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Saccharification of newspaper waste after ammonia fiber expansion or extractive ammonia

Saccharification of newspaper waste after ammonia fiber expansion or extractive ammonia The lignocellulosic fractions of municipal solid waste (MSW ) can be used as renewable resources due to the wide- spread availability, predictable and low pricing and suitability for most conversion technologies. In particular, after the typical paper recycling loop, the newspaper waste (NW ) could be further valorized as feedstock in biorefinering industry since it still contains up to 70 % polysaccharides. In this study, two different physicochemical methods— ammonia fiber expansion (AFEX) and extractive ammonia (EA) were tested for the pretraetment of NW. Further - more, based on the previously demonstrated ability of the recombinant enzymes endocellulase rCelStrep, α-l - arabinofuranosidase rPoAbf and its evolved variant rPoAbf F435Y/Y446F to improve the saccharification of different lignocellulosic pretreated biomasses (such as corn stover and Arundo donax), in this study these enzymes were tested for the hydrolysis of pretreated NW, with the aim of valorizing the lignocellulosic fractions of the MSW. In particular, a mixture of purified enzymes containing cellulases, xylanases and accessory hemicellulases, was chosen as reference mix and rCelStrep and rPoAbf or its variant were replaced to EGI and Larb. The results showed that these enzymatic mixes are not suitable for the hydrolysis of NW after AFEX or EA pretreatment. On the other hand, when the enzymes rCelStrep, rPoAbf and rPoAbf F435Y/Y446F were tested for their effect in hydrolysis of pretreated NW by addition to a commercial enzyme mixture, it was shown that the total polysaccharides conversion yield reached 37.32 % for AFEX pretreated NW by adding rPoAbf to the mix whilst the maximum sugars conversion yield for EA pretreated NW was achieved 40.80 % by adding rCelStrep. The maximum glucan conversion yield obtained (45.61 % for EA pretreated NW by adding rCelStrep to the commercial mix) is higher than or comparable to those reported in recent manuscripts adopting hydrolysis conditions similar to those used in this study. Keywords: AFEX pretreatment, Arabinofuranosidase, Biorefining, Cellulase, EA pretreatment, Hemicellulase, Municipal solid waste, Newspaper waste water by leachate, as well as air pollution by their burn- Introduction ing. Monitoring of pollution from different waste man - The world municipal solid waste (MSW) volume is agement options is crucial and environmental concerns expected to reach 2.2 billion tonnes by 2025, due to the are increasing the focus on the reuse and the recycling increase of urban residents to 4.3 billion. Improperly managed urban solid waste is one of the main causes of MSW and on processes for adding value to wastes. In of environmental pollution and a serious health haz particular, the lignocellulosic fractions of MSW can be a source of a wide range of high added value products by ard, due to contamination of groundwater and surface biotechnological “tailor made” processes (Liguori et  al. 2013) for the biorefineries development (Menon and Rao *Correspondence: vfaraco@unina.it 2012; Fava et  al. 2015; Esposito and Antonietti 2015). Department of Chemical Sciences, University of Naples ‘‘Federico II’’, The future of biorefining industry depends mostly on the Complesso Universitario Monte S. Angelo, Via Cintia, 4, 80126 Naples, Italy availability of cheap, sustainable and abundant biomasses Full list of author information is available at the end of the article © 2016 Montella et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Montella et al. AMB Expr (2016) 6:18 Page 2 of 10 as feedstock. The lignocellulosic fractions from MSW are We have previously shown that tailor-made enzy- more suitable to achieve this objective than dedicated matic cocktails including the endocellulase rCelStrep agricultural crops (avoiding the displacement of food from Streptomyces sp. G12 recombinantly expressed crops and minimizing the conflict food versus fuel) or in Escherichia coli (Amore et  al. 2012b), the α-l- forest resources (avoiding the issues concerning a mas- arabinofuranosidase rPoAbf from the fungus Pleu- sive deforestation). Moreover the change of land use rotus ostreatus recombinantly expressed in Pichia and the removal of forest trees in large areas can have pastoris (Amore et  al. 2012a) and/or its evolved variant a negative impact on the ecosystem. The lignocellulosic rPoAbf F435Y/Y446F (Giacobbe et  al. 2014) are suitable fractions from MSW show further advantages such as to improve the hydrolysis yields of AFEX pretreated corn widespread availability, predictable and low pricing, and stover and Arundo donax (Giacobbe et  al. 2015). Based suitability for most conversion technologies. on these results, these enzymes were chosen in this study Among the lignocellulosic fractions of MSW, paper to investigate their ability to improve the saccharification waste represents approximately 30 % of MSW and it is the of AFEX- and EA-treated NW, with the aim of valorizing second most abundant lignocellulose fraction after humid this lignocellulosic MSW fraction. fraction (U.S. Environmental Protection Agency—EPA 2015). The major method of paper waste management is Materials and methods recycling: according to EPA, the paper waste can typically Feedstock be recycled from 5 to 7 times, before papermaking fibers NW was used as feedstock for the saccharification become too short and weak to hold together; each recy- experiments. The NW was collected in the recycle sta - cling requires de-inking with chemicals processing and tion of the Office for International Student and Scholar by adding virgin wood fibers. The newspaper waste (NW) (OISS) at Michigan State University (MSU), East Lan- alone constitutes up to 14  % of MSW (Subhedar et  al. sing, Michigan and it was mainly composed of free 2015), and it can be recycled fewer times that office paper weekly newspaper distributed in Lansing area. After due to the fact that it is usually made by shorter fibers. shredding into pieces of 1  cm in wide and 20  cm in After the typical paper recycling loop, the NW can be fur- length, the NW was milled with a 2 mm diameter sieve ther valorized as feedstock in biorefinering industry since and stored under dry conditions at room temperature it still contains up to 70  % polysaccharides which can be until use. hydrolysed to fermentable sugars (Wang et al. 2012a). For an effective saccharification, the NW, as all the lig - Compositional analysis nocellulose biomasses, requires a physical, chemical or The composition analyses of NW were performed by acid enzymatic pretreatment to break down the recalcitrant hydrolysis according to the laboratory analytical pro- lignin and increase the polysaccharides accessibility for cedures (LAPs) developed by the National Renewable the following hydrolysis. The latter one can be performed Energy Laboratory (NREL) (Sluiter et  al. 2010; Temple- by enzymatic method, that is more eco-friendly than ton et al. 2010): chemical conversion, but it is not yet economically com- petitive, mainly due the high cost of the needed enzymes. • “Preparation of samples for compositional analysis” Development of enzymes with improved performances (Hames et al. 2008), by enhancing their stability and specific activity is • “Determination of structural carbohydrates and therefore pursued. Unlike dedicated energy crops and lignin in biomass” (Sluiter et al. 2008c), agro-industrial waste, the NW saccharification yield is • “Determination of total solids in biomass and total generally low probably due to high lignin content, dense dissolved solids in liquid process samples” (Sluiter structure and the additional physical barrier constituted et al. 2008a), by toner’s ink and inorganic coating linked to the lignin • “Determination of ash in biomass” (Sluiter et  al. (Chu and Feng 2013; Kim et al. 2006; Kuhad et al. 2010). 2008b). In order to overcome this bottleneck, the evaluation of the most efficient pretreatment method and of a suitable A moisture analyser was used to evaluate the mois- tailor-made enzymatic mixture are the crucial steps. ture content. The acid insoluble lignin (Klason lignin) This study is aimed at evaluating the feasibility of was detected by weighting the dried residue after total NW as feedstock for the fermentable sugars production removal of the sugars. Monomeric sugars were quanti- by testing two different physicochemical pretreatment fied using a Biorad Aminex HPX-87H high-performance methods, ammonia fiber expansion (AFEX) and extrac - liquid chromatography (HPLC) column using 5 mM sul- tive ammonia fiber expansion (EA). phuric acid as mobile phase. Montella et al. AMB Expr (2016) 6:18 Page 3 of 10 AFEX pretreatment Cellic HTec3 from Novozymes (Denmark) were used NW was subjected to AFEX pretreatment by varying in this study. Moreover, mixes of the following purified reaction temperature (65–75  °C), moisture (10.7–25  % enzymes were tested. The four core fungal cellulases were on dry weight basis), ammonia to biomass ratio (2.0:1 cellobiohydrolase I (CBH I; glycoside hydrolase—GH— and 2.8:1) at fixed residence time of 15  min. AFEX was family 7A), cellobiohydrolase II (CBH II; GH family 6A), done in a high pressure stainless steel Parr reactor. Bio- endoglucanase I (EG I; GH family 7B) and β-glucosidase mass was sprayed with water to reach appropriate mois- (βG; GH family 3). CBH I, CBH II and EG I were purified ture content after taking into consideration the moisture from Spezyme CP (Danisco US Inc., Genencor Division, content of original biomass. Then the reactor was closed Rochester, NY) using four different chromatography meth - and vacuum applied to remove residual air in the reac- ods: size exclusion, anion and cation exchange, hydropho- tor. The required amount of liquid ammonia was loaded bic interaction and affinity (Gao et  al. 2010a , b); βG was using a ammonia delivery pump into a reactor pre-heated purified from Novo 188 (Novozyme, Davis, CA, USA), by by external heating mantle. The biomass was mixed dur - using anion and cation exchange chromatography (Gao ing AFEX pretreatment process for 15  min. As the tem- et  al. 2010a, b). The bacterial hemicellulases added to the perature of reactor was increased, the pressure in the core cellulases were xylanases (LX3, GH family 10; LX4, vessel increased (between 200 and 400  psi) relying on GH family 11), β-xylosidase (LβX; GH family 52) and the ammonia to biomass loading. After the completion α-arabinofuranosidase (LArb, GH