Isolation of new cellulase and xylanase producing strains and application to lignocellulosic biomasses hydrolysis and succinic acid production

Accepted Manuscript Isolation of new cellulase and xylanase producing strains and application to lignocellulosic biomasses hydrolysis and succinic acid production Anna Pennacchio, Valeria Ventorino, Donatella Cimini, Olimpia Pepe, Chiara Schiraldi, Michela Inverso, Vincenza Faraco PII: DOI: Reference:

S0960-8524(18)30365-1 https://doi.org/10.1016/j.biortech.2018.03.027 BITE 19666

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

1 December 2017 3 March 2018 5 March 2018

Please cite this article as: Pennacchio, A., Ventorino, V., Cimini, D., Pepe, O., Schiraldi, C., Inverso, M., Faraco, V., Isolation of new cellulase and xylanase producing strains and application to lignocellulosic biomasses hydrolysis and succinic acid production, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.03.027

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Isolation of new cellulase and xylanase producing strains and application to lignocellulosic biomasses hydrolysis and succinic acid production

Anna Pennacchio1§, Valeria Ventorino2§, Donatella Cimini3, Olimpia Pepe2, Chiara Schiraldi3, Michela Inverso3, Vincenza Faraco1*

1

Department of Chemical Sciences, University of Naples Federico II, Naples, Italy

2

Department of Agricultural Sciences, University of Naples Federico II, Portici

(Naples), Italy; 3

Department of Experimental Medicine, School of Medicine, University of Campania

"Luigi Vanvitelli", via L.de Crecchio 7, 80138 Naples, Italy

*Correspondence: Prof. Vincenza Faraco, University of Naples Federico II, Department of Chemical Sciences, Complesso Universitario Monte S. Angelo, via Cintia 4, 80126, Naples, Italy, [email protected]. §: These authors contributed equally to this work.

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Abstract 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 lignocelllulose 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.

Keywords: Streptomyces; strain identification; endo-cellulase production; lignocellulose; saccharification; succinic acid fermentation

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1. Introduction Among the compounds that are currently produced by fermentation, succinic acid has received a great deal of attention because it was classified as one of the main value added bio-based chemicals with enormous market potential by United States Department of Energy (Choi et al., 2015). Biotechnological succinic acid production processes have being applied by several companies such as Myriant, Reverdia, BioAmber, and Succinity that established biobased production platforms from purified sugars (Becker et al., 2015). As an alternative, the use of lignocellulosic feedstock as raw material for biotechnological production of succinic acid is a crucial priority for cost reduction. As a matter of fact, lignocellulose -that is the main component of woody and non-woody plants including the huge amounts of lignocellulosic wastes- stands out as the main renewable resource for biofuels and other added value bioproducts (Howard et al., 2003; Liguori and Faraco 2016a; Liguori et al., 2016b). The high production costs of enzymes for (hemi)cellulose saccharification -representing one of the main bottlenecks for the lignocellulose conversion- lead the research to discovering more effective (hemi)cellulolytic enzymes and applying on-site plants for enzyme production (Singhania et al., 2015; Li et al., 2017). In this context, this work was aimed at developing new biocatalysts with increased yield of hydrolysis of the (hemi)cellulose into fermentable sugars for succinic acid production, in order to overcome the limitations that prevent a competitive production of biochemicals from lignocellulosic biomasses. Eucalyptus camaldulensis and Populus nigra biomasses after natural biodeterioration had been previously proven appropriate sources for new microorganisms and/or enzymes relevant for lignocellulose conversion (Ventorino et al., 2015; Ventorino et al., 2016a; Montella et al., 2017). The main objectives of this

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work were therefore to screen six actinobacteria strains isolated from those biomasses for their cellulase and xylanase activities, to test the enzymatic mixtures of the strains Streptomyces flavogriseus AE63X and AE64X producing the highest levels of cellulase and xylanase activities in hydrolysis of pretreated Arundo donax, Populus nigra and Panicum virgatum by substituting components of a commercial enzymatic mixture, and to demonstrate the fermentability of the enzymatically obtained hydrolysates for succinic acid production using the strain Basfia succiniciproducens BPP7. 2. Materials and Methods 2.1. Polyphasic characterization of bacterial strains Six bacterial strains from of Eucalyptus camaldulensis and Populus nigra (Ventorino et al., 2015) were grown on starch casein agar medium (Pepe et al., 2013) and characterized on the basis of their colony morphology, microscopic features (phasecontrast microscopy, shape, and presence of spores) and biochemical characteristics (Gram-stains and catalase activity). Molecular identification was performed by 16S rRNA gene sequencing as reported in Amore et al. (2013). The DNA sequences were analyzed as previously reported (Ventorino et al., 2016b). Multiple nucleotide alignments of nearly full-length 16S rRNA sequences of isolated strains were carried out and phylogenetic analysis was also performed (Ventorino et al., 2016c). 2.2. Screening of bacterial strains on solid media Bacterial cell suspensions (0.5 of McFarland Turbidity Standard corresponding to approximately 1.5  108 CFU mL-1) were spotted in triplicate on selective solid media for the detection of endo- and exo-cellulase, cellobiase, xylanase and pectinase activities by qualitative and semi-quantitative agar spot methods as previously described (Ventorino et al., 2015). Endo-cellulase and pectinase activity were recorded as the

