Dataset Information

Bioprospecting for fungal enzymes for applications in microalgal-biomass based biorefineries

ABSTRACT: Microalgal biomass is a promising feedstock for biofuels, feed/food and biomaterials. However, while production and commercialization of single-product commodities is still not economically viable, obtaining multiple products in a biomass biorefinery faces several techno- economic challenges. The aim of this study was to identify a suitable source of hydrolytic enzymes for algal biomass saccharification. Screening of twenty-six fungal isolates for secreted enzymes activity on Chlamydomonas reinhardtii biomass resulted in the identification of Aspergillus niger IB-34 as a candidate strain. Solid state fermentation on wheat bran produced the most active enzyme preparations. From sixty-five proteins identified by LC-MS, the majority corresponded to predicted secreted proteins belonging to the Gene Ontology categories of catalytic activity/hydrolase activity on glycosyl and O-glycosyl compounds. Defatted biomass of the more biotechnologically relevant strains towards the production of commodities, Chlorella sorokiniana and Scenedesmus obliquus, was fully saccharified after a mild pretreatment at 80 °C for 10 min, at a high biomass load of 10 % (w/v). Deffated and 2 saccharified biomass of both strains was further converted into ethanol by fermentation with Saccharomyces cerevisiae at a theoretical maximum efficiency, either by separated or simultaneous sccharification and fermentation. The resulting insoluble protein after biomass defatting with an organic solvent and enzymatic saccharification resulted in a high digestibility in an in vitro digestion assay. Proof-of-concept is presented for an enzyme-assisted biomass biorefinery which recovered 81% of the main biomass fractions in a likely active form for the conversion of lipids and carbohydrates into biofuels and proteins into feed/food.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Aspergillus Niger Cbs 101883

SUBMITTER: Araceli Bader

LAB HEAD: Fabiana Consolo

PROVIDER: PXD034998 | Pride | 2023-03-10

REPOSITORIES: Pride

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Bioprospecting for fungal enzymes for applications in microalgal biomass biorefineries.

Bader Araceli Natalia AN Sanchez Rizza Lara L Consolo Verónica Fabiana VF Curatti Leonardo L

Applied microbiology and biotechnology 20221217 2-3

Microalgal biomass is a promising feedstock for biofuels, feed/food, and biomaterials. However, while production and commercialization of single-product commodities are still not economically viable, obtaining multiple products in a biomass biorefinery faces several techno-economic challenges. The aim of this study was to identify a suitable source of hydrolytic enzymes for algal biomass saccharification. Screening of twenty-six fungal isolates for secreted enzymes activity on Chlamydomonas rein ...[more]

PMID: 36527478

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Project description:<h4>Background</h4>Bioconversion of wood into bioproducts and biofuels is hindered by the recalcitrance of woody raw material to bioprocesses such as enzymatic saccharification. Targeted modification of the chemical composition of the feedstock can improve saccharification but this gain is often abrogated by concomitant reduction in tree growth.<h4>Results</h4>In this study, we report on transgenic hybrid aspen (Populus tremula × tremuloides) lines that showed potential to increase biomass production both in the greenhouse and after 5 years of growth in the field. The transgenic lines carried an overexpression construct for Populus tremula × tremuloides vesicle-associated membrane protein (VAMP)-associated protein PttVAP27-17 that was selected from a gene-mining program for novel regulators of wood formation. Analytical-scale enzymatic saccharification without any pretreatment revealed for all greenhouse-grown transgenic lines, compared to the wild type, a 20-44% increase in the glucose yield per dry weight after enzymatic saccharification, even though it was statistically significant only for one line. The glucose yield after enzymatic saccharification with a prior hydrothermal pretreatment step with sulfuric acid was not increased in the greenhouse-grown transgenic trees on a dry-weight basis, but increased by 26-50% when calculated on a whole biomass basis in comparison to the wild-type control. Tendencies to increased glucose yields by up to 24% were present on a whole tree biomass basis after acidic pretreatment and enzymatic saccharification also in the transgenic trees grown for 5 years on the field when compared to the wild-type control.<h4>Conclusions</h4>The results demonstrate the usefulness of gene-mining programs to identify novel genes with the potential to improve biofuel production in tree biotechnology programs. Furthermore, multi-omic analyses, including transcriptomic, proteomic and metabolomic analyses, performed here provide a toolbox for future studies on the function of VAP27 proteins in plants.

Project description:Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5M-bM-^@M-^Qhydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts. E.coli ethanologen strain GLBRCE1 or GLBRCE1 lacking the plasmid-borne PET cassette (GLBRCE1_pBBR) was grown in AFEX corn stover hydrolysate (ACSH) Fermentations were carried out in 3 L bioreactors (Applikon Biotechnology) containing 2.45 L of ACSH, and cultures were diluted into ACSH with initial OD at 0.2, grown anaerobically overnight, and then inoculated into bioreactors to a starting OD600 of 0.2. One biological replicate was grown in each medium. RNA samples were obtained at 3 time points, corresponding to exponential (Exp), transitional (Trans), and stationary (Stat) growth phases.

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Dataset Information

Bioprospecting for fungal enzymes for applications in microalgal-biomass based biorefineries

Publications

Bioprospecting for fungal enzymes for applications in microalgal biomass biorefineries.

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