxidation coupled to H2 formation and the generation of a transmembrane electrochemical ion gradient ([Formula: see text]). Inverted membrane vesicles (IMVs) prepared from CO-grown cells also produced H2 and ATP from CO (via a loosely attached CO dehydrogenase) or a chemical reductant. Finally, we show that Ech activity led to the translocation of both H+ and Na+ across the membrane of the IMVs. The H+ gradient was then used by the ATP synthase for energy conservation. These data demonstrate that the energy-converting hydrogenase in concert with an ATP synthase may be the simplest form of respiration; it combines carbon dioxide fixation with the synthesis of ATP in an ancient pathway.

Enhanced depolymerization and utilization of raw lignocellulosic material by co-cultures of Ruminiclostridium thermocellum with hemicellulose-utilizing partners.

Ruminiclostridium thermocellum is one of the most promising candidates for consolidated bioprocessing (CBP) of low-cost lignocellulosic materials to biofuels but it still shows poor performance in its ability to deconstruct untreated lignocellulosic substrates. One promising approach to increase R. thermocellum's rate of hydrolysis is to co-culture this cellulose-specialist with partners that possess synergistic hydrolysis enzymes and metabolic capabilities. We have created co-cultures of R. thermocellum with two hemicellulose utilizers, Ruminiclostridium stercorarium and Thermoanaerobacter thermohydrosulfuricus, both of which secrete xylanolytic enzymes and utilize the pentose oligo- and monosaccharides that inhibit R. thermocellum's hydrolysis and metabolism. When grown on milled wheat straw, the co-cultures were able to solubilize up to 58% more of the total polysaccharides than the R. thermocellum mono-culture control. Repeated passaging of the co-cultures on wheat straw yielded stable populations with reduced R. thermocellum cell numbers, indicating competition for cellodextrins released from cellulose hydrolysis, although these stabilized co-cultures were still able to outperform the mono-culture controls. Repeated passaging on Avicel cellulose also yielded stable populations. Overall, the observed synergism suggests that co-culturing R. thermocellum with other members is a viable option for increasing the rate and extent of untreated lignocellulose deconstruction by R. thermocellum for CBP purposes.

Expression of engineered carbonyl reductase from Ogataea minuta in Rhodococcus opacus and its application to whole-cell bioconversion in anhydrous solvents.

The carbonyl reductase from the methylotrophic yeast Ogataea minuta can catalyze the regio- and enantio-selective reduction of prochiral ketones to chiral alcohols, and is available for industrial manufacturing of statin drugs. We previously conducted a directed evolution experiment of the enzyme, and obtained a mutant (OCR_V166A) with improved tolerance to organic solvents. This expanded the applicability of the enzyme to the bioconversion of water-insoluble compounds (Honda et al., J. Biosci. Bioeng., 123, 673-678, 2017). In the present study, we expressed OCR_V166A in Rhodococcus opacus cells, which have a highly lipophilic surface structure and are dispersible in anhydrous organic solvents, and developed a whole-cell biocatalyst which can function in an organic-solvent-based reaction medium. The secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (TeADH) was employed as an NADPH-regenerating enzyme and co-expressed with OCR_V166A in R. opacus. The whole-cell bioconversion of 2,2,2-trifluoroacetophenone to α-(trifluoromethyl)benzyl alcohol was performed in organic solvents, including isopropanol, isobutanol, and cyclohexanol, which served both as reaction media and as substrates for TeADH. The type of organic solvents markedly affected not only the product titer but also the enantio-purity of the product. When isobutanol was used as the reaction medium, the whole-cell biocatalyst showed higher stability than the isolated enzyme. Consequently, a high concentration (1 M) of the substrate was converted to the product with an overall conversion yield of 81% (mol/mol) in 24 h.

Thermoanaerobacter species differ in their potential to reduce organic acids to their corresponding alcohols.

The reduction of organic acids to their corresponding alcohols has been shown for some bacterial species within the Firmicutes super-phylum and a genetically modified strain of the hyperthermophilic archaeon Pyrococcus furiosus. In the latter strain, an aldehyde:ferredoxin oxidoreductase (AOR) catalyzed the reduction of a variety of organic acids to their corresponding aldehydes, as shown by the deletion of the corresponding aor gene. Here, we found that the genomes of a few thermophilic bacterial species within the genus Thermoanaerobacter which have been described to efficiently ferment sugars to ethanol harbor a copy of aor, while others do not. Specific AOR activity was only found in strains with aor, and the gene was highly expressed in Thermoanaerobacter sp. strain X514. The reduction of a variety of organic acids was observed for several Thermoanaerobacter sp.; however, strains with aor reduced, e.g., isobutyrate at much higher rates of up to 5.1 mM h-1 g-1. Organic acid reduction also led to increased growth rates in Thermoanaerobacter sp. strain X514 and in Thermoanaerobacter pseudethanolicus. Organic acid activation may proceed via acyl-CoA with subsequent NADH-dependent reduction by an aldehyde dehydrogenase (ALDH), or via direct reduction by AOR. Cell-free extracts of Thermoanaerobacter sp. strain X514 exhibited both enzyme activities at comparable rates. Therefore, the biochemistry of organic acid reduction to alcohols in Thermoanaerobacter sp. remains to be elucidated; however, relatively high specific activities and the correlation of AOR specific activities with alcohol production rates suggest a role for AOR.