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“Indices of Relative Enzyme Activity, (ICMC or IPEC) = diameter of clearing or halo zone/colony diameter” using carboxymethylcellulose (CMC) agar or Pectin agar media, respectively (Pepe et al., 2013; Giacobbe et al., 2014; Ventorino et al., 2015). Exo-cellulase activity was detected by observing the development of bacterial colonies after incubation on Avicel agar (Ventorino et al., 2015). Cellobiase and xylanase activities were estimated by observing a clear zone around the colonies after incubation on cellobiose agar (Ventorino et al., 2015) or Luria-Bertani (LB) agar medium (Oxoid, Milan, Italy) supplemented with 0.05% Remazol brilliant blue-R and a 0.5% solution of sonicated xylan (Sigma-Aldrich, Milan, Italy) as described by Ko et al. (2012). 2.3. Screening of bacterial strains in liquid medium For quantifying the endo-cellulase and xylanase activities, the Streptomyces strains were cultivated in 100 mL plugged Erlenmeyer flasks, by incubation for ten days at 28°C on a rotary shaker (150xg) as reported in Ventorino et al. (2016a). The liquid medium adopted for analysis of cellulase and xylanase production levels contained 1% CMC or xylan, respectively. Samples of liquid cultures were withdrawn every 24 hours, centrifuged at 5000xg for 30 minutes at 4°C and used to measure optical density (OD600nm) and extracellular cellulase and xylanase activities. Endo-1,4-ß-glucanase and xylanase activities produced in liquid culture were assayed using Azo-CMC (Megazyme, Ireland) and xylan (Megazyme, Ireland) as substrates, respectively, following supplier's instructions, and determined by referring to a standard curve. Absorbance of the reaction solutions was measured at 590 nm. One unit of enzyme is defined as the amount of enzyme catalyzing the release of 1 µmol of glucose or xylose equivalent per min. The reported results correspond to mean values of three independent experiments, each one performed in three replicates.

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2.4. Partial characterization of enzymatic extracts To obtain enzymatic extract, liquid cultures of the two selected Streptomyces flavogriseus strains were centrifuged at 5000xg for 30 minutes at 4°C to remove cells and the supernatant containing cellulase and xylanase activity was concentrated by using the stirred ultrafiltration Amicon® system (Millipore Corporation, Bedford, MA, USA) with a 10 kDa polyethersulfone membrane. The concentrated enzymatic extract was used to determine the optimum temperature and pH and thermo-resistance of the cellulase and xylanase activity. The optimum temperature and thermoresistance of cellulase activity were assessed at 40, 45, 50 and 55 °C by using Azo-CMC (Megazyme, Ireland) dissolved in 50 mM Na citrate at pH 5.0 as substrate, following supplier’s instructions. The optimum pH of cellulase activity was assayed at 50°C in 50 mM Na-citrate buffer, at pH 4.0, 5.0, 6.0, 7.0, and 8.0 using Azo-CMC (Megazyme, Ireland) as substrate dissolved in the above mentioned buffer. To determine the optimum temperature and thermoresistance of xylanase activity, the substrate of the activity assay (birch-wood xylan) was dissolved in 50 mmol L−1 Na citrate at pH 5.0 and the incubation (10 min) was performed at 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C and 70 °C. The optimum pH of xylanase activity was determined using the substrate birchwood xylan dissolved in 50 mmol L−1 citrate phosphate buffers, with pH values between 4.0 and 8.0 and performing the incubation (10 min) at 50 °C. The samples withdrawn were assayed for residual xylanase activity as described above. The reported results correspond to mean values of the three independent experiments, each one performed in three replicates. 2.5. Enzymatic hydrolysis of biomasses and quantification of released sugars

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Arundo donax, Populus nigra and Panicum virgatum pretreated according to Garbero et al. (2010) and De Bari et al. (2013) were utilized as substrates as slurry without any downstream process in biotransformation experiments carried out in capped tubes, on the rotary shaker ThermoMixer C (Eppendorf, Milan, Italy) at 6000 rpm. The hydrolysis of pretreated lignocellulosic materials with the enzymatic cocktails was carried out at a concentration of pretreated biomass of 10% (w/v) in a total volume of 2.5 mL of 50 mM sodium citrate buffer pH 5.0. The components of the enzymatic commercial cocktail adopted as benchmark were Accelerase1500, Accelerase BG and Accelerase XY provided by Genencor and were prepared at the amounts expressed as units per gram of pretreated biomass: 5.4, 145 and 4000, respectively. The two commercial enzymes were the best available among those most widely used (MaitanAlfenas et al., 2015; Cardona et al., 2014). As a further check of the use of the same amounts of new enzymatic activities of this work and commercial enzymes, Filter Paper Activity (FPA) assay was also performed in addition to AZO-CMC assay. The FPA assay was carried out in a mixture containing 0.5 mL of enzyme solution diluted in 50 mM citrate buffer (pH 4.8) and 50 mg of Whatman No. 1 filter paper. The mixture was then incubated at 50 °C for 1 h and the released reducing sugars were determined using the DNS method measuring absorbance at 405 nm. FPU was defined as 0.37 divided by the amount of enzyme that produced 2.0 mg glucose equivalents in 1 h from 50 mg of filter paper. All experiments were carried out in triplicates. Saccharification performances of the tested enzymatic extracts were evaluated replacing Accelerase 1500 and/or Accelerase XY by each of the tested extracts. The samples were withdrawn at different time intervals (0, 24, 48 and 72 h), cooled on ice and centrifuged at 16.500xg for 30 min at 4°C. The supernatants were analysed to quantify the amount of sugars

