Miscanthus straw as substrate for biosuccinic acid production: Focusing on pretreatment and downstream processing

Bioresource Technology 278 (2019) 82–91

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Miscanthus straw as substrate for biosuccinic acid production: Focusing on pretreatment and downstream processing

T

⁎

Katarzyna Dąbkowskaa, Merlin Alvarado-Moralesb, Mariusz Kuglarzc, , Irini Angelidakib a

Faculty of Chemical and Process Engineering, Warsaw University of Technology, 00-645 Warsaw, Waryńskiego 1, Poland Department of Environmental Engineering, Technical University of Denmark, Building 113, DK-2800 Lyngby, Denmark c Faculty of Materials, Civil and Environmental Engineering, University of Bielsko-Biala, Willowa 2, 43-309 Bielsko-Biala, Poland b

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO

A B S T R A C T

Keywords: Succinic acid Miscanthus biomass Glycerol pretreatment Delignification Succinic acid recovery

The main aim of this study was to optimize pretreatment strategies of Miscanthus × giganteus for biosuccinic acid production. A successful pretreatment with organosolv method (80% w/w of glycerol, 1.25% of H2SO4), prevented sugars conversion to furfurals and organic acids, and thereby resulted in high sugar recovery (glucan > 98%, xylan > 91%) and biomass delignification (60%). Pretreated biomass was subjected to hydrolysis with various cellulolytic enzyme cocktails (Viscozyme® L, Carezyme 1000L®, β-Glucanase, Cellic® CTec2, Cellic® HTec2). The most effective enzymes mixture composed of Cellic® CTec2 (10% w/w), β-Glucanase (5% w/w) and Cellic® HTec2 (1% w/w) resulted in high glucose (93.1%) and xylose (69.2%) yields after glycerolbased pretreatment. Succinic acid yield of 75–82% was obtained after hydrolysates fermentation, using Actinobacillus succinogenes 130Z. Finally a successful downstream concept for succinic acid purification was proposed. The succinic acid recovery with high purity (> 98%) was developed.

1. Introduction Effective and cost-efficient production of succinic acid (buildingblock chemical) from lignocellulosic biomass, including perennial C4 crop, is strictly connected with the amount of sugars released during pre-treatment and subsequent enzymatic hydrolysis. Miscanthus, and specifically the species of Miscanthus × giganteus, is of particular

⁎

interest, because it ensures high biomass production and high carbohydrates content in dry matter, which decreases competition with food and feed crops for arable land (Brosse et al., 2012, Ge et al., 2016). The most commonly applied pretreatment methods used for Miscanthus biomass disruption, are based on steam treatment facilitated by addition of acids (0.3–0.7% H2SO4 at 150–180 °C for 6–10 min or 1–4% H2SO4 at 120 °C for 13–20 min (Yoo et al., 2016, Xu et al., 2012).

Corresponding author. E-mail address: [email protected] (M. Kuglarz).

https://doi.org/10.1016/j.biortech.2019.01.051 Received 31 October 2018; Received in revised form 10 January 2019; Accepted 11 January 2019 Available online 14 January 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.

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losses and sugar/lignin by-products generation (furfural, 5-hydroxymethyl-2-furaldehyde (HMF), phenolic compounds, organic acids). The aim of the study was to identify optimal pretreatment conditions, applying glycerol-based method with catalyst addition (1.0–1.5% H2SO4) as well as establish the optimal combination and dosage of various commercially available cellulolytic enzyme preparations (Cellic® CTec2, Cellic® HTec2, Viscozyme® L, Carezyme 1000L®, and βglucanase) for effective hydrolysis of pretreated miscanthus. According to our best knowledge, mixtures of cellulolytic cocktails have never been applied for the enhancement of glucose and xylose yields of miscanthus biomass, especially after organosolv pretreatment. Finally, an effective concept of succinic broth purification and succinic acid recovery, ensuring crystals purity > 98%, was proposed.

During acid-based pretreatment, hemicellulose is hydrolyzed into its pentose monomers. However, a significant drawback of steam treatment with acids addition is the formation of inhibitors (e.g. furfural, hydroxymethylfurfural), which can significantly decrease sugar utilization as well as negatively affect subsequent processes, i.e. enzymatic hydrolysis or/and fermentation. Moreover, dilute-acid methods are not effective in lignin removal, which negatively affects subsequent enzymatic hydrolysis. Application of alkaline or oxidative pretreatment has proved to be one of the most effective for softening the biomass structure and removing the lignin (Kumar and Sharma, 2017, Zhao et al., 2017). Recently, alkali-based methods, including potassium/sodium hydroxide (50 °C, 7 days), sodium carbonate (80 °C, 3 h) and hydrogen peroxide (1% H2O2 at 11.5 pH for 60 min at room temperature) proved to be effective in biomass delignification (Rodrigues et al., 2016, Yeh et al., 2016). However, significantly higher reaction times are required compared to dilute-acid methods or thermal-based processes. In these cases, downstream processing of residues after pretreatment, connected with biomass washing (e.g. removal of calcium and sodium salts) should also be considered. Taking into account the above, the development of effective pretreatment methods is still the key component of the whole process of bio-succinic acid production. Pretreatments based on organic solvents, especially using glycerol, have recently gained a lot of attention (Zhang et al., 2016). Glycerol, characterized by polar polyalcohol structure, can easily penetrate into the biomass and provides an effective reagent for biomass fractionation and improving enzymatic hydrolysis of cellulose. The main advantages of using glycerol for lignocellulosic biomass pretreatment is associated with the possibility of lignin separation and recovery without significant chemical modifications, which can be used as feedstock for direct or indirect production of industrial commodities, such as: adhesives, resins, aromatics, carbon fibers (Zhang et al., 2016, Choi et al., 2019). Moreover, this type of pretreatment is usually connected with low carbohydrate degradation into by-products, while, solvent (glycerol) and be recovered and recycled back to biomass pretreatment (Zhang et al., 2016). Compared to other pretreatments (acid-, alkaliand oxidative-based), glycerol has only been used as solvent during pretreatment of a very limited range of agricultural residues, e.g. wheat straw (Zhang et al., 2016), corn straws, rice husk (Diaz et al., 2015) or sugarcane bagasse (Lv et al., 2018). According to best of our knowledge, succinic acid has never been produced from Miscanthus × giganteus pretreated with glycerol as delignification agent. The pre-treated material is usually enzymatically hydrolysed, in order to obtain fermentable sugars and this process represents one of the most essential step in the processing of lignocellulosic materials into valuable bio-products, including succinic acid (Pino et al., 2018). Due to the complex, rigid and heterogenic structure of lignocellulosic polysaccharides, their complete breakdown into monosaccharides requires several hydrolytic enzymes with diverse action modes and working in a synergic manner (Van Dyk and Pletschke, 2012). The available enzymatic cocktails show various levels of effectiveness, depending on the characteristics of the feedstock used. Up to now, Celluclast® 1.5L, supplemented with Novozyme 188 containing β-glucosidase (both Novozymes, Denmark) has been the most widely used. These multi-enzyme products were applied in the hydrolysis of various lignocellulosic feedstocks, including miscanthus biomass (PieprzykKokocha et al., 2016). Recently, the use of newer generation enzymatic mixtures, e.g. Cellic® CTec2 (Novozyme, Denmark) or Accelerase 1500 (DuPont) is becoming more and more popular. Concept of succinic production via biotechnological processes as well as succinic broths pre-purification and treatment ensuring a final product purity suitable for chemical upgrading still remains a challenge. An effective and cost-efficient production of succinic acid is strictly connected with the amount of sugars released during pretreatment and/or subsequent enzymatic hydrolysis. This is the first study evaluating Miscanthus × giganteus pretreatment with organosolv method (using waste glycerol), on the biomass delignification, sugar

