ALACEN: A Holistic Herbaceous Biomass Fractionation Process Attaining a Xylose-Rich Stream for Direct Microbial Conversion to Bioplastics.

Lignocellulose biorefining is a promising technology for the sustainable production of chemicals and biopolymers. Usually, when one component is focused on, the chemical nature and yield of the others are compromised. Thus, one of the bottlenecks in biomass biorefining is harnessing the maximum value from all of the lignocellulosic components. Here, we describe a mild stepwise process in a flow-through setup leading to separate flow-out streams containing cinnamic acid derivatives, glucose, xylose, and lignin as the main components from different herbaceous sources. The proposed process shows that minimal degradation of the individual components and conservation of their natural structure are possible. Under optimized conditions, the following fractions are produced from wheat straw based on their respective contents in the feed by the ALkaline ACid ENzyme process: (i) 78% ferulic acid from a mild ALkali step, (ii) 51% monomeric xylose free of fermentation inhibitors by mild ACidic treatment, (iii) 82% glucose from ENzymatic degradation of cellulose, and (iv) 55% native-like lignin. The benefits of using the flow-through setup are demonstrated. The retention of the lignin aryl ether structure was confirmed by HSQC NMR, and this allowed monomers to form from hydrogenolysis. More importantly, the crude xylose-rich fraction was shown to be suitable for producing polyhydroxybutyrate bioplastics. The direct use of the xylose-rich fraction by means of the thermophilic bacteria Schlegelella thermodepolymerans matched 91% of the PHA produced with commercial pure xylose, achieving 138.6 mgPHA/gxylose. Overall, the ALACEN fractionation method allows for a holistic valorization of the principal components of herbaceous biomasses.

Microbial lipid biosynthesis from lignocellulosic biomass pyrolysis products.

Lipids are a biorefinery platform to prepare fuel, food and health products. They are traditionally obtained from plants, but those of microbial origin allow for a better use of land and C resources, among other benefits. Several (thermo)chemical and biochemical strategies are used for the conversion of C contained in lignocellulosic biomass into lipids. In particular, pyrolysis can process virtually any biomass and is easy to scale up. Products offer cost-effective, renewable C in the form of readily fermentable molecules and other upgradable intermediates. Although the production of microbial lipids has been studied for 30 years, their incorporation into biorefineries was only described a few years ago. As pyrolysis becomes a profitable technology to depolymerize lignocellulosic biomass into assimilable C, the number of investigations on it raises significantly. This article describes the challenges and opportunities resulting from the combination of lignocellulosic biomass pyrolysis and lipid biosynthesis with oleaginous microorganisms. First, this work presents the basics of the individual processes, and then it shows state-of-the-art processes for the preparation of microbial lipids from biomass pyrolysis products. Advanced knowledge on separation techniques, structure analysis, and fermentability is detailed for each biomass pyrolysis fraction. Finally, the microbial fatty acid platform comprising biofuel, human food and animal feed products, and others, is presented. Literature shows that the microbial lipid production from anhydrosugars, like levoglucosan, and short-chain organic acids, like acetic acid, is straightforward. Indeed, processes achieving nearly theoretical yields form the latter have been described. Some authors have shown that lipid biosynthesis from different lignin sources is biochemically feasible. However, it still imposes major challenges regarding strain performance. No report on the fermentation of pyrolytic lignin is yet available. Research on the microbial uptake of pyrolytic humins remains vacant. Microorganisms that make use of methane show promising results at the proof-of-concept level. Overall, despite some issues need to be tackled, it is now possible to conceive new versatile biorefinery models by combining lignocellulosic biomass pyrolysis products and robust oleaginous microbial cell factories.

From agro-waste to tool: biotechnological characterization and application of Ganoderma lucidum E47 laccase in dye decolorization.

The culture of fungal species from agro-waste allows for the sustainable preparation of valuable biotechnological products and contributes to establish the Circular Economy concept. The Ganoderma lucidum species is well known as producer of laccases (EC 1.10.3.2), which serves as a tool to oxidize chemicals. When producing G. lucidum E47 basidiomes with edible purposes out of rice crop residues, its laccase remains as by-product. In this work, we report the biotechnological characterization and application of the laccase recovered from spent cultures of the G. lucidum E47 strain. We detected at least one polypeptide (ca. 59 kDa) which displays attractive activity and stability values when used in the range of 18-45 °C in mildly acidic environment (pH 4.8-5.8). These parameters can be enhanced in the presence of organic cosolvents such as butyl acetate and methyl iso-butyl ketone, but the opposite effect is observed with solvents of lower log P. The best activity-stability performance is reached when the biocatalyst is used in pH 4.8 buffer with 5% (v/v) butyl acetate at 37 °C. The laccase was capable of decolorizing xanthene, azo and triarylmethane dyes, exhibiting excellent selectivity on bromocresol green and bromocresol purple. Furthermore, the biocatalyst displayed an attractive activity when assessed for the decolorization of bromocresol green in a proof-of-concept effluent biotreatment.

