Microbial and metabolic diversity of anaerobic D-galacturonate fermentation

Over the past decade the demand for technology which can make our global economy more sustainable has increased exponentially. In the field of biotechnology, one of the many advancements is focussed on the conversion of agricultural waste streams towards commodity chemicals. Plant biomass can be an environmental and sustainable alternative for the use of petrochemical substrates, with a special interest towards processes which use plant biomass in waste streams rather than currently used feedstocks, such as corn and sugarcane molasses that are competing with food production.BT/Industrial Microbiolog

Effective soil-stiffness validation: Shaker excitation of an in-situ monopile foundation

In an attempt to decrease the modelling uncertainty associated with the soil-structure interaction of large-diameter monopile foundations, a hydraulic shaker was used to excite a real-sized, in-situ monopile foundation in stiff, sandy soil in a near-shore wind farm. The response in terms of natural frequency and damping of a pile-only system is significantly more influenced by the soil than a full offshore wind turbine structure, and therefore ensures a higher degree of certainty regarding the assessment of the soil reaction. Steady-state vibration amplitudes with frequencies between 1 and 9 Hz were retrieved from strain gauges vertically spaced along the embedded pile, and accelerometers attached to the top of the pile and to the shaker. The measured response is used to validate an effective 1D stiffness method, which is applied as a smart initial guess for a model-based identification of the effective soil-structure interaction properties in terms of stiffness, damping and soil inertia. The performance of the stiffness method is compared to the currently employed p-y stiffness design method. While the effective stiffness method seems to overestimate the actual low-frequency stiffness with about 20%, the p-y method appears to underestimate this stiffness with 140%. The assumption of linear soil behaviour for most of the occurring pile displacements is shown to be acceptable. A damping ratio of 20% (critical) is identified as effective soil damping for the monopile, which is estimated to correspond to a 0.14% damping ratio contribution from the soil for the full structure. The unique measurement setup yielded a ‘first-off’ opportunity to validate a soil-structure interaction model for a rigidly behaving pile. We have shown that indeed such a pile reacts stiffer than predicted by the p-y curve method, and that its response can be modeled more accurately with our recently developed effective stiffness method.Offshore EngineeringApplied MechanicsDynamics of StructuresEngineering Structure

A Novel D-Galacturonate Fermentation Pathway in Lactobacillus suebicus Links Initial Reactions of the Galacturonate-Isomerase Route With the Phosphoketolase Pathway

D-galacturonate, a key constituent of pectin, is a ubiquitous monomer in plant biomass. Anaerobic, fermentative conversion of D-galacturonate is therefore relevant in natural environments as well as in microbial processes for microbial conversion of pectin-containing agricultural residues. In currently known microorganisms that anaerobically ferment D-galacturonate, its catabolism occurs via the galacturonate-isomerase pathway. Redox-cofactor balancing in this pathway strongly constrains the possible range of products generated from anaerobic D-galacturonate fermentation, resulting in acetate as the predominant organic fermentation product. To explore metabolic diversity of microbial D-galacturonate fermentation, anaerobic enrichment cultures were performed at pH 4. Anaerobic batch and chemostat cultures of a dominant Lactobacillus suebicus strain isolated from these enrichment cultures produced near-equimolar amounts of lactate and acetate from D-galacturonate. A combination of whole-genome sequence analysis, quantitative proteomics, enzyme activity assays in cell extracts, and in vitro product identification demonstrated that D-galacturonate metabolism in L. suebicus occurs via a novel pathway. In this pathway, mannonate generated by the initial reactions of the canonical isomerase pathway is converted to 6-phosphogluconate by two novel biochemical reactions, catalyzed by a mannonate kinase and a 6-phosphomannonate 2-epimerase. Further catabolism of 6-phosphogluconate then proceeds via known reactions of the phosphoketolase pathway. In contrast to the classical isomerase pathway for D-galacturonate catabolism, the novel pathway enables redox-cofactor-neutral conversion of D-galacturonate to ribulose-5-phosphate. While further research is required to identify the structural genes encoding the key enzymes for the novel pathway, its redox-cofactor coupling is highly interesting for metabolic engineering of microbial cell factories for conversion of pectin-containing feedstocks into added-value fermentation products such as ethanol or lactate. This study illustrates the potential of microbial enrichment cultivation to identify novel pathways for the conversion of environmentally and industrially relevant compounds.BT/Industrial MicrobiologyOLD BT/Cell Systems EngineeringBT/Environmental BiotechnologyBT/Biotechnolog

“Candidatus Galacturonibacter soehngenii” Shows Acetogenic Catabolism of Galacturonic Acid but Lacks a Canonical Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase Complex

Acetogens have the ability to fixate carbon during fermentation by employing the Wood-Ljungdahl pathway (WLP), which is highly conserved across Bacteria and Archaea. In a previous study, product stoichometries in galacturonate-limited, anaerobic enrichment cultures of “Candidatus Galacturonibacter soehngenii,” from a novel genus within the Lachnospiraceae, suggested the simultaneous operation of a modified Entner-Doudoroff pathway for galacturonate fermentation and a WLP for acetogenesis. However, a draft metagenome-assembled genome (MAG) based on short reads did not reveal homologs of genes encoding a canonical WLP carbon-monoxide-dehydrogenase/acetyl-Coenzyme A synthase (CODH/ACS) complex. In this study, NaH13CO3 fed to chemostat-grown, galacturonate-limited enrichment cultures of “Ca. G. soehngenii” was shown to be incorporated into acetate. Preferential labeling of the carboxyl group of acetate was consistent with acetogenesis via a WLP in which the methyl group of acetate was predominately derived from formate. This interpretation was further supported by high transcript levels of a putative pyruvate-formate lyase gene and very low transcript levels of a candidate gene for formate dehydrogenase. Reassembly of the “Ca. G. soehngenii” MAG with support from long-read nanopore sequencing data produced a single-scaffold MAG, which confirmed the absence of canonical CODH/ACS-complex genes homologs. However, high CO-dehydrogenase activities were measured in cell extracts of “Ca. G. soehngenii” enrichment cultures, contradicting the absence of corresponding homologs in the MAG. Based on the highly conserved amino-acid motif associated with anaerobic Ni-CO dehydrogenase proteins, a novel candidate was identified which could be responsible for the observed activities. These results demonstrate operation of an acetogenic pathway, most probably as a yet unresolved variant of the Wood-Ljungdahl pathway, in anaerobic, galacturonate-limited cultures of “Ca. G. soehngenii.”BT/Industrial MicrobiologyBT/Environmental BiotechnologyBT/Biotechnolog