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released by high-performance liquid chromatography (HPLC; Dionex, Sunnyvale, CA, USA) using the protocol described in Ventorino et al. (2016a). 2.6. Determination of protein concentration Protein concentration of crude enzyme preparation was evaluated using Bradford reactive of Biorad (München, Germany) following supplier’s instructions using Bovine Serum Albumin (BSA) as the standard protein. 2.7. Accession numbers The 16S rRNA gene sequences obtained from bacterial strains were deposited in the GenBank nucleotide database under accession numbers from KY646457 to KY646462 (http://www.ncbi.nlm.nih.gov). 2.8 Statistical analyses One-way ANOVA followed by Tukey’s HSD post-hoc for pair-wise comparison of means (at P < 0.05) was used to assess the difference in the enzymatic activities of bacterial strains such as ICMC and IPEC. Statistical analyses were performed using SPSS19.0 statistical software package (SPSS Inc., Cary, NC, USA). 2.9 Small scale bottle fermentation experiments Small scale bottle fermentation experiments were performed at 37°C and 100 rpm, in a rotary shaker incubator (model Minitron, Infors, Bottmingen, Switzerland) in 0.25 L bottles filled with 0.25 L of glucose-free MH medium containing 5 g L-1 yeast extract, 2 g L-1 (NH4)2SO4, 0.2 g L-1 CaCl2∙H2O, 0.2 g L-1 MgCl2∙6H2O, 2 g L-1NaCl, 3 g L-1 K2HPO4, 10 g L-1 MgCO3,1 mg L-1 Na2S∙9H2O as described in Cimini et al. (2016). The enzymatic hydrolysates of A. donax, P. nigra and P. virgatum, diluted to 50% (v/v) in the final working volume, were used as C sources. All bottle experiments were repeated two times, and data are reported as means ± standard deviations.

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2.10 Batch fermentations on 3 L scale Batch experiments were performed on a Biostat CT bioreactor (3 L total volume) with a working volume of 2.4 L (Sartorius Stedim; Melsungen, Germany). Before inoculating the main tank, B. succiniciproducens BPP7 (Ventorino et al., 2017) was grown for 16h on MH medium as described in Cimini et al. (2016). Fermentations were carried out at 37°C on glucose-free MH medium supplemented with enzymatic hydrolysates of P. nigra and P. virgatum diluted to 50 % (v/v) and run for 26 to 43 h. The experiments were repeated two times, and data are reported as means ± standard deviations. 2.11 Analysis of substrates and products of fermentation Broth samples were collected during cultivations to follow substrate consumption and metabolic products formation. The determination of glucose, xylose and acids was evaluated as reported in Cimini et al. (2016). All hydrolysates (substrates) were also analysed for potentially inhibitory compounds such as furfural, hydroxymethylfurfural (HMF), 4-hydroxic benzoic acid and vanillin using the protocol previously reported (Cimini et al., 2016). 3. Results and discussion 3.1. Polyphasic characterization and screening of bacterial strains With the aim to isolate and characterize novel microorganisms producing biocatalysts for enhanced hydrolysis of (hemi)cellulose into monosaccharides, six bacterial strains from Eucalyptus camaldulensis and Populus nigra (Ventorino et al., 2015) were subjected to phenotypic analyses showing a colony morphology typical of actinobacteria -with a spore-bearing aerial mycelium and a black/gray, irregular, slightly raised and lobate colony- and Gram and catalase positive behaviors.

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Sequencing of 16S rRNA gene of strains showed that their nearly full-length gene sequence (> 1,400 bp) presents an identity of 99-100% with Streptomyces (S.) flavogriseus (synonym S. flavovirens) using BLAST software (Table 1). The phylogenetic tree, created including the 16S rRNA sequences of type strains that also comprised species belonging to the ‘Streptomyces griseus clade’ (Figure 1) indicated that the closest relative species was S. flavogriseus, demonstrating that the strains AE63X, AP63X, AE64X, AE52X, AP65X and AP67X can be classified as belonging to this species (Figure 1). These strains were assembled in a cluster with high bootstrap values (> 80%) indicating numerous branching points in the phylogenetic tree (Figure 1). The S. flavogriseus strains were tested on solid media for various lignocellulose degrading enzymatic activities (Table 1). It is worth of noting that all strains showed multiple (endo- and exo-cellulase as well as pectinase and xylanase) enzymatic activities, that lends them interesting as sources of biocatalysts for lignocellulose conversion. Because of this and the requirement of discovering more effective (hemi)cellulolytic enzymes, the six Streptomyces strains were further analysed in liquid culture for their ability to secrete cellulase and xylanase activities, adopting 1% CMC and 1% xylan as inducer substrate, respectively. As shown in Table 1, where the results of the maximum activity and the time of its production are listed, the two strains S. flavogriseus AE63X and S. flavogriseus AE64X revealed to produce the highest levels of cellulase and xylanase activities. Very few works investigated so far cellulase production in Streptomyces flavogriseus (Saini et al. 2015), even if cellulolytic activity has been reported in different actinobacteria, such as Streptomyces, Thermoactinomyces and Cellulomonas. It is worth of noting that the highest endo-1,4-ß-glucanase activity