2. Materials and methods 2.1. Feedstock Biomass of Miscanthus (M. × giganteus), used in this study, was obtained from a farm located in the north of Poland (Pomeranian province). The biomass was planted on plowed soil. Seedlings from domestic plantations were put to a depth of 3–5 cm. The biomass was harvested in spring 2015 and originated from 5 years plantation. The biomass was dried indoors for 2 months at approx. 18 °C to simulate field-drying. Dry biomass was fragmented to pieces between 1 and 2 cm, using Retsch crusher (BB200, Germany). The dry matter (DM) content was 93–94% (w/w). The biomass contained: cellulose (42.5 ± 1.8%), hemicellulose (25.0 ± 1.2%), including 23.2 ± 1.1% of xylan as well as insoluble lignin (23.0 ± 1.5%) and 2.75 ± 0.1% of ash. 2.2. Biomass pretreatment and enzymatic hydrolysis Firstly, the biomass was treated with glycerol fraction without dilution (100% glycerol fraction, including about 90% w/w of glycerol) and glycerol:water mixtures, containing: 80% and 70% of glycerol (w/ w). Secondly, glycerol-water mixture of 80:20 (w/w %) was supplemented with H2SO4 (1.0; 1.25; 1.5% w/v) (Table 1). Pretreatment was conducted at 160 °C for 10 min and biomass loading of 10% w/v. Separated solids were washed with 2.0% NaOH solution to remove the adsorbed lignin from its surface. Next, solid fraction was washed with distilled water until reaching a neutral pH value. Solid fraction recovered after glycerol-based pretreatment, in conditions considered as the most effective (80:20 glycerol-water mixture with 1.25% of H2SO4), was used as feedstock for enzymatic hydrolysis. Enzymatic hydrolysis was also performed on untreated biomass (nonpretreated by glycerol) to compare with enzymatic assays of pretreated biomass (Table 2). The hydrolysis was conducted at a solid loading of 5% in a 50 mM sodium citrate buffer, pH 5.4. Each reaction was performed in a 300 mL. Erlenmeyer flask (working volume of 100 mL) at 50 °C for 96 h. Five commercial cellulolytic enzyme cocktails acting on lignocellulosic carbohydrates were used in this study: i) Viscozyme® L - a blend of multiple cellulolytic and hemicellulolytic enzyme from Aspergillus aculeatus, 26.5 FPU/g (V2010, Sigma-Aldrich); ii) Carezyme 1000L® – a blend of enzymes secreted by Aspergillus niger – 30.1 FPU/g (C2605, Sigma-Aldrich); iii) β-Glucanase from Trichoderma longibrachiatum – a mixture of enzymes with mainly β-1-3 and β-1-4-glucanase, xylanase, and cellulase activities, 42.1 FPU/g (G4423, Sigma-Aldrich); iv) Cellic® CTec2 - a blend of aggressive cellulases, hemicellulases and β-glucosidases from Trichoderma sp., 64.6 FPU/g (Novozymes, Denmark); v) Cellic® HTec2 – reach in endoxylanases with high specificity toward soluble hemicellulose, 10.3 FPU/g (Novozymes, Denmark). Cellulase activity expressed as filter paper unit (FPU) per gram of enzyme solution was determined by the Ghose method, established by International Union of Pure and Applied Chemistry (IUPAC). 83

Bioresource Technology 278 (2019) 82–91

– 0.50 0.73 0.70 0.87 1.17 1.27 – 1.17 2.17 2.83 3.50 3.83 5.83

± ± ± ± ± ±

0.2d 0.3 cd 0.3bc 0.3bc 0.3b 0.8a

TPC, g L−1

glycerol fraction without dilution (100% glycerol fraction, including about 90% w/w of glycerol), glycerol:water mixtures containing 80% w/w of glycerol, glycerol:water mixtures containing 70% w/w of glycerol, FIS – water insoluble fraction, GL – waste glycerol after biodiesel production, W – water, DM – dry matter.