Fixation of CO2 and CO on a diverse range of carbohydrates using anaerobic, non-photosynthetic mixotrophy.

Biological CO2 fixation is an important technology that can assist in combating climate change. Here, we show an approach called anaerobic, non-photosynthetic mixotrophy can result in net CO2 fixation when using a reduced feedstock. This approach uses microbes called acetogens that are capable of concurrent utilization of both organic and inorganic substrates. In this study, we investigated the substrate utilization of 17 different acetogens, both mesophilic and thermophilic, on a variety of different carbohydrates and gases. Compared to most model acetogen strains, several non-model mesophilic strains displayed greater substrate flexibility, including the ability to utilize disaccharides, glycerol and an oligosaccharide, and growth rates. Three of these non-model strains (Blautia producta, Clostridium scatologenes and Thermoanaerobacter kivui) were chosen for further characterization, under a variety of conditions including H2- or syngas-fed sugar fermentations and a CO2-fed glycerol fermentation. In all cases, CO2 was fixed and carbon yields approached 100%. Finally, the model acetogen C. ljungdahlii was engineered to utilize glucose, a non-preferred sugar, while maintaining mixotrophic behavior. This work demonstrates the flexibility and robustness of anaerobic, non-photosynthetic mixotrophy as a technology to help reduce CO2 emissions.

Physiological, metabolic and biotechnological features of extremely thermophilic microorganisms.

The current upper thermal limit for life as we know it is approximately 120°C. Microorganisms that grow optimally at temperatures of 75°C and above are usually referred to as 'extreme thermophiles' and include both bacteria and archaea. For over a century, there has been great scientific curiosity in the basic tenets that support life in thermal biotopes on earth and potentially on other solar bodies. Extreme thermophiles can be aerobes, anaerobes, autotrophs, heterotrophs, or chemolithotrophs, and are found in diverse environments including shallow marine fissures, deep sea hydrothermal vents, terrestrial hot springs-basically, anywhere there is hot water. Initial efforts to study extreme thermophiles faced challenges with their isolation from difficult to access locales, problems with their cultivation in laboratories, and lack of molecular tools. Fortunately, because of their relatively small genomes, many extreme thermophiles were among the first organisms to be sequenced, thereby opening up the application of systems biology-based methods to probe their unique physiological, metabolic and biotechnological features. The bacterial genera Caldicellulosiruptor, Thermotoga and Thermus, and the archaea belonging to the orders Thermococcales and Sulfolobales, are among the most studied extreme thermophiles to date. The recent emergence of genetic tools for many of these organisms provides the opportunity to move beyond basic discovery and manipulation to biotechnologically relevant applications of metabolic engineering. WIREs Syst Biol Med 2017, 9:e1377. doi: 10.1002/wsbm.1377 For further resources related to this article, please visit the WIREs website.

"Hot" acetogenesis.

Thermophilic microorganisms as well as acetogenic bacteria are both considered ancient. Interestingly, only a few species of bacteria, all belonging to the family Thermoanaerobacteraceae, are described to conserve energy from acetate formation with hydrogen as electron donor and carbon dioxide as electron acceptor. This review reflects the metabolic differences between Moorella spp., Thermoanaerobacter kivui and Thermacetogenium phaeum, with focus on the biochemistry of autotrophic growth and energy conservation. The potential of these thermophilic acetogens for biotechnological applications is discussed briefly.

Growth and metabolic profiling of the novel thermophilic bacterium Thermoanaerobacter sp. strain YS13.

A strictly anaerobic, thermophilic bacterium, designated strain YS13, was isolated from a geothermal hot spring. Phylogenetic analysis using the 16S rRNA genes and cpn60 UT genes suggested strain YS13 as a species of Thermoanaerobacter. Using cellobiose or xylose as carbon source, YS13 was able to grow over a wide range of temperatures (45-70 °C), and pHs (pH 5.0-9.0), with optimum growth at 65 °C and pH 7.0. Metabolic profiling on cellobiose, glucose, or xylose in 1191 medium showed that H2, CO2, ethanol, acetate, and lactate were the major metabolites. Lactate was the predominant end product from glucose or cellobiose fermentations, whereas H2 and acetate were the dominant end products from xylose fermentation. The metabolic balance shifted away from ethanol to H2, acetate, and lactate when YS13 was grown on cellobiose as temperatures increased from 45 to 70 °C. When YS13 was grown on xylose, a metabolic shift from lactate to H2, CO2, and acetate was observed in cultures as the temperature of incubation increased from 45 to 65 °C, whereas a shift from ethanol and CO2 to H2, acetate, and lactate was observed in cultures incubated at 70 °C.