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levels achieved by S. flavogriseus AE64X and AE63X in the present work (0.87 and 0.451 U mL-1 respectively) were similar to or higher than the activity from S. argenteolus AE58P (0.41 ± 0.05 U mL-1) (Ventorino et al., 2016a), that from Streptomyces sp. G12 (0.1 U/mL ± 0.09) (Amore et al., 2012) and that from S. lividans source (0.409 ± 0.020 U mL-1) (Tomotsune et al., 2014). Furthermore, S. flavogriseus AE63X showed also a higher xylanase activity (0.95 U mL-1 after one day) than that of Streptomyces strain C1-3 (0.6 U mL−1) (Meryandini et al., 2007) and that of Streptomyces strain AMT-3 (0.17 ±0.02U mL−1) (Nascimiento et al., 2002). These results led us to apply the extracellular enzymatic mixtures from S. flavogriseus AE64X and AE63X in pretreated lignocellulose saccharification substituting both the cellulase and xylanase of commercial source. 3.2. Partial characterization of (hemi)cellulose converting enzymes In order to establish the optimal conditions for application of the selected enzymatic mixtures to the biomasses conversion, the optimal temperature and pH and thermoresistance of cellulase and xylanase activities produced by S. flavogriseus AE63X and AE64X were estimated. Cellulase activity secreted by both Streptomyces strains was maximum at 50 °C (Figure 2A) and pH 5 (Figure 2B) and showed a half-life of around 48 h at 40°C and 24 h at 50°C. The optimum temperature of xylanase activity from the investigated Streptomyces strains (tested in the range 40–70 °C) is 65 °C (Figure 2C). The optimal pH of Streptomyces strains for xylanase activity (tested in the range 4-8) is pH 5 (Figure 2D). The xylanase activity of our Streptomyces strains showed a half-life of 5 min at 70 °C, 2 h at 50 °C and 60 °C, and 2 h at 40 °C. The enzymes immediately lose activity at temperatures higher than 70 °C. 3.3. Arundo donax, Populus nigra and Panicum virgatum saccharification

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Saccharification experiments of the pretreated biomasses Arundo donax, Populus nigra and Panicum virgatum were carried out by adopting the enzymatic extracts from the selected strains S. flavogriseus AE64X and AE63X and from the strain S. argenteolus AE58P, whose enzymatic crude extract had been applied to the saccharification of pretreated Arundo donax in Ventorino et al. (2016a) and that was hereby adopted as a reference. A. donax has macromolecular composition in terms of percentage of glucans and xylans of 36% and 20% respectively, whilst the macromolecular composition of Populus nigra in terms of percentage of glucans and xylans was 39% and 13% respectively and Panicum virgatum has 11% of glucans and 9% of xylans. The monosaccharide yields were estimated on samples collected at 72 h and expressed in gL-1. In order to test the saccharification yield of the selected enzymatic extracts, different experiments were performed, replacing Accelerase 1500 (Mix 1) or Accelerase XY (Mix 2) or both the commercial enzymes (Mix 3) with the same amounts of cellulase activities of the enzymatic crude extracts from S. flavogriseus AE63X or S. flavogriseus AE64X or S. argenteolus AE58P. The monosaccharide yields of enzyme saccharification were estimated on samples collected at 72 h and expressed in g L-1 (Table 2). The bioconversions in which the Accelerase 1500 or Accelerase XY were substituted with the new enzymatic mixtures were conducted at the optimal pH and temperature measured for the enzymatic activities cellulase and xylanase discovered in this work and reported for the commercial enzymes- towards Azo-CMC e Azo-xylan, respectively. On the other hand, the bioconversions in which both Accelerase 1500 and Accelerase XY were replaced with the enzymatic extracts from S. flavogriseus AE63X, S. flavogriseus AE64X or S. argenteolus AE58P were performed at 50 °C, that is the

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optimal temperature of both commercial and new cellulases since this activity is very low at 65 °C, whilst the xylanase activity is almost maximum at 50 °C. As shown in Table 2, when the enzymatic extracts from S. flavogriseus AE64X, S. flavogriseus AE63X and S. argenteolus AE58P replaced both the Accellerase 1500 and Accelerase XY (Mix 3), the glucose and xylose yields were higher than those obtained when the enzymatic extract from S. flavogriseus AE64X, S. flavogriseus AE63X and S. argenteolus AE58P replaced the Accellerase 1500 (Mix 1) or the Accelerase XY (Mix 2). This could be due to a better synergy between the cellulase and xylanase activities from the same microorganism in the absence of commercial enzymes. It is worth of noting that the enzymes from S. flavogriseus AE64X led better results for A. donax, P. nigra and P. virgatum saccharification than those obtained with the commercial enzymes tested in our experiments. Moreover, our results were even better than those obtained with other previously adopted enzymes. Giacobbe et al. (2016) and Marcolongo et al. (2014) tested the cellulase rCelStrep from Streptomyces sp. G12, recombinantly expressed in Escherichia coli and purified, in saccharification of pretreated A. donax adding the purified cellulase rCelStrep to the commercial mixture Cellic®Ctec3/Htec3 of Novozymes or using it in substitution of Accellerase®1500, respectively, and observed a decrease of the glucose and xylose yield. Moreover, our experiments were performed on 10% (w/v) of biomass as described in Scordia et al. (2010) and Samayan and Schall (2010) whilst experiments developed in our previous works (Marcolongo et al., 2014; Giacobbe et al., 2016; Ventorino et al., 2016a) were carried out on 5% (w/v) of biomass, revealing a better sugar yield with more biomass. Furthermore, it is worth of noting that the enzymes from S. flavogriseus AE64X and AE63X led better bioconversion results even in comparison to those previously