Hydrolysate obtained after hydrolysis with the most effective enzymatic mixture (10% w/w Cellic® CTec2, 5% w/w β-Glucanase and 1% w/w Cellic® HTec2) was used as feedstock for succinic acid production. Fermentation was performed in a batch mode, using 200 mL sealed anaerobic bottles with 100 mL working volume. Before fermentation, hydrolysates were autoclaved at 121 °C for 20 min. The fermentation was conducted for sample without synthetic medium (nutrients) as well as for hydrolysate mixed with sterile hydrolysate at 75:25 (v/v), which was selected as the most optimal, based on our previous studies (Gunnarsson et al., 2015). Medium composition and start-up of the fermenters were previously described (Gunnarsson et al., 2015). In each case, about 5% (v/v) of exponentially growing inoculum (OD660 = 4.6) was added. The strain of A. succinogenes 130Z (DSM 22257) was obtained from DSMZ (German Collection of Microorganisms and Cell Cultures). 1 g of solid MgCO3 per 1 g of total sugar was supplied and acted as an indirect CO2 source and pH buffer for fermentation medium. Samples of 1 mL were taken (after 0, 3, 6, 12, 15, 18, 24, 36, 48 h) and used for analysis of sugars (glucose, xylose), acids (succinic, acetic, formic, lactic) and optical density at 660 nm (OD660) measurements. 2.4. Downstream separation and purification processes for succinic acid recovery The fermentation broth was centrifuged for 20 min at 10,000 rpm to separate the cell biomass and filtered via ultrafiltration membranes (polyacrylonitrile membrane 12%, 0.2 μm pore size), using AMICON® unit (RC800, USA). The membrane was prepared by phase inversion method (Fryczkowska et al., 2017). Next, broths were pre-purified using activated carbon (1% w/v, reaction time: 1 h) to remove the organic impurities that contributed to the dark color of the broth. After pre-purification (centrifugation, ultrafiltration and activated carbon treatment), the broth was treated with the following combinations of unit operation processes: resin-based method followed by vacuum distillation and crystallization. After crystallization (the last step of purification and recovery method), the slurry was filtered (Whatman paper no. 1) and the obtained crystals were dried at 70 °C for 24 h. Amberlite IR 120H cation-exchange resin was used and the clear aqueous fermentation broth (after pre-purification) was passed through the resin packed in a glass column at a rate of 10 mL min−1. The pH of the effluent after ion-exchange resin was about 2.0. After each cycle, resin regeneration by using 5% HCl was performed as the resin after the process was in a dissociated state. It was then followed by elution with deionized water until the effluent reached a neutral value. Secondly, broths were treated using vacuum distillation to separate volatile acids (acetic and formic acid) and water. Heldolph instrument, type Basis Hei-VAP HL (supply power 1400 W, No. 563-01100-00, Germany), was used for vacuum distillation. The temperature was about 65 °C and pressure of vacuum filtration amounted to 30 mm Hg. During this process, the filtrate (liquid used for succinic acid separation) was concentrated to about 10% of its original volume. Crystallization of succinic acid was performed at 4 °C for 12 h.

c

b

a

– 1.39 ± 0.4d 4.39 ± 0.6c 3.51 ± 0.5c 9.47 ± 0.6b 13.75 ± 0.8a 10.8 ± 1.0b 1.2d 1.0c 1.0c 0.8b 1.4a 2.1b ± ± ± ± ± ± – 2.89 7.54 6.43 15.4 22.7 16.7 – 4.87 7.57 5.52 6.00 14.7 22.0 – – – – – – 0.87 ± 0.20 ± ± ± ± ± ± – 85 81 79 73 63 60 ± ± ± ± ± ± ± 23.0 24.7 21.0 21.1 14.3 9.18 11.1 ± ± ± ± ± ± ± 25.0 22.7 22.2 22.4 24.9 21.0 5.00 ± ± ± ± ± ± ± 42.5 48.3 52.7 52.2 56.0 68.5 67.5 Untreated GL (100%)a GL:W (80:20)b GL:W (70:30)c GL:W (80:20)b + 1.0% H2SO4 GL:W (80:20)b + 1.25% H2SO4 GL:W (80:20)b + 1.50% H2SO4

Glucan, % DM

1.8d 1.7c 1.7bc 1.5bc 1.2b 1.3a 1.2a

Xylan, % DM

1.2a 2.2a 1.7a 1.3a 1.7a 1.4a 0.5b

Lignin, % DM

1.5a 1.5a 2.2a 2.0a 1.5b 0.8c 0.7bc

FIS, %

1.2a 1.1b 1.0b 1.0c 1.5d 2.1d

Xylose, g L−1

± ± ± ± ± ±

0.8c 0.6c 0.3c 1.00c 1.1b 2.0a

Lignin fraction, g L−1

Lignin fraction, g 100 g−1

Acetic acid, g L−1

± ± ± ± ± ±

0.1b 0.2ab 0.3ab 0.1ab 0.3a 0.3a

Firstly, the efficiency of commercially available cellulolytic enzyme preparations such as Cellic® CTec2, Viscozyme® L, Carezyme 1000L®, and β-glucanase (dosage of 15% w/w expressed as percentage of substrate dry matter; determined during our previous results, data not shown) was investigated. Secondly, Cellic® CTec2 and β-glucanase, considered as the most efficient, were added in a mixture, using various percentage dosage of these two enzymatic cocktails (i.e. 10 and 5% w/ w; 7.5 and 7.5% w/w; 5 and 10% w/w, respectively). Finally, the mixture of Cellic® CTec2 (10% w/w) and β-glucanase (5% w/w) found to be the most efficient was supplemented with Cellic® HTec2 (dosage of 1 or 2% w/w) to study its influence on glucan and xylan saccharification. 2.3. Succinic acid production procedure

Glucose, g L−1

Pretreatment liquor Pretreated solid fraction (FIS) Pretreatment/catalysts (% w/w)

Table 1 Characteristics of untreated and pretreated miscanthus by glycerol-based method (solid fractions and pretreatment liquors) (average values n = 4, ± standard deviations, the same letters represent data equivalent statistically p > 0.05, FIS – water insoluble fraction, GL – waste glycerol after biodiesel production, W – water, DM – dry matter).

