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International audience; Ferredoxin-NAD(P) + oxidoreductases are important enzymes for redox balancing in n-butanol production by Clostridium acetobutylicum, but the encoding genes remain unknown. Here, the authors identify the long sought-after genes and increase n-butanol production by optimizing the levels of the two enzymes.Clostridium acetobutylicum is a promising biocatalyst for the renewable production of n-butanol. Several metabolic strategies have already been developed to increase butanol yields, most often based on carbon pathway redirection. However, it has previously demonstrated that the activities of both ferredoxin-NADP(+) reductase and ferredoxin-NAD(+) reductase, whose encoding genes remain unknown, are necessary to produce the NADPH and the extra NADH needed for butanol synthesis under solventogenic conditions. Here, we purify, identify and partially characterize the proteins responsible for both activities and demonstrate the involvement of the identified enzymes in butanol synthesis through a reverse genetic approach. We further demonstrate the yield of butanol formation is limited by the level of expression of CA_C0764, the ferredoxin-NADP(+) reductase encoding gene and the bcd operon, encoding a ferredoxin-NAD(+) reductase. The integration of these enzymes into metabolic engineering strategies introduces opportunities for developing a homobutanologenic C. acetobutylicum strain.

Clostridium acetobutylicum has received renewed interest worldwide as a promising producer of biofuels and bulk chemicals such as n-butanol, 1,3-propanediol, 1,3-butanediol, isopropanol, and butyrate. To develop commercial processes for the production of bulk chemicals via a metabolic engineering approach it is necessary to better characterize both the primary metabolism and metabolic regulation of C. acetobutylicum. Here, we review the history of the development of omics studies of C. acetobutylicum, summarize the recent application of quantitative/integrated omics approaches to the physiological analysis and metabolic engineering of this bacterium, and provide directions for future studies to address current challenges.

Clostridium acetobutylicum possesses two homologous buk genes, buk (or buk1) and buk2, which encode butyrate kinases involved in the last step of butyrate formation. To investigate the contribution of buk in detail, an in-frame deletion mutant was constructed. However, in all the Delta buk mutants obtained, partial deletions of the upstream ptb gene were observed, and low phosphotransbutyrylase and butyrate kinase activities were measured. This demonstrates that i) buk (CA_C3075) is the key butyrate kinase-encoding gene and that buk2 (CA_C1660) that is poorly transcribed only plays a minor role; and ii) strongly suggests that a Delta buk mutant is not viable if the ptb gene is not also inactivated, probably due to the accumulation of butyryl-phosphate, which might be toxic for the cell. One of the Delta buk Delta ptb mutants was subjected to quantitative transcriptomic (mRNA molecules/cell) and fluxomic analyses in acidogenic, solventogenic and alcohologenic chemostat cultures. In addition to the low butyrate production, drastic changes in metabolic fluxes were also observed for the mutant: i) under acidogenic conditions, the primary metabolite was butanol and a new metabolite, 2-hydroxy-valerate, was produced ii) under solventogenesis, 58% increased butanol production was obtained compared to the control strain under the same conditions, and a very high yield of butanol formation (0.3 g g-(1)) was reached; and iii) under alcohologenesis, the major product was lactate. Furthermore, at the transcriptional level, adhE2, which encodes an aldehyde/alcohol dehydrogenase and is known to be a gene specifically expressed in alcohologenesis, was surprisingly highly expressed in all metabolic states in the mutant. The results presented here not only support the key roles of buk and ptb in butyrate formation but also highlight the metabolic flexibility of C. acetobutylicum in response to genetic alteration of its primary metabolism.

Background: Clostridium acetobutylicum and Clostridium saccharobutylicum are Gram-positive, spore-forming, anaerobic bacterium capable of converting various sugars and polysaccharides into solvents (acetone, butanol, and ethanol). The sequencing of their genomes has prompted new approaches to genetic analysis, functional genomics, and metabolic engineering to develop industrial strains for the production of biofuels and bulk chemicals.Results: The method used in this paper to knock-out, knock-in, or edit genes in C. acetobutylicum and C. saccharobutylicum combines an improved electroporation method with the use of (i) restrictionless Δupp (which encodes uracil phosphoribosyl-transferase) strains and (ii) very small suicide vectors containing a markerless deletion/insertion cassette, an antibiotic resistance gene (for the selection of the first crossing-over) and upp (from C. acetobutylicum) for subsequent use as a counterselectable marker with the aid of 5-fluorouracil (5-FU) to promote the second crossing-over. This method was successfully used to both delete genes and edit genes in both C. acetobutylicum and C. saccharobutylicum. Among the edited genes, a mutation in the spo0A gene that abolished solvent formation in C. acetobutylicum was introduced in C. saccharobutylicum and shown to produce the same effect.Conclusions: The method described in this study will be useful for functional genomic studies and for the development of industrial strains for the production of biofuels and bulk chemicals.

