Concrete is the most widely used construction material worldwide, yet Portland-cement production accounts for approximately 8 _2 emissions. Bio-cementation processes based on calcium carbonate precipitation offer a promising low-carbon alternative, but their engineering application is limited by a lack of predictive models for coupled transport, reaction, and cementation processes. This study presents a 1D numerical model for bio-concrete production using urease-active calcite powder (UACP). Unlike previous models based on in-situ bacterial growth, urease is initially and homogeneously distributed by mixing UACP with sand, reducing model complexity in terms of the number of degrees of freedom. The model captures the coupled evolution of fluid flow, ureolysis-driven carbonate precipitation, and porosity–permeability reduction, with a novel conceptual formulation to limit the ureolysis rate for agreement with experiments. Parameters are calibrated using a selected quasi-1D experiment and validated against four additional setups. The model predicts whether cementation is complete or incomplete and reproduces final average porosities with errors below 7.5

Seit mehr als 20 Jahren beschäftigt sich das Institut für Leichtbau Entwerfen und Konstruieren (ILEK) der Universität Stuttgart in interdisziplinären Forschungsarbeiten mit dem scheinbaren Paradoxon „Leicht bauen mit Beton“. In einem zweiteiligen Beitrag werden nun Auszüge aus der aktuellen Forschung des ILEK zu diesem Thema vorgestellt. Der erste Teil betrachtet die Entwicklungen auf der Materialebene (Materialleichtbau). Hier geht es um das Ziel der Reduktion der Verwendung von primären Ressourcen, der materialbedingten Emissionen und des Abfalls in den verschiedenen Nutzungsphasen eines Bauwerks. Zu den hier vorgestellten Entwicklungen gehören: die Substitution der natürlichen Gesteinskörnung mit Rezyklaten, biomineralisiertes Calciumcarbonat als innovativer Ersatz für Portlandzement, Basaltfaserverbundbewehrungen als Alternative zum konventionellen Beton‐ und Spannstahl sowie rezyklierbare Sandschalungen zur abfallfreien Herstellung leichter Betonstrukturen.

For more than 20 years, the Institute for Lightweight Structures and Conceptual Design (ILEK) has been engaged in interdisciplinary research on the supposed paradox of "lightweight construction with concrete". Excerpts from the current research are now presented in a two-part article. This first part considers developments at the material level (lightweight materials) with the aim of minimizing use of primary resources, material-related emissions, and waste in different phases of building life cycle. These include: the substitution of natural aggregates with recycled ones, biomineralized calcium carbonate as an innovative substitute for cement, basalt fiber composite reinforcement as an alternative to conventional reinforcing and prestressing steel, and recyclable sand formworks for zero-waste production of lightweight concrete structures.

The conversion of rac-phenylglycinonitrile by different variants of the nitrilase from Pseudomonas fluorescens EBC191 (EC 3.5.5.1) was studied and the amounts and chiral composition of the formed phenylglycine and phenylglycine amide compared. Muteins that converted rac-phenylglycinonitrile to extraordinarily high amounts of phenylglycine or phenylglycine amide were tested for the chemoenzymatic enantioselective one-pot synthesis of (R)- and (S)-phenylglycine and (R)- and (S)-phenylglycine amide. The chemoenzymatic synthesis combined the initial step in the traditional chemical Strecker synthesis which results in the formation of rac-phenylglycinonitrile from benzaldehyde, cyanide, and ammonia with the enzymatic conversion of the formed nitrile by the nitrilase variants. The aminonitrile synthesis was optimized in order to obtain conditions which allowed under mildly alkaline conditions (pH 9.5) maximal yields of phenylglycinonitrile and the in-situ racemization of the compound. The racemic phenylglycinonitrile was directly converted under the alkaline conditions without any interposed purification step by cells of Escherichia coli overexpressing recombinant nitrilase variants. The application of a mutant of E. coli defect in a (S)-phenylglycine amide hydrolysing peptidase (E. coli JM109ΔpepA) expressing a highly reaction- and (R)-specific nitrilase variant allowed the synthesis of (R)-phenylglycine with ee-values ≥ 95% in yields up to 81% in relation to the initially added benzaldehyde. These yields indicated a dynamic kinetic resolution which involved the racemization of (S)- to (R)-phenylglycinonitrile under the used alkaline conditions with the concurrent hydrolysis of (R)-phenylglycinonitrile to (R)-phenylglycine. The addition of resting cells of E. coli JM109ΔpepA synthesizing an amide forming nitrilase variant to the final product of the Strecker synthesis and/or using E. coli strains with an intact aminopeptidase gene resulted in the preferred formation of (S)-phenylglycine amide, (R)-phenylglycine amide or (S)-phenylglycine.