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obtained with the commercial enzymes. For A. donax conversion with commercial enzymes, the best results obtained until today are glucose yield of 18 g L−1 (Scordia et al., 2010; Ventorino et al., 2016a) and xylose yield of 6.8 g L−1 (Ventorino et al. 2016a) and 7.2 g L−1 (Marcolongo et al., 2014). Samayan and Schall (2010) obtained the glucose and xylose yield of 16 g L−1 and 4.9 g L−1, respectively, for P. nigra conversion in the presence of commercial enzymes and 4.5 g L−1 and 3.4 g L−1, for P. virgatum conversion. Inhibitory compounds, such as HMF, furfural, 4-hydroxibenzoic acid and vanillin, are released during hydrolytic treatments performed at a high temperature (50 °C) over prolonged time periods (3 d) (Saha et al., 2005; Sassner et al., 2006; Aliberti et al., 2017) and were therefore measured in our hydrolysates (Table 3). Different values of inhibitors concentrations were observed among the different biomasses due to their different compositions. Slight differences were also detected for the same substrate resulting from bioconversions with the different enzymes possibly due to different biotransformations correspondingly to the applied enzymatic extracts. The concentration of inhibitory compounds found in all A. donax hydrolysates were lower compared to those previously reported (Cimini et al., 2016). Overall, no significant differences were found between the hydrolysates obtained from commercial and the enzymes investigated in this work. Interestingly higher concentrations of HMF, Furfural and 4-hydroxibenzoic acid were released from P. nigra biomass, compared to the others. 3.4 Production of succinic acid from the Arundo donax, Populus nigra and Panicum virgatum hydrolysates

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In order to test the fermentability for succinic acid production on the hydrolysates of A. donax, P. nigra and P. virgatum obtained by the new enzymatic mixtures, fermentation experiments (in 0.25 L bottles) with the strain Basfia succiniciproducens BPP7 (Ventorino et al., 2017) were performed. The experiments on A. donax were performed by diluting the hydrolysate to 50 % (v/v) in the final growth medium since it was previously observed that a lower dilution (90% v/v) dramatically reduced the productivity (Cimini et al., 2016). In the present work, biomass hydrolyses were conducted by replacing sodium acetate with citrate in order to reduce the initial concentration of acetate in the medium. It is worth of noting that B. succiniciproducens BPP7 achieved similar succinic acid yields and productivity on the A. donax (50% v/v) hydrolysed with the commercial enzymatic mix and on those obtained with enzymatic mixtures from S. flavogriseus AE63X and AE64X (Table 4). In the search for convenient lignocellulosic biomasses for the production of added value compounds, the biomasses of P. nigra and P. virgatum were also used as main carbon sources for small scale fermentation experiments. For the first time to the best of our knowledge, B. succiniciproducens was proven to be able to grow and produce succinic acid on this kind of hydrolysates, that is relevant due to the need to expand the feedstock availability for the developed process. A high yield of succinic acid on consumed sugars was obtained on P. nigra hydrolysed with the novel biocatalysts, although lower compared to that obtained in the presence of commercial enzymes. The latter was quite high considering the maximal theoretical yield of succinic acid on pure glucose and xylose, probably due to the additional carbon present in the complex nutrients together with the hydrolysate in the MH medium.

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Succinic acid productivity was maximal during the first 24h of growth with a maximum value of 0.23±0.01 g/L∙h obtained with the S. flavogriseus AE64X hydrolysate that was very similar to that observed on the control lot as shown in Table 4. B. succiniciproducens BPP7 growing on P. virgatum reached a maximal productivity ranging from 0.123±0.02 g/L∙h to 0.163±0.02 g/L∙h (control hydrolysates 0.15 g/L∙h) during the first 24h of cultivation. High succinic acid yields on P. virgatum treated with the newly developed mixtures of hydrolytic enzymes were also demonstrated, as reported in table 4, showing a slight improvement compared to results obtained on hydrolysates treated with commercial enzymes (0.91 g/g),. Such a high yield of succinic acid was recently described by Dessie et al. (2017) growing Actinobacillus succinogenes on fruit and vegetable wastes. 3.5 Production of succinic acid on 3L bioreactors Succinic acid production on hydrolysates of P. nigra and P. virgatum was scaled up to a 3L bioreactor to perform batch fermentation experiments with pH adjustment and constant sparging of CO2. Comparing succinic acid titers, productivity and yield on consumed sugars obtained in the small scale fermentations with the different enzymatic hydrolysates, the best performing ones, namely those from S. argenteolus AE58P for P. virgatum and from S. flavogriseus AE64X for P. nigra, were selected for the proof of principle step. Overall, improved sugar consumption and succinic acid production rates were obtained in environmentally controlled conditions. In particular, the complete conversion of glucose (18-20 g L−1) and xylose (2-3 g L−1) from hydrolysed P. nigra into succinic acid was achieved within about 30h, with a final concentration of succinic acid ranging from 15 to 18 g L−1, and an average yield on consumed sugars of about 0.75 g/g (figure 3). The batch processes conducted on P. virgatum hydrolysates lasted in

