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Agricultural crop residues serve as a renewable source for production of bioethanol and other value added chemicals. Optimization of enzymatic saccharification may ensure cost-effective production of bioethanol and other industrially important products. Here, we attempted optimization of various process variables affecting enzymatic hydrolysis of microwave-assisted acid pretreated chili post-harvest residue was evaluated by adopting statistical design experiments. The optimum conditions of enzymatic hydrolysis were solid loading of 15% (w/w), cellulase loading of 20 FPU per gram of pretreated biomass and incubation time of 12 h. The high solid loading, low cellulase loading and low incubation time may lead to better process economics. Maximum reducing sugar yield of 0.205 g/g was observed. Fermentation inhibitors, such as furfural and 5-hydroxymethylfurfural were absent in the hydrolysate obtained after enzymatic saccharification of pretreated biomass and were found suitable for the production of various value added products like xylanases, bioethanol and biopolymer (poly-3-hydroxybutyrate).

Biodiversity of soil microbial populations could be altered and affected by anthropogenic pressures due to the release of organic and inorganic xenobiotic compounds and/or the application of remediation practices. Therefore, the assessment of the response of microbiota to environmental pollution and to bioremediation techniques is a critical issue in soil ecology. In this study a culture- independent approach was used to investigate the indigenous bac- terial community structure in two contaminated soils of a National Interest Priority Site in Campania (southern Italy) and to monitor the impact of different remediation technologies. Our results show that bacterial populations shifted in the polluted soils over time after the application of compost and microbial inoculum. Statistical analyses based on the similarity of DGGE profiles show that the bacterial community structure and diversity was not affected by contamination. Hence the main change in similarity levels was induced by sampling time and by the interaction between soil eco-friendly bioremediation treatments.

Spent mushroom compost is the residual compost waste generated by the mushroom production industry. Annually 3 million tons of these residues are generated in Europe resulting in disposal costs of up to 150 million Euro. The BIOrescue project aims to develop an innovative biorefinery strategy to valorise this promising source of biomass together with other underutilised lignocellulosic feedstocks. This paper shows the main results obtained during the first half of BIOrescue project., Proceedings of the 26th European Biomass Conference and Exhibition, 14-17 May 2018, Copenhagen, Denmark, pp. 1084-1088

Mushroom production generates three million tonnes of compost each year in the European Union which is currently landfilled or used for landscaping purposes even though it contains valuable organic components. In view of transforming this compost into a new income stream for mushroom producers, the BIOrescue project aims to develop and demonstrate a new innovative biorefinery concept based on the cascading use of spent mushroom substrate (SMS) supplemented by wheat straw (and other seasonal underutilized lignocellulosic feedstocks). This new concept will avoid disposal and allow for the production of some biodegradable bio-based products and bioactive compounds that will help to replace the existing ones based on fossil resources. The research will help to expand the business opportunities of the mushroom cultivation farms, and the know-how and business opportunities of all the partners involved. The main scientific innovations are: improved methods for the lab-based rapid (NIR) (1) analysis of biomass, innovative two step fractionation of SMS, synergistic effects for complete SMS glucan hydrolysis, innovative enzyme immobilisation strategy, development of highly efficient glucan-enzymes, novel lignin based nano- and micro-carriers and biopesticide production from monomeric sugars derived from SMS and their packaging into nanocarriers., Proceedings of the 25th European Biomass Conference and Exhibition, 12-15 June 2017, Stockholm, Sweden, pp. 2015-2018

Fossil fuel reserves depletion, global warming, costly and problematic waste recycling and population growth greatly induce to fi nd renewable energy sources. Second generation bioethanol produced from lignocellulosic materials exhibits great potential as liquid biofuel to substitute gasoline. Production costs of enzymes involved in cellulose hydrolysis into fermentable sugars represent the main obstacle to achieve competitive production of cellulosic ethanol. Cheaper and more effi cient biocatalysts for the saccharifi cation step are, therefore, required for making the whole process more competitive. The biodiversity of natural niches has been so far exploited for the isolation of new cellulolytic microorganisms whose enzymes are naturally evolved for an effi cient conversion of cellulose into fermentable sugars. This review discusses advances in isolation of bacteria, namely Bacillus spp., from several natural habitats and their ability to produce cellulase activity.