family 51). LX3 and of pretreatment process, the pressure was released from LX4 from Clostridium thermocellum, LβX from Geoba- the vessel and ammonia was vented in the hood. Subse- cillus stearothermophilus and LArb from Geobacillus sp. quently, the biomass was transferred to a tray and dried G11MC16 were recombinantly expressed in E. coli BL21 in the hood overnight to remove residual ammonia. The (DE3) and purified using HIS-select nickel affinity chro - dry pretreated biomass was stored in a sealed polythene matography (Gao et al. 2010a, b, 2011). The endoglucanase bag at 4 °C until use. rCelStrep (GH family 12) was from Streptomyces sp. G12 and recombinantly expressed in E. coli (Amore et  al. Extractive ammonia (EA) pretreatment 2012b); the α-l -arabinofuranosidases rPoAbf wild type NW was subjected to EA pretreatment (reaction tem- (GH family 51) from P. ostreatus and its mutant rPoAbf perature: 120  °C; ammonia to biomass ratio: 3:1  kg/kg F435Y/Y446F were recombinantly expressed in P. pasto- dry biomass; moisture content: 10 % on dry weight basis; ris (Amore et  al. 2012a). These enzymes were purified by fixed residence time: 15 min). EA was done in a high pres - ammonium sulphate precipitation followed by hydropho- sure stainless steel Parr reactor. Biomass was first sprayed bic interaction chromatography (Amore et al. 2012a, b). with water to reach appropriate moisture content after taking into consideration the moisture content of origi- Enzymatic hydrolysis nal biomass. The required amount of liquid ammonia was The enzymatic hydrolyses were carried in 5  mL vials. loaded using a ammonia delivery pump into a reactor An amount of pretreated biomass was added as to load pre-heated by external heating mantle. The biomass was 1  % (w/w) glucan in 2  mL total volume with the desired mixed during EA pretreatment process for 15  min. As enzymes. The buffer solution was 50 mM citrate, pH 4.8. the temperature of reactor was increased, the pressure in Microbial and fungal contaminations were prevented by the vessel raised (between 600 and 800 psi) depending on adding sodium azide 0.5 mM. The hydrolysis parameters the ammonia to biomass loading. After the completion of were: 50 °C, 250 rpm, 72 h. Sampling was collected every pretreatment process, extractives (mostly lignin) gener- 24 h to evaluate carbohydrates hydrolysis. ated during the process along with liquid ammonia was Fifteen milligram per gram of glucan of the commercial collected via a sintered frit into another high pressure enzymatic preparation Novozymes Cellic (60  % CTec3 reactor. The collection pressure was released from the and 40  % HTec3) was used for hydrolysis experiment to vessel and ammonia was vented in the hood to recover select the best pretreatment conditions. the extractives. The pretreated biomass was transferred The following enzymes were used for the hydrolysis of to a tray and dried in the hood overnight to remove AFEX and EA pretreated NW. The MIX A was prepared residual ammonia present in the biomass. Then dry EA including: CBH I, CBH II and EG I (3.32  mg/g glucan treated biomass was stored in a sealed polythene bag in a each), βG (2 mg/g glucan), LX3 and LX4 (1.66 mg/g glu- refrigerator until further use. can each), LβX and LArb (0.6 mg/g glucan each). In the mix B, the EG I was replaced by an equal Commercial and purified enzymes and their sources amount of endoglucanase rCelStrep; in the mix C the The commercially available complex of cellulases and LArb was replaced by an equal amount of the α-l- hemicellulases Cellic CTec3 and the enzyme solution arabinofuranosidase rPoAbf; in the mix D the LArb Montella et al. AMB Expr (2016) 6:18 Page 4 of 10 was replaced by the same amount of the mutant rPoAbf Arabinan conversion (%) F435Y/Y446F. Arabinose concentration in hydrolysate g L Moreover, rCelStrep, rPoAbf or rPoAbf F435Y/ Arabinan concentration in loaded biomass g L Y446F were added alternatively or in combination to the MIX 1 containing 15  mg/g glucan of commercial ® ® 1.136 preparation mix (60  % Cellic CTec3 and 40  % Cellic HTec3 from Novozymes). In particular the following Mannan conversion (%) enzymes were added: in the MIX 2, the endoglucanase rCelStrep (3.32  mg/g glucan); in the MIX 3 the α-l- Mannose concentration in hydrolysate g L arabinofuranosidase rPoAbf (0.6  mg/g glucan); in the Mannan concentration in loaded biomass g L MIX 4 the mutant rPoAbf F435Y/Y446F (0.6  mg/g glu- can); in the MIX 5 both the endoglucanase rCelStrep × 1.11 (3.32  mg/g glucan) and the α-l-arabinofuranosidase rPoAbf (0.6  mg/g glucan); in the MIX 6 both the endo- where 1.11 is the ratio of MW (180.16  g/ glucose (or mannose) glucanase rCelStrep (3.32  mg/g glucan) and the mutant mol) to MW (162.14 g/mol) and 1.11 is the glucan (or mannan) α-l-arabinofuranosidase rPoAbf F435Y/Y446F (0.6 mg/g ratio of MW (150.13 g/mol) to MW xylose (or arabinose) xylan (or glucan). (132.13 g/mol). arabinan) Sugar analysis Results Monomeric sugars concentration were determined by Composition analysis and pretreatment of newspaper high performance liquid chromatography (HPLC). All waste experiments were performed in triplicate. Composition analysis of untreated NW was carried out About 200  μl hydrolysate were collected in a centri- and the results reported in Table 1 revealed a glucan con- fuge tube, heated to 100 °C for 10 min (to inactivate the tent of ~44  %, a hemicellulose content of ~15  % and a enzymes), then spun down at 8000  rpm for 10  min and Klason lignin content of ~25 %. the supernatant was stored in a HPLC vial at −20  °C The macromolecular composition of AFEX pre - until further use. Monomeric sugars concentration in treated NW is assumed to have the same composition the hydrolysate was determined by HPLC using Biorad of untreated NW (Table  1), due to the fact that, as pre- Aminex HPX-87P. Shimadzu HPLC Prominence system viously reported, the AFEX pretreatement preserves the (Columbia, MD, USA) with a refractive index detec- macrostructure of the lignocellulosic biomasses, reducing tor (RID) were used for analyzing the sugars. Water was the degree of polymerization of (hemi)cellulose minimiz- used as the mobile phase at a fixed flow rate of 0.6  ml/ ing the degradation of the original carbohydrates (Holt- min, with isocratic elution. The column temperature was zapple et  al. 1991; Kumar et  al. 2009). In order to select maintained at 60 °C and the HPLC sample injection vol- the best AFEX conditions to be used during the further ume was 20 μl. Standard curves were generated using dif- saccharification experiments, three different sets of con - ferent concentrations of mixed sugars. A guard column ditions were tested on NW (Table  2), varying ammonia with similar packing was used throughout the chroma- loading, temperature and moisture content. These con - tography experiments. ditions were chosen based on the previous published The sugars conversion is calculated according to the screening of AFEX pretreatments on NW (Holtzapple following equations: et al. 1991, 1992). The AFEX pretreated NW was subjected to enzymatic Glucan conversion (%) hydrolysis using the mix composed of 60 % Cellic CTec3 and 40  % Cellic HTec3 from Novozymes. As shown Glucose concentration in hydrolysate g L in Fig.  1, the maximum glucose and xylose yields were Glucan concentration in loaded biomass g L achieved after 72 h of hydrolysis with the AFEX pretreat- ment condition N1 (2.8  kg ammonia/kg dry biomass, 1.11 65  °C, and 10.7  % of moisture content) and N2 (2.8  kg ammonia/kg dry biomass, 75  °C, and 25  % of moisture Xylan conversion (%) content) and the maximum total sugars conversion was Xylose concentration in hydrolysate g L respectively 29.58  ±  0.35 and 29.02  ±  0.35  % (Table  2). The condition N1 was selected for the further hydrolysis Xylan concentration in loaded biomass g L experiments due to the milder reaction temperature of pretreatment (10 °C less than condition N2). 1.136 Montella et al. AMB Expr (2016) 6:18 Page 5 of 10 Table 1 Macromolecular composition of newspaper waste that the NW still contains significant percentages of struc - before and after EA pretreatment tural sugars after EA pretreatment (~58  %). In particular, the percentages of glucan content is ~41  %, the percent- Newspaper waste age of mannan content is ~8 % and the percentage of xylan Untreated EA pretreated content is ~5 %; the total lignin content is ~31 % (Table 1). Moisture content 7.00 ± 1.00 9.75 ± 0.07 Hydrolysis of newspaper waste by using the enzymes Ash 7.04 ± 0.14 7.25 ± 0.15 rCelStrep, rPoAbf and its variant in comparison with the Structural carbohydrate enzymes EGI or LArb Glucan 44.21 ± 4.02 41.36 ± 0.01 As shown in several papers, a mix of cellulases and acces- Xylan 5.11 ± 0.19 5.20 ± 0.22 sory hemicellulases is crucial to enhance hydrolysis of Galactan 1.81 ± 0.13 1.84 ± 0.03 pretreated lignocellulosic biomasses (Gao et al. 2010a, b; Arabinan 1.09 ± 0.09 1.73 ± 0.03 Jørgensen et al. 2007). The MIX A, previously optimized Mannan 9.83 ± 0.47 8.16 ± 0.05 for corn stover saccharification after AFEX pretreatment Lignin (Gao et al. 2011), was chosen as reference mix. This mix - Acid insoluble lignin 25.88 ± 2.48 30.03 ± 0.89 ture contains the cellobiohydrolases CBHI and CBHII, Acid soluble lignin 0.96 ± 0.01 1.05 ± 0.19 the endo-glucanase EGI, the beta-glucosidase βG, the Composition closure 95.92 96.63 xylanases LX3 and LX4, the beta-xylosidase LβX and the α-l-arabinofuranosidase LArb. In order to evalu - ate the hydrolysis yield by using the enzymes rCelStrep, As an alternative pretreatment method, the EA was also rPoAbf and rPoAbf F435Y/Y446F, these enzymes were tested on NW. The EA pretreatment converts native cel - replaced to EGI (MIX B) and to LArb (MIX C and MIX lulose I to cellulose III, delignifying the biomass simulta- D), respectively. neously (da Costa et al. 2015). Composition analysis of EA The highest glucan, xylan, mannose and arabinose pretreated NW was carried out and the results showed conversions were reached after 72  h of hydrolysis for Table 2 AFEX conditions tested on newspaper waste AFEX conditions Ammonia loading Reaction Moisture Fixed residence Sugars conversion (kg:kg dry biomass) temperature (°C) content (%) time (min) (after 72 h hydrolysis) (%) (g/L) N1 2.8:1 65 10.7 15 29.58 4.62 N2 2.8:1 75 25 29.02 4.53 N3 2.0:1 75 25 26.14 4.08 Fig. 1 Glucan and xylan conversion during the hydrolysis of newspaper waste after three different AFEX pretreatment conditions: N1 (ammonia loading 2.8:1; 65 °C; 10.7 % moisture content; 15 min), N2 (ammonia loading 2.8:1; 75 °C; 25 % moisture content; 15 min) and N3 (ammonia loading 2.0:1; 65 °C; 10.7 % moisture content; 15 min) Montella et al. AMB Expr (2016) 6:18 Page 6 of 10 all the tested biomasses, pretreatment methods and undetectable for all mixes. In conclusion, there was no enzyme mixes. In Figs.  2 and 3 the conversion data for substantial improvement by replacing rCelStrep, rPoAbf the most abundant polysaccharides (glucan and xylan) or its variant to the corresponding enzymatic activities in are reported. the reference mix. For AFEX pretreated NW, as shown in Fig.  2, the glu- As shown in Fig.  3, for EA pretreated NW, the glucan can conversion reached 7.3  % for MIX A and did not conversion reached 14.3 % for MIX A and decreased for increase significantly by using the other mixes. The xylan other three mixes (up to 13.1  % for MIX B, 10.7  % for conversion was 13.59  % for MIX A and increased up to MIX C and 13.4 % for MIX D). The xylan conversion was 14.44  % only for MIX B (corresponding to the substitu- 19.2  % for MIX A and increased only for MIX B (up to tion of rCelStrep to EGI), decreasing for the other mixes 20.3  %), decreasing for MIX C (16.9  %) and for MIX D (12.2  % for MIX C and 13.1  % for MIX D). The arab - (15.4  %). The arabinose conversion reached 12.5  % for inose conversion reached 7.61  % for MIX A, increased MIX A and decreased to 11.1  % for MIX B, 9.9  % for up to 8.86 % for MIX B and decreased to 4.4 % for MIX MIX C and 8.70  % for MIX D. The mannose conversion C and to 5.3 % for MIX D. The mannose conversion was was less than 1  % for all the mixes. In conclusion, the Fig. 2 Glucan and xylan conversion during the hydrolysis of AFEX pretreated newspaper waste by using MIX A: CBH I, CBH II and EG I (3.32 mg/g glucan each), βG (2 mg/g glucan), LX3 and LX4 (1.66 mg/g glucan each), LβX and LArb (0.6 mg/g glucan each); MIX B (replacing EG I with endoglu- canase rCelStrep); MIX C (replacing LArb with the α-l -arabinofuranosidase rPoAbf ); MIX D (replacing LArb with mutant rPoAbf F435Y/Y446F) Fig. 3 Glucan and xylan conversion during the hydrolysis of EA pretreated newspaper waste by using MIX A: CBH I, CBH II and EG I (3.32 mg/g glucan each), βG (2 mg/g glucan), LX3 and LX4 (1.66 mg/g glucan each), LβX and LArb (0.6 mg/g glucan each); MIX B (replacing EG I with endoglu- canase rCelStrep); MIX C (replacing LArb with the α-l -arabinofuranosidase rPoAbf ); MIX D (replacing LArb with mutant rPoAbf F435Y/Y446F) Montella et al. AMB Expr (2016) 6:18 Page 7 of 10 maximum polysaccharides conversion yield (12.4 %) was was obtained by adding rPoAbf F435Y/Y446F (0.6  mg/g obtained for MIX A. glucan) to MIX 1. As shown in Fig. 5, for the EA pretreated NW, the glu- Evaluation of synergism between commercial enzyme can conversion reached 35.6  % for MIX 1 and increased preparation and rCelstrep and/or rPoAbf and its mutant up to 45.6 % for MIX 2, 40.1 % for MIX 3, 43.6 % for MIX The enzymes rCelStrep, rPoAbf or its evolved variant 4, 42.657  % for MIX 5 and 38.5  % for MIX 6. The xylan rPoAbf F435Y/Y446F were added alternatively or in com- conversion was 44.9  % for MIX 1 and increased up to bination to 15 mg/g glucan of MIX 1 (60 % Cellic CTec3 55.6  % for MIX 2, 51.3  % for MIX 3, 58.1  % for MIX 4, and 40 % Cellic HTec3 from Novozymes) for the hydrol- 53.7  % for MIX 5 and 49.2  % for MIX 6. The arabinose ysis reactions of pretreated NW. The amount of enzyme conversion reached 49.0  % for MIX 1 and increased up loading was chosen based on the data obtained by Gao to 52.1  % for MIX 2, 49.2  % for MIX 3, 53.7  % for MIX et  al. (2011) and due to the promising results previously 4, 51.0 % for MIX 5 and 50.2 % for MIX 6. The mannose achieved for the hydrolysis of AFEX pretreated Corn conversion reached 17.2  % for MIX 1 and decreased for stover and A. donax (Giacobbe et al. 2015). all the other mixes (13.6 % for MIX 2, 12.8 % for MIX 3, The highest glucan, xylan and arabinose conversion 11.4 % for MIX 4, 13.0 % for MIX 5 and 10.0 % for MIX 6. were reached after 72  h of hydrolysis for all tested bio- In conclusion, the maximum polysaccharides conversion masses, pretreatment methods and enzyme mixes. In yield (40.80  %) was obtained for MIX 2, by adding rCel- Figs.  4 and 5 the conversion data for the most abundant Strep (3.32 mg/g glucan) to MIX 1. polysaccharides (glucan and xylan) are reported. As shown in Fig.  4, for AFEX pretreated NW, the glu- Discussion can conversion reached 34.0  % for MIX 1 and increased Composition analysis of untreated NW, assumed equal up to 39.4 % for MIX 2, 41.2 % for MIX 3, 42.7 % for MIX to the macromolecular composition of AFEX pretreated 4, 41.22  % for MIX 5 and 41.0  % for MIX 6. The xylan NW, as aforementioned, was in agreement with the val- conversion was 46.7  % for MIX 1 and increased up to ues previously reported by Wang et  al. (2012b, 2013), 53.3  % for MIX 2, 57.5  % for MIX 3, 58.7  % for MIX 4, Subhedar et  al. (2015), Sangkharak (2011) and Orozco 57.50  % for MIX 5 and 56.7  % for MIX 6. The arabinose et  al. (2013) related to the untreated biomass. Fur- conversion reached 80.7  % for MIX 1 and decreased thermore, composition analysis of EA pretreated NW for all other mixes (67.3 % for MIX 2, 71.6 % for MIX 3, revealed that this technique, similarly to the AFEX pre- 70.8 % for MIX 4, 71. 7 % for MIX 5 and 70.0 % for MIX treatment, does not significantly change the composition. 6). The mannose conversion was undetectable for MIX 1 Furthermore, the cellulose percentage after acid pretreat- and reached 4.5 % for MIX 2, 6.1 % for MIX 3, 4.7 % for ment, reported by Guerfali et al. (2015), and the holocel- mix 4, 4.6  % for MIX 5 and 4.5  % for MIX 6. In conclu- lulose percentage, after alkaline pretreatment, reported sion, the maximum polysaccharides conversion (37.32 %) by Wu et  al. (2014), were comparable to data obtained Fig. 4 Glucan and xylan conversion during the hydrolysis of AFEX pretreated newspaper waste by using MIX 1 (15 mg/g of glucan of commercial enzymatic preparation Novozymes Cellic —60 % CTec3 and 40 % HTec3; MIX 2 (adding 3.32 mg/g glucan of endoglucanase rCelStrep to MIX 1); MIX 3 (adding 0.6 mg/g glucan of the α-l -arabinofuranosidase rPoAbf to MIX 1); MIX 4 (adding 0.6 mg/g glucan of the mutant rPoAbf F435Y/Y446F to MIX 1); MIX 5 (adding 3.32 mg/g glucan of endoglucanase rCelStrep and 0.6 mg/g glucan of the α-l -arabinofuranosidase rPoAbf to MIX 1); MIX 6 (adding 3.32 mg/g glucan of endoglucanase rCelStrep and 0.6 mg/g glucan of the mutant rPoAbf F435Y/Y446F to MIX 1) Montella et al. AMB Expr (2016) 6:18 Page 8 of 10 Fig. 5 Glucan and xylan conversion during the hydrolysis of EA pretreated newspaper waste by using MIX 1 (15 mg/g of glucan of commercial enzymatic preparation Novozymes Cellic —60 % CTec3 and 40 % HTec3; MIX 2 (adding 3.32 mg/g glucan of endoglucanase rCelStrep to MIX 1); MIX 3 (adding 0.6 mg/g glucan of the α-l -arabinofuranosidase rPoAbf to MIX 1); MIX 4 (adding 0.6 mg/g glucan of the mutant rPoAbf F435Y/Y446F to MIX A’); MIX 5 (adding 3.32 mg/g glucan of endoglucanase rCelStrep and 0.6 mg/g glucan of the α-l -arabinofuranosidase rPoAbf to MIX 1); MIX 6 (adding 3.32 mg/g glucan of endoglucanase rCelStrep and 0.6 mg/g glucan of the mutant rPoAbf F435Y/Y446F to MIX 1) after AFEX or EA method; contrariwise, the alpha cel- barrier constituted by toner’s ink and inorganic coating lulose and holocellulose percentage, reported by Sang- that remain linked to the lignin after pretreatment. kharak (2011), after pretreatment by using NaOH are On the other hand, the AFEX and EA pretreatments both higher than those reported in this study, but the had comparable effects on the polysaccharides conversion reaction conditions used for AFEX reaction are milder yield after the hydrolysis of NW by using Cellic CTec3 (65 versus 100  °C and residence time 15  min versus 4  h and HTec3 and the addition of rCelStrep, rPoAbf or its for AFEX and basic pretreatment respectively). evolved variant rPoAbf F435Y/Y446F to the commercial The hydrolysis of pretreated NW obtained by using mix improved the sacharification process. In particular, mixes of purified enzymes showed that the EA method the maximum glucan conversion yields reached for AFEX improve the saccharification in comparison with the pretreated NW and EA pretreated NW were obtained by AFEX, although the hydrolysis yields remained low in an additive effect of the α-l-arabinofuraosidase rPoAbf comparison to the data previously reported in literature. F435Y/Y446F and the endocellulase rCelStrep, respec- The maximum total polysaccharides conversion yield (for tively, in addition of the array of (hemi)cellulase activi- EA-pretreated-NW hydrolyzed by enzymatic MIX A) ties present in the commercial mix. The increase of the was similar to the yields reported by Ali and Khan Mohd glucan conversion yield was respectively of 25 and 28  % (2011) after basic pretreatment and six days of micro- more than the yields obtained by hydrolysis with com- bial hydrolysis by Aspergillus niger and lower than the mercial enzymes without any addition. The best obtained most recent reported results (Wua et  al. 2014; Chu and sugars conversion yield was higher than or compara- Feng 2013; Wang et  al. 2012b; Guerfali et  al. 2015; Sub- ble to those reported in recent manuscripts adopting hedar and Gogate 2014; Kim et al. 2006; Xin et al. 2010). hydrolysis conditions similar to those used in this study. Moreover, the glucan conversion yield was very low (not In particular, the maximum obtained glucan conversion exceeding 7.3 %). This is the major drawback as glucose is yields were comparable to those described by Wang et al. the main carbon source for a wide range of industrial fer- (2012b) after enzymatic hydrolysis using 5  % (w/w) bio- mentation processes by using well-known microorgan- mass and 64  mg/g glucan of commercial enzymes (~3 isms capable of using mainly hexoses (Jang et  al. 2012). times more than those loaded in this study). Moreover, These results showed that the enzymatic mix previously Wu et al. (2014) obtained sugar conversion yields compa- optimized for pretreated corn stover saccharification is rable to this study after basic pretreatment and combin- not suitable for the hydrolysis of NW after AFEX or EA ing acid and enzymatic hydrolysis. Subhedar and Gogate pretreatment. Moreover, the replacement of the enzymes (2014) obtained ~31  % of total sugars yield (lower than rCelStrep, rPoAbf and rPoAbf F435Y/Y446F respectively the results of this study) by using enzymatic hydrolysis to EGI and LArb did not change significantly the sac - and the yield increased only after an ultrasound-assisted charification yield. These data suggested that the purified enzymatic hydrolysis. However, the best obtained