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An in-depth examination of the effects of a pulsed laser on the additively manufactured 316L stainless steel by laser powder bed fusion (L-PBF) was conducted in this work. It demonstrated that dense parts can be obtained over a broad spectrum of parameters during pulsed laser L-PBF. The results show that a pulsed laser can significantly refine the sub-micron cellular structure and microsize grain structure and hence enhance the mechanical properties of L-PBF 316L SS. Simulations conducted via FLOW-3D validate the distinctions in thermal history between various printing parameters. The improved cooling rate during the laser process led to microstructure refinement. The Hall–Petch relationship of the AM 316L SS at the sub-micron scale was determined and could provide a guideline for Hall–Petch strengthening of AM stainless steel with cellular structure refinement.

This work reports on employing X-ray computed tomography (XCT) to develop a predictive model aimed at optimizing laser process parameters for laser powder bed fusion. A commercially available statistical analysis software was successfully combined with XCT obtained porosity data obtained from 316L stainless steel to develop an accurate model that predicted the parameter sets and ranges with the lowest porosity. The predictions indicated that laser velocity and hatch spacing had a numerically linear relationship with laser power and can be combined to minimize porosity at any selected laser power in a specific range. In fact, the predictions indicated that the minimum porosity at any laser power is associated with a specific line energy input of approximately 0.13 J/mm for this alloy. The lowest predicted porosity at each laser power was fabricated and tested with the 85- and 92-W powers confirming ultra-low porosity. Lower laser powers, however, exhibited significantly higher porosity in contrast with the prediction. This resulted from the lower hatch spacing and velocity causing higher energy density and metallurgical defects from macro-balling. Thermodynamic calculations in the optimum laser power range yielded a line energy of 0.131 J/mm, which agrees rather well with the XCT predicted line energy and indicates that porosity generation is governed by the thermo-physical behavior of the alloy. A parameter space in the optimum range was fabricated and confirmed that the lowest porosities exist along a line energy of 0.13 J/mm, where melt pool temperature was predicted to be between 2526 and 2785 °C.

Moorella thermoacetica is one of the model acetogenic bacteria for the resolution of the Wood–Ljungdahl (acetyl-CoA) pathway in which CO 2 is autotrophically assimilated yielding acetyl-CoA as central intermediate. Its further conversion into acetate relies on subsequent phosphotransacetylase (PTA) and acetate kinase reactions. However, the genome of M. thermoacetica contains no pta homologous gene. It has been speculated that the moth_ 0864 and moth_ 1181 gene products sharing similarities with an evolutionarily distinct phosphotransacylase involved in 1,2-propanediol utilization (PDUL) of Salmonella enterica act as PTAs in M. thermoacetica . Here, we demonstrate specific PTA activities with acetyl-CoA as substrate of 9.05 and 2.03 U/mg for the recombinant enzymes PDUL1 (Moth_1181) and PDUL2 (Moth_0864), respectively. Both showed maximal activity at 65 °C and pH 7.6. Native proteins (90 kDa) are homotetramers composed of four subunits with apparent molecular masses of about 23 kDa. Thus, one or both PDULs of M. thermoacetica might act as PTAs in vivo catalyzing the penultimate step of the Wood–Ljungdahl pathway toward the formation of acetate. In silico analysis underlined that up to now beside of M. thermoacetica, only Sporomusa ovata contains only PDUL like class III -PTAs but no other phosphotransacetylases or phosphotransbutyrylases (PTBs).

Summary In a continuous culture under phosphate limitation the metabolism of Clostridium acetobutylicum depends on the external pH level. By comparing seven steady‐state conditions between pH 5.7 and pH 4.5 we show that the switch from acidogenesis to solventogenesis occurs between pH 5.3 and pH 5.0 with an intermediate state at pH 5.1. Here, an integrative study is presented investigating how a changing external pH level affects the clostridial acetone–butanol–ethanol (ABE) fermentation pathway. This is of particular interest as the biotechnological production of n‐butanol as biofuel has recently returned into the focus of industrial applications. One prerequisite is the furthering of the knowledge of the factors determining the solvent production and their integrative regulations. We have mathematically analysed the influence of pH‐dependent specific enzyme activities of branch points of the metabolism on the product formation. This kinetic regulation was compared with transcriptomic regulation regarding gene transcription and the proteomic profile. Furthermore, both regulatory mechanisms were combined yielding a detailed projection of their individual and joint effects on the product formation. The resulting model represents an important platform for future developments of industrial butanol production based on C. acetobutylicum. The industrial‐relevant bacterium Clostridium acetobutylicum, grown in a phosphate‐limited continuous culture, shifts its metabolism in response to changing pH levels. This results in a transition from the formation of the acids acetate and butyrate to the solvents acetone and butanol. In this study, we investigate how pH‐dependent kinetic and transcriptional regulatory mechanisms trigger this pH‐induced metabolic switch. We demonstrate that these two forms of regulation can either independently or jointly affect the observed metabolic phase transition. Whereas previous studies have elucidated pH‐induced changes on the transcriptomic and the proteomic level, our investigation shows that information about pH‐dependent kinetic enzyme properties, in particular specific catalytic activities, is required for an improved understanding of the pH‐induced alterations to the metabolic flux through the network.