Agricultural crop residues serve as a renewable source for production of bioethanol and other value added chemicals. Optimization of enzymatic saccharification may ensure cost-effective production of bioethanol and other industrially important products. Here, we attempted optimization of various process variables affecting enzymatic hydrolysis of microwave-assisted acid pretreated chili post-harvest residue was evaluated by adopting statistical design experiments. The optimum conditions of enzymatic hydrolysis were solid loading of 15% (w/w), cellulase loading of 20 FPU per gram of pretreated biomass and incubation time of 12 h. The high solid loading, low cellulase loading and low incubation time may lead to better process economics. Maximum reducing sugar yield of 0.205 g/g was observed. Fermentation inhibitors, such as furfural and 5-hydroxymethylfurfural were absent in the hydrolysate obtained after enzymatic saccharification of pretreated biomass and were found suitable for the production of various value added products like xylanases, bioethanol and biopolymer (poly-3-hydroxybutyrate).

The enzymatic extracellular mixtures of two new microorganisms – Streptomyces flavogriseus AE64X and AE63X – isolated from Eucalyptus camaldulensis and Populus nigra and producing cellulase and xylanase, were characterized and applied to hydrolysis of pretreated Arundo donax, Populus nigra and Panicum virgatum (10% w/v) replacing the commercial enzymes Accelerase 1500 and Accelerase XY (5.4 and 145 U/g of pretreated biomass, respectively). It is worth of noting that the newly developed extracellular enzymatic mixtures, without any purification step and at the same dosage, presented saccharification yields that are higher (86% for S. flavogriseus AE64X) than those of commercial enzymes (81%). Moreover, these enzymatic mixes allowed us to hydrolyse both cellulose and xylan within the different lignocellulose biomasses substituting both the cellulase and xylanase of commercial source. The produced sugars were also fermentable by Basfia succiniciproducens BPP7 into succinic acid with high yield.

Brewer’s spent grain (BSG), the solid residue of beer production, is attracting significant attention as raw material for the production of added value substances, since until recently it was mainly used as animal feed or deposited in landfills, causing serious environmental problems. Therefore, this work aimed at developing a bioprocess using BSG as a substrate for the production of cellulases and xylanases for waste saccharification and bioenergy production. Different fungi were analyzed for their cellulolytic and xylanolytic abilities, through a first screening on solid media by assessment of fungal growth and enzyme production on agar containing carboxylmethylcellulose or xylan as the sole carbon source, respectively. The best cellulase and xylanase producers were subjected to quantitative evaluation of enzyme production in liquid cultures. Aspergillus niger LPB-334 was selected for its ability to produce cellulase and xylanase at high levels and it was cultivated on BSG by solid state fermentation. The cellulase production reached a maximum of 118.04 ± 8.4 U/g of dry substrate after 10 days of fermentation, while a maximum xylanase production of 1315.15 ± 37.5 U/g of dry substrate was reached after 4 days. Preliminary characterization of cellulase and xylanase activities and identification of the enzymes responsible were carried out.

Background: Due to its wide range of applications in the food, pharmaceutical and chemical fields, microbial syn- thesis of succinic acid is receiving growing attention, generating already relevant industrial results, as well as fueling constant research for improvements. In order to develop a sustainable process, a special focus is now set on the exploitation and conversion of lignocellulosic biomasses into platform chemicals. Results: In the present work we used Basfia succiniciproducens BPP7 in separated hydrolysis and fermentation experi- ments with Arundo donax as starting material. Fed-batch strategies showed a maximal production of about 37 g/L of succinic acid after 43 h of growth and a productivity of 0.9 g/L h on the pilot scale. Global mass balance calculations demonstrated a hydrolysis and fermentation efficiency of about 75%. Moreover, the application of a material flow analysis showed the obtainment of 88.5 and 52 % of succinic acid, per kg of virgin biomass and on the total generated output, respectively. Conclusions: The use of fed-batch strategies for the growth of B. succiniciproducens on A. donax improved the titer and productivity of succinic acid on pre-pilot scale. Process evaluation through material flow analysis showed successful results and predicted a yield of succinic acid of about 30% in a fed-batch process that uses A. donax as only carbon source also in the feed. Preliminary considerations on the possibility to achieve an energetic valorization of the residual solid coming from the fermentation process were also carried out. Keywords: Basfia succiniciproducens, Arundo donax, Fed-batch, Succinic acid, Pre-pilot scale, Inhibition, Material flow analysis