results enzymes are not probably able to overcome the physical in this study are lower than those reported by Guerfali Montella et al. AMB Expr (2016) 6:18 Page 9 of 10 Chu KH, Feng X. Enzymatic conversion of newspaper and office paper to et  al. (2015) and Kim et  al. (2006) that add the action of fermentable sugars. Process Saf Environ Prot. 2013;91:123–30. surfactant agents to the enzymatic hydrolysis. da Costa Sousa L, Jina M, Chundawata S, Bokade V, Tangd X, Azarpira A, Lu F, In conclusion, the feasibility of NW pretreatment Avcif U, Humpula J, Uppugundlaa N, Gunawana C, Pattathilf S, Chehg A, Kotharih N, Kumarh R, Ralph J, Hahnf MG, Wymanh CE, Singh S, Simmonsi by both AFEX and EA were demonstrated. The results BA, Dale BE, Balan V. Next-generation ammonia pretreatment enhances showed no substantial differences between the two tested biofuel production from biomass via simultaneous cellulose decrystalliza- methods on hydrolysis yield. However, the AFEX was tion and lignin extraction. Accepted: Energy and Environmental Science; the best pretreatment technique mainly due to the mild Environmental Protection Agency—EPA (2015). http://www3.epa.gov/epa- reaction conditions. Moreover, the best sugars conver- waste/nonhaz/municipal/ Accessed 15 Jan 2016. sion yield obtained by adding the recombinant enzymes Esposito D, Antonietti M. Redefining biorefinery: the search for unconventional building blocks for materials. Chem Soc Rev. 2015;44:5821–35. to the commercial mixture was higher than or compara- Fava F, Totaro G, Diels L, Reis M, Duarte J, Carioca OB, Poggi-Varaldo HM, Fer- ble to those reported in previous studies adopting similar reira BS. Biowaste biorefinery in Europe: opportunities and research and hydrolysis conditions. These promising but not optimal development needs. New Biotechnol. 2015;32(1):100–8. Gao D, Chundawat SP, Krishnan C, Balan V, Dale BE. Mixture optimization of results suggest that the process can be optimized in order six core glycosyl hydrolases for maximizing saccharification of ammonia to further enhance the NW hydrolysis yield. fiber expansion (AFEX) pretreated corn stover. Bioresour Technol. 2010a;101(8):2770–81. Authors’ contributions Gao D, Chundawat SP, Liu T, Hermanson S, Gowda K, Brumm P, Dale BE, Balan V. SM wrote the main manuscript text and prepared figures and tables. LCS Strategy for identification of novel fungal and bacterial glycosyl hydrolase designed and carried out the AFEX and EA pretreatment experiments. VB hybrid mixtures that can efficiently saccharify pretreated lignocellulosic designed the hydrolysis experiments and contributed to analyze the results. biomass. Bioenerg Res. 2010b;3(1):67–81. SM and SG carried out the hydrolysis experiments and analyzed all the results. Gao D, Uppugundla N, Chundawat SP, Yu X, Hermanson S, Gowda K, Brumm CG carried out the HPLC analysis. VF contributed to conceiving the study and P, Mead D, Balan V, Dale BE. Hemicellulases and auxiliary enzymes for participated in its design and coordination and drafted the manuscript. OP improved conversion of lignocellulosic biomass to monosaccharides. contributed to analyze all the results and to draft the manuscript. VF is the cor- Biotechnol Biofuels. 2011;4:5. doi:10.1186/1754-6834-4-5. responding author. All authors read and approved the final manuscript. Giacobbe S, Balan V, Montella S, Fagnano M, Mori M, Faraco V. Assessment of bacterial and fungal (hemi)cellulose-degrading enzymes in sacchari- Author details fication of ammonia fibre expansion-pretreated Arundo donax. Appl Department of Chemical Sciences, University of Naples ‘‘Federico II’’, Microbiol Biotechnol. 2015. doi 10.1007/s00253-015-7066-3. Complesso Universitario Monte S. Angelo, Via Cintia, 4, 80126 Naples, Italy. Giacobbe S, Vincent F, Faraco V. Development of an improved variant of GH51 Department of Chemical Engineering and Materials Science, DOE Great α-l -arabinofuranosidase from Pleurotus ostreatus by directed evolution. Lakes Bioenergy Research Center, Michigan State University, East Lansing, MI New Biotechnol. 2014;31(3):230–6. 48823, USA. Department of Agriculture, University of Naples “Federico II”, Guerfali M, Saidi A, Gargouri A, Belghith H. Enhanced enzymatic hydrolysis of Portici, Naples, Italy. waste paper for ethanol production using separate saccharification and fermentation. Appl Biochem Biotechnol. 2015;175:25–42. Acknowledgements Hames B, Ruiz R, Scarlata C, Sluiter A, Sluiter J, Templeton D. Preparation of sam- This research was supported by a Marie Curie International Research Staff ples for compositional analysis laboratory analytical procedure (LAP). 2008. Exchange Scheme Fellowship within the 7th European Community Frame- Holtzapple MT, Jun J-H, Ashok G, Patibandla SL, Dale BE. The ammo- work Programme: ‘Improvement of technologies and tools, e.g., biosystems nia freeze explosion (AFEX) process. Appl Biochem Biotechnol. and biocatalysts, for waste conversion to develop an assortment of high 1991;28–29(1):59–74. added value eco-friendly and cost-effective bio-products’ BIOASSORT (grant Holtzapple MT, Lundeen JE, Sturgis R, Lewis HE, Dale BE. Pretreatment of number 318931). We gratefully acknowledge support given to VB by the lignoceilulosic municipal solid waste by ammonia fiber explosion (AFEX). office of Biological and Environmental Research in the DOE Office of Science Appl Biochem Botechnol. 1992;34–35(1):5–21. through the Great Lakes Bioenergy Research Centre (GLBRC) (Grant DE-FC02- Jang YS, Kim B, Shin JH, Choi YJ, Choi S, Song CW, Lee J, Park HG, Lee SY. 07ER64494). We also thank Lucigen Enzyme Company for supplying the Bio-based production of C2–C6 platform chemicals. Biotechnol Bioeng. research enzymes and Novozyme for supplying the enzyme solutions Cellic 2012;109(10):2437–59. CTec3 and the Cellic HTec3 for this work. Jørgensen H, Kristensen JB, Felby C. Enzymatic conversion of lignocellulose into fermentable sugars: challenges and opportunities. Biofuels Bioprod Competing interests Biorefin. 2007;1:119–34. The authors declare that they have no competing interests. Kim SB, Kim HJ, Kim CJ. Enhancement of the enzymatic digest- ibility of waste newspaper using Tween. Appl Biochem Biotechnol. Received: 5 December 2015 Accepted: 23 February 2016 2006;129–132:486–95. Kuhad RC, Mehta G, Gupta R, Sharma KK. Fed batch enzymatic saccharification of newspaper cellulosics improves the sugar content in the hydrolysates and eventually the ethanol fermentation by Saccharomyces cerevisiae. Biomass Bioenergy. 2010;34:1189–94. Kumar R, Mago G, Balan V, Wyman CE. Physical and chemical characterizations References of corn stover and poplar solids resulting from leading pretreatment Ali MN, Khan Mohd M. Production of bioethanol fuel from renewable technologies. Bioresour Technol. 2009;100:3948–62. agrobased cellulosic wastes and waste news papers. Int J Eng Sci Tech- Liguori R, Amore A, Faraco V. Waste valorization by biotechnological nol. 2011;3:884. conversion into added value products. Appl Microbiol Biotechnol. Amore A, Amoresano A, Birolo L, Henrissat B, Leo G, Palmese A, Faraco V. A 2013;97(14):6129–47. family GH51 α-l -arabinofuranosidase from Pleurotus ostreatus: identi- Menon V, Rao M. Trends in bioconversion of lignocellulose: biofuels plat- fication recombinant expression and characterization. Appl Microbiol form chemicals and biorefinery concept. Prog Energy Combust Sci. Biotechnol. 2012a;94(4):995–1006. 2012;38(4):522–50. Amore A, Pepe O, Ventorino V, Birolo L, Giangrande C, Faraco V. Cloning and Orozco AM, Al-Muhtaseb AH, Rooney D, Walker GM, Aiouache F, Ahmad M. recombinant expression of a cellulase from the cellulolytic strain Strepto- Fermentable sugars recovery from lignocellulosic waste-newspaper by myces sp.12 isolated from compost. Microb Cell Fact. 2012b;11:164–75. catalytic hydrolysis. Environ Technol. 2013;34:3005–16. Montella et al. AMB Expr (2016) 6:18 Page 10 of 10 Sangkharak K. Optimization of enzymatic hydrolysis for ethanol production Templeton DW, Scarlata CJ, Sluiter JB, Wolfrum EJ. Compositional analysis of by simultaneous saccharification and fermentation of wastepaper. Waste lignocellulosic feedstocks. 2. Method uncertainties. J Agric Food Chem. Manage Res. 2011;29:1134–44. 2010;58(16):9054–62. Sluiter A, Hames B, Hyman D, Payne C, Ruiz R, Scarlata C, Sluiter J, Templeton Wang L, Sharifzadeh M, Templerb R, Murphy RJ. Technology performance and D, Wolfe J. Determination of total solids in biomass and total dissolved economic feasibility of bioethanol production from various waste papers. solids in liquid process samples. Laboratory analytical procedure (LAP). Energy Environ Sci. 2012a;2:5717–30. 2008a. Wang L, Sharifzadeh M, Templer R, Murphy RJ. Bioethanol production from Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, and Templeton D. Deter- various waste papers: economic feasibility and sensitivity analysis. Appl mination of ash in biomass. Laboratory analytical procedure (LAP). Energy. 2013;111:1172–82. 2008b. Wang L, Templer R, Murphy RJ. High-solids loading enzymatic hydrolysis of Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D. Deter- waste papers for biofuel production. Appl Energy. 2012b;99:23–31. mination of structural crbohydrates and lignin in biomass. Laboratory Wua F-C, Huangb S-S, Shih I-L. Sequential hydrolysis of waste newspaper analytical procedure (LAPc). 2008c. and bioethanol production from the hydrolysate. Bioresour Technol. Sluiter JB, Ruiz RO, Scarlat CJ, Sluiter AD, Templeton DW. Compositional analy- 2014;167:159–68. sis of lignocellulosic feedstocks. 1. Review and description of methods. J Wu F-C, Huang S-S, Shih I-L. Sequential hydrolysis of waste newspaper Agric Food Chem. 2010;58(16):9043–53. and bioethanol production from the hydrolysate. Bioresour Technol. Subhedar PB, Babu NR, Gogate PR. Intensification of enzymatic hydrolysis of 2014;167:159–68. waste newspaper using ultrasound for fermentable sugar production. Xin F, Geng A, Chen ML, Gum MJM. Enzymatic hydrolysis of sodium dodecyl Ultrason Sonochem. 2015;22(3):26–32. sulphate (SDS)-pretreated newspaper for cellulosic ethanol production Subhedar PB, Gogate PR. Alkaline and ultrasound assisted alkaline pretreat- by Saccharomyces cerevisiae and Pichia stipitis. Appl Biochem Biotechnol. ment for intensification of delignification process from sustainable raw- 2010;162:1052–64. material. Ultrason Sonochem. 2014;21(1):216–25. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png AMB Express Springer Journals

Saccharification of newspaper waste after ammonia fiber expansion or extractive ammonia

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Copyright © 2016 by Montella et al.