We proposed a novel electromagnetic-based chemical sensor that is realized by using a metamaterial absorber. The metamaterial absorber comprises a split-ring-cross resonator (SRCR) and a microfluidic channel. The SRCR can generate LC resonance that is very sensitive to changes in the effective dielectric constant around the capacitive gap. In addition, microfluidic channels can change the effective dielectric constant of the dielectric substrate by using an infinitesimal quantity of a liquid on the order of microliters. The proposed chemical sensor can detect the electrical properties of any unidentified liquids injected into the channels, as well as concentration changes in the liquids. The performance of the proposed sensor is demonstrated using the absorption measurements of a fabricated prototype sample with waveguides. In addition, the relationship between the absorption frequency and chemical concentration is demonstrated.

In this study, we demonstrated a new class of frequency-switchable metamaterial absorber in the X-band. Eutectic gallium-indium (EGaIn), a liquid metal alloy, was injected in a microfluidic channel engraved on polymethyl methacrylate (PMMA) to achieve frequency switching. Numerical simulation and experimental results are presented for two cases: when the microfluidic channels are empty, and when they are filled with liquid metal. To evaluate the performance of the fabricated absorber prototype, it is tested with a rectangular waveguide. The resonant frequency was successfully switched from 10.96 GHz to 10.61 GHz after injecting liquid metal while maintaining absorptivity higher than 98%.

In this paper, we present three ultra-thin frequency selective surface (FSS) microwave absorbers on a single layer, that have three remarkable features. First, the various absorbing bandwidths can be obtained in our absorber by adjusting the two absorbing peaks, which are produced by two different resonance effects on the FSS. By manipulating the dimension parameters, we design the dual-band, 3-dB and 10-dB band absorbers. We achieved the absorber's thickness of $0.039\lambda_{\rm g}$ with the single layer due to high loss of inter-digital capacitor patterns on the FSS. Additionally, the proposed absorbers are insensitive to polarized electromagnetic (EM) waves because of the symmetric structure of the FSS. In this study, the absorber mechanism and transmission line (TL) modeling for the absorber design are studied. Each of the proposed absorbers is analyzed by using the TL equivalent circuit model and full-wave analysis. The loss factor is studied in order to demonstrate the origin of losses, and a parametric study is conducted to explain how to vary the absorbing bandwidths. The performances of each proposed absorber are also experimentally demonstrated.

In this letter, a novel frequency tunable metamaterial absorber is proposed for wireless humidity sensor applications made with natural cork. Natural cork has excellent hygroscopicity and its electrical properties can be changed depending on the moisture content. In this work, the absorption ratio of the proposed absorber are simulated and measured in different moisture content ( ${\rm M} = 0\% $ , 1.44%, 5.88%, 13.32%, 18.8%) of the cork substrate. The resonant frequency changed from 4.92 GHz in the dry ( ${\rm M} = 0\% $ ) state to 3.49 GHz in the wet ( ${\rm M} = 18.8\% $ ) state; the measured absorptivity is higher than 90% in all moisture content values. To the best of our knowledge, it is the first metamaterial absorber using natural cork.

In this letter, a novel, flexible inkjet-printed metamaterial absorber is proposed. The unit cell consists of a miniaturized Jerusalem-cross resonator and a completely conductive bottom. Because the conductive patterns are inkjet-printed using silver nanoparticle inks on paper, the proposed metamaterial absorber is flexible. It is also eco-friendly because it does not produce any chemical waste. In this letter, the working principle of the proposed miniaturized unit cell is explained, and parametric simulations are described. The inkjet-printing process is delineated, and the silver nanoparticle inks and paper used are characterized. The performance of the proposed absorber is demonstrated with a full-wave simulation and measurements at the $X$ -band. The experimental results show that the absorption rate at 9.09 GHz is greater than 99% at the normal incidence. Angular and polarization insensitivity are also experimentally demonstrated.

A polarization-independent ultrawideband metamaterial absorber is proposed for X-band applications. High absorptivity over an ultrawide spectrum is achieved by the combination of an artificial impedance surface (AIS) and a resistor-capacitor (RC) layer. In addition, the unique hexagonal shape of an AIS and RC layer enables polarization insensitivity. A circuit analysis is introduced based on a transmission-line model and shows good agreement with the full-wave analysis. Fabrication tolerance issues are considered with parametric studies in the electromagnetic simulation. The proposed absorber is fabricated on low-cost FR4 substrates, and its absorption performance is experimentally demonstrated at different angles and polarizations of incident electric fields.