Background To establish bioplastics as a real alternative to conventional plastics, high production costs must be constrained by using different kinds of wastewater streams as organic substrates and novel microbial strains as material-accumulating bacteria with high performance. Volatile fatty acids (VFAs) from the effluent of dairy wastewater biodigestion represent a new and inexpensive feedstock, which was used in this study for biopolymer production through microbial processes. Results Cupriavidus necator DSM 13513 was particularly able to accumulate PHAs when operating in fed-batch mode by limiting the oxygen level together with intermittent feeding of a carbon source; maximum poly-β-hydroxybutyrate (PHB) accumulation was achieved in 48 h without compromising microbial growth. The complex VFAs mixture from the digestate did not influence PHA homopolymer accumulation. In fact, structural characterization by NMR analysis revealed PHB synthesis by C. necator DSM 13513 grown with different VFAs mixtures. Moreover, the bioplastic disk obtained from C. necator DSM 13513 cells grown on VFAs from digested dairy wastewater effluent presented good thermic properties and low affinity to water. Conclusions Overall, the results make digested dairy wastewater effluent suitable for PHB production for specific biobased industrial applications.

Lactic acid (LA) is an important biomolecule applied in food, pharmaceutical and chemical areas, mainly to produce biodegradable polymers, such as poly–lactic acid (PLA). In this work, an efficient fermentative process for LA production was developed using potato processing waste (PPW) hydrolysate with Lactobacillus pentosus. After optimization and kinetics studies, LA production reached 150 g/L with a productivity of 1.6 g/L.h in Erlenmeyer flasks. LA production was also conducted in STR where 110 g/L were reached with a productivity of 2.4 g/L.h. LA recovery consisted of a clarification step, with powdered activated carbon, with further precipitation at low temperature and acidification of calcium lactate for conversion to LA. The process was effective for contaminants' removal and clarification, and LA concentration to 416 g/L. Good perspectives for LA production, recovery and clarification were observed. Future studies will be carried out for LA purification and polymerization for PLA synthesis.

International audience; The need to develop competitive and eco-friendly processes in the cosmetic industry leads to the search for new enzymes with improved properties for industrial bioconversions in this sector. In the present study, a complete methodology to generate, express and screen diversity for the type C feruloyl esterase from Fusarium oxysporium FoFaeC was set up in a high-throughput fashion. A library of around 30,000 random mutants of FoFaeC was generated by error prone PCR of fofaec cDNA and expressed in Yarrowia lipolytica. Screening for enzymatic activity towards the substrates 5-bromo-4-chloroindol-3-yl and 4-nitrocatechol-1-yl ferulates allowed the selection of 96 enzyme variants endowed with improved enzymatic activity that were then characterized for thermo- and solvent- tolerance. The five best mutants in terms of higher activity, thermo- and solvent- tolerance were selected for analysis of substrate specificity. Variant L432I was shown to be able to hydrolyze all the tested substrates, except methyl sinapate, with higher activity than wild type FoFaeC towards methyl p-coumarate, methyl ferulate and methyl caffeate. Moreover, the E455D variant was found to maintain completely its hydrolytic activity after two hour incubation at 55 °C, whereas the L284Q/V405I variant showed both higher thermo- and solvent- tolerance than wild type FoFaeC. Small molecule docking simulations were applied to the five novel selected variants in order to examine the binding pattern of substrates used for enzyme characterization of wild type FoFaeC and the evolved variants.