K. Dąbkowska et al.

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Table 2 Results of enzymatic hydrolysis obtained after 72 h of reactions carried out with various enzymatic cocktails used as catalysts (average values n = 3, ± standard deviation, the same letters represent data equivalent statistically p > 0.05). Assay

Enzymatic cocktail/mixture (% w/wa)

Glucose, g L−1

Glucose, yield, %

Xylose, g L−1

Xylose yield, %

Total sugar, g L−1

Total yield, %

R1 R2 R3 R4 R5 R6 R7 R8 R9 R10

CTec2 (15%) CTec2 (15%) VZL (15%) CZ1000L (15%) βGlu (15%) CTec2 (10%) + βGlu (5%) CTec2 (7.5%) + βGlu (7.5%) CTec2 (5%) + βGlu (10%) CTec2 (10%) + βGlu (5%) + HTec2 (1%) CTec2 (10%) + βGlu (5%) + HTec2 (2%)

2.97 30.2 8.16 12.4 25.9 32.8 30.5 30.1 35.6 35.8

12.5 78.7 21.4 32.4 67.7 85.8 79.8 78.8 93.1 93.7

0.58 7.41 2.74 1.56 8.11 8.16 7.73 7.26 8.25 8.26

4.20 59.8 22.7 12.9 68.0 68.5 64.8 64.3 69.2 68.5

3.56 37.2 10.9 13.9 34.0 40.9 38.2 37.4 43.8 44.2

9.47 74.3 21.7 27.9 67.8 81.6 76.3 74.5 87.5 87.9

± ± ± ± ± ± ± ± ± ±

0.3 g 0.8c 0.5f 0.7e 0.9d 0.4b 0.2c 0.5c 0.7a 0.7a

± ± ± ± ± ± ± ± ± ±

1.1 g 2.0c 1.4f 1.8e 1.1d 1.0b 0.5c 1.4c 2.0a 1.9a

± ± ± ± ± ± ± ± ± ±

0.1f 0.2bc 0.3d 0.2e 0.3ab 0.3ab 0.5ab 0.5c 0.3a 0.3a

± ± ± ± ± ± ± ± ± ±

0.5e 1.8b 2.3c 1.6d 2.3a 2.4a 2.1ab 5.1ab 2.3a 2.4a

± ± ± ± ± ± ± ± ± ±

0.3 g 1.0c 0.4f 0.7e 1.1d 0.5b 0.6c 0.7c 0.7a 0.5a

± ± ± ± ± ± ± ± ± ±

0.6 g 1.8c 0.8f 1.3e 2.3d 0.5b 0.9c 1.5c 0.9a 0.9a

a enzymatic cocktails weight expressed as substrate dry matter used during experiment (% w/w), CTec2 – Cellic® CTec2, VZL – Viscozyme® L, CZ1000L – Carezyme 1000L®, βGlu – β-glucanase, HTec2 – Cellic® HTec2, R1 – untreated biomass, R2-10 – solid fraction after glycerol based pretreatment GL:W (80:20) + 1.25% H2SO4.

2.5. Calculations

SA − precipitation yield (%) =

2.5.1. Biomass pretreatment and hydrolysis Effectiveness of biomass pretreatment was based on FIS (fraction insoluble) recovery (solids after pretreatment), increase of cellulose content in pretreated biomass, biomass delignification as well as enzymatic digestibility of pretreated biomass. The chemical composition of pretreated biomass (cellulose, hemicellulose, lignin) was presented as percentage of dry matter as well as the values were related to its initial content in untreated biomass (% of increase/decrease). The amount of FIS (fraction insoluble solids after pretreatment) was calculated according to Kuglarz et al. (2016). Biomass delignification was based on acid-insoluble lignin in untreated and pretreated biomass and calculated according to (Eq. (1)):

Delign. (%) =

(

FIS

LigninUntreated − LigninPret . solids · 100 LigninUntreated

) ·100

(3)

Overall yield (%) =

(4)

SA in crystals recovered (g) – represents the amount of succinic acid recovered after downstream processing of 1 L of fermentation broth, measured after unit dry weight of crystals diluted in distilled water. SA in liquor before precipitation (g) – amount of succinic acid in 1 L of liquor before precipitation. SA in fermentation broth (g) – initial amount of succinic acid in 1 L of fermentation broth.

(1)

Crystals purity was defined as follows (Eq. (5)):

Purity (%) =

Ligninuntreated – lignin content in biomass before pretreatment, g. LigninPret.solids – lignin content in dried solid fraction, recovered after pretreatment, g.

SA in crystals recovered ·100 Total crystals recovered

(5)

Total amount of crystals recovered (g) – dry weight of crystals recovered after downstream processing of 1 L fermentation broth. 2.6. Analytical methods

Effectiveness of enzymatic hydrolysis was based on glucose, xylose and total sugar yields calculated according to our previous studies (Kuglarz et al., 2016).

Total solids (TS), volatile solids (VS), ash and protein content were determined according to standards methods (APHA, 2005). pH was measured using a standard pH meter (Aldrich® glass pH electrode, Z113077-1EA). The content of cellulose, hemicellulose and lignin in raw material as well as solid residues after pretreatment were determined according to the National Renewable Energy Laboratory (NREL) analytical methods for biomass characterization (Sluiter et al., 2008). After the pretreatment, the liquors subjected to lignin precipitation, using 5% (v/v) solution of H2SO4 to decrease pH to ∼2. After precipitation, samples were filtered through a pre-weighted filter paper using a vacuum filtration unit. The precipitated lignin was washed twice with 1 L of hot distilled water (pH = 6.6–6.8) in order to remove impurities. Concentrations of sugars and organic acids (succinic, acetic, formic) were measured by using high performance liquid chromatography HPLC (Agilent 1260 Infinity, Germany) equipped with a BioRad Aminex HPX-87H column at 63 °C and ultraviolet (UV) and refractive index (RI) detector (67162A, Germany), using 4 mM H2SO4 as eluent at 0.6 mL∙min−1 flow rate. All chemicals used in this study were of analytical grade. Glycerol used in the study as solvent for biomass fractionation was characterized as technical class and its chemical composition was as follows: glycerol 90 ± 1.5%; inorganic components: 2.55 ± 0.5%, methanol: < 0.02%; non-glycerol organic substances: < 2.0%. Density of the glycerol fraction amounted to 1235 g L−1 at 20 °C; pH 6.3–6.4. The glycerol fraction was obtained

2.5.2. Succinic acid fermentation and recovery 2.5.2.1. Succinic yield. Succinic acid yield (YSA) was calculated as the amount of succinic acid (g) obtained per 1 g of sugars (glucose + xylose) consumed (Eq. (2)).

