A Two-Compartment Fermentation System to Quantify Strain-Specific Interactions in Microbial Co-Cultures.

To fulfil the growing interest in investigating microbial interactions in co-cultures, a novel two-compartment bioreactor system was developed, characterised, and implemented. The system allowed for the exchange of amino acids and peptides via a polyethersulfone membrane that retained biomass. Further system characterisation revealed a Bodenstein number of 18, which hints at backmixing. Together with other physical settings, the existence of unwanted inner-compartment substrate gradients could be ruled out. Furthermore, the study of Damkoehler numbers indicated that a proper metabolite supply between compartments was enabled. Implementing the two-compartment system (2cs) for growing Streptococcus thermophilus and Lactobacillus delbrueckii subs. bulgaricus, which are microorganisms commonly used in yogurt starter cultures, revealed only a small variance between the one-compartment and two-compartment approaches. The 2cs enabled the quantification of the strain-specific production and consumption rates of amino acids in an interacting S. thermophilus-L. bulgaricus co-culture. Therefore, comparisons between mono- and co-culture performance could be achieved. Both species produce and release amino acids. Only alanine was produced de novo from glucose through potential transaminase activity by L. bulgaricus and consumed by S. thermophilus. Arginine availability in peptides was limited to S. thermophilus' growth, indicating active biosynthesis and dependency on the proteolytic activity of L. bulgaricus. The application of the 2cs not only opens the door for the quantification of exchange fluxes between microbes but also enables continuous production modes, for example, for targeted evolution studies.

Identifying and Engineering Bottlenecks of Autotrophic Isobutanol Formation in Recombinant C. ljungdahlii by Systemic Analysis.

Clostridium ljungdahlii (C. ljungdahlii, CLJU) is natively endowed producing acetic acid, 2,3-butandiol, and ethanol consuming gas mixtures of CO2, CO, and H2 (syngas). Here, we present the syngas-based isobutanol formation using C. ljungdahlii harboring the recombinant amplification of the "Ehrlich" pathway that converts intracellular KIV to isobutanol. Autotrophic isobutanol production was studied analyzing two different strains in 3-L gassed and stirred bioreactors. Physiological characterization was thoroughly applied together with metabolic profiling and flux balance analysis. Thereof, KIV and pyruvate supply were identified as key "bottlenecking" precursors limiting preliminary isobutanol formation in CLJU[KAIA] to 0.02 g L-1. Additional blocking of valine synthesis in CLJU[KAIA]:ilvE increased isobutanol production by factor 6.5 finally reaching 0.13 g L-1. Future metabolic engineering should focus on debottlenecking NADPH availability, whereas NADH supply is already equilibrated in the current generation of strains.

Electron availability in CO2 , CO and H2 mixtures constrains flux distribution, energy management and product formation in Clostridium ljungdahlii.

Acetogens such as Clostridium ljungdahlii can play a crucial role reducing the human CO2 footprint by converting industrial emissions containing CO2 , CO and H2 into valuable products such as organic acids or alcohols. The quantitative understanding of cellular metabolism is a prerequisite to exploit the bacterial endowments and to fine-tune the cells by applying metabolic engineering tools. Studying the three gas mixtures CO2  + H2 , CO and CO + CO2  + H2 (syngas) by continuously gassed batch cultivation experiments and applying flux balance analysis, we identified CO as the preferred carbon and electron source for growth and producing alcohols. However, the total yield of moles of carbon (mol-C) per electrons consumed was almost identical in all setups which underlines electron availability as the main factor influencing product formation. The Wood-Ljungdahl pathway (WLP) showed high flexibility by serving as the key NAD+ provider for CO2  + H2, whereas this function was strongly compensated by the transhydrogenase-like Nfn complex when CO was metabolized. Availability of reduced ferredoxin (Fdred ) can be considered as a key determinant of metabolic control. Oxidation of CO via carbon monoxide dehydrogenase (CODH) is the main route of Fdred formation when CO is used as substrate, whereas Fdred is mainly regenerated via the methyl branch of WLP and the Nfn complex utilizing CO2  + H2 . Consequently, doubled growth rates, highest ATP formation rates and highest amounts of reduced products (ethanol, 2,3-butanediol) were observed when CO was the sole carbon and electron source.

Continuous Adaptive Evolution of a Fast-Growing Corynebacterium glutamicum Strain Independent of Protocatechuate.