In this study, a novel ultra-wideband antenna with a tri-band notch capability is proposed. The proposed antenna can reject worldwide interoperability for microwave access (WiMAX, 3.3–3.7 GHz), wireless local area network (WLAN) IEEE802.11a/n (5.15–5.825 GHz), and international telecommunication union (ITU, 8.025–8.4 GHz) bands. The band notch capability is achieved by merely a split ring resonator (SRR) and complementary-SRR (CSRR) patterns. The SRR under the radiating patch, the CSRR loaded on the radiating patch, and the CSRRs on the ground of the CPW feeding reject the WiMAX, WLAN, and ITU bands, respectively. The simulation and measurement results demonstrate the performance of the proposed antenna.

Background Clostridium acetobutylicum possesses two homologous adhE genes, adhE1 and adhE2, which have been proposed to be responsible for butanol production in solventogenic and alcohologenic cultures, respectively. To investigate their contributions in detail, in-frame deletion mutants of each gene were constructed and subjected to quantitative transcriptomic (mRNA molecules/cell) and fluxomic analyses in acidogenic, solventogenic, and alcohologenic chemostat cultures. Results Under solventogenesis, compared to the control strain, only ΔadhE1 mutant exhibited significant changes showing decreased butanol production and transcriptional expression changes in numerous genes. In particular, adhE2 was over expressed (126-fold); thus, AdhE2 can partially replace AdhE1 for butanol production (more than 30 % of the in vivo butanol flux) under solventogenesis. Under alcohologenesis, only ΔadhE2 mutant exhibited striking changes in gene expression and metabolic fluxes, and butanol production was completely lost. Therefore, it was demonstrated that AdhE2 is essential for butanol production and thus metabolic fluxes were redirected toward butyrate formation. Under acidogenesis, metabolic fluxes were not significantly changed in both mutants except the complete loss of butanol formation in ΔadhE2, but numerous changes in gene expression were observed. Furthermore, most of the significantly up- or down-regulated genes under this condition showed the same pattern of change in both mutants. Conclusions This quantitative system-scale analysis confirms the proposed roles of AdhE1 and AdhE2 in butanol formation that AdhE1 is the key enzyme under solventogenesis, whereas AdhE2 is the key enzyme for butanol formation under acidogenesis and alcohologenesis. Our study also highlights the metabolic flexibility of C. acetobutylicum to genetic alterations of its primary metabolism. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0507-0) contains supplementary material, which is available to authorized users.

In this paper, a novel ultra wide band(UWB) antenna with tri-band notch capability is proposed. The proposed antenna can reject WiMAX(3.3~3.7 GHz), WLAN IEEE 802.11a/n(5.15~5.825 GHz), and ITU(8.025~8.4 GHz) bands. Band rejection capability is achieved only split ring resonators(SRRs) and complementary SRRs(CSRRs). The SRR under the radiating patch, the CSRR loaded on the radiating patch, and the CSRRs on the ground of the CPW feeding reject the WiMAX, WLAN, and ITU bands, respectively. The simulation and measurement results demonstrate the performances of the proposed antenna.

Engineering industrial microorganisms for ambitious applications, for example, the production of second-generation biofuels such as butanol, is impeded by a lack of knowledge of primary metabolism and its regulation. A quantitative system-scale analysis was applied to the biofuel-producing bacterium Clostridium acetobutylicum, a microorganism used for the industrial production of solvent. An improved genome-scale model, iCac967, was first developed based on thorough biochemical characterizations of 15 key metabolic enzymes and on extensive literature analysis to acquire accurate fluxomic data. In parallel, quantitative transcriptomic and proteomic analyses were performed to assess the number of mRNA molecules per cell for all genes under acidogenic, solventogenic, and alcohologenic steady-state conditions as well as the number of cytosolic protein molecules per cell for approximately 700 genes under at least one of the three steady-state conditions. A complete fluxomic, transcriptomic, and proteomic analysis applied to different metabolic states allowed us to better understand the regulation of primary metabolism. Moreover, this analysis enabled the functional characterization of numerous enzymes involved in primary metabolism, including (i) the enzymes involved in the two different butanol pathways and their cofactor specificities, (ii) the primary hydrogenase and its redox partner, (iii) the major butyryl coenzyme A (butyryl-CoA) dehydrogenase, and (iv) the major glyceraldehyde-3-phosphate dehydrogenase. This study provides important information for further metabolic engineering of C. acetobutylicum to develop a commercial process for the production of n-butanol., IMPORTANCE Currently, there is a resurgence of interest in Clostridium acetobutylicum, the biocatalyst of the historical Weizmann process, to produce n-butanol for use both as a bulk chemical and as a renewable alternative transportation fuel. To develop a commercial process for the production of n-butanol via a metabolic engineering approach, it is necessary to better characterize both the primary metabolism of C. acetobutylicum and its regulation. Here, we apply a quantitative system-scale analysis to acidogenic, solventogenic, and alcohologenic steady-state C. acetobutylicum cells and report for the first time quantitative transcriptomic, proteomic, and fluxomic data. This approach allows for a better understanding of the regulation of primary metabolism and for the functional characterization of numerous enzymes involved in primary metabolism.