SAProd . ·100 SugarConsumed

SA in crystals recovered ·100 SA in fermentation broth

where:

where:

YSA (%) =

SA in crystals recovered ·100 SA in liquior before precipitation

(2)

where: SAProd. – concentration of succinic acid produced (g L−1); SugarConsumed – amount of glucose and xylose consumed during succinic acid fermentation (g L−1). 2.5.2.2. Crystallization. Effectiveness of the crystallization performed as the last step of broth downstream processing was presented as an unit yield, expressed as the amount of succinic crystals recovered with respect to the succinic acid content in liquor before precipitation (Eq. (3)). Whilst, the overall yields included succinic acid losses during previous steps of separation (pre-purification, resin treatment, vacuum distillation) and were referred to initial succinic acid concentration in fermentation broths (Eq. (4)). 85

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A

from biodiesel production, based on rapeseed (Podlasie Province, Poland). High resolution 1H NMR spectrum of the obtained succinic acid crystals was recorded with a Brucker Ascend 600 MHz spectrometer (USA). In case of succinic acid fermentation, cell growth (OD660) was monitored by measuring the optical density at 660 nm, using a spectrophotometer (Hach Lange DR 5000, A23778, Germany). Before OD analyses, insoluble MgCO3 was removed, by mixing the sample with 7% (w/v) HCl at the ratio of 1:1.

Process loss

Pretreatment liquor

Solid fraction

100% 90% 80%

Hemicellulose

70%

2.7. Statistical analysis

60% 50% 40% 30%

All results are presented as average values (n = 3 or 4) with standard deviations ( ± ). The characteristics of biomass (untreated, pretreated) as well as results obtained during enzymatic hydrolysis were compared statistically. One-way ANOVA test followed by Tukey’s HSD tests were used for multiple comparisons between samples, with the level of significant set at 0.05. The same letters represent data equivalent statistically (p > 0.05).

20% 10%

B

3. Results and discussion

Process loss

Pretreatment liquor

GL(80%)+ 1.50% acid

GL(80%)+ 1.25% acid

GL(80%)+ 1.0% acid

GL(70%)

GL(80%)

GL(100%)

0%

Solid fraction

100%

3.1. Biomass pretreatment

90% 80%

Lignin fraction

All pretreatment methods applied in this study showed a significant influence on the carbohydrate content of pretreated material. Glucan content increased by 59–61% after biomass pretreatment with glycerol method in acidic conditions (1.0–1.25% H2SO4) (data equivalent statistically, p > 0.05, Table 1). Further increase of catalyst (1.5% H2SO4) did not have a positive influence on glucan content in pretreated biomass. Pretreatment based on glycerol addition (without acid) resulted in significantly lower increase of glucan content, i.e. 14–24% (Table 1). In all tested pretreatments, between 96% and 99% of cellulose was recovered in pretreated biomass (water insoluble fraction) (data not shown). This proves the effectiveness of using glycerol in preserving cellulose fraction in pretreated biomass. Only insignificant amounts of glucose were released after pretreatment with the highest acid addition (1.5% H2SO4) (Table 1). This was also associated with no glucose degradation products (5-hydroxymethyl-2-furaldehyde, HMF) released into pretreatment liquor. The latter compound can further degrade into levulinic acid. However, no levulinic acid was observed in liquids after biomass pretreatment (data not shown). Compared to cellulose, structure of hemicellulose is more susceptible to being hydrolyzed into its constituents sugars (Gunnarsson et al., 2015, Kuglarz et al., 2016). The conditions of pretreatment influenced significantly the biomass susceptibility to hemicellulose hydrolysis. The highest hemicellulose release into xylose (30% of initial hemicellulose fraction) was obtained after glycerol pretreatment in acidic conditions (80% of glycerol, 1.25 H2SO4). Relatively lower release of xylose (14–17% of initial hemicellulose fraction) into pretreatment liquor was recorded for glycerol pretreatment with lower (1.0 H2SO4) and the highest acid addition in this study (1.5% H2SO4) (Fig. 1). In all cases, arabinose was observed in pretreatment liquors in small concentrations (< 1.0 g L−1, data not shown), which was connected with insignificant content of initial arabinan in untreated biomass (1.8% of dry matter). Glycerol treatment of miscanthus in acidic conditions (≤1.25% H2SO4) resulted in more than twice lower C5 sugar loss compared to most commonly used pretreatments, i.e. dilute acid method or alkaline method (Gunnarsson et al., 2015). This is a valuable advantage of using glycerol for lignocellulosic biomass pretreatment (Kumar and Sharma, 2017, Zhao et al., 2017, Zhang et al., 2016). Effective biomass pretreatments enable lignin dissolution and separation, which results in obtaining the biomass with lower lignin content. The highest lignin solubilization, i.e. 40–60% were obtained after glycerol pretreatment (80% glycerol) in acidic conditions (1.0–1.25% H2SO4), which was equivalent to 9.5–13.8 g lignin/100 g initial biomass treated (Table 1). In these cases, pretreated biomass

70% 60% 50% 40% 30% 20% 10%

GL(80%)+ 1.50% acid

GL(80%)+ 1.25% acid

GL(80%)+ 1.0% acid

GL(70%)

GL(80%)

GL(100%)

0%

Fig. 1. Influence of glycerol-based pretreatment on hemicellulose and lignin recovery (A –hemicellulose, B – lignin, GL – glycerol, acid – sulfuric acid used for biomass pretreatment).

contained between 9 and 11% dry matter of lignin (Table 1), which is at the level considered as beneficial for subsequent enzymatic hydrolysis (Chang and Holtzapple, 2000, Novo et al., 2011). Key factors responsible for lignin dissolution during organosolv pretreatment are associated with hydrolysis of the internal bonds in lignin as well as lignin-hemicellulose bonds. Cleavage of ether linkages is considered as a primary agent responsible for lignin fragmentation, while, glycerol is responsible for solubilizing the lignin fragments produced by cleavage of the ether linkages. Lignin dissolved in pretreatment liquors is usually recovered via precipitation (Nitsos et al., 2018, Meng et al., 2019). Similarly to hemicellulosic fraction, further increase of acid used as catalyst did not impact the biomass delignification (Fig. 1). Whilst, application of glycerol without water addition as solvent for biomass disruption and delignification allowed to solubilize only 9% of hemicellulose and 6% of lignin fraction (Fig. 1). Effective miscanthus disruption required application of glycerol:water mixtures containing 80% of glycerol (Table 1, Fig. 1). This is in agreement with previous results stating that water autoionization at high temperatures, coming from glycerol-water mixtures, helps to depolymerize hemicellulose via selective hydrolysis of glycosidic bonds and acetyl groups. Whilst, H3O+ ions from ionization of acetic acid released due to hydrolysis of hemicellulose fraction (Table 1), enhance hydrolytic cleavage of ether bonds, present in lignin. Consequently, water plays a very important 86