In response to changing extracellular pH levels, phosphate-limited continuous cultures of Clostridium acetobutylicum reversibly switches its metabolism from the dominant formation of acids to the prevalent production of solvents. Previous experimental and theoretical studies have revealed that this pH-induced metabolic switch involves a rearrangement of the intracellular transcriptomic, proteomic and metabolomic composition of the clostridial cells. However, the influence of the population dynamics on the observations reported has so far been neglected. Here, we present a method for linking the pH shift, clostridial growth and the acetone–butanol–ethanol fermentation metabolic network systematically into a model which combines the dynamics of the external pH and optical density with a metabolic model. Furthermore, the recently found antagonistic expression pattern of the aldehyde/alcohol dehydrogenases AdhE1/2 and pH-dependent enzyme activities have been included into this combined model. Our model predictions reveal that the pH-induced metabolic shift under these experimental conditions is governed by a phenotypic switch of predominantly acidogenic subpopulation towards a predominantly solventogenic subpopulation. This model-driven explanation of the pH-induced shift from acidogenesis to solventogenesis by population dynamics casts an entirely new light on the clostridial response to changing pH levels. Moreover, the results presented here underline that pH-dependent growth and pH-dependent specific enzymatic activity play a crucial role in this adaptation. In particular, the behaviour of AdhE1 and AdhE2 seems to be the key factor for the product formation of the two phenotypes, their pH-dependent growth, and thus, the pH-induced metabolic switch in C. acetobutylicum .

Transport in topological matter has shown a variety of novel phenomena over the past decade. Although numerous transport studies have been conducted on three-dimensional topological insulators (TIs), study of ballistic motion and thus exploration of potential landscapes on a hundred nanometer scale is for the prevalent TI materials almost impossible due to their low carrier mobility. Therefore, it is unknown whether helical Dirac electrons in TIs, bound to interfaces between topologically distinct materials, can be manipulated on the nanometer scale by local gates or locally etched regions. Here we impose a submicron periodic potential onto a single surface of Dirac electrons in high-mobility strained mercury telluride (HgTe), which is a strong TI. Pronounced geometric resistance resonances constitute the clear-cut observation of a ballistic effect in three-dimensional TIs. Ballistic motion on nanometer scale of topological surface states has rarely been studied. Here, Maier et al. report pronounced geometric resistance resonances of high-mobility Dirac electrons in strained HgTe, suggesting a ballistic effect in three-dimensional topological insulators.

As a member of the saccharolytic clostridia, a variety of different carbohydrates like glucose, fructose, or mannose can be used as carbon and energy source by Clostridium acetobutylicum ATCC 824. Thirteen phosphoenolpyruvate-dependent phosphotransferase systems (PTS) have been identified in C. acetobutylicum, which are likely to be responsible for the uptake of hexoses, hexitols, or disaccharides. Here, we focus on three PTS which are expected to be involved in the uptake of fructose, PTSFʳᵘ, PTSᴹᵃⁿᴵ, and PTSᴹᵃⁿᴵᴵ. To analyze their individual functions, each PTS was inactivated via homologous recombination or insertional mutagenesis. Standardized comparative batch fermentations in a synthetic medium with glucose, fructose, or mannose as sole carbon source identified PTSFʳᵘ as primary uptake system for fructose, whereas growth with fructose was not impaired in PTSᴹᵃⁿᴵ and slightly altered in PTSᴹᵃⁿᴵᴵ-deficient strains of C. acetobutylicum. The inactivation of PTSᴹᵃⁿᴵ resulted in slower growth on mannose whereas the loss of PTSᴹᵃⁿᴵᴵ revealed no phenotype during growth on mannose. This is the first time that it has been shown that PTSFʳᵘ and PTSᴹᵃⁿᴵ of C. acetobutylicum are directly involved in fructose and mannose uptake, respectively. Moreover, comprehensive comparison of the fermentation products revealed that the loss of PTSFʳᵘ prevents the solvent shift as no butanol and only basic levels of acetone and ethanol could be determined.