Microbial utilization of lignin: available biotechnologies for its degradation and valorization.

Lignocellulosic biomasses, either from non-edible plants or from agricultural residues, stock biomacromolecules that can be processed to produce both energy and bioproducts. Therefore, they become major candidates to replace petroleum as the main source of energy. However, to shift the fossil-based economy to a bio-based one, it is imperative to develop robust biotechnologies to efficiently convert lignocellulosic streams in power and platform chemicals. Although most of the biomass processing facilities use celluloses and hemicelluloses to produce bioethanol and paper, there is no consolidated bioprocess to produce valuable compounds out of lignin at industrial scale available currently. Usually, lignin is burned to provide heat or it remains as a by-product in different streams, thus arising environmental concerns. In this way, the biorefinery concept is not extended to completion. Due to Nature offers an arsenal of biotechnological tools through microorganisms to accomplish lignin valorization or degradation, an increasing number of projects dealing with these tasks have been described recently. In this review, outstanding reports over the last 6 years are described, comprising the microbial utilization of lignin to produce a variety of valuable compounds as well as to diminish its ecological impact. Furthermore, perspectives on these topics are given.

Biotransformation of dehydro-epi-androsterone by Aspergillus parasiticus: Metabolic evidences of BVMO activity.

The research on the synthesis of steroids and its derivatives is of high interest due to their clinical applications. A particular focus is given to molecules bearing a D-ring lactone like testolactone because of its bioactivity. The Aspergillus genus has been used to perform steroid biotransformations since it offers a toolbox of redox enzymes. In this work, the use of growing cells of Aspergillus parasiticus to study the bioconversion of dehydro-epi-androsterone (DHEA) is described, emphasizing the metabolic steps leading to D-ring lactonization products. It was observed that A. parasiticus is not only capable of transforming bicyclo[3.2.0]hept-2-en-6-one, the standard Baeyer-Villiger monooxygenase (BVMO) substrate, but also yielded testololactone and the homo-lactone 3ß-hydroxy-17a-oxa-D-homoandrost-5-en-17-one from DHEA. Moreover, the biocatalyst degraded the lateral chain of cortisone by an oxidative route suggesting the action of a BVMO, thus providing enough metabolic evidences denoting the presence of BVMO activity in A. parasiticus. Furthermore, since excellent biotransformation rates were observed, A. parasiticus is a promising candidate for the production of bioactive lactone-based compounds of steroidal origin in larger scales.

Self-sufficient redox biotransformation of lignin-related benzoic acids with Aspergillus flavus.

Aromatic carboxylic acids are readily obtained from lignin in biomass processing facilities. However, efficient technologies for lignin valorization are missing. In this work, a microbial screening was conducted to find versatile biocatalysts capable of transforming several benzoic acids structurally related to lignin, employing vanillic acid as model substrate. The wild-type Aspergillus flavus growing cells exhibited exquisite selectivity towards the oxidative decarboxylation product, 2-methoxybenzene-1,4-diol. Interestingly, when assaying a set of structurally related substrates, the biocatalyst displayed the oxidative removal of the carboxyl moiety or its reduction to the primary alcohol whether electron withdrawing or donating groups were present in the aromatic ring, respectively. Additionally, A. flavus proved to be highly tolerant to vanillic acid increasing concentrations (up to 8 g/L), demonstrating its potential application in chemical synthesis. A. flavus growing cells were found to be efficient biotechnological tools to perform self-sufficient, structure-dependent redox reactions. To the best of our knowledge, this is the first report of a biocatalyst exhibiting opposite redox transformations of the carboxylic acid moiety in benzoic acid derivatives, namely oxidative decarboxylation and carboxyl reduction, in a structure-dependent fashion.

Selection of a new whole cell biocatalyst for the synthesis of 2-deoxyribose 5-phosphate.

2-deoxyribose 5-phosphate (DR5P) is a key intermediate in the biocatalyzed preparation of deoxyribonucleosides. Therefore, DR5P production by means of simpler, cleaner, and economic pathways becomes highly interesting. One strategy involves the use of bacterial whole cells containing DR5P aldolase as biocatalyst for the aldol addition between acetaldehyde and D: -glyceraldehyde 3-phosphate or glycolytic intermediates that in situ generate the acceptor substrate. In this work, diverse microorganisms capable of synthesizing DR5P were selected by screening several bacteria genera. In particular, Erwinia carotovora ATCC 33260 was identified as a new biocatalyst that afforded 14.1-mM DR5P starting from a cheap raw material like glucose.