In this paper, we propose a novel microfluidic tunable metamaterial (MM) absorber printed on a paper substrate in silver nanoparticle ink. The metamaterial is designed using a periodic array consisting of square patches. The conductive patterns are inkjet-printed on paper using silver nanoparticle inks. The microfluidic channels are laser-etched on polymethyl methacrylate (PMMA). The conductive patterns on paper and the microfluidic channels on PMMA are bonded by an SU-8 layer that is also inkjet-printed on the conductive patterns. The proposed MM absorber provides frequency-tuning capability for different fluids in the microfluidic channels. We performed full-wave simulations and measurements that confirmed that the resonant frequency decreased from 4.42 GHz to 3.97 GHz after the injection of distilled water into the microfluidic channels. For both empty and water-filled channels, the absorptivity is higher than 90% at horizontal and vertical polarizations.

This paper describes a tunable metamaterial (MM) absorber that incorporates novel microfluidic channels and is realized using inkjet printing on a photo-paper substrate. The fabricated sample demonstrated frequency-switching capability owing to distilled water flowing in the microfluidic channels. In addition, the resonant frequency was changed from 4.42 to 3.97 GHz when the empty channels were filled with de-ionized water. An analysis of the results suggests that microfluidic technology is a simpler and more effective way to achieve tuning functionality. The proposed structure is the first microfluidic absorber based on a photo-paper substrate.

In this paper, a novel inkjet-printed metamaterial absorber is proposed for the first time. The proposed absorber consists of circular Jerusalem-Cross resonators and it is printed on a thin paper substrate. The performance of the proposed absorber is validated through EM simulation and measurement. Flexibility and high absorptivity are achieved in X-band.

In this paper, we present a novel electronically switchable metasurface that provides absorption and reflection modes depending on bias voltages of pin diodes. The metasurface is designed by a periodic split ring resonator loading the pin diode. When the pin diodes turn on, the metasurface is working as the absorber. When the pin diodes turn off, the metasurface is working as the reflector. For a reliable operation, bias networks are designed as well. The performance of the proposed metasurface is demonstrated from both full-wave simulation and measurement. At 10.97 GHz, the metasurface shows 99.4% absorptivity and 100% reflectivity at the absorptivity and reflection modes, respectively.

In this paper, a novel wideband metamaterial (MM) absorber is proposed. The proposed absorber is composed of a hexagonal MM structure and a resistor-capacitor (RC) layer, with an air gap between the two layers. The additional RC layer comprises a series resistor and capacitor; this layer and an air gap have an important effect on the wider bandwidth. The proposed absorber shows absorbing properties over a very wide bandwidth. The performance of the proposed absorber is demonstrated by simulation and measurement results.

In this paper, three different unit cells are designed on the basis split-ring-cross resonators, and each unit cell has an absorption rate greater than 90% at incident angles of 0 degrees, 30 degrees, and 45 degrees, respectively. They are non-periodically placed in three different zones on the curved surface. Therefore, the proposed conformal metamaterial absorber can achieve a high absorption rate. The performance of the proposed absorber is compared with that of a metallic curved surface and a conformal metamaterial absorber with the same unit cells. (C) 2013 Optical Society of America

In this paper, we present a novel electromagnetic metamaterial which is able to electronically switch a reflector and absorber. For the absorbing mode, a split ring resonator is employed as an electric LC resonator. The reflector mode is achieved by switching the current path off. The performance is successfully demonstrated both by a full-wave simulation and measurement. At 10 GHz, high absorption is obtained when the switch is the on-state, while the perfect reflection is obtained when the switch on the off-state.

A novel active metasurface is proposed in this paper. The proposed metasurface can be electronically switched between the reflection and the absorption modes by a tunable component, such as a PIN diode. When the PIN diode is in the on-state, the proposed metasurface exhibits near-perfect absorptivity at 10 GHz. On the other hand, when the PIN diode is in the off-state, the proposed metasurface exhibits near-perfect reflectivity over a wide bandwidth at 10 GHz. A unit cell is designed to include a DC bias network, which yields reliable performance.

Additional file 1. Supplementary experimental procedures and results.

Additional file 1. Supplementary experimental procedures and results.