Integrated production of cellulosic bioethanol and succinic acid from industrial hemp in a biorefinery concept

Accepted Manuscript Integrated production of cellulosic bioethanol and succinic acid from industrial hemp in a biorefinery concept Mariusz Kuglarz, Merlin Alvarado-Morales, Dimitar Karakashev, Irini Angelidaki PII: DOI: Reference:

S0960-8524(15)01484-4 http://dx.doi.org/10.1016/j.biortech.2015.10.081 BITE 15706

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

11 September 2015 20 October 2015 23 October 2015

Please cite this article as: Kuglarz, M., Alvarado-Morales, M., Karakashev, D., Angelidaki, I., Integrated production of cellulosic bioethanol and succinic acid from industrial hemp in a biorefinery concept, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.10.081

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Integrated production of cellulosic bioethanol and succinic acid from industrial hemp in a biorefinery concept

Mariusz Kuglarza, Merlin Alvarado-Moralesb, Dimitar Karakashevb, Irini Angelidakib∗ a

Faculty of Materials, Civil and Environmental Engineering, University of Bielsko-

Biala, Willowa 2, 43-309 Bielsko-Biala, Poland b

Department of Environmental Engineering, Technical University of Denmark, DK-

2800 Kongens Lyngby, Denmark

∗

Corresponding author. Tel: +45 4525 1429; fax: +45 4593 2850. E-mail:

[email protected] (I. Angelidaki)

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Abstract: The aim of this study was to develop integrated biofuel (cellulosic bioethanol) and biochemical (succinic acid) production from industrial hemp (Cannabis sativa L.) in a biorefinery concept. Two types of pretreatments were studied (dilute-acid and alkaline oxidative method). High cellulose recovery (> 95%) as well as significant hemicelluloses solubilization (49-59%) after acid-based method and lignin solubilization (35-41%) after alkaline H2O2 method were registered. Alkaline pretreatment showed to be superior over the acid-based method with respect to the rate of enzymatic hydrolysis and ethanol productivity. With respect to succinic acid production, the highest productivity was obtained after liquid fraction fermentation originated from steam treatment with 1.5% of acid. The mass balance calculations clearly showed that 149 kg of EtOH and 115 kg of succinic acid can be obtained per 1 ton of dry hemp. Results obtained in this study clearly document the potential of industrial hemp for a biorefinery.

Keywords: cellulosic bioethanol, succinic acid, industrial hemp, pretreatment, biorefinery

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1.

Introduction Bioethanol is considered as one of the most promising biofuels as it can be easily

incorporated into existing fuel systems and can partially substitute fossil fuels used in transportation (Talebnia et al., 2010). First generation bioethanol based on starch-based plants (maize, sugar cane) is not sustainable in many parts of the world where land utilization for energy crops cultivation is not possible. Therefore, second generation cellulose-based bioethanol production from agricultural residues or dedicated crop plantations is a more promising approach. However, one of the challenges of ethanol production from lignocellulosic biomass is the lack of overall utilization of by-products generated during the conversion process into valuable products. This can be achieved by applying the “biorefinery concept”, which includes production of: fuels, heat, electricity, feed and bio-chemicals (IEA, 2008). Industrial hemp (Cannabis sativa L.) known as a non-food crop is considered as a particularly promising alternative to currently used energy crops, e.g. energetic willow (Finnan and Styles, 2013). The plant can produce high biomass yields even in cold environmental conditions, resulting in high area-efficiency, which decreases competition with food and feed crops for arable land (Prade et al., 2011). The hemp is used for various purposes, including use as isolation material as well as ropes and cloth made from its fibers. However, hemp has lost partially its importance as a raw material in textile industry, being replaced by cotton and synthetic fibers (Finnan and Styles, 2013). The crop has been tested as solid fuel and as a biomass feedstock in either biogas, biohydrogen, bioethanol (Rehman et al., 2013; Kuglarz et al., 2014) or succinic and lactic acid production alone (Gandolfi et al., 2015; Gunnarsson et al., 2015). There is only one report evaluating the usage of hemp for integrated biofuels (cellulosic bioethanol and biogas) production (Kreuger et