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Biotechnology
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Abstract

The lignocellulosic fractions of municipal solid waste (MSW ) can be used as renewable resources due to the wide- spread availability, predictable and low pricing and suitability for most conversion technologies. In particular, after the typical paper recycling loop, the newspaper waste (NW ) could be further valorized as feedstock in biorefinering industry since it still contains up to 70 % polysaccharides. In this study, two different physicochemical methods— ammonia fiber expansion (AFEX) and extractive ammonia (EA) were tested for the pretraetment of NW. Further - more, based on the previously demonstrated ability of the recombinant enzymes endocellulase rCelStrep, α-l - arabinofuranosidase rPoAbf and its evolved variant rPoAbf F435Y/Y446F to improve the saccharification of different lignocellulosic pretreated biomasses (such as corn stover and Arundo donax), in this study these enzymes were tested for the hydrolysis of pretreated NW, with the aim of valorizing the lignocellulosic fractions of the MSW. In particular, a mixture of purified enzymes containing cellulases, xylanases and accessory hemicellulases, was chosen as reference mix and rCelStrep and rPoAbf or its variant were replaced to EGI and Larb. The results showed that these enzymatic mixes are not suitable for the hydrolysis of NW after AFEX or EA pretreatment. On the other hand, when the enzymes rCelStrep, rPoAbf and rPoAbf F435Y/Y446F were tested for their effect in hydrolysis of pretreated NW by addition to a commercial enzyme mixture, it was shown that the total polysaccharides conversion yield reached 37.32 % for AFEX pretreated NW by adding rPoAbf to the mix whilst the maximum sugars conversion yield for EA pretreated NW was achieved 40.80 % by adding rCelStrep. The maximum glucan conversion yield obtained (45.61 % for EA pretreated NW by adding rCelStrep to the commercial mix) is higher than or comparable to those reported in recent manuscripts adopting hydrolysis conditions similar to those used in this study. Keywords: AFEX pretreatment, Arabinofuranosidase, Biorefining, Cellulase, EA pretreatment, Hemicellulase, Municipal solid waste, Newspaper waste water by leachate, as well as air pollution by their burn- Introduction ing. Monitoring of pollution from different waste man - The world municipal solid waste (MSW) volume is agement options is crucial and environmental concerns expected to reach 2.2 billion tonnes by 2025, due to the are increasing the focus on the reuse and the recycling increase of urban residents to 4.3 billion. Improperly managed urban solid waste is one of the main causes of MSW and on processes for adding value to wastes. In of environmental pollution and a serious health haz particular, the lignocellulosic fractions of MSW can be a source of a wide range of high added value products by ard, due to contamination of groundwater and surface biotechnological “tailor made” processes (Liguori et  al. 2013) for the biorefineries development (Menon and Rao *Correspondence: vfaraco@unina.it 2012; Fava et  al. 2015; Esposito and Antonietti 2015). Department of Chemical Sciences, University of Naples ‘‘Federico II’’, The future of biorefining industry depends mostly on the Complesso Universitario Monte S. Angelo, Via Cintia, 4, 80126 Naples, Italy availability of cheap, sustainable and abundant biomasses Full list of author information is available at the end of the article © 2016 Montella et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Montella et al. AMB Expr (2016) 6:18 Page 2 of 10 as feedstock. The lignocellulosic fractions from MSW are We have previously shown that tailor-made enzy- more suitable to achieve this objective than dedicated matic cocktails including the endocellulase rCelStrep agricultural crops (avoiding the displacement of food from Streptomyces sp. G12 recombinantly expressed crops and minimizing the conflict food versus fuel) or in Escherichia coli (Amore et  al. 2012b), the α-l- forest resources (avoiding the issues concerning a mas- arabinofuranosidase rPoAbf from the fungus Pleu- sive deforestation). Moreover the change of land use rotus ostreatus recombinantly expressed in Pichia and the removal of forest trees in large areas can have pastoris (Amore et  al. 2012a) and/or its evolved variant a negative impact on the ecosystem. The lignocellulosic rPoAbf F435Y/Y446F (Giacobbe et  al. 2014) are suitable fractions from MSW show further advantages such as to improve the hydrolysis yields of AFEX pretreated corn widespread availability, predictable and low pricing, and stover and Arundo donax (Giacobbe et  al. 2015). Based suitability for most conversion technologies. on these results, these enzymes were chosen in this study Among the lignocellulosic fractions of MSW, paper to investigate their ability to improve the saccharification waste represents approximately 30 % of MSW and it is the of AFEX- and EA-treated NW, with the aim of valorizing second most abundant lignocellulose fraction after humid this lignocellulosic MSW fraction. fraction (U.S. Environmental Protection Agency—EPA 2015). The major method of paper waste management is Materials and methods recycling: according to EPA, the paper waste can typically Feedstock be recycled from 5 to 7 times, before papermaking fibers NW was used as feedstock for the saccharification become too short and weak to hold together; each recy- experiments. The NW was collected in the recycle sta - cling requires de-inking with chemicals processing and tion of the Office for International Student and Scholar by adding virgin wood fibers. The newspaper waste (NW) (OISS) at Michigan State University (MSU), East Lan- alone constitutes up to 14  % of MSW (Subhedar et  al. sing, Michigan and it was mainly composed of free 2015), and it can be recycled fewer times that office paper weekly newspaper distributed in Lansing area. After due to the fact that it is usually made by shorter fibers. shredding into pieces of 1  cm in wide and 20  cm in After the typical paper recycling loop, the NW can be fur- length, the NW was milled with a 2 mm diameter sieve ther valorized as feedstock in biorefinering industry since and stored under dry conditions at room temperature it still contains up to 70  % polysaccharides which can be until use. hydrolysed to fermentable sugars (Wang et al. 2012a). For an effective saccharification, the NW, as all the lig - Compositional analysis nocellulose biomasses, requires a physical, chemical or The composition analyses of NW were performed by acid enzymatic pretreatment to break down the recalcitrant hydrolysis according to the laboratory analytical pro- lignin and increase the polysaccharides accessibility for cedures (LAPs) developed by the National Renewable the following hydrolysis. The latter one can be performed Energy Laboratory (NREL) (Sluiter et  al. 2010; Temple- by enzymatic method, that is more eco-friendly than ton et al. 2010): chemical conversion, but it is not yet economically com- petitive, mainly due the high cost of the needed enzymes. • “Preparation of samples for compositional analysis” Development of enzymes with improved performances (Hames et al. 2008), by enhancing their stability and specific activity is • “Determination of structural carbohydrates and therefore pursued. Unlike dedicated energy crops and lignin in biomass” (Sluiter et al. 2008c), agro-industrial waste, the NW saccharification yield is • “Determination of total solids in biomass and total generally low probably due to high lignin content, dense dissolved solids in liquid process samples” (Sluiter structure and the additional physical barrier constituted et al. 2008a), by toner’s ink and inorganic coating linked to the lignin • “Determination of ash in biomass” (Sluiter et  al. (Chu and Feng 2013; Kim et al. 2006; Kuhad et al. 2010). 2008b). In order to overcome this bottleneck, the evaluation of the most efficient pretreatment method and of a suitable A moisture analyser was used to evaluate the mois- tailor-made enzymatic mixture are the crucial steps. ture content. The acid insoluble lignin (Klason lignin) This study is aimed at evaluating the feasibility of was detected by weighting the dried residue after total NW as feedstock for the fermentable sugars production removal of the sugars. Monomeric sugars were quanti- by testing two different physicochemical pretreatment fied using a Biorad Aminex HPX-87H high-performance methods, ammonia fiber expansion (AFEX) and extrac - liquid chromatography (HPLC) column using 5 mM sul- tive ammonia fiber expansion (EA). phuric acid as mobile phase. Montella et al. AMB Expr (2016) 6:18 Page 3 of 10 AFEX pretreatment Cellic HTec3 from Novozymes (Denmark) were used NW was subjected to AFEX pretreatment by varying in this study. Moreover, mixes of the following purified reaction temperature (65–75  °C), moisture (10.7–25  % enzymes were tested. The four core fungal cellulases were on dry weight basis), ammonia to biomass ratio (2.0:1 cellobiohydrolase I (CBH I; glycoside hydrolase—GH— and 2.8:1) at fixed residence time of 15  min. AFEX was family 7A), cellobiohydrolase II (CBH II; GH family 6A), done in a high pressure stainless steel Parr reactor. Bio- endoglucanase I (EG I; GH family 7B) and β-glucosidase mass was sprayed with water to reach appropriate mois- (βG; GH family 3). CBH I, CBH II and EG I were purified ture content after taking into consideration the moisture from Spezyme CP (Danisco US Inc., Genencor Division, content of original biomass. Then the reactor was closed Rochester, NY) using four different chromatography meth - and vacuum applied to remove residual air in the reac- ods: size exclusion, anion and cation exchange, hydropho- tor. The required amount of liquid ammonia was loaded bic interaction and affinity (Gao et  al. 2010a , b); βG was using a ammonia delivery pump into a reactor pre-heated purified from Novo 188 (Novozyme, Davis, CA, USA), by by external heating mantle. The biomass was mixed dur - using anion and cation exchange chromatography (Gao ing AFEX pretreatment process for 15  min. As the tem- et  al. 2010a, b). The bacterial hemicellulases added to the perature of reactor was increased, the pressure in the core cellulases were xylanases (LX3, GH family 10; LX4, vessel increased (between 200 and 400  psi) relying on GH family 11), β-xylosidase (LβX; GH family 52) and the ammonia to biomass loading. After the completion α-arabinofuranosidase (LArb, GH family 51). LX3 and of pretreatment process, the