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3.2.2. Enzymatic hydrolysis with mixtures of commercial cellulolytic cocktails The results of hydrolysis performed with simultaneous use of Cellic® CTec2 and β-glucanase, (i.e. enzymatic cocktails which exhibited the highest affinity towards glucose and xylose during testing various commercial cellulolytic cocktails), namely, application of Cellic® CTec2 (10% w/w) and β-glucanase (5% w/w) mixture led to slightly higher (approx. 7–8%) final glucose yield than the value observed after usage of Cellic® CTec2 (15% w/w) (R2 and R6, Table 2, data different statistically, p > 0.05). Glucan saccharification was significantly reduced after replacement of > 33% of Cellic® CTec2 dosage by β-glucanase (R7-8, Table 2), in comparison to hydrolysis catalyzed by Cellic® CTec2. Xylose yield was almost unaffected by supplementation of Cellic® CTec2 with β-glucanase (Table 2). In the next experiments, Cellic® CTec2 (10% w/w) and β-glucanase (5% w/w) mixture was supplemented with reach in endoxylanases - Cellic® HTec2 (Table 2, R9-R10). Irrespective of Cellic® HTec2 dose (1% or 2% w/w), addition of third enzyme (Cellic® HTec2) had a positive influence on both the course of reaction as well as on final glucan saccharification yield (Table 2). Glucose conversion showed a gradual increase reaching a plateau after about 48 h of hydrolysis reaction, while, final glucose yield was almost the same in both reactions (catalyzed by addition of 1% or 2% w/w Cellic® HTec2, data equivalent statistically p > 0.05). These values (93–94% after 72 h) were about 14–15% and 7–8% higher compared to yields obtained with Cellic® CTec2 (15%) and Cellic® CTec2 (10%)/βglucanase (5%) mixture (Table 2, data statistically different p < 0.05), respectively. Improvement of glucan hydrolysis by Cellic® HTec2 is in accordance with our previous research (Kuglarz et al., 2018). The increased final glucan saccharification yield observed for reaction catalyzed by the mixture of Cellic® CTec2 (10%) and β-Glucanase (5%) in comparison to Cellic® CTec2 (15%) could have been caused not only by the cellulolytic enzymes present in β-glucanase but also by its xylanase activity. As regards xylan saccharification, the results show that the combination of different enzymatic cocktails only slightly affected the reaction course as well as final yield of xylose released from the biomass after glycerol pretreatment. All enzymatic mixtures of Cellic® CTec2 and β-Glucanase or Cellic® CTec2/β-Glucanase/Cellic® HTec2 did not improve xylose yields compared to xylose yield obtained for β-Glucanase used as the only enzymatic cocktail (Table 2, data statistically equivalent, p > 0.05). Whilst, Cellic® CTec2/β-Glucanase/Cellic® HTec2 mixtures allowed to achieve higher xylose yields than xylose values obtained for Cellic® CTec2 as the only cocktail (Table 2). The enzymatic mixture composed of Cellic® CTec2, β-Glucanase and Cellic® HTec2 (dosage of 10% w/w, 5% w/w and 1% w/w respectively), was considered as the most effective for miscanthus, pretreated with glycerol at acidic conditions and thus was selected for subsequent experiments (succinic acid–based fermentation). The analyzed method of mixing different enzymatic cocktails (Cellic® CTec2, β-Glucanase and Cellic® HTec2) allowed to achieve the highest total sugar yield (88%), which was significantly higher compared to conversion yields obtained after usage of single enzymatic cocktails or Cellic® CTec2 and βGlucanase mixtures (Table 2). These results are superior compared to other studies on enzymatic hydrolysis of miscanthus pretreated with various methods. For example, hydrolysis yields below 60% were obtained, when M. giganteus was pretreated with ammonia fiber expansion method before subsequent hydrolysis with mixture of Spezyme SP and Novozyme 188 (Murnen et al., 2007). Our results indicate that the strategy of mixing commercially available cellulolytic enzyme preparations may in the future contribute to the development of new and improved enzymatic mixtures. It is relevant to consider that this novel approach can be taken into account among other enzymes requiring technologies endeavoring to enhance economic efficiency and fermentation yields of valuable chemicals, including production of succinic acid from pretreated miscanthus.

role in glycerol:warer reaction mixtures used for biomass pretreatment, which is connected with its nucleophilic behavior during ether bonds hydrolysis, while, glycerol is responsible for solubilizing lignin fragments obtained by cleavage of the ether bonds and transporting them from biomass cells into the solution (Novo et al., 2011). Considering effectiveness of the pretreatment, miscanthus pretreated with glycerol (80% of glycerol content) in acidic conditions (1.25% H2SO4) allowed to reach the highest total content of carbohydrates in biomass dry matter, i.e. 88–89% (Table 1). Taking into account the fact that Actinobacillus succinogenes consumes both sugars C5 and C6 (Gunnarsson et al., 2015), the pretreated biomass constitutes a valuable feedstock for biochemical production. Results obtained in this study clearly showed that glycerol pretreatment of miscanthus in acidic conditions (< 1.5% H2SO4) did not cause significant sugar losses as well as significant amounts of degradation products, which is a benefit of this method.