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al., 2011). However a biorefinery concept for integrated biochemical and biofuel production from this crop has never been developed. Such process integration can deliver efficient platform of hemp biomass utilization for high- value low- volume product (for ex. succinate) and low- value high- volume fuel product (for ex. ethanol). Using ordinary baker’s yeast, Saccharomyces cerevisiae, only C6 sugars from biomass can be converted to ethanol, and the ethanol fermentation of C5 sugars from hemicellulosic fraction still remains a challenge due to low conversions yields (Kricka et al., 2015). The problem can be solved by using the bacterium A. succinogenes, which is considered as one of the most promising strains for the industrial production of succinic acid as it can ferment a wide range of carbon sources: glucose, xylose, arabinose, galactose, maltose, fructose, sucrose, cellobiose, lactose, mannitol, arabitol, sorbitol. Moreover, succinic acid production via fermentation consumes CO2, which can definitely improve the sustainability of the biorefinery process. Therefore, it is evident that biosuccinic acid production could contribute to the abatement of CO2 emissions (Cheng et al., 2012b). Nevertheless, costs of cellulosic bioethanol and biosuccinic acid production from lignocellulosic materials are still too high to fully compete with the market price of the petroleum-derived chemicals (Talebnia et al., 2010; Cheng et al., 2012b). Cellulosic bioethanol production integrated with the coproduction of biochemical such as succinic acid can reduce biofuel production costs and maximize the degree of hemp biomass utilization. However, lignocellulosic biomass contains cellulose, hemicelluloses and lignin tightly bonded, forming a complex structure. Thus, one of the most challenging processes in lignocellulosic biomass utilization for fuels and bio-chemicals is the pretreatment employed to lose the lignocellulosic structure prior to saccharification (Talebnia et al., 2010). One of the most promising and currently

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used pretreatment approaches for different types of feedstock are acid- and alkali-based methods. The method based on steam treatment in presence of sulfuric acid, which is considered as effective catalyst, proved to be effective in hemicelluloses solubilization. Whilst, alkaline oxidative treatment has shown to be one of the most effective methods for softening the biomass structure and removing the lignin (Taherzadeh and Karimi, 2008; Talebnia et al., 2010). Up to our knowledge so far, the influence of hemp pretreatment on the combined cellulosic bioethanol and biosuccinic acid production has never been investigated. In the present study, the biofuel (cellulosic bioethanol) and succinic acid (bio-based chemical) production potential of hemp was evaluated in a biorefinery concept. The aim was to investigate: the effect of biomass pretreatment (dilute-acid versus alkaline oxidative-based method) on sugar and ethanol yield after hydrolysis, and after fermentation respectively; the potential of by-products generated during ethanol production (liquid fraction after pre-treatment and stillage after fermentation) as feedstock for biosuccinic acid production using A. succinogenes 130Z. Furthermore, the effect of nutrients addition to liquid fractions after pretreatment and after ethanol distillation (stillage), on the efficiency of succinic acid production was studied. 2. Materials and Methods 2.1. Feedstock Industrial hemp (C. sativa L., Fedora 17 strain) was cultivated, at Lönnstorp experimental farm, Swedish University of Agricultural Sciences. The average annual precipitation and temperature of the cropping site were about 500.0 mm and 8.0 °C respectively. The hemp was sown in late April, on a loamy soil with 15% of clay and 3% of organic matter. The biomass was harvested in October 2012 and the average