pressure was released from LX4 from Clostridium thermocellum, LβX from Geoba- the vessel and ammonia was vented in the hood. Subse- cillus stearothermophilus and LArb from Geobacillus sp. quently, the biomass was transferred to a tray and dried G11MC16 were recombinantly expressed in E. coli BL21 in the hood overnight to remove residual ammonia. The (DE3) and purified using HIS-select nickel affinity chro - dry pretreated biomass was stored in a sealed polythene matography (Gao et al. 2010a, b, 2011). The endoglucanase bag at 4 °C until use. rCelStrep (GH family 12) was from Streptomyces sp. G12 and recombinantly expressed in E. coli (Amore et  al. Extractive ammonia (EA) pretreatment 2012b); the α-l -arabinofuranosidases rPoAbf wild type NW was subjected to EA pretreatment (reaction tem- (GH family 51) from P. ostreatus and its mutant rPoAbf perature: 120  °C; ammonia to biomass ratio: 3:1  kg/kg F435Y/Y446F were recombinantly expressed in P. pasto- dry biomass; moisture content: 10 % on dry weight basis; ris (Amore et  al. 2012a). These enzymes were purified by fixed residence time: 15 min). EA was done in a high pres - ammonium sulphate precipitation followed by hydropho- sure stainless steel Parr reactor. Biomass was first sprayed bic interaction chromatography (Amore et al. 2012a, b). with water to reach appropriate moisture content after taking into consideration the moisture content of origi- Enzymatic hydrolysis nal biomass. The required amount of liquid ammonia was The enzymatic hydrolyses were carried in 5  mL vials. loaded using a ammonia delivery pump into a reactor An amount of pretreated biomass was added as to load pre-heated by external heating mantle. The biomass was 1  % (w/w) glucan in 2  mL total volume with the desired mixed during EA pretreatment process for 15  min. As enzymes. The buffer solution was 50 mM citrate, pH 4.8. the temperature of reactor was increased, the pressure in Microbial and fungal contaminations were prevented by the vessel raised (between 600 and 800 psi) depending on adding sodium azide 0.5 mM. The hydrolysis parameters the ammonia to biomass loading. After the completion of were: 50 °C, 250 rpm, 72 h. Sampling was collected every pretreatment process, extractives (mostly lignin) gener- 24 h to evaluate carbohydrates hydrolysis. ated during the process along with liquid ammonia was Fifteen milligram per gram of glucan of the commercial collected via a sintered frit into another high pressure enzymatic preparation Novozymes Cellic (60  % CTec3 reactor. The collection pressure was released from the and 40  % HTec3) was used for hydrolysis experiment to vessel and ammonia was vented in the hood to recover select the best pretreatment conditions. the extractives. The pretreated biomass was transferred The following enzymes were used for the hydrolysis of to a tray and dried in the hood overnight to remove AFEX and EA pretreated NW. The MIX A was prepared residual ammonia present in the biomass. Then dry EA including: CBH I, CBH II and EG I (3.32  mg/g glucan treated biomass was stored in a sealed polythene bag in a each), βG (2 mg/g glucan), LX3 and LX4 (1.66 mg/g glu- refrigerator until further use. can each), LβX and LArb (0.6 mg/g glucan each). In the mix B, the EG I was replaced by an equal Commercial and purified enzymes and their sources amount of endoglucanase rCelStrep; in the mix C the The commercially available complex of cellulases and LArb was replaced by an equal amount of the α-l- hemicellulases Cellic CTec3 and the enzyme solution arabinofuranosidase rPoAbf; in the mix D the LArb Montella et al. AMB Expr (2016) 6:18 Page 4 of 10 was replaced by the same amount of the mutant rPoAbf Arabinan conversion (%) F435Y/Y446F. Arabinose concentration in hydrolysate g L Moreover, rCelStrep, rPoAbf or rPoAbf F435Y/ Arabinan concentration in loaded biomass g L Y446F were added alternatively or in combination to the MIX 1 containing 15  mg/g glucan of commercial ® ® 1.136 preparation mix (60  % Cellic CTec3 and 40  % Cellic HTec3 from Novozymes). In particular the following Mannan conversion (%) enzymes were added: in the MIX 2, the endoglucanase rCelStrep (3.32  mg/g glucan); in the MIX 3 the α-l- Mannose concentration in hydrolysate g L arabinofuranosidase rPoAbf (0.6  mg/g glucan); in the Mannan concentration in loaded biomass g L MIX 4 the mutant rPoAbf F435Y/Y446F (0.6  mg/g glu- can); in the MIX 5 both the endoglucanase rCelStrep × 1.11 (3.32  mg/g glucan) and the α-l-arabinofuranosidase rPoAbf (0.6  mg/g glucan); in the MIX 6 both the endo- where 1.11 is the ratio of MW (180.16  g/ glucose (or mannose) glucanase rCelStrep (3.32  mg/g glucan) and the mutant mol) to MW (162.14 g/mol) and 1.11 is the glucan (or mannan) α-l-arabinofuranosidase rPoAbf F435Y/Y446F (0.6 mg/g ratio of MW (150.13 g/mol) to MW xylose (or arabinose) xylan (or glucan). (132.13 g/mol). arabinan) Sugar analysis Results Monomeric sugars concentration were determined by Composition analysis and pretreatment of newspaper high performance liquid chromatography (HPLC). All waste experiments were performed in triplicate. Composition analysis of untreated NW was carried out About 200  μl hydrolysate were collected in a centri- and the results reported in Table 1 revealed a glucan con- fuge tube, heated to 100 °C for 10 min (to inactivate the tent of ~44  %, a hemicellulose content of ~15  % and a enzymes), then spun down at 8000  rpm for 10  min and Klason lignin content of ~25 %. the supernatant was stored in a HPLC vial at −20  °C The macromolecular composition of AFEX pre - until further use. Monomeric sugars concentration in treated NW is assumed to have the same composition the hydrolysate was determined by HPLC using Biorad of untreated NW (Table  1), due to the fact that, as pre- Aminex HPX-87P. Shimadzu HPLC Prominence system viously reported, the AFEX pretreatement preserves the (Columbia, MD, USA) with a refractive index detec- macrostructure of the lignocellulosic biomasses, reducing tor (RID) were used for analyzing the sugars. Water was the degree of polymerization of (hemi)cellulose minimiz- used as the mobile phase at a fixed flow rate of 0.6  ml/ ing the degradation of the original carbohydrates (Holt- min, with isocratic elution. The column temperature was zapple et  al. 1991; Kumar et  al. 2009). In order to select maintained at 60 °C and the HPLC sample injection vol- the best AFEX conditions to be used during the further ume was 20 μl. Standard curves were generated using dif- saccharification experiments, three different sets of con - ferent concentrations of mixed sugars. A guard column ditions were tested on NW (Table  2), varying ammonia with similar packing was used throughout the chroma- loading, temperature and moisture content. These con - tography experiments. ditions were chosen based on the previous published The sugars conversion is calculated according to the screening of AFEX pretreatments on NW (Holtzapple following equations: et al. 1991, 1992). The AFEX pretreated NW was subjected to enzymatic Glucan conversion (%) hydrolysis using the mix composed of 60 % Cellic CTec3 and 40  % Cellic HTec3 from Novozymes. As shown Glucose concentration in hydrolysate g L in Fig.  1, the maximum glucose and xylose yields were Glucan concentration in loaded biomass g L achieved after 72 h of hydrolysis with the AFEX pretreat- ment condition N1 (2.8  kg ammonia/kg dry biomass, 1.11 65  °C, and 10.7  % of moisture content) and N2 (2.8  kg ammonia/kg dry biomass, 75  °C, and 25  % of moisture Xylan conversion (%) content) and the maximum total sugars conversion was Xylose concentration in hydrolysate g L respectively 29.58  ±  0.35 and 29.02  ±  0.35  % (Table  2). The condition N1 was selected for the further hydrolysis Xylan concentration in loaded biomass g L experiments due to the milder reaction temperature of pretreatment (10 °C less than condition N2). 1.136 Montella et al. AMB Expr (2016) 6:18 Page 5 of 10 Table 1 Macromolecular composition of newspaper waste that the NW still contains significant percentages of struc - before and after EA pretreatment tural sugars after EA pretreatment (~58  %). In particular, the percentages of glucan content is ~41  %, the percent- Newspaper waste age of mannan content is ~8 % and the percentage of xylan Untreated EA pretreated content is ~5 %; the total lignin content is ~31 % (Table 1). Moisture content 7.00 ± 1.00 9.75 ± 0.07 Hydrolysis of newspaper waste by using the enzymes Ash 7.04 ± 0.14 7.25 ± 0.15 rCelStrep, rPoAbf and its variant in comparison with the Structural carbohydrate enzymes EGI or LArb Glucan 44.21 ± 4.02 41.36 ± 0.01 As shown in several papers, a mix of cellulases and acces- Xylan 5.11 ± 0.19 5.20 ± 0.22 sory hemicellulases is crucial to enhance hydrolysis of Galactan 1.81 ± 0.13 1.84 ± 0.03 pretreated lignocellulosic biomasses (Gao et al. 2010a, b; Arabinan 1.09 ± 0.09 1.73 ± 0.03 Jørgensen et al. 2007). The MIX A, previously optimized Mannan 9.83 ± 0.47 8.16 ± 0.05 for corn stover saccharification after AFEX pretreatment Lignin (Gao et al. 2011), was chosen as reference mix. This mix - Acid insoluble lignin 25.88 ± 2.48 30.03 ± 0.89 ture contains the cellobiohydrolases CBHI and CBHII, Acid soluble lignin 0.96 ± 0.01 1.05 ± 0.19 the endo-glucanase EGI, the beta-glucosidase βG, the Composition closure 95.92 96.63 xylanases LX3 and LX4, the beta-xylosidase LβX and the α-l-arabinofuranosidase LArb. In order to evalu - ate the hydrolysis yield by using the enzymes rCelStrep, As an alternative pretreatment method, the EA was also rPoAbf and rPoAbf F435Y/Y446F, these enzymes were tested on NW. The EA pretreatment converts native cel - replaced to EGI (MIX B) and to LArb (MIX C and MIX lulose I to cellulose III, delignifying the biomass simulta- D), respectively. neously (da Costa et al. 2015). Composition analysis of EA The highest glucan, xylan, mannose and arabinose pretreated NW was carried out and the results showed conversions were reached after 72  h of hydrolysis for Table 2 AFEX conditions tested on newspaper waste AFEX conditions Ammonia loading Reaction Moisture Fixed residence Sugars conversion (kg:kg dry biomass) temperature (°C) content (%) time (min) (after 72 h hydrolysis) (%) (g/L) N1 2.8:1 65 10.7 15 29.58 4.62 N2 2.8:1 75 25 29.02 4.53 N3 2.0:1 75 25 26.14 4.08 Fig. 1 Glucan and xylan conversion during the hydrolysis of newspaper waste after three different AFEX pretreatment conditions: N1 (ammonia loading 2.8:1; 65 °C; 10.7 % moisture content; 15 min), N2 (ammonia loading 2.8:1; 75 °C; 25 % moisture content; 15 min) and N3 (ammonia loading 2.0:1; 