Gentisate 1,2-dioxygenases (GDOs) are non-heme iron enzymes that catalyze the oxidation of dihydroxylated aromatic substrate, gentisate (2,5-dihydroxybenzoate). Salicylate 1,2-dioxygenase (SDO), a member of the GDO family, performs the ring scission of monohydroxylated substrates such as salicylate, thereby oxidizing a broader range of substrates compared to GDOs. Although the two types of enzymes share a high degree of sequence similarity, the origin of substrate specificity between SDO and GDOs is not understood. We present electron paramagnetic resonance (EPR) investigation of ferrous-nitrosyl complexes of SDO and a GDO from the bacterium Corynebacterium glutamicum (GDOCg). The EPR spectra of these complexes, which mimic the Fe-substrate-O2 intermediates in the catalytic cycle, show unexpected differences in the substrate binding mode and the coordination geometry of the metal cofactor in the two enzymes. Binding of substrate to the ferrous center increases the symmetry of the Fe(II)–NO complex in SDO, while a reverse trend is observed in GDOCg where substrate ligation reduces the symmetry of the nitrosyl complex. Identical EPR spectra were obtained for the NO derivatives of a variant of GDOCg(A112G), which can oxidize salicylate, and wild-type GDOCg revealing that the A112G mutation does not alter the nature of the Fe-substrate-O2 ternary complex.

The conversion of phenylglycinonitrile (2-aminophenylacetonitrile) by Escherichia coli strains was studied, which recombinantly expressed the arylacetonitrilase (NitA) from Pseudomonas fluorescens EBC191 and different nitrilase variants with altered reaction specificities. The whole-cell catalysts which formed the wild-type nitrilase converted (R,S)-phenylglycinonitrile preferentially to (S)-phenylglycine with a low degree of enantioselectivity. A recombinant strain which formed a variant of NitA produced mainly (S)-phenylglycine amide from (R,S)-phenylglycinonitrile and a second variant showed an almost complete enantioconversion and produced (R)-phenylglycine and left (S)-phenylglycinonitrile. The microbial-produced (S)-phenylglycinonitrile was used to study the chemical racemisation of (S)-phenylglycinonitrile at alkaline pH values in order to establish a dynamic kinetic resolution of the substrate. Subsequently, the conversion of (R,S)-phenylglycinonitrile by the whole-cell catalysts was studied at a pH of 10.8 which allowed a sufficient racemisation rate of phenylglycinonitrile. Surprisingly, under these conditions, strongly increased amounts of (S)-phenylglycine were formed by the recombinant E. coli cells expressing the amide-forming nitrilase variant. The aminopeptidase PepA from E. coli was identified by the construction of a deletion mutant and subsequent complementation as responsible amidase activity, which converted (S)-phenylglycine amide to (S)-phenylglycine.

The conversion of aliphatic nitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191 (NitA) was analyzed. The nitrilase hydrolysed a wide range of aliphatic mono- and dinitriles and showed a preference for unsaturated aliphatic substrates containing 5–6 carbon atoms. In addition, increased reaction rates were also found for aliphatic nitriles carrying electron withdrawing substituents (e.g. chloro- or hydroxy-groups) close to the nitrile group. Aliphatic dinitriles were attacked only at one of the nitrile groups and with most of the tested dinitriles the monocarboxylates were detected as major products. In contrast, fumarodinitrile was converted to the monocarboxylate and the monocarboxamide in a ratio of about 65:35. Significantly different relative amounts of the two products were observed with two nitrilase variants with altered reaction specifities. NitA converted some aliphatic substrates with higher rates than 2-phenylpropionitrile, which is one of the standard substrates for arylacetonitrilases. This indicated that the traditional classification of nitrilases as “arylacetonitrilases”, “aromatic” or “aliphatic” nitrilases might require some corrections. This was also suggested by the construction of some variants of NitA which were modified in an amino acid residue which was previously suggested to be essential for the conversion of aliphatic substrates by a homologous nitrilase.

