Within the context of the circular economy, the transformation of agri-food waste or by-products into valuable products is essential to promoting a transition towards more sustainable and efficient utilisation of resources. Whey is a very abundant by-product of dairy manufacturing. Apart from partial reutilisation in animal feed or some food supplements, the sustainable management and disposal of whey still represent significant environmental challenges. In this work, whey is considered a valuable resource for producing high-value products, specifically 2,3-butanediol (2,3-BDO), which was produced through fermentation using the bacterial strain Lactococcus lactis 43103. The described process yielded a >90% purity of 2,3-BDO, which was evaluated as a potential chain extender in the synthesis of bio-based waterborne polyurethane dispersions (PUDs). The incorporation of whey-derived 2,3-BDO led to the development of PUDs with up to 90% bio-based content without detrimental effects on the process or liquid-phase properties. The combination of 100% bio-based polyether polyols with partially renewable L-lysine ethyl ester diisocyanate and whey-derived 2,3-BDO as a chain extender generated totally stable, low-particle-size water dispersions of amorphous polymers characterised by similar structure and molecular weight compared to those of alternative petroleum-based PUDs. These results open up the possibility of incorporating fermentation-derived 2,3-BDO as a totally renewable component in bio-based PUDs as potential sustainable resinous systems for further formulation of water-based coatings or adhesives.

Cheese manufacturing generates huge amounts of whey, part of it being disposed of as waste, posing serious environmental problems. However, whey is a valuable resource that can be valorized in a cost-effective and sustainable way, being fermentation processes a suitable option. The non-engineered mutant strain Lactococcus lactis 43103, a non-pathogenic microorganism, was found to efficiently produce 2,3-butanediol (2,3-BDO) from lactose. Culture of this strain in a whey-based medium adapted to its nutritional requirements under optimized fermentation conditions resulted in 2,3-BDO productions close to 18 g/L, representing 76.8 % of the maximum theoretical yield, which reached 94.7 % when acetoin, its immediate precursor in the metabolic pathway, was included. The fermentation performance was not very far from that of the best producers (natural or engineered) and was the best one among the non-pathogenic and non-engineered bacteria. These results open up the possibility of valorizing whey through fermentation to 2,3-BDO, a valuable platform chemical.

A downstream process for the purification and concentration of formic acid (FA) from FA/gluconic acid (GA) mixtures, obtainable by a coupled biocatalytic reaction of CO2 reduction and glucose oxidation, has been developed. The process involved two technologies: (i) a first nanofiltration (NF) step to separate FA and GA, and (ii) a second reactive liquid-liquid extraction (RLLE) step to concentrate FA. The NF process, using a Synder NFX membrane, consisted of three NF steps separated into two divergent lines, named permeate and retentate pathways. The first NF was common for both pathways, resulting in a permeate strongly enriched in FA and depleted in GA, and a retentate with opposite characteristics. In the permeate pathway, this first permeate was subjected to a second NF to obtain a 99.6% pure FA permeate. In the retentate pathway, an additional NF step on the first retentate resulted in a concentrated 99.4% pure GA retentate. The final diluted FA permeate was concentrated by RLLE using tri-N-octylamine as extractant in n-octanol, and a final back-extraction with NaOH. The optimized RLLE process involved a 100-fold volume decrease and resulted in a final FA solution (as sodium formate) of 174.5 g/L, 78 times more concentrated than the feed.

The aim of this work has been to find a scalable process to purify lignin and separate the cellulose-rich fraction from a poplar wood hydrolysis rest produced at a bioethanol pilot-plant. At initial laboratory optimization, a mild extraction of lignin was performed with 1 M NaOH at 90 degrees C to dissolve the lignin, that was further precipitated with sulfuric acid and this formed an easily filtrable powder. The main sub-product was a solid residue enriched in cellulose, which was successfully saccharified and tested for its fermentability. Further upscaling of the method was done at Bio Based Europe Pilot Plant (BBEPP) in Belgium. In a first run 14.2 kg of purified poplar lignin was obtained and in a second run 128.6 kg was obtained. The wet fiber residue was processed by washing and enzymatic hydrolysis to obtain, 26.7 kg of concentrated sugar solution with 509.4 g/L of glucose.

Flexible 2,3-butanediol and methyl ethyl ketone production by acetoin electroreduction in aqueous media at room temperature in flow cells.

Precursors have been satisfactorily synthesized from lignocellulosic biomass for later use in the synthesis of polyurethanes resulting in competitive final properties with those of petroleum derived polyurethanes.