The complex changes in the life cycle of Clostridium acetobutylicum, a promising biofuel producer, are not well understood. During exponential growth, sugars are fermented to acetate and butyrate, and in the transition phase, the metabolism switches to the production of the solvents acetone and butanol accompanied by the initiation of endospore formation. Using phosphate-limited chemostat cultures at pH 5.7, C. acetobutylicum was kept at a steady state of acidogenic metabolism, whereas at pH 4.5, the cells showed stable solvent production without sporulation. Novel proteome reference maps of cytosolic proteins from both acidogenesis and solventogenesis with a high degree of reproducibility were generated. Yielding a 21% coverage, 15 protein spots were specifically assigned to the acidogenic phase, and 29 protein spots exhibited a significantly higher abundance in the solventogenic phase. Besides well-known metabolic proteins, unexpected proteins were also identified. Among these, the two proteins CAP0036 and CAP0037 of unknown function were found as major striking indicator proteins in acidogenic cells. Proteome data were confirmed by genome-wide DNA microarray analyses of the identical cultures. Thus, a first systematic study of acidogenic and solventogenic chemostat cultures is presented, and similarities as well as differences to previous studies of batch cultures are discussed.

The hetero-dimeric CoA-transferase CtfA/B is believed to be crucial for the metabolic transition from acidogenesis to solventogenesis in Clostridium acetobutylicum as part of the industrial-relevant acetone-butanol-ethanol (ABE) fermentation. Here, the enzyme is assumed to mediate re-assimilation of acetate and butyrate during a pH-induced metabolic shift and to faciliate the first step of acetone formation from acetoacetyl-CoA. However, recent investigations using phosphate-limited continuous cultures have questioned this common dogma. To address the emerging experimental discrepancies, we investigated the mutant strain Cac-ctfA398s::CT using chemostat cultures. As a consequence of this mutation, the cells are unable to express functional ctfA and are thus lacking CoA-transferase activity. A mathematical model of the pH-induced metabolic shift, which was recently developed for the wild type, is used to analyse the observed behaviour of the mutant strain with a focus on re-assimilation activities for the two produced acids. Our theoretical analysis reveals that the ctfA mutant still re-assimilates butyrate, but not acetate. Based upon this finding, we conclude that C. acetobutylicum possesses a CoA-tranferase-independent butyrate uptake mechanism that is activated by decreasing pH levels. Furthermore, we observe that butanol formation is not inhibited under our experimental conditions, as suggested by previous batch culture experiments. In concordance with recent batch experiments, acetone formation is abolished in chemostat cultures using the ctfa mutant.

As a member of the saccharolytic clostridia, a variety of different carbohydrates like glucose, fructose, or mannose can be used as carbon and energy source by Clostridium acetobutylicum ATCC 824. Thirteen phosphoenolpyruvate-dependent phosphotransferase systems (PTS) have been identified in C. acetobutylicum, which are likely to be responsible for the uptake of hexoses, hexitols, or disaccharides. Here, we focus on three PTS which are expected to be involved in the uptake of fructose, PTS(Fru), PTS(ManI), and PTS(ManII). To analyze their individual functions, each PTS was inactivated via homologous recombination or insertional mutagenesis. Standardized comparative batch fermentations in a synthetic medium with glucose, fructose, or mannose as sole carbon source identified PTS(Fru) as primary uptake system for fructose, whereas growth with fructose was not impaired in PTS(ManI) and slightly altered in PTS(ManII)-deficient strains of C. acetobutylicum. The inactivation of PTS(ManI) resulted in slower growth on mannose whereas the loss of PTS(ManII) revealed no phenotype during growth on mannose. This is the first time that it has been shown that PTS(Fru) and PTS(ManI) of C. acetobutylicum are directly involved in fructose and mannose uptake, respectively. Moreover, comprehensive comparison of the fermentation products revealed that the loss of PTS(Fru) prevents the solvent shift as no butanol and only basic levels of acetone and ethanol could be determined.