The gentisate 1,2-dioxygenases (GDOs) from Corynebacterium glutamicum and various other organisms oxidatively cleave the aromatic nucleus of gentisate (2,5-dihydroxybenzoate), but are not able to convert salicylate (2-hydroxybenzoate). In contrast, the α-proteobacterium Pseudaminobacter salicylatoxidans synthesises an enzyme ('salicylate dioxygenase', SDO) which cleaves gentisate, but also (substituted) salicylate(s). Sequence comparisons showed that the SDO belongs to a group of GDOs mainly originating from Gram-positive bacteria which also include the GDO from C. glutamicum ATCC 13032. The combination of sequence comparisons with previously performed structural and mutational analyses of the SDO allowed to identify an amino acid residue (Ala112) which might prevent the oxidation of (substituted) salicylate(s) by the GDO from C. glutamicum Therefore, the relevant mutation (Ala→Gly) was introduced into the GDO from C. glutamicum The GDO variant obtained gained the ability to oxidise salicylate and several other monohydroxylated substrates. In order to screen a broader range of enzyme variants a chromogenic assay was developed which allowed the detection of bacterial colonies converting salicylate. The applicability of this test system was proven by screening a set of GDO variants obtained by saturation mutagenesis at different positions. This demonstrated that also GDO variants carrying the mutations Ala112→Ser, Ala112→Ile and Ala112→Asp converted salicylate.

The genome of the α-proteobacterium Pseudaminobacter salicylatoxidans codes for a ferrous iron containing ring-fission dioxygenase which catalyzes the 1,2-cleavage of (substituted) salicylate(s), gentisate (2,5-dihydroxybenzoate), and 1-hydroxy-2-naphthoate. Sequence alignments suggested that the "salicylate 1,2-dioxygenase" (SDO) from this strain is homologous to gentisate 1,2-dioxygenases found in bacteria, archaea and fungi. In the present study the catalytic mechanism of the SDO and gentisate 1,2-dioxygenases in general was analyzed based on sequence alignments, mutational and previously performed crystallographic studies and mechanistic comparisons with "extradiol- dioxygenases" which cleave aromatic nuclei in the 2,3-position. Different highly conserved amino acid residues that were supposed to take part in binding and activation of the organic substrates were modified in the SDO by site-specific mutagenesis and the enzyme variants subsequently analyzed for the conversion of salicylate, gentisate and 1-hydroxy-2-naphthoate. The analysis of enzyme variants which carried exchanges in the positions Arg83, Trp104, Gly106, Gln108, Arg127, His162 and Asp174 demonstrated that Arg83 and Arg127 were indispensable for enzymatic activity. In contrast, residual activities were found for variants carrying mutations in the residues Trp104, Gly106, Gln108, His162, and Asp174 and some of these mutants still could oxidize gentisate, but lost the ability to convert salicylate. The results were used to suggest a general reaction mechanism for gentisate-1,2-dioxygenases and to assign to certain amino acid residues in the active site specific functions in the cleavage of (substituted) salicylate(s).

The alpha-Proteobacterium Pseudaminobacter salicylatoxidans BN12 forms a peculiar gentisate 1,2-dioxygenase (SDO) that oxidatively cleaves gentisate (2,5-dihydroxybenzoate) and additionally 1-hydroxy-2-naphthoate, salicylate and various amino-, chloro-, fluoro-, hydroxy- and methylsalicylates. In the present study, the conversion of 5-fluorosalicylate by this enzyme was analysed using various analytical techniques. Spectrophotometric assays showed that the conversion of 5-fluorosalicylate by the purified enzyme resulted in the formation of a new unstable intermediate showing an absorbance maximum at λmax = 292 nm. The analysis of the enzymatic reaction by HPLC showed that two main products with absorbance maxima at λmax = 292-296 nm were formed from 5-fluorosalicylate. The same two products (although in different relative proportions) were also formed when the SDO transformed 5-chlorosalicylate or when a purified 5-nitrosalicylate 1,2-dioxygenase from Bradyrhizobium sp. JS329 oxidized 5-nitrosalicylate. A whole cell system with recombinant Escherichia coli cells overexpressing the SDO activity was established in order to produce larger amounts of the reaction products. The reaction products were subsequently identified by (1)H-NMR and mass spectrometry as stereoisomers of 2-oxo-3-(5-oxofuran-2-ylidine)propanoic acid. The release of fluoride in the course of the dioxygenolytic cleavage reaction was confirmed by ion-chromatography and (19)F-NMR.