With the aim of applying biotechnology to produce acetoin, a chemical that can be used as an aroma and as a building block for other compounds, several putative mutants with reduced lactic acid synthesis were obtained from a wild-type homolactic strain of L. lactis subjected to chemical mutagenesis. Among these mutants, a strain was isolated, CML B4, that showed reduced lactate dehydrogenase (LDH) and increased NADH oxidase (NOX) activities. Shaken flask cultures of this mutant strain mainly produced acetoin, increasing the levels produced compared to the wild-type strain by 15-fold. A point mutation detected in the ldh gene encoding LDH was probably the genetic defect responsible for this phenotype. In pH-controlled aerobic batch fermentation, the CML B4 strain produced more than 40 gL(-1) acetoin, which was increased by up to 59 in fed-batch fermentations, with yields close to 88 and 74%, respectively, and productivities exceeding 2 gL(-1) h(-1). These results indicate that this strain could be used industrially as a cell factory for the production of acetoin from bioresources.

Glyoxal oxidase of P. chrysosporium is a radical copper oxidase that catalyzes oxidation of aldehydes to carboxylic acids coupled to dioxygen reduction to H(2)O(2). In addition to known substrates, glycerol is also found to be a substrate for glyoxal oxidase. During enzyme turnover, glyoxal oxidase undergoes a reversible inactivation, probably caused by loss of the active site free radical, resulting in short-lasting enzyme activities and undetectable substrate conversions. Enzyme activity could be extended by including two additional enzymes, horseradish peroxidase and catalase, in addition to a redox chemical activator, such as Mn(III) (or Mn(II)+H(2)O(2)) or hexachloroiridate. Using this three-enzyme system glycerol was converted in glyceric acid in a two-step reaction, with glyceraldehyde as intermediate. A possible operation mechanism is proposed in which the three enzymes would work coordinately allowing to maintain a sustained glyoxal oxidase activity. In the course of its catalytic cycle, glyoxal oxidase alternates between two functional and interconvertible reduced and oxidized forms resulting from a two-electron transfer process. However, glyoxal oxidase can also undergo an one-electron reduction to a catalytically inactive form lacking the active site free radical. Horseradish peroxidase could use glyoxal oxidase-generated H(2)O(2) to oxidize Mn(II) to Mn(III) which, in turn, would reoxidize and reactivate the inactive form of glyoxal oxidase. Catalase would remove the excess of H(2)O(2) generated during the reaction. In spite of the improvement achieved using the three-enzyme system, glyoxal oxidase inactivation still occurred, which resulted in low substrate conversions. Possible causes of inactivation, including end-product inhibition, are discussed.

The high molecular weight of chitosan, which results in a poor solubility at neutral pH values and high viscosity aqueous solutions, limits its potential uses in the fields of food, health and agriculture. However, most of these limitations are overcome by chitosan oligosaccharides obtained by enzymatic hydrolysis of the polymer. Several commercial enzymes with different original specificities were assayed for their ability to hydrolyze a 93% deacetylation degree chitosan and compared with a chitosanase. According to the patterns of viscosity decrease and reducing end formation, three enzymes--cellulase, pepsin and lipase A--were found to be particularly suitable for hydrolyzing chitosan at a level comparable to that achieved by chitosanase. Unlike the appreciable levels of both 2-amino-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-glucose monomers released from chitosan by the other enzymes after a 20h-hydrolysis (4.6-9.1% of the total product weight), no monomer could be detected following pepsin cleavage. As a result, pepsin produced a higher yield of chitosan oligosaccharides than the other enzymes: 52% versus as much as 46%, respectively. Low molecular weight chitosans accounted for the remaining 48% of hydrolysis products. The calculated average polymerization degree of the products released by pepsin was around 16 units after 20h of hydrolysis. This product pattern and yield are proposed to be related to the bond cleavage specificity of pepsin and the high deacetylation degree of chitosan used as substrate. The optimal reaction conditions for hydrolysis of chitosan by pepsin were 40 degrees C and pH 4.5, and an enzyme/substrate ratio of 1:100 (w/w) for reactions longer than 1h.

Asexual spores or conidia are dispersive propagules produced as an alternative to vegetative growth by a diverse group of filamentous fungi. The cellular development programmes which govern conidiation have been intensely studied in the last few decades, although important gaps stand in the way of our understanding of this phenomenon, namely in the areas of the environmental sensing mechanisms and signal transduction pathways. The aim of this review is to summarize the current advances in conidiation induction in the genus Penicillium, and to put them into context with the state of our knowledge stemming from work in widely studied fungal model systems.