65 °C; 10.7 % moisture content; 15 min) Montella et al. AMB Expr (2016) 6:18 Page 6 of 10 all the tested biomasses, pretreatment methods and undetectable for all mixes. In conclusion, there was no enzyme mixes. In Figs.  2 and 3 the conversion data for substantial improvement by replacing rCelStrep, rPoAbf the most abundant polysaccharides (glucan and xylan) or its variant to the corresponding enzymatic activities in are reported. the reference mix. For AFEX pretreated NW, as shown in Fig.  2, the glu- As shown in Fig.  3, for EA pretreated NW, the glucan can conversion reached 7.3  % for MIX A and did not conversion reached 14.3 % for MIX A and decreased for increase significantly by using the other mixes. The xylan other three mixes (up to 13.1  % for MIX B, 10.7  % for conversion was 13.59  % for MIX A and increased up to MIX C and 13.4 % for MIX D). The xylan conversion was 14.44  % only for MIX B (corresponding to the substitu- 19.2  % for MIX A and increased only for MIX B (up to tion of rCelStrep to EGI), decreasing for the other mixes 20.3  %), decreasing for MIX C (16.9  %) and for MIX D (12.2  % for MIX C and 13.1  % for MIX D). The arab - (15.4  %). The arabinose conversion reached 12.5  % for inose conversion reached 7.61  % for MIX A, increased MIX A and decreased to 11.1  % for MIX B, 9.9  % for up to 8.86 % for MIX B and decreased to 4.4 % for MIX MIX C and 8.70  % for MIX D. The mannose conversion C and to 5.3 % for MIX D. The mannose conversion was was less than 1  % for all the mixes. In conclusion, the Fig. 2 Glucan and xylan conversion during the hydrolysis of AFEX pretreated newspaper waste by using MIX A: CBH I, CBH II and EG I (3.32 mg/g glucan each), βG (2 mg/g glucan), LX3 and LX4 (1.66 mg/g glucan each), LβX and LArb (0.6 mg/g glucan each); MIX B (replacing EG I with endoglu- canase rCelStrep); MIX C (replacing LArb with the α-l -arabinofuranosidase rPoAbf ); MIX D (replacing LArb with mutant rPoAbf F435Y/Y446F) Fig. 3 Glucan and xylan conversion during the hydrolysis of EA pretreated newspaper waste by using MIX A: CBH I, CBH II and EG I (3.32 mg/g glucan each), βG (2 mg/g glucan), LX3 and LX4 (1.66 mg/g glucan each), LβX and LArb (0.6 mg/g glucan each); MIX B (replacing EG I with endoglu- canase rCelStrep); MIX C (replacing LArb with the α-l -arabinofuranosidase rPoAbf ); MIX D (replacing LArb with mutant rPoAbf F435Y/Y446F) Montella et al. AMB Expr (2016) 6:18 Page 7 of 10 maximum polysaccharides conversion yield (12.4 %) was was obtained by adding rPoAbf F435Y/Y446F (0.6  mg/g obtained for MIX A. glucan) to MIX 1. As shown in Fig. 5, for the EA pretreated NW, the glu- Evaluation of synergism between commercial enzyme can conversion reached 35.6  % for MIX 1 and increased preparation and rCelstrep and/or rPoAbf and its mutant up to 45.6 % for MIX 2, 40.1 % for MIX 3, 43.6 % for MIX The enzymes rCelStrep, rPoAbf or its evolved variant 4, 42.657  % for MIX 5 and 38.5  % for MIX 6. The xylan rPoAbf F435Y/Y446F were added alternatively or in com- conversion was 44.9  % for MIX 1 and increased up to bination to 15 mg/g glucan of MIX 1 (60 % Cellic CTec3 55.6  % for MIX 2, 51.3  % for MIX 3, 58.1  % for MIX 4, and 40 % Cellic HTec3 from Novozymes) for the hydrol- 53.7  % for MIX 5 and 49.2  % for MIX 6. The arabinose ysis reactions of pretreated NW. The amount of enzyme conversion reached 49.0  % for MIX 1 and increased up loading was chosen based on the data obtained by Gao to 52.1  % for MIX 2, 49.2  % for MIX 3, 53.7  % for MIX et  al. (2011) and due to the promising results previously 4, 51.0 % for MIX 5 and 50.2 % for MIX 6. The mannose achieved for the hydrolysis of AFEX pretreated Corn conversion reached 17.2  % for MIX 1 and decreased for stover and A. donax (Giacobbe et al. 2015). all the other mixes (13.6 % for MIX 2, 12.8 % for MIX 3, The highest glucan, xylan and arabinose conversion 11.4 % for MIX 4, 13.0 % for MIX 5 and 10.0 % for MIX 6. were reached after 72  h of hydrolysis for all tested bio- In conclusion, the maximum polysaccharides conversion masses, pretreatment methods and enzyme mixes. In yield (40.80  %) was obtained for MIX 2, by adding rCel- Figs.  4 and 5 the conversion data for the most abundant Strep (3.32 mg/g glucan) to MIX 1. polysaccharides (glucan and xylan) are reported. As shown in Fig.  4, for AFEX pretreated NW, the glu- Discussion can conversion reached 34.0  % for MIX 1 and increased Composition analysis of untreated NW, assumed equal up to 39.4 % for MIX 2, 41.2 % for MIX 3, 42.7 % for MIX to the macromolecular composition of AFEX pretreated 4, 41.22  % for MIX 5 and 41.0  % for MIX 6. The xylan NW, as aforementioned, was in agreement with the val- conversion was 46.7  % for MIX 1 and increased up to ues previously reported by Wang et  al. (2012b, 2013), 53.3  % for MIX 2, 57.5  % for MIX 3, 58.7  % for MIX 4, Subhedar et  al. (2015), Sangkharak (2011) and Orozco 57.50  % for MIX 5 and 56.7  % for MIX 6. The arabinose et  al. (2013) related to the untreated biomass. Fur- conversion reached 80.7  % for MIX 1 and decreased thermore, composition analysis of EA pretreated NW for all other mixes (67.3 % for MIX 2, 71.6 % for MIX 3, revealed that this technique, similarly to the AFEX pre- 70.8 % for MIX 4, 71. 7 % for MIX 5 and 70.0 % for MIX treatment, does not significantly change the composition. 6). The mannose conversion was undetectable for MIX 1 Furthermore, the cellulose percentage after acid pretreat- and reached 4.5 % for MIX 2, 6.1 % for MIX 3, 4.7 % for ment, reported by Guerfali et al. (2015), and the holocel- mix 4, 4.6  % for MIX 5 and 4.5  % for MIX 6. In conclu- lulose percentage, after alkaline pretreatment, reported sion, the maximum polysaccharides conversion (37.32 %) by Wu et  al. (2014), were comparable to data obtained Fig. 4 Glucan and xylan conversion during the hydrolysis of AFEX pretreated newspaper waste by using MIX 1 (15 mg/g of glucan of commercial enzymatic preparation Novozymes Cellic —60 % CTec3 and 40 % HTec3; MIX 2 (adding 3.32 mg/g glucan of endoglucanase rCelStrep to MIX 1); MIX 3 (adding 0.6 mg/g glucan of the α-l -arabinofuranosidase rPoAbf to MIX 1); MIX 4 (adding 0.6 mg/g glucan of the mutant rPoAbf F435Y/Y446F to MIX 1); MIX 5 (adding 3.32 mg/g glucan of endoglucanase rCelStrep and 0.6 mg/g glucan of the α-l -arabinofuranosidase rPoAbf to MIX 1); MIX 6 (adding 3.32 mg/g glucan of endoglucanase rCelStrep and 0.6 mg/g glucan of the mutant rPoAbf F435Y/Y446F to MIX 1) Montella et al. AMB Expr (2016) 6:18 Page 8 of 10 Fig. 5 Glucan and xylan conversion during the hydrolysis of EA pretreated newspaper waste by using MIX 1 (15 mg/g of glucan of commercial enzymatic preparation Novozymes Cellic —60 % CTec3 and 40 % HTec3; MIX 2 (adding 3.32 mg/g glucan of endoglucanase rCelStrep to MIX 1); MIX 3 (adding 0.6 mg/g glucan of the α-l -arabinofuranosidase rPoAbf to MIX 1); MIX 4 (adding 0.6 mg/g glucan of the mutant rPoAbf F435Y/Y446F to MIX A’); MIX 5 (adding 3.32 mg/g glucan of endoglucanase rCelStrep and 0.6 mg/g glucan of the α-l -arabinofuranosidase rPoAbf to MIX 1); MIX 6 (adding 3.32 mg/g glucan of endoglucanase rCelStrep and 0.6 mg/g glucan of the mutant rPoAbf F435Y/Y446F to MIX 1) after AFEX or EA method; contrariwise, the alpha cel- barrier constituted by toner’s ink and inorganic coating lulose and holocellulose percentage, reported by Sang- that remain linked to the lignin after pretreatment. kharak (2011), after pretreatment by using NaOH are On the other hand, the AFEX and EA pretreatments both higher than those reported in this study, but the had comparable effects on the polysaccharides conversion reaction conditions used for AFEX reaction are milder yield after the hydrolysis of NW by using Cellic CTec3 (65 versus 100  °C and residence time 15  min versus 4  h and HTec3 and the addition of rCelStrep, rPoAbf or its for AFEX and basic pretreatment respectively). evolved variant rPoAbf F435Y/Y446F to the commercial The hydrolysis of pretreated NW obtained by using mix improved the sacharification process. In particular, mixes of purified enzymes showed that the EA method the maximum glucan conversion yields reached for AFEX improve the saccharification in comparison with the pretreated NW and EA pretreated NW were obtained by AFEX, although the hydrolysis yields remained low in an additive effect of the α-l-arabinofuraosidase rPoAbf comparison to the data previously reported in literature. F435Y/Y446F and the endocellulase rCelStrep, respec- The maximum total polysaccharides conversion yield (for tively, in addition of the array of (hemi)cellulase activi- EA-pretreated-NW hydrolyzed by enzymatic MIX A) ties present in the commercial mix. The increase of the was similar to the yields reported by Ali and Khan Mohd glucan conversion yield was respectively of 25 and 28  % (2011) after basic pretreatment and six days of micro- more than the yields obtained by hydrolysis with com- bial hydrolysis by Aspergillus niger and lower than the mercial enzymes without any addition. The best obtained most recent reported results (Wua et  al. 2014; Chu and sugars conversion yield was higher than or compara- Feng 2013; Wang et  al. 2012b; Guerfali et  al. 2015; Sub- ble to those reported in recent manuscripts adopting hedar and Gogate 2014; Kim et al. 2006; Xin et al. 2010). hydrolysis conditions similar to those used in this study. Moreover, the glucan conversion yield was very low (not In particular, the maximum obtained glucan conversion exceeding 7.3 %). This is the major drawback as glucose is yields were comparable to those described by Wang et al. the main carbon source for a wide range of industrial fer- (2012b) after enzymatic hydrolysis using 5  % (w/w) bio- mentation processes by using well-known microorgan- mass and 64  mg/g glucan of commercial enzymes (~3 isms capable of using mainly hexoses (Jang et  al. 2012). times more than those loaded in this study). Moreover, These results showed that the enzymatic mix previously Wu et al. (2014) obtained sugar conversion yields compa- optimized for pretreated corn stover saccharification is rable to this study after basic pretreatment and combin- not suitable for the hydrolysis of NW after AFEX or EA ing acid and enzymatic hydrolysis. Subhedar and Gogate pretreatment. Moreover, the replacement of the enzymes (2014) obtained ~31  % of total sugars yield (lower than rCelStrep, rPoAbf and rPoAbf F435Y/Y446F respectively the results of this study) by using enzymatic hydrolysis to EGI and LArb did not change significantly the sac - and the yield increased only after an ultrasound-assisted charification yield. These data