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26h (figure 4) and due to the lower amount of available sugars in the fermentation media (glucose 6-7 g L−1; xylose 1.7-2.4 g L−1), the final titer of succinic acid in the broth was on average of about 7.4 g L−1, resulting 50% lower compared to that achieved on P. nigra based media (figure 3). However, the Ysucc/gluc+xyl (0.89g/g) and the volumetric productivity observed during the exponential phase (0.77g/L∙h) were 20 and 17% higher, respectively, compared to those attained by using P. nigra as starting feedstock. Several renewable resources have been investigated for succinic acid production up to date to reduce process costs and develop sustainable processes, and, those that offer an alternative to food-based feedstocks are of particular interest. Actinobacillus succinogenes synthesised between 15 and 50 g L−1 of succinic acid in batch conditions growing on corn waste materials with a productivity of 0.6-1 g/Lh and a yield ranging between 0.7 and 1.2 g/g on total sugars (Li et al., 2010; Liu et al., 2008; Chen et al., 2010). The same strain produced about 19 g L−1 of target product in 24h 1L fed-batch fermentations on carob pods, with a yield on total sugars of 0.94 g/g (Carvalho et al., 2016). Growth of B. succiniciproducens CCUG57335 was also previously described on corn stover and although 30 g L−1 of product were released in the medium at the end of the batch process, a lower yield of about 0.69 g/g on consumed sugar was achieved (Salvachau et al., 2016). A recent research described the use of immobilized cell cultures for the production of succinic acid from spent sulphite liquor (Alexandri et al., 2017). The authors developed an efficient fed-batch process allowing B. succiniciproducens to produce above 40 g L−1 of succinic acid in 80 h of growth. Yield and productivity, however, were slightly lower compared to those described in the present batch process. Also immobilized A. succinogenes cells were used for repeated

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fed-batch processes on dextran fermentation wastewaters resulting in valuable data, namely an average succinic acid concentration, yield, and productivity of about 56.5 g L−1, 0.82 g/g and 1.28 g/Lh respectively (Chen et al ., 2010). In the present study, B. succiniciproducens BPP7 grown in simple batch experiments on hydrolysates of P. virgatum and P. nigra overall demonstrated competitive yields and productivities in comparison to the above mentioned processes, using waste materials, for the development of sustainable succinic acid production routes. Moreover, improved or at least comparable results to those obtained by growing the strain on A. donax in the same conditions, were achieved. This indicates potential lying both in the selected raw materials and in the developed biocatalysts.

4. Conclusions Two new isolates -Streptomyces flavogriseus AE64X and AE63X- were shown able to produce extracellular enzymatic mixtures whose application in saccharification without any purification step provided yields higher (of 86% and 82%, respectively, -averaged on pretreated Arundo donax, Populus nigra and Panicum virgatum) than those of commercial enzymes (81%). Moreover, these mixes hydrolysed both cellulose and xylan substituting both the commercial cellulase and xylanase. The produced sugars were fermentable by Basfia succiniciproducens BPP7 into succinic acid with competitive yields and productivities. These results underlined the biotechnological importance of the new Streptomyces strains as biocatalyst-producing bacteria for production of biochemicals from lignocellulose.

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“E-supplementary data of this work can be found in online version of the paper”

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Acknowledgements This work was supported by the Ministero dell’Università e della Ricerca Scientifica[PON03PE_00107_1/1 “Development of green technologies for production of BIOchemicals and their use in preparation and industrial application of POLImeric materials from agricultural biomasses cultivated in a sustainable way in Campania region - BioPoliS”] funded in frame of the Operative National Programme Research and Competitiveness 2007–2013 D. D. Prot. n. 713/Ric. del 29.10.2010. VF conceived the work, wrote the manuscript for the part of screening of the strains in liquid culture, enzymatic saccharifications and the related analytical determinations and edited all the manuscript; AP performed the experiments of screening of the strains in liquid culture, enzymatic saccharifications and the related analytical determinations; VV performed the experiments of identification and biotechnological characterization of the strains and contributed to write the manuscript for this part; DC and MI performed bottle and fermentation experiments and the related analytical determinations; OP contributed to write the manuscript for the part of identification and biotechnological characterization of the strains and provided the strains; CS and DC contributed to write the manuscript for the part of bottle and fermentation experiments and related analytical determinations.

20

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31. Samayan, I.P., Schall, C.A., 2010. Saccharification ionic liquid pretreated biomass with commercial enzyme mixtures. Bioresour Technol. 101, 3561-3566. 32. Sassner, P., Galbe, M., Zacchi, G., 2006. Bioethanol production based on simultaneous saccharification and fermentation of steam-pretreated Salix at high dry-matter content. Enzyme Microb. Technol. 39, 756–762. 33. Scordia, D., Cosentino, S.L., Jeffries, T.W., 2010. Second generation bioethanol production from Saccharum spontaneum L. ssp. aegyptiacum (Willd) Hack. Bioresour Technol. 101, 5358–5365. 34. Singhania, R.R., Saini, R., Adsul, M., Saini, J.K., Mathur, A., Tuli, D., 2015. An integrative process for bio-ethanol production employing SSF produced cellulase without extraction. Biochem. Eng J. 102, 45–48. 35. Tomotsune, K., Kasuga, K., Tsuchida, M., Shimura, Y., Kobayashi, M., Agematsu, H., Ikeda, H., Ishikawa, J., Kojima, I., 2014. Cloning and sequence analysis of the cellulase genes isolated from two cellulolytic Streptomycetes and their heterologous expression in Streptomyces lividans. Int. J. Soc. Mater. Eng. Resour. 20, 213–218. 36. Ventorino, V., Aliberti, A., Faraco, V., Robertiello, A., Giacobbe, S., Ercolini, D., Amore, A., Fagnano, M., Pepe O., 2015. Exploring the microbiota dynamics related to vegetable biomasses degradation and study of lignocellulose-degrading bacteria for industrial biotechnological application. Sci. Rep. 5, 8161. 37. Ventorino, V., Ionata, E., Birolo, E., Montella, S., Marcolongo, L., De Chiaro, A., Espresso, F., Faraco, V., Pepe, O., 2016a. Lignocellulose-adapted endo-cellulase producing Streptomyces strains for bioconversion of cellulose-based materials. Front. Microbiol. 7, 2061.