Structural and Functional Evidence Indicates Selective Oxygen Signaling in Caldanaerobacter subterraneus H-NOX.

Acute and specific sensing of diatomic gas molecules is an essential facet of biological signaling. Heme nitric oxide/oxygen binding (H-NOX) proteins are a family of gas sensors found in diverse classes of bacteria and eukaryotes. The most commonly characterized bacterial H-NOX domains are from facultative anaerobes and are activated through a conformational change caused by formation of a 5-coordinate Fe(II)-NO complex. Members of this H-NOX subfamily do not bind O2 and therefore can selectively ligate NO even under aerobic conditions. In contrast, H-NOX domains encoded by obligate anaerobes do form stable 6-coordinate Fe(II)-O2 complexes by utilizing a conserved H-bonding network in the ligand-binding pocket. The biological function of O2-binding H-NOX domains has not been characterized. In this work, the crystal structures of an O2-binding H-NOX domain from the thermophilic obligate anaerobe Caldanaerobacter subterraneus (Cs H-NOX) in the Fe(II)-NO, Fe(II)-CO, and Fe(II)-unliganded states are reported. The Fe(II)-unliganded structure displays a conformational shift distinct from the NO-, CO-, and previously reported O2-coordinated structures. In orthogonal signaling assays using Cs H-NOX and the H-NOX signaling effector histidine kinase from Vibrio cholerae (Vc HnoK), Cs H-NOX regulates Vc HnoK in an O2-dependent manner and requires the H-bonding network to distinguish O2 from other ligands. The crystal structures of Fe(II) unliganded and NO- and CO-bound Cs H-NOX combined with functional assays herein provide the first evidence that H-NOX proteins from obligate anaerobes can serve as O2 sensors.

Manufacturing demonstration of microbially mediated zinc sulfide nanoparticles in pilot-plant scale reactors.

The thermophilic anaerobic metal-reducing bacterium Thermoanaerobacter sp. X513 efficiently produces zinc sulfide (ZnS) nanoparticles (NPs) in laboratory-scale (≤ 24-L) reactors. To determine whether this process can be up-scaled and adapted for pilot-plant production while maintaining NP yield and quality, a series of pilot-plant scale experiments were performed using 100-L and 900-L reactors. Pasteurization and N2-sparging replaced autoclaving and boiling for deoxygenating media in the transition from small-scale to pilot plant reactors. Consecutive 100-L batches using new or recycled media produced ZnS NPs with highly reproducible ~2-nm average crystallite size (ACS) and yields of ~0.5 g L(-1), similar to the small-scale batches. The 900-L pilot plant reactor produced ~320 g ZnS without process optimization or replacement of used medium; this quantity would be sufficient to form a ZnS thin film with ~120 nm thickness over 0.5 m width × 13 km length. At all scales, the bacteria produced significant amounts of acetic, lactic, and formic acids, which could be neutralized by the controlled addition of sodium hydroxide without the use of an organic pH buffer, eliminating 98 % of the buffer chemical costs. The final NP products were characterized using XRD, ICP-OES, TEM, FTIR, PL, DLS, HPLC, and C/N analyses, which confirmed that the growth medium without organic buffer enhanced the ZnS NP properties by reducing carbon and nitrogen surface coatings and supporting better dispersivity with similar ACS.

A biological process effective for the conversion of CO-containing industrial waste gas to acetate.

Acetogens have often been observed to be inhibited by CO above an inhibition threshold concentration. In this study, a two-stage culture consisting of carboxydotrophic archaea and homoacetogenic bacteria is found to be effective in converting industrial waste gas derived from a steel mill process. In the first stage, Thermococcus onnurineus could grow on the Linz-Donawitz converter gas (LDG) containing ca. 56% CO as a sole energy source, converting the CO into H2 and CO2. Then, in the second stage, Thermoanaerobacter kivui could grow on the off-gas from the first stage culture, consuming the H2 and CO in the off-gas completely and producing acetate as a main product. T. kivui alone could not grow on the LDG gas. This work represents the first demonstration of acetate production using steel mill waste gas by a two-stage culture of carboxydotrophic hydrogenogenic microbes and homoacetogenic bacteria.