Corynebacterium glutamicum is a commonly applied host for the industrial production of amino acids. While valued for its robustness, it is somewhat inferior to competing strains such as Escherichia coli because of the relatively low growth rate of 0.40 h-1 in synthetic, industrial media. Accordingly, adaptive laboratory evolution (ALE) experiments were performed in continuous cultivation mode to select for a growth-improved host. To ensure industrial attractiveness, this ALE study aimed at a reduction of dependency on costly growth-boosting additives such as protocatechuate (PCA) or complex media supplements. Consequently, double selection pressures were installed consisting of a steady increase in growth rate demands and a parallel reduction of complex medium fractions. Selection yielded C. glutamicum EVO5 achieving 0.54 h-1 and 1.03 gGlc gCDW -1 h-1 in minimal medium without abovementioned supplements. Sequencing revealed 10 prominent mutations, three of them in key regulator genes.

Transcriptional response of Escherichia coli to ammonia and glucose fluctuations.

In large-scale production processes, metabolic control is typically achieved by limited supply of essential nutrients such as glucose or ammonia. With increasing bioreactor dimensions, microbial producers such as Escherichia coli are exposed to changing substrate availabilities due to limited mixing. In turn, cells sense and respond to these dynamic conditions leading to frequent activation of their regulatory programmes. Previously, we characterized short- and long-term strategies of cells to adapt to glucose fluctuations. Here, we focused on fluctuating ammonia supply while studying a continuously running two-compartment bioreactor system comprising a stirred tank reactor (STR) and a plug-flow reactor (PFR). The alarmone ppGpp rapidly accumulated in PFR, initiating considerable transcriptional responses after 70 s. About 400 genes were repeatedly switched on/off when E. coli returned to the STR. E. coli revealed highly diverging long-term transcriptional responses in ammonia compared to glucose fluctuations. In contrast, the induction of stringent regulation was a common feature of both short-term responses. Cellular ATP demands for coping with fluctuating ammonia supply were found to increase maintenance by 15%. The identification of genes contributing to the increased ATP demand together with the elucidation of regulatory mechanisms may help to create robust cells and processes for large-scale application.

Switching between nitrogen and glucose limitation: Unraveling transcriptional dynamics in Escherichia coli.

Transcriptional control under nitrogen and carbon-limitation conditions have been well analyzed for Escherichia coli. However, the transcriptional dynamics that underlie the shift in regulatory programs from nitrogen to carbon limitation is not well studied. In the present study, cells were cultivated at steady state under nitrogen (ammonia)-limited conditions then shifted to carbon (glucose) limitation to monitor changes in transcriptional dynamics. Nitrogen limitation was found to be dominated by sigma 54 (RpoN) and sigma 38 (RpoS), whereas the "housekeeping" sigma factor 70 (RpoD) and sigma 38 regulate cellular status under glucose limitation. During the transition, nitrogen-mediated control was rapidly redeemed and mRNAs that encode active uptake systems, such as ptsG and manXYZ, were quickly amplified. Next, genes encoding facilitators such as lamB were overexpressed, followed by high affinity uptake systems such as mglABC and non-specific porins such as ompF. These regulatory programs are complex and require well-equilibrated and superior control. At the metabolome level, 2-oxoglutarate is the likely component that links carbon- and nitrogen-mediated regulation by interacting with major regulatory elements. In the case of dual glucose and ammonia limitation, sigma 24 (RpoE) appears to play a key role in orchestrating these complex regulatory networks.

Engineering E. coli for large-scale production - Strategies considering ATP expenses and transcriptional responses.

Microbial producers such as Escherichia coli are evolutionarily trained to adapt to changing substrate availabilities. Being exposed to large-scale production conditions, their complex, multilayered regulatory programs are frequently activated because they face changing substrate supply due to limited mixing. Here, we show that E. coli can adopt both short- and long-term strategies to withstand these stress conditions. Experiments in which glucose availability was changed over a short time scale were performed in a two-compartment bioreactor system. Quick metabolic responses were observed during the first 30s of glucose shortage, and after 70s, fundamental transcriptional programs were initiated. Since cells are fluctuating under simulated large-scale conditions, this scenario represents a continuous on/off switching of about 600 genes. Furthermore, the resulting ATP maintenance demands were increased by about 40-50%, allowing us to conclude that hyper-producing strains could become ATP-limited under large-scale production conditions. Based on the observed transcriptional patterns, we identified a number of candidate gene deletions that may reduce unwanted ATP losses. In summary, we present a theoretical framework that provides biological targets that could be used to engineer novel E. coli strains such that large-scale performance equals laboratory-scale expectations.