3.2. Enzymatic hydrolysis 3.2.1. Hydrolysis with various commercial cellulolytic cocktails In all the cases, glucose and xylose were the major monomeric sugars determined in the hydrolysates, though trace amounts of arabinose (< 1% of monosaccharides present in hydrolysates) were also detected (data not shown). As expected, the enzymatic hydrolysis of the raw (untreated) miscanthus was a low-efficient process with the final glucose and xylose yields equaled to 12.5% and 4.2%, respectively (R1, Table 2). This is in accordance with previous studies (Yang et al., 2015). The use of glycerol for pretreatment improved biomass susceptibility to enzymatic saccharification. The highest yields of glucose (79%) were obtained after hydrolysis catalyzed by Cellic® CTec2. In this case, total sugar concentration was more than ten times higher compared to its value determined for untreated biomass (R2, Table 2), which can be explained by relatively high activity of carbohydrate degrading enzymes present in this enzyme blend. Both glucose and xylose reaction course reached a plateau state after 48–72 h and further hydrolysis has no positive effect on sugar release. Application of β-glucanase resulted in higher xylose yield compared to results obtained for Cellic® CTec2 (R2 and R5, Table 2, data statistically different p < 0.05). The least satisfactory results (glucose and xylose yields < 32%) were found for reactions catalyzed by Viscozyme® L and Carezyme® 1000L (R3-R4, Table 2). Hydrolysis results of miscanthus (Cellic® CTec2), pretreated with glycerol in acidic conditions, are superior to most of previous studies on miscanthus pretreatment by various methods. For example, M. giganteus pretreated in 5% NaOH at 121 °C and hydrolysed with Celluclast® 1.5L exhibited glucan saccharification yields equaled to 52%, while, almost all hemicellulose (94.6%) was degraded during the pretreatment step (Michalska et al., 2015). In another study concerning dilute acid and hydrophilic ionic liquids pretreatment, the maximum glucose yield obtained in reaction catalyzed by Cellulase from Trichoderma reesei did not exceed 75% (Auxenfans et al., 2014). This value is similar to the best values of glucose yield obtained in current study, i.e. for the process catalyzed by Cellic® CTec2 (Table 2). There are also some studies showing higher enzymatic digestibility (with Cellic® CTec2) of M. giganteus (glucan and xylan yields reaching values close to 100% after 72 h) compared to glucose and xylose yields obtained in the present study. However, the biomass was pretreated in more severe conditions (i.e. gaseous ammonia and subsequent hot water processing at high temperatures up to 150 °C in the 1st stage and up to 220 °C in the 2nd stage) compared to present study (Cayetano and Kim, 2018). To the best of our knowledge, there is no available reports evaluating the influence of organosolv pretreatment, using glycerol, on effectiveness of miscanthus hydrolysis catalyzed by Cellic® CTec2.

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Fig. 2. Course of succinic acid fermentation (A – hydrolysate after glycerol pretreatment with 1.25% H2SO4 and fermented without nutrients addition, B - hydrolysate after glycerol pretreatment with 1.25% H2SO4 and fermented at 75:25 hydrolysate: nutrients mixing ratio, sugar util. (%) = [(initial sugar content - sugar content after succinic acid production)/initial sugar content)]∙100, succinic yield (%) = calculated as [g succinic acid produced/g sugar consumed]∙100).

xylose (1.2–3.5 g L−1, Fig. 2) was recorded after 48 h of succinic fermentation. This is in accordance with previous results showing that A. Succinogenes prefers glucose as substrate rather than C5 sugars (e.g. xylose) (Salvachua et al., 2016). Fermentation of hydrolysate at 75:25 mixing ratio (hydrolysate:medium) allowed to utilize > 96% of initial sugar content (glucose and xylose). Comparing the time course of fermentation, it was observed that cell density (shown as DCW in Fig. 2) reached higher maximum in shorter time when fermenting a more diluted hydrolysate (75:25 v/v hydrolysate to medium mixing ratio). This indicates that nitrogen source and minerals could be a limiting factor in succinic acid production from hydrolysates after glycerol pretreatment (Du et al., 2007). Depending on fermentation conditions e.g. type of feedstock used and pretreatment method other metabolites, such as acetic, formic, lactic acid or ethanol, can be produced in different amounts during fermentation (McKinlay et al., 2005). In the initial growth phase, succinic, acetic and formic acids were simultaneously produced. After 24 h of the process, the production of acetic and formic acids ceased, which is connected with low cell-biomass generation during stationary phase and consequently lower energy requirements (Fig. 2). In this case, carbon flux shifts towards succinic acid production (Pateraki et al., 2016).

3.3. Succinic fermentation Succinic yields of 75 ± 2.0% (0.75 g∙g−1) and 82 ± 1.8% (0.82 g∙g−1) were achieved for hydrolysate without and with nutrients addition (75:25 v/v hydrolysate to medium mixing ratio), respectively (Fig. 2). The maximal theoretical molar yield for succinic acid per mole of glucose corresponds to 1.71 (12/7) (Eq. (6)) and this equation equals to 1.12 g g−1 when expressed as a mass unit. This theoretical maximal yield would be achieved anaerobically and represents a theoretical situation where the microorganisms do not use substrate for growth, maintenance or an external electron acceptor.

C6 H12 O6 +

6 12 6 CO2 → C4 H6 O4 + H2 O 7 7 7

(6)

Succinic acid is produced anaerobically by naturally producing organisms, including A. succinogenes, via the reverse TCA (tricarboxylic acid) cycle starting with a phosphoenolpyruvate carboxylation route (Pateraki et al., 2016). The corresponding standard free Gibbs energy of reaction (ΔGr0 ) corresponds to −229 kJ mol−1 and its negative value indicates that product reaction could allow production of biochemical useful energy (ATP, Adenozyno-5′-trifosforan). Glucose and xylose were simultaneously consumed during succinic acid fermentation. There was no glucose detected at the end of the fermentation, while, residual 88

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Fig. 3. Succinic acid production and succinic broth pre-purification and downstream processes aimed at maximizing the production of high purity product (A – simplified mass balance of Miscanthus biomass, B – concept of succinic broth pre-purification and downstream processes, a - biomass pretreated with 80% glycerol with 1.25% of H2SO4, b – enzymatic hydrolysis with mixture of 10% Cellic® CTec2 + 5% β-Glucanase + 1% Cellic® HTec2, c – succinic acid production at 75:25 v/v hydrolysate to medium mixing ratio, d - broth pre-purification and downstream treatment, e – after pre-purification: centrifugation, ultrafiltration and activated carbon treatment, f – the residue after ion-exchange was diluted due to additional column washing with 250 mL of distilled water, g – concentrated residue after vacuum distillation to about 5% of initial broth volume, h – water, acetic and formic acids, DM – dry matter, SA. – succinic acid; AA – acetic acid; FA – formic acid).