The isolation and structure determination of conidiogenol and conidiogenone, tetracyclic diterpenes with a novel carbon skeleton, from extracts of the fermentation broth of Penicillium cyclopium is reported. Conidiogenol and conidiogenone are potent and selective inducers of conidiogenesis in P. cyclopium in liquid culture, and relay information about the environmental conditions to the producing organism.

ABSTRACT The filamentous fungus Penicillium cyclopium conidiates in the presence of calcium ions in submerged culture without nutrient limitation according to a precisely timed program. Conidiation could be prematurely induced in a nutritionally sufficient medium which had previously supported growth, suggesting that a metabolite which influenced the process was produced. A diterpenoid with conidiation-inducing activity, conidiogenone, was purified from the culture medium, along with conidiogenol, a putative derivative with delayed activity. Contrary to previous thought, the presence of calcium was demonstrated to only decrease the threshold concentration of conidiogenone required for the induction to proceed. In light of these results, a mechanism of conidiation induction is presented according to which the mycelium produces a conidiation inducer (conidiogenone) that accumulates extracellularly. When a threshold concentration is reached, induction likely takes place by interaction with a specific cellular receptor. The results indicate that conidiogenone is both sufficient and necessary to induce conidiation.

In response to alkaline ambient pH, the Aspergillus nidulans PacC transcription factor mediating pH regulation of gene expression is activated by proteolytic removal of a negative‐acting C‐terminal domain. We demonstrate interactions involving the ∼150 C‐terminal PacC residues and two regions located immediately downstream of the DNA binding domain. Our data indicate two full‐length PacC conformations whose relative amounts depend upon ambient pH: one ‘open’ and accessible for processing, the other ‘closed’ and inaccessible. The location of essential determinants for proteolytic processing within the two more upstream interacting regions probably explains why the interactions prevent processing, whereas the direct involvement of the C‐terminal region in processing‐preventing interactions explains why C‐terminal truncating mutations result in alkalinity mimicry and pH‐independent processing. A mutant PacC deficient in pH signal response and consequent processing behaves as though locked in the ‘closed’ form. Single‐residue substitutions, obtained as mutations bypassing the need for pH signal transduction, identify crucial residues in each of the three interactive regions and overcome the processing deficiency in the ‘permanently closed’ mutant.

Addition of Ca2+ (1 to 10 mM) to submerged cultures of Penicillium cyclopium induces conidiation. Ca2+ induced an increase in cytosolic pH from approximately 7.00 to > 7.60 in less than 10 min, as determined with the fluorescent pH probe fluorescein. Measurement of the H(+)-ATPase activity in total membrane fractions did not show any stable activation in vivo as a result of Ca2+ treatment. By fluorescence ratio imaging microscopy, it was observed that vegetative hyphae exhibit a tip-to-base pH gradient, with the tip being more acidic. Ca2+ caused this gradient to dissipate within 10 min. The effect of several agents that are supposed to cause internal acidification, by different means, on conidiation was tested. Concentrations of these agents that did not significantly affect growth but inhibited Ca(2+)-induced conidiation also prevented the intracellular alkalinization observed after exposure to the cation. Calcium channel blockers (lanthanum, cobalt, verapamil, and nifedipine) were not able to inhibit Ca(2+)-induced conidiation, although their effect on calcium uptake was not evaluated. However, the combined results point towards externally bound Ca2+ as the primary agent of conidiation induction, causing changes in plasma membrane function which disrupt the pH gradient observed during apical growth.

Summary: A new simple procedure for the production of protoplasts of Penicillium cyclopium with high regeneration rates and efficient transport activity is described, involving the use of a preparation of Novozym 234 with very low protease activity. The combination of a heat pretreatment at 55 °C for 15 min with the use of the protease inhibitor aprotinin resulted in a 97% reduction of Novozym 234 protease activity with respect to untreated controls. Polysaccharide-hydrolysing activity was inhibited much less, to 60% of the untreated Novozym 234 level. Protoplasts could be successfully produced with the new low-protease Novozym 234 preparation, showing a threefold increase in regeneration capacity compared to control protoplasts obtained with the original preparation. The rates of 3-O-methylglucose uptake and the capacity to accumulate this sugar analogue were also higher in protoplasts obtained by the new method.