suggested that the purified enzymatic hydrolysis. However, the best obtained results enzymes are not probably able to overcome the physical in this study are lower than those reported by Guerfali Montella et al. AMB Expr (2016) 6:18 Page 9 of 10 Chu KH, Feng X. Enzymatic conversion of newspaper and office paper to et  al. (2015) and Kim et  al. (2006) that add the action of fermentable sugars. Process Saf Environ Prot. 2013;91:123–30. surfactant agents to the enzymatic hydrolysis. da Costa Sousa L, Jina M, Chundawata S, Bokade V, Tangd X, Azarpira A, Lu F, In conclusion, the feasibility of NW pretreatment Avcif U, Humpula J, Uppugundlaa N, Gunawana C, Pattathilf S, Chehg A, Kotharih N, Kumarh R, Ralph J, Hahnf MG, Wymanh CE, Singh S, Simmonsi by both AFEX and EA were demonstrated. The results BA, Dale BE, Balan V. Next-generation ammonia pretreatment enhances showed no substantial differences between the two tested biofuel production from biomass via simultaneous cellulose decrystalliza- methods on hydrolysis yield. However, the AFEX was tion and lignin extraction. Accepted: Energy and Environmental Science; the best pretreatment technique mainly due to the mild Environmental Protection Agency—EPA (2015). http://www3.epa.gov/epa- reaction conditions. Moreover, the best sugars conver- waste/nonhaz/municipal/ Accessed 15 Jan 2016. sion yield obtained by adding the recombinant enzymes Esposito D, Antonietti M. Redefining biorefinery: the search for unconventional building blocks for materials. Chem Soc Rev. 2015;44:5821–35. to the commercial mixture was higher than or compara- Fava F, Totaro G, Diels L, Reis M, Duarte J, Carioca OB, Poggi-Varaldo HM, Fer- ble to those reported in previous studies adopting similar reira BS. Biowaste biorefinery in Europe: opportunities and research and hydrolysis conditions. These promising but not optimal development needs. New Biotechnol. 2015;32(1):100–8. Gao D, Chundawat SP, Krishnan C, Balan V, Dale BE. Mixture optimization of results suggest that the process can be optimized in order six core glycosyl hydrolases for maximizing saccharification of ammonia to further enhance the NW hydrolysis yield. fiber expansion (AFEX) pretreated corn stover. Bioresour Technol. 2010a;101(8):2770–81. Authors’ contributions Gao D, Chundawat SP, Liu T, Hermanson S, Gowda K, Brumm P, Dale BE, Balan V. SM wrote the main manuscript text and prepared figures and tables. LCS Strategy for identification of novel fungal and bacterial glycosyl hydrolase designed and carried out the AFEX and EA pretreatment experiments. VB hybrid mixtures that can efficiently saccharify pretreated lignocellulosic designed the hydrolysis experiments and contributed to analyze the results. biomass. Bioenerg Res. 2010b;3(1):67–81. SM and SG carried out the hydrolysis experiments and analyzed all the results. Gao D, Uppugundla N, Chundawat SP, Yu X, Hermanson S, Gowda K, Brumm CG carried out the HPLC analysis. VF contributed to conceiving the study and P, Mead D, Balan V, Dale BE. Hemicellulases and auxiliary enzymes for participated in its design and coordination and drafted the manuscript. OP improved conversion of lignocellulosic biomass to monosaccharides. contributed to analyze all the results and to draft the manuscript. VF is the cor- Biotechnol Biofuels. 2011;4:5. doi:10.1186/1754-6834-4-5. responding author. All authors read and approved the final manuscript. Giacobbe S, Balan V, Montella S, Fagnano M, Mori M, Faraco V. Assessment of bacterial and fungal (hemi)cellulose-degrading enzymes in sacchari- Author details fication of ammonia fibre expansion-pretreated Arundo donax. Appl Department of Chemical Sciences, University of Naples ‘‘Federico II’’, Microbiol Biotechnol. 2015. doi 10.1007/s00253-015-7066-3. Complesso Universitario Monte S. Angelo, Via Cintia, 4, 80126 Naples, Italy. Giacobbe S, Vincent F, Faraco V. Development of an improved variant of GH51 Department of Chemical Engineering and Materials Science, DOE Great α-l -arabinofuranosidase from Pleurotus ostreatus by directed evolution. Lakes Bioenergy Research Center, Michigan State University, East Lansing, MI New Biotechnol. 2014;31(3):230–6. 48823, USA. Department of Agriculture, University of Naples “Federico II”, Guerfali M, Saidi A, Gargouri A, Belghith H. Enhanced enzymatic hydrolysis of Portici, Naples, Italy. waste paper for ethanol production using separate saccharification and fermentation. Appl Biochem Biotechnol. 2015;175:25–42. Acknowledgements Hames B, Ruiz R, Scarlata C, Sluiter A, Sluiter J, Templeton D. Preparation of sam- This research was supported by a Marie Curie International Research Staff ples for compositional analysis laboratory analytical procedure (LAP). 2008. Exchange Scheme Fellowship within the 7th European Community Frame- Holtzapple MT, Jun J-H, Ashok G, Patibandla SL, Dale BE. The ammo- work Programme: ‘Improvement of technologies and tools, e.g., biosystems nia freeze explosion (AFEX) process. Appl Biochem Biotechnol. and biocatalysts, for waste conversion to develop an assortment of high 1991;28–29(1):59–74. added value eco-friendly and cost-effective bio-products’ BIOASSORT (grant Holtzapple MT, Lundeen JE, Sturgis R, Lewis HE, Dale BE. Pretreatment of number 318931). We gratefully acknowledge support given to VB by the lignoceilulosic municipal solid waste by ammonia fiber explosion (AFEX). office of Biological and Environmental Research in the DOE Office of Science Appl Biochem Botechnol. 1992;34–35(1):5–21. through the Great Lakes Bioenergy Research Centre (GLBRC) (Grant DE-FC02- Jang YS, Kim B, Shin JH, Choi YJ, Choi S, Song CW, Lee J, Park HG, Lee SY. 07ER64494). We also thank Lucigen Enzyme Company for supplying the Bio-based production of C2–C6 platform chemicals. Biotechnol Bioeng. research enzymes and Novozyme for supplying the enzyme solutions Cellic 2012;109(10):2437–59. CTec3 and the Cellic HTec3 for this work. Jørgensen H, Kristensen JB, Felby C. Enzymatic conversion of lignocellulose into fermentable sugars: challenges and opportunities. Biofuels Bioprod Competing interests Biorefin. 2007;1:119–34. The authors declare that they have no competing interests. Kim SB, Kim HJ, Kim CJ. Enhancement of the enzymatic digest- ibility of waste newspaper using Tween. Appl Biochem Biotechnol. Received: 5 December 2015 Accepted: 23 February 2016 2006;129–132:486–95. Kuhad RC, Mehta G, Gupta R, Sharma KK. Fed batch enzymatic saccharification of newspaper cellulosics improves the sugar content in the hydrolysates and eventually the ethanol fermentation by Saccharomyces cerevisiae. Biomass Bioenergy. 2010;34:1189–94. Kumar R, Mago G, Balan V, Wyman CE. Physical and chemical characterizations References of corn stover and poplar solids resulting from leading pretreatment Ali MN, Khan Mohd M. Production of bioethanol fuel from renewable technologies. Bioresour Technol. 2009;100:3948–62. agrobased cellulosic wastes and waste news papers. Int J Eng Sci Tech- Liguori R, Amore A, Faraco V. Waste valorization by biotechnological nol. 2011;3:884. conversion into added value products. Appl Microbiol Biotechnol. Amore A, Amoresano A, Birolo L, Henrissat B, Leo G, Palmese A, Faraco V. A 2013;97(14):6129–47. family GH51 α-l -arabinofuranosidase from Pleurotus ostreatus: identi- Menon V, Rao M. Trends in bioconversion of lignocellulose: biofuels plat- fication recombinant expression and characterization. Appl Microbiol form chemicals and biorefinery concept. Prog Energy Combust Sci. Biotechnol. 2012a;94(4):995–1006. 2012;38(4):522–50. Amore A, Pepe O, Ventorino V, Birolo L, Giangrande C, Faraco V. Cloning and Orozco AM, Al-Muhtaseb AH, Rooney D, Walker GM, Aiouache F, Ahmad M. recombinant expression of a cellulase from the cellulolytic strain Strepto- Fermentable sugars recovery from lignocellulosic waste-newspaper by myces sp.12 isolated from compost. Microb Cell Fact. 2012b;11:164–75. catalytic hydrolysis. Environ Technol. 2013;34:3005–16. Montella et al. AMB Expr (2016) 6:18 Page 10 of 10 Sangkharak K. Optimization of enzymatic hydrolysis for ethanol production Templeton DW, Scarlata CJ, Sluiter JB, Wolfrum EJ. Compositional analysis of by simultaneous saccharification and fermentation of wastepaper. Waste lignocellulosic feedstocks. 2. Method uncertainties. J Agric Food Chem. Manage Res. 2011;29:1134–44. 2010;58(16):9054–62. Sluiter A, Hames B, Hyman D, Payne C, Ruiz R, Scarlata C, Sluiter J, Templeton Wang L, Sharifzadeh M, Templerb R, Murphy RJ. Technology performance and D, Wolfe J. Determination of total solids in biomass and total dissolved economic feasibility of bioethanol production from various waste papers. solids in liquid process samples. Laboratory analytical procedure (LAP). Energy Environ Sci. 2012a;2:5717–30. 2008a. Wang L, Sharifzadeh M, Templer R, Murphy RJ. Bioethanol production from Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, and Templeton D. Deter- various waste papers: economic feasibility and sensitivity analysis. Appl mination of ash in biomass. Laboratory analytical procedure (LAP). Energy. 2013;111:1172–82. 2008b. Wang L, Templer R, Murphy RJ. High-solids loading enzymatic hydrolysis of Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D. Deter- waste papers for biofuel production. Appl Energy. 2012b;99:23–31. mination of structural crbohydrates and lignin in biomass. Laboratory Wua F-C, Huangb S-S, Shih I-L. Sequential hydrolysis of waste newspaper analytical procedure (LAPc). 2008c. and bioethanol production from the hydrolysate. Bioresour Technol. Sluiter JB, Ruiz RO, Scarlat CJ, Sluiter AD, Templeton DW. Compositional analy- 2014;167:159–68. sis of lignocellulosic feedstocks. 1. Review and description of methods. J Wu F-C, Huang S-S, Shih I-L. Sequential hydrolysis of waste newspaper Agric Food Chem. 2010;58(16):9043–53. and bioethanol production from the hydrolysate. Bioresour Technol. Subhedar PB, Babu NR, Gogate PR. Intensification of enzymatic hydrolysis of 2014;167:159–68. waste newspaper using ultrasound for fermentable sugar production. Xin F, Geng A, Chen ML, Gum MJM. Enzymatic hydrolysis of sodium dodecyl Ultrason Sonochem. 2015;22(3):26–32. sulphate (SDS)-pretreated newspaper for cellulosic ethanol production Subhedar PB, Gogate PR. Alkaline and ultrasound assisted alkaline pretreat- by Saccharomyces cerevisiae and Pichia stipitis. Appl Biochem Biotechnol. ment for intensification of delignification process from sustainable raw- 2010;162:1052–64. material. Ultrason Sonochem. 2014;21(1):216–25.

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Published: Mar 2, 2016

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