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Figure Captions Figure 1. Neighbor-Joining tree based on the comparison of 16S rRNA gene sequences of bacterial strains (AE63X, AP63X, AE64X, AE52X, AP65X and AP67X) and 17 type strains of ‘Streptomyces griseus clade’ sequences from RDP. Bootstrap values (expressed as percentages of 1000 replications) are given at the nodes. The sequence accession numbers used for the phylogenetic analysis are shown in parentheses following the species name. The scale bar estimates the number of substitutions per site. Figure 2. Effect of different temperature (A) and pH (B) on endo-cellulase activity produced by the selected strains S. flavogriseus AE64X and AE63X and of different temperature (C) and pH (D) on xylanase activity produced by these strains. Bars indicate ± SD of three replicates of three independent experiments. Fig 3. Growth of B. succiniciproducens BPP7 in P. nigra hydrolysed with S. flavogriseus AE64X in batch conditions. Curves indicate glucose and xylose consumption and succinic and acetic acid production in 3 L bioreactor experiments. Experiments were performed on 2 different hydrolysate lots (A and B). Fig 4. Growth of B. succiniciproducens BPP7 in P. virgatum hydrolysed with S. argenteolus AE58P in batch conditions. Curves indicate glucose and xylose consumption and succinic and acetic acid production in 3 L bioreactor experiments. Experiments were performed on 2 different hydrolysate lots (A and B).

27

Table 1. Strain

Identification (% identity)

Accession number

*

C

§

A

†

CE

*

P

†

X

Cellulase activity (U/mL)

Xylanase Time activity (days) (U/mL)

Time (days)

Streptomyces 37 ± flavogriseus KY646457 + 10 ± 1.0B ++ 0.87±0.19 7 0.6±0.09 3 1.0A (100%) Streptomyces 33 ± 0.389±0.11 9 AE52X flavogriseus KY646460 + 9 ± 0.3B + 0.1±0.31 4 0.5B (100%) Streptomyces 21 ± 12 ± 0.313±0.23 10 AP67X flavogriseus KY646458 + + ++ 0.35±0.17 2 0.5C 1.0A (99%) Streptomyces 21 ± 12 ± AE63X flavogriseus KY646459 + ++ 0.451±0.09 4 0.95±0.13 1 C 1.0 1.0A (99%) Streptomyces 20 ± 12 ± 0.398±0.12 9 AP65X flavogriseus KY646461 + + + 0.12±0.10 2 0.7C 0.5A (100%) Streptomyces 20 ± 13 ± 0.313±0.17 9 AP63X flavogriseus KY646462 + + ++ 0.4±0.24 2 0.9C 1.0A (100%) Identification, enzymatic activities and maximum value of cellulase and xylanase activity of six Actinobacteria strains isolated from lignocellulosic biomasses of Eucalyptus camaldulensis and Populus nigra. Enzymatic activities: C, endo-cellulase; A, exo-cellulase; CE, cellobiase; X, xylanase; P, pectinase; *ICMC o IPEC index, values represent the means ± SD of three replicates. Different letters after values indicate significant differences (P < 0.05); §growth; † − negative; + low intensity; ++ middle intensity. AE64X