et al., 2011). Effectiveness of crystallization applied as the last step of broth treatment, calculated as an unit process, was 68.0 ± 1.5%. Whilst, overall yield, including losses during previous purification steps, amounted to 50.6 ± 2.1%. However, a lower recovery was compensated by the highest purity of succinic crystals, i.e. 98.9 ± 0.5% (Fig. 3), which allows to use the separated succinic acid for chemical transformations (Cheng et al., 2012). Whilst, omitting ultrafiltration after broth centrifugation resulted in lower succinic crystals purity (86–90%) (data not shown). The effectiveness of applied method of succinic broth purification and downstreaming (resin-based treatment + vacuum distillation + crystallization) resulted in significantly better yields and product purity compared to conventional precipitation methods (based on calcium succinate precipitation) as well as via direct crystallization method with broth acidification (Luque et al., 2009). The quality of crystals precipitated was confirmed by NMR analyses. The mother liquor after succinic acid precipitation contained between 32 and 34 g L−1 of succinic acid, which is strictly connected with specified succinic acid solubility (Li et al., 2010). For improved output of the separation processes, attempts aimed at recovering the residual succinic acid from mother liquor can be developed, e.g. concentrating the succinic content above its solubility and performing the second step crystallization.

3.4. Succinic broths purification and product recovery The initial step of the downstream process is usually connected with removal of biomass, color as well as protein residues. In our case, prepurification (centrifugation, ultrafiltration, activated carbon treatment) resulted in a completely clear broth (Filtrate I). The succinic acid loss during pre-purification amounted to about 8.0 ± 1.5% of its initial concentration in the fermentation broth (Fig. 3). Succinic broth (Filtrate I) was treated with following combinations of downstream processes: resin-based treatment + vacuum distillation + crystallization). In first step, pre-purified broths were filtered through the Amberlite IR 120H, cation-exchange resin. This resin exhibited high affinity to both sodium and potassium ions and allowed to change succinate into succinic acid. The pH of fermentation broth after passing through the resin was about 2.0. In our case, 75 ± 2.5% of the succinic acid present in the fermentation broth (in the form of succinate, pH = 6.8–7.0) was recorded in the effluent after resin treatment (Filtrate II, Fig. 3). About 17 ± 1.5% of initial succinic acid was lost using ion-exchange treatment. No change in resin capacity was observed after 15 cycles. However, regeneration of resin by using HCl was necessary after each cycle as the resin was in a fully dissociated state. The loss of other broth components amounted to 18 ± 1.5% and 22 ± 2.0% for acetic and formic acid, respectively. The residue after ion-exchange treatment was concentrated to about 5–6% of its original volume via vacuum distillation, (concentrated residue, Fig. 3). In this part of succinic broth downstream processing, acetic and formic acids were completely removed, while, there was no significant loss of succinic acids compared to its content after previous treatment steps. Acetic and formic acids are characterized by significantly lower boiling points than succinic acid and thus they vaporize together with the majority of water (Vlysidis

3.5. Mass balance of the overall process and future outlook Organosolv pretreatment of miscanthus in acidic conditions (80% w/ w glycerol, 1.25% H2SO4) and enzymatic hydrolysis of pretreated biomass at optimized dosage of commercially available cellulolytic preparations (10% w/w Cellic® CTec2, 5% w/w β-glucanase, 1% w/w 89

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References

Cellic® HTec2) resulted in high values of glucose (93%) and xylose (69%) yields as well a high total release of sugars, i.e. 87.5% (pretreatment + enzymatic hydrolysis) (Table 2). Future investigations are required to evaluate the effectiveness of the new proposed enzymatic mixture for various biomass lignocellulosic materials, including those from developing countries, rice straw or husk, sugarcane bagasse, maize straw (Ullah et al., 2015). Using A. Succinogenes, > 96% of initial sugar content (glucose and xylose) was utilized, which resulted in 422 kg of succinic acid as the main product. In the present study, acetic (84 kg) and formic (43 kg) acids were produced as the main fermentation byproducts (Fig. 3). The developed concept of broth purification and downstreaming (resin-based treatment + vacuum distillation + crystallization) resulted in 213 kg succinic acid (> 98%) per 1 Mg of biomass treated (Fig. 3). Pretreatment liquors containing glycerol added as solvent during biomass treatment were identified as the main by-products generated during biomass processing into succinic acid. Such liquors after lignin recovery can be recycled back to the biomass pretreatment step. Alternatively, pretreatment liquors can be used as feedstock for anaerobic digestion (AD). This fraction exhibited a high biodegradability (90%) and allowed to produce 410 dm3 CH4·kg VS−1 (data not shown). Additionally, the economy of a succinic acid production from miscanthus can be improved by effective utilization of lignin fraction. Lignin solubilization at optimized pretreatment conditions amounted to 60% (Table 1), which was equivalent to 129 kg lignin per 1 Mg of biomass treated (Fig. 3). Separated lignin can be used for heat or heat and power generation and integrated with lignocellulosic biorefinery for energy-intensive processes, such as: biomass organosolv pretreatment, purification of succinic broths. It should be mentioned that the present study is a simplified analysis, which is focusing on process development of products and main by-products. Meanwhile, the results obtained can be used as an important input for extended environmental and economic analyses. However, it is worth to mention that current LCA analysis clearly showed that bio-succinic acid production is a promising industrial alternative to the currently used petroleum counterparts. Moreover, competitiveness of bio-succinic acid production can be improved by optimization of fermentative process as well as upgrading the obtained succinic crystals to high added value chemicals (Moussa et al., 2016).

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4. Conclusions The results obtained in this study clearly confirmed that Miscanthus × giganteus after glycerol pretreatment in acidic conditions (1.25% H2SO4) and enzymatic hydrolysis can be considered as a promising feedstock for succinic acid production (succinic yield: 82%). Mixing different enzymatic cocktails (Cellic® CTec2, β-Glucanase and Cellic® HTec2) allowed to achieve a total sugar yield of 88%, which was significantly higher compared to conversion yields obtained for single enzymatic cocktails. Developed concept of pre-purification and downstream processes allowed to obtained high quality succinic acid (> 98%), which can be used for further chemical transformations. Acknowledgements This work was partially supported by Polish Ministry of Science and Higher Education via Juventus Plus program for young scientists “The concept succinic acid production from lignocellulosic biomass using waste CO2 (0395/IP2/2016/74)” (public funds for science in the years 2016–2019), conducting research at a high level and having outstanding scientific achievements. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.01.051. 90

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