CO2 /HCO3⁻ perturbations of simulated large scale gradients in a scale-down device cause fast transcriptional responses in Corynebacterium glutamicum.

The exploration of scale-down models to imitate the influence of large scale bioreactor inhomogeneities on cellular metabolism is a topic with increasing relevance. While gradients of substrates, pH, or dissolved oxygen are often investigated, oscillating CO2/HCO3 (-) levels, a typical scenario in large industrial bioreactors, is rarely addressed. Hereby, we investigate the metabolic and transcriptional response in Corynebacterium glutamicum wild type as well as the impact on L-lysine production in a model strain exposed to pCO2 gradients of (75-315) mbar. A three-compartment cascade bioreactor system was developed and characterized that offers high flexibility for installing gradients and residence times to mimic industrial-relevant conditions and provides the potential of accurate carbon balancing. The phenomenological analysis of cascade fermentations imposed to the pCO2 gradients at industry-relevant residence times of about 3.6 min did not significantly impair the process performance, with growth and product formation being similar to control conditions. However, transcriptional analysis disclosed up to 66 differentially expressed genes already after 3.6 min under stimulus exposure, with the overall change in gene expression directly correlateable to the pCO2 gradient intensity and the residence time of the cells.

mer-Bis[2-(1,3-benzothiazol-2-yl)phenyl-κC,N][3-phenyl-5-(2-pyridyl)-1,2,4-triazol-1-ido-κN,N]iridium(III) deuterochloro-form 3.5-solvate.

In the title compound, [Ir(C(13)H(9)N(4))(C(13)H(8)NS)(2)]·3.5CDCl(3), the coordination at iridium is octa-hedral, but with narrow ligand bite angles. The bond lengths at iridium show the expected trans influence, with the Ir-N bonds trans to C being appreciably longer than those trans to N. The chelate rings are mutually perpendicular, the inter-planar angles between them all lying within 6° of 90°. All ligands are approximately planar; the maximum inter-planar angles within ligands are ca 10°. The three ordered deuterochloro-form mol-ecules are all involved in C⋯D-A contacts that can be inter-preted as hydrogen bonds of various types. The fourth deuterochloroform is disordered over an inversion centre.

mer-[3-Phenyl-5-(2-pyridyl-κN)-1,2,4-triazol-1-ido-κN]bis-(2-quinolylphenyl-κC,N)iridium(III) deuterochloro-form disolvate.

In the title compound, [Ir(C(13)H(9)N(4))(C(15)H(10)N)(2)]·2CDCl(3), the coordination at iridium is octa-hedral, but with narrow ligand bite angles ranging from 74.85 (8) to 83.99 (8)°. The bond lengths at iridium show the expected trans influence, with Ir-N trans to C being appreciably longer than trans to N. The chelate rings are mutually perpendicular to a reasonable approximation [interplanar angles ranging from 77.79 (6) to 83.93 (7)°]. All ligands are approximately planar; the maximum inter-planar angles within ligands are ca 12°. One CDCl(3) solvent molecule is severly disordered and was excluded from the refinement.

Efficient and long-time stable red iridium(III) complexes for organic light-emitting diodes based on quinoxaline ligands.