28

Tables 2. Glucose yield

Commercial enzyme

Arundo Donax biomass Mix 1 Mix 2 25±0.5 25±0.78

Mix 3 27±0.3

Populus nigra biomass Mix 1 Mix 2 23±0.09 25±0.58

Mix 3 25±0.15

Panicum virgatum biomass Mix 1 Mix 2 Mix 3 4±0.10 3.7±0.09 4.9±0.18

S. argenteolus AE58P

13±0.28

25±0.45

25.6±0.32

19±0.92 26±0.18

27±0.53

3.8±0.58

3.6±0.22

4.7±0.55

S. flavogriseus AE64X

27.3±0.23

18.5±0.05

29±0.25

18±0.7

30±0.31

4.1±0.13

4.1±0.18

5.4±0.04

S. flavogriseus AE63X Xylose yield

26±0.08

22±0.09

28.5±0.1

23±0.65 18±0.23

25.2±0.56 3.9±0.17

3.8±0.62

5.2±0.31

26.5±0.21

Commercial enzyme

Arundo Donax biomass Mix 1 Mix 2 Mix 3 6±0.34 11±0.087 15±0.37

Populus nigra biomass Mix 1 Mix 2 6±0.16 11±0.52

S. argenteolus AE58P

2.5±0.56 5.8±0.73

14.2±0.68

3.8±0.6

2.8±0.63 8.1±0.24

1.84±0.34

1.1±0.77

4.7±0.17

S. flavogriseus AE64X

3.1±0.83 4.97±1.02

17.6±0.42

4.9±0.31

3.1±0.31 17.6±0.14

2.1±0.35

1.8±0.75

6.3±0.35

S. flavogriseus AE63X

2.1±0.29 3.5±0.48

16±0.29

3.1±0.15

2.5±0.87 16±0.3

3.2±0.22

3.1±0.089

6.6±0.45

Mix 3 15±0.20

Panicum virgatum biomass Mix 1 Mix 2 Mix 3 1.58±0.32 1.2±0.087 6±0.52

Glucose and xylose yields (g L-1) during enzymatic hydrolysis of pretreated Arundo donax, Populus nigra and Panicum virgatum biomasses by using commercial enzymes and cellulase and xylanase from the two selected strains S. flavogriseus AE64X and S. flavogriseus AE63X and S. argenteolus AE58P. The values represent the means ± SD of three replicates. Composition of mix enzymes used: Mix 1, Accellerase 1500 or cellulase from or S. flavogriseus AE64X or S. flavogriseus AE63X or S. argenteolus AE58P (5.4 U/g of pretreated biomass, 0.6 U Azo-CMC, 1.8 FPU), Accellerase BG (145 U/g), Accellerase XY (4000 U/g); Mix 2, Accellerase 1500 (5.4 U/g), Accellerase BG (145 U/g), Accellerase XY or xylanase from or S. flavogriseus AE64X or S. flavogriseus AE63X or S. argenteolus AE58P (4000 U/g); Mix 3, Accellerase 1500 or cellulase from or S. flavogriseus AE64X or S. flavogriseus AE63X or S. argenteolus AE58P (5.4 U/g, 0.6 U Azo-CMC, 1.8 FPU), Accellerase BG (145 U/g), Accellerase XY or xylanase from or S. flavogriseus AE64X or S. flavogriseus AE63X or S. argenteolus AE58P (4000 U/g).

29

Table 3.

A. donax

P. nigra

P. virgatum

HMF

Furfural

4-hydroxibenzoic acid

Vanillin

Acetic acid

(mg/L)

(mg/L)

(mg/L)

(mg/L)

(g/L)

Commercial enzymes

2.3±0.3

1.00±0.3

3.7±0.7

1.2±0.3

1.9±0.1

S. argenteolus AE58P

1.7±0.1

1.62±0.4

3.5±1.0

1.5±0.3

1.9±0.2

S. flavogriseus AE64X

4.0±0.2

1.0±0.3

2.4±0.8

3.9±1.0

1.8±0.2

S. flavogriseus AE63X

3.4±0.8

0.5±0.0

3.6±0.8

4.4±1.4

1.8±0.3

Commercial enzymes

16

1.2

66.6

6.6

0.8

S. argenteolus AE58P

15.1±3.4

1.4±0.1

62.7±22.2

7.0±2.1

1.2±0.2

S. flavogriseus AE64X

17.7±0.4

1.2±0.0

73.7±2.6

8.2±0.6

1.4±0.1

S. flavogriseus AE63X

14.7±2.6

1.1±0.3

60.0±24.1

6.9±2.5

1.2±0.3

Commercial enzymes

0.9

0.4

0.5

1.50

0.7

S. argenteolus AE58P

0.6±0.1

0.2±0.0

1.2±0.4

1.2±0.2

0.4±0.0

S. flavogriseus AE64X

1.0±0.1

0.6±0.1

0.6±0.0

1.5±0.1

0.6±0.0

S. flavogriseus AE63X

0

0.5±0.1

40.0±10.0

2.6±0.6

0.4±0,1

Determination of inhibitory compounds present in A. donax, P. nigra and P. virgatum enzymatic hydrolysates used to perform bottle and fermentation experiments on the 3L bioreactor.

30

Table 4. YCsucc/C(gluc+xyl) Enzyme mix

Succinic acid

rsucc24h

rsucc48h

rsucc64h/72h*

24h

(%)

(g L-1)

(g/L∙h)

(g/L∙h)

(g/L∙h)

(g/g)

Commercial

5.68±0.40 0.190±0.019

0.114±0.002

0.087±0.006

0.830±0.037

5.60±0.29

0.189±0.008

0.118±0.004

0.085±0.002

0.906±0.051

6.01±0.16

0.191±0.030

0.115±0.007

0.087±0.006

0.856±0.067

6.03±0.21

0.199±0.016

0.118±0.015

0.093±0.013

0.875±0.091

6.70±0.40

0.22±0.008

0.12±0.007

0.09±0.004

1.53±0.059

6.35±0.65

0.231±0.009

0.125±0.013

0.087±0.001

1.07±0.066

6.43±0.23

0.231±0.006

0.134±0.0048 0.085±0.0045 0.98±0.015

6.10±0.28

0.209±0.01

0.120±0.0046 0.082±0.007

1.22±0.049

3.62±0.28

0.151±0.020

0.075±0.008

-

0.91±0.070

4.31±0.00

0.123±0.022

0.063±0.003

-

1.33±0.087

4.03±0.30

0.163±0.020

0.088±0.012

-

1.15±0.097

4.03±0.63

0.148±0.016

0.084±0.013

-

1.11±0.092

enzymes S. argenteolus AE58P

A. donax

S. flavogriseus AE64X S. flavogriseus AE63X Commercial enzymes S. argenteolus AE58P S. flavogriseus P. nigra

AE64X S. flavogriseus AE63X Commercial enzymes S. argenteolus AE58P P. virgatum

S. flavogriseus AE64X S. flavogriseus AE63X

Performance of B. succiniciproducens BPP7 on A. donax, P.nigra and P.virgatum hydrolysed with different biocatalysts. Y indicates the g of succinic acid produced on the total g of glucose and xylose consumed . r24h, r48h and r64h indicate the volumetric production rates of succinic acid after 24, 48 h and 64 h of growth, respectively. *Experiments performed on A.donax and P.nigra lasted 64 and 72h, respectively.

31

Figure 1

32

Figure 2 A

C

B

D

33

Figure 3A

Figure 3B

34

Figure 4A

Figure 4B

35

Highlights    

Six Streptomyces with lignocellulose conversion activities were identified Cellulases and xylanases of two strains were tested in lignocellulose hydrolysis These enzymes led to better saccharification yield than commercial enzymes Fermentability of the enzymatic hydrolysates for succinate production was proven