The protein glycosylation is a post-translational modification of crucial importance for its involvement in molecular recognition, protein trafficking, regulation, and inflammation. Indeed, abnormalities in protein glycosylation are correlated with several disease states such as cancer, inflammatory diseases, and congenial disorders. The understanding of cellular mechanisms through the elucidation of glycan composition encourages researchers to find analytical solutions for their detection. Actually, the multiplicity and diversity of glycan structures bond to the proteins, the variations in polarity of the individual saccharide residues, and the poor ionization efficiencies make their detection much trickier than other kinds of biopolymers. An overview of the most prominent techniques based on mass spectrometry (MS) for protein glycosylation (glycoproteomics) studies is here presented. The tricks and pre-treatments of samples are discussed as a crucial step prodromal to the MS analysis to improve the glycan ionization efficiency. Therefore, the different instrumental MS mode is also explored for the qualitative and quantitative analysis of glycopeptides and the glycans structural composition, thus contributing to the elucidation of biological mechanisms.

The risk for human health and environment due to the presence in agricultural soils of organic and inorganic contaminants derived from illegal waste disposal, posed the need to develop a sustainable strategy for the restoration of soils with high agricultural potential. In this study an eco-compatible method as soil tillage and compost amendment was applied in two sites of a National Interest Priority Site of Campania Region (Italy) known as “Terra dei Fuochi”. After one year, the concentration of heavy hydrocarbons (C > 12) significantly reduced in a range from 84 to 95%, probably due to the aeration causing photo-oxidation processes and an improvement of microbial activity able to degrade organic pollutants. Although a reduction trend was observed, the potentially toxic elements (PTEs) concentration in soils was unmodified indicating a potential risk of plant uptake. The high-throughput sequencing approach used to explore the response of native microbial populations following in situ treatments showed that microbiota dynamic was differently affected in the two sites. Therefore, the characteristics of the sites could differently interact with compost determining a different microbial response. Alphaproteobacteria (incidence range of 14–18%), Actinobacteria (incidence range of 10–22%) and Sordariomycetes (incidence range of 23–57%) were the dominant autochthonous populations in all soils indicating their high adaptability to different environments and high resistance to environmental stress as well as their high potential in degradation of organic xenobiotic compounds. The remediation practices based on soil tillage and compost amendment promoted the restoration of the microbial biofertility of soils affected by spotted multi-contamination, to bring them back to agricultural use.

Twenty-eight fungal feruloyl esterases (FAEs) were evaluated for their synthetic abilities in a ternary system of n-hexane: t-butanol: 100 mM MOPS-NaOH pH 6.0 forming detergentless microemulsions. Five main derivatives were synthesized, namely prenyl ferulate, prenyl caffeate, butyl ferulate, glyceryl ferulate, and l-arabinose ferulate, offering, in general, higher yields when more hydrophilic alcohol substitutions were used. Acetyl xylan esterase-related FAEs belonging to phylogenetic subfamilies (SF) 5 and 6 showed increased synthetic yields among tested enzymes. In particular, it was shown that FAEs belonging to SF6 generally transesterified aliphatic alcohols more efficiently while SF5 members preferred bulkier l-arabinose. Predicted surface properties and structural characteristics were correlated with the synthetic potential of selected tannase-related, acetyl-xylan-related, and lipase-related FAEs (SF1-2, -6, -7 members) based on homology modeling and small molecular docking simulations.

The type C feruloyl esterase FoFaeC from Fusarium oxysporum is a newly discovered enzyme with high potential for use in the hydrolysis of lignocellulosic biomass but it shows low activity towards sinapates. In this work, small molecule docking simulations were employed in order to identify important residues for the binding of the four model methyl esters of hydroxycinnamic acids, methyl ferulate/caffeate/sinapate/p-coumarate, to the predicted structure of FoFaeC. Subsequently rational redesign was applied to the enzyme' active site in order to improve its specificity towards methyl sinapate. A double mutation (F230H/T202V) was considered to provide hydrophobic environment for stabilization of the methoxy substitution on sinapate and a larger binding pocket. Five mutant clones and the wild type were produced in Pichia pastoris and biochemically characterized. All clones showed improved activity, substrate affinity, catalytic efficiency and turnover rate compared to the wild type against methyl sinapate, with clone P13 showing a 5-fold improvement in catalytic efficiency. Although the affinity of all mutant clones was improved against the four model substrates, the catalytic efficiency and turnover rate decreased for the substrates containing a hydroxyl substitution.