We report the design and characterization of three heteroleptic orange-red phosphorescent iridium(III) complexes bearing two 2-(4-fluorophenyl)-3-methyl-quinoxaline (fpmqx) cyclometalated ligands combined with three different ancillary ligands, triazolylpyridine (trz), picolinate (pic), and acetylacetonate (acac). All of these complexes emit an orange to red color in the spectral range of 605-628 nm in dichloromethane. Strong spin-orbit coupling of the iridium atom allows the formally forbidden mixing of singlet and triplet states. Because of the structureless phosphorescent line shapes and low Stokes shifts between triplet metal-to-ligand charge-transfer ((3)MLCT) absorption and phosphorescent emission, we propose that emission originates predominantly from the (3)MLCT state with a lesser admixture of totally ligand-based (3)(pi-pi*) states. The influence of 5d-electron densities of the iridium center on highest occupied molecular orbitals leads to high emission quantum yields in toluene (Phi(p) = 0.39-0.42) and to short triplet lifetimes. Cyclovoltammetry measurements show reversible oxidation peaks from 0.74 to 0.92 V and reversible reduction waves with potentials ranging from -1.58 to -2.05 V versus Cp(2)Fe/Cp(2)Fe(+). All complexes have been applied in simple test devices and also in stable, long-living devices to evaluate their electroluminescent device performances, for which we especially report the influence of the chosen ancillary ligands on emission colors, efficiencies, and device lifetimes. We obtained narrowband emission ranging from 613 to 630 nm with a full width at half-maximum of 64-71 nm, and a maximum in power efficiency of eta(p) = 14.6 lm/W at a current density of J = 0.01 mA/cm(2) for [(fpmqx)(2)Ir(pic)]. The operating lifetimes of [(fpmqx)(2)Ir(trz)] in both neat and mixed matrixes were longer than that of the established stable tris(1-phenylisoquinolinato)iridium(III) [Ir(piq)(3)]. From the lifetime measurements, it becomes clear that the stability is strongly correlated to the type of ancillary ligand. An extrapolated lifetime of 58 000 h with an initial brightness of 1000 cd/m(2), together with a very low voltage increase of 0.2 V over a time period of 1000 h (starting voltage of 4.1 V), was achieved. Such a high device lifetime is attributed to the chemical stability of all materials toward both charge carriers and excitons.

Dynamic response of the expression of hxt1, hxt5 and hxt7 transport proteins in Saccharomyces cerevisiae to perturbations in the extracellular glucose concentration.

Glucose transport in Saccharomyces cerevisiae relies on a multi-factorial uptake system. The modulation of its efficiency depends on the differential expression of various sets of hexose transport-related proteins whose glucose affinity differs considerably. The expression of three different glucose transport proteins (HXT1, HXT5 and HXT6/7 with low-, intermediate- and high-affinity, respectively) was monitored as a result of modified extracellular glucose concentrations. Cultivation at glucose-limited (continuous) conditions was instantly replaced by a batch (and thus, non-limited) mode. Further, to mimic concentration gradients in large-scale production bioreactors, multiple and rapid transient glucose pulses were applied to chemostat cultivation. Antibodies against the HXT-proteins were used to monitor the proteins' expression levels prior to and after perturbing the external glucose concentrations. HXT5 and HXT6/7 were either expressed during the starvation-like steady-state phases in the chemostat cultivations, whereas HXT1 could not be detected at all. HXT1, however, is subsequently expressed during the excess of glucose in the batch mode, while the HXT5 and HXT6/7 transporters were at least found to decline. These findings coincide well with the transporters' affinity profiles. As a result of repeated and rapid transient glucose pulses during continuous fermentation, especially HXT6/7 pointed out to alter the protein expression pattern.

Highly efficient enzymatic synthesis of 2-monoacylglycerides and structured lipids and their production on a technical scale.

We report here a two-step process for the high-yield enzymatic synthesis of 2-monoacylglycerides (2-MAG) of saturated as well as unsaturated fatty acids with different chain lengths. The process consists of two steps: first the unselective esterification of fatty acids and glycerol leading to a triacylglyceride followed by an sn1,3-selective alcoholysis reaction yielding 2-monoacylglycerides. Remarkably, both steps can be catalyzed by lipase B from Candida antarctica (CalB). The whole process including esterification and alcoholysis was scaled up in a miniplant to a total volume of 10 l. With this volume, a two-step process catalyzed by CalB for the synthesis of 1,3-oleoyl-2-palmitoylglycerol (OPO) using tripalmitate as starting material was established. On a laboratory scale, we obtained gram quantities of the synthesized 2-monoacylglycerides of polyunsaturated fatty acids such as arachidonic-, docosahexaenoic- and eicosapentaenoic acids and up to 96.4% of the theoretically possible yield with 95% purity. On a technical scale (>100 g of product, >5 l of reaction volume), 97% yield was reached in the esterification and 73% in the alcoholysis and a new promising process for the enzymatic synthesis of OPO was established.

Diamond planar refractive lenses for third- and fourth-generation X-ray sources.

The fabrication and testing of planar refractive hard X-ray lenses made from bulk CVD diamond substrates is reported. The lens structures were generated by electron-beam lithography and transferred by reactive-ion etching into the diamond. Various lens designs were fabricated and tested at 12.4 and 17.5 keV photon energy. Efficiencies of up to 71% and gains of up to 26 were achieved. A line focus of 3.2 micro m (FWHM) was measured. These lenses should be able to withstand the extreme flux densities expected at the planned fourth-generation X-ray sources.