International audience; The chemical syntheses currently employed for industrial purposes, including in the manufacture of cosmetics, present limitations such as unwanted side reactions and the need for harsh chemical reaction conditions. In order to overcome these drawbacks, novel enzymes are developed to catalyze the targeted bioconversions. In the present study, a methodology for the construction and the automated screening of evolved variants library of a Type B feruloyl esterase from Myceliophthora thermophila (MtFae1a) was developed and applied to generation of 30,000 mutants and their screening for selecting the variants with higher activity than the wild-type enzyme. The library was generated by error-prone PCR of mtfae1a cDNA and expressed in Saccharomyces cerevisiae. Screening for extracellular enzymatic activity towards 4-nitrocatechol-1-yl ferulate, a new substrate developed ad hoc for high-throughput assays of feruloyl esterases, led to the selection of 30 improved enzyme variants. The best four variants and the wild-type MtFae1a were investigated in docking experiments with hydroxycinnamic acid esters using a model of 3D structure of MtFae1a. These variants were also used as biocatalysts in transesterification reactions leading to different target products in detergentless microemulsions and showed enhanced synthetic activities, although the screening strategy had been based on improved hydrolytic activity.

Bio-based succinic acid production from lignocellulosic biomass is one of the attractive and prominent alternative technologies to overcome issues associated with the utilization of fossil sources. In this context, it is necessary to find new microorganisms that are able to efficiently ferment this recalcitrant feedstock. The ecological approach developed in this study enabled the isolation of Basfia succiniciproducens BPP7 from a complex rumen ecosystem. This new wild-type strain was able to synthesize up to 6.06 ± 0.05 g/L of succinate (corresponding to 0.84 ± 0.017 g of succinate per gram of consumed glucose + xylose and to 0.14 ± 0.001 g of succinate per gram of glucans + xylans present in the biomass before hydrolysis) from Arundo donax hydrolysate in separate hydrolysis and fermentation (SHF) experiments. Higher titers of succinic acid were obtained through the optimization of growth conditions. The optimal medium composition identified on the smaller scale was then used for 2.5-L batch experiments, which used A. donax hydrolysate and yeast extract as the main C and N sources, respectively. A maximal titer of 9.4 ± 0.4 g/L of succinic acid was obtained after 24 h. The overall results clearly demonstrate the potential of B. succiniciproducens BPP7 for succinate production.

Cellulases are a important family of hydrolytic enzymes which catalyze the bond of cellulose and other related cello-oligosaccharide derivates. Industrial applications require enzymes highly stable and economically viable in terms of reusability. These costs can be reduced by immobilizing the cellulases, offering a potential solution through enzyme recycling and easy recovery. The covalent immobilization of enzymes is reported here: one is commercial cellulase from Aspergillus Niger and other one is recombinant enzyme, named CelStrep it because was isolated from a new cellulolytic strain, Streptomyces sp. G12,. The optimal pH for binding is 4.6 for both cellulases and the optimal enzyme concentrations are 1 mg/mL and 5 mg/mL respectively. The support for immobilization is a poliacrylic matrix. Experiments carried out in this work show positive results of enzyme immobilization in terms of efficiency and stability and confirm the economic and biotechnical advantages of enzyme immobilization for a wide range of industrial applications.

This work was aimed at identifying and at characterizing new Pleurotus ostreatus laccases, in order to individuate the most suitable biocatalysts for specific applications. The existence of a laccase gene clustering was demonstrated in this basidiomycete fungus, and three new laccase genes were cloned, taking advantage of their closely related spatial organization on the fungus genome. cDNAs coding for two of the new laccases were isolated and expressed in the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis, in order to optimize their production and to characterize the recombinant proteins. Analysis of the P. ostreatus laccase gene family allowed the identification of a “laccase subfamily” consisting of three genes. A peculiar intron–exon structure was revealed for the gene of one of the new laccases, along with a high instability of the recombinant enzyme due to lability of its copper ligand. This study allowed enlarging the assortment of P. ostreatus laccases and increasing knowledge to improve laccase production.