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yield of dry matter amounted to 12 Mg ha-1. Hemp harvested was dried indoors at approx. 18°C to simulate field-drying. Dry hemp was chopped using a shredder (2-3 cm length) and ground to particles of 2 mm; using a cutting mill (Retsch SM 2000). 2.2. Biomass pretreatment and hydrolysis Biomass was pretreated with dilute-acid or alkaline oxidative-based method. The dilute-acid pretreatment was based on temperature (180 °C) and H2SO4 addition (1% or 1.5% w/v). After acid addition, the mixture was steam treated in a batch reactor for 10 min. The oxidative-based method involved the addition of 3% H2O2 (w/v) and pH value adjustment to 11.5. In this cases, the mixture was heated at 90 °C for 1 h or 2 h. Both types of pretreatment were conducted at solid content of 10% (w/v) feedstock/water and repeated four times. After pretreatment, the slurry was separated into solid fraction (fraction insoluble solids, FIS) and liquid fraction, using a commercial filtration unit (Buchner unit). Solid fraction after pretreatment (dilute-acid or alkaline oxidative-based method) was washed with deionized water until neutral pH was reached, dried in at 60 °C for 24 h, and stored in sealed plastic bags at 4 °C for further usage. The liquid fraction was neutralized using 2M NaOH (after acid-based method) or 50% H3PO4 (after alkaline H2O2 method). Untreated biomass and solid fractions recovered after all pretreatments tested (by acid-based or alkaline H2O2 method) were used as feedstock for enzymatic hydrolysis. The process was conducted at a solid loading of 7.5% in a 50 mM sodium citrate buffer, pH 4.8. Hydrolysis was performed at 50 °C for 48 h. Celluclast 1.5 L® (Celluclast, 20 FPU/g glucan) derived from Trichoderma ressei and Novozyme 188 (15 IU/g glucan) from Aspergillus niger were used for enzymatic hydrolysis. 2.3. Ethanol fermentation

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Cellulosic ethanol was produced from enzymatically hydrolyzed untreated hemp (non-pretreated by acid- or alkaline oxidative-based method) and solid fractions after all tested pretreatments. The slurry after enzymatic hydrolysis was supplemented with the following amounts of minerals (g L-1): (NH4)2SO4, (3.75); K2HPO4, (2.11); MgSO4·7H2O, (0.375) and CaCl2·2H2O, (0.5). For fermentation, 5% (v/v) of Saccharomyces cerevisiae inoculum was added. The fermentation was performed at 35 °C for 48 h in 300 ml Pyrex flasks equipped with air locks. Pure nitrogen gas was sparged into the media to provide anaerobic conditions. Samples of one milliliter were taken periodically (after 0, 3, 6, 12, 24, 36 and 48 h), immediately centrifuged at 10.000 g for 10 min. and then the supernatants were filtered (0.2 µm pore size filters) before sugars and ethanol determination. After the fermentation, ethanol was distilled to a purity of 95-96% (Büchi Rotavapor equipped with vacuum pomp) leaving stillage as a by-product. The stillage was tested as feedstock for succinic acid production. However, bioethanol used as fuel should not contain more than 0.5% of water, which can be achieved by molecular sieve adsorption, pervaporation or vapor permeation (Huang et al., 2008). In a present study, complete ethanol purification was not performed, as the main aim of distillation was to separate stillage. The separated stillage was stored at - 4 °C and used as feedstock for succinic production. 2.4. Succinic acid production 2.4.1. Feedstock preparation Stillage (originated from biomass pretreated by alkaline H2O2 method) and liquid fractions after acid-based pretreatments, were used as feedstock for succinic acid production. Liquid fractions after alkaline hydrogen peroxide pretreatment and stillage after fermentation of biomass pretreated by steam with acid were not used as feedstock

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for succinic acid production, due to a very low sugar content (Table 3). Before using as feedstock for succinic acid production, pH value of liquid fractions and stillage were adjusted to about 6.5 using 2 M NaOH or 50% phosphoric acid (H3PO4) and autoclaved at 121 °C for 20 min. 2.4.2. Succinic acid tests A. succinogenes 130Z (DSM 22257) was obtained from DSMZ and the stock culture was stored in glycerol at −80 °C prior use. Seed culture medium composition and way of preparation were previously described (Gunnarsson et al., 2015). Two separate tests of succinic acid production were performed. Firstly, succinic acid production tests were performed in 200 mL sealed anaerobic bottles with 100 mL working volume. Bottles were flushed with pure nitrogen and incubated at 37 °C and 150 rpm for 48 h. The following ratios of feedstock (liquid fraction or stillage): medium were tested (% vol.): 100:0; 75:25 and 50:50. Secondly, batch fermentation of liquid fractions was conducted in two identical 3-L fermenters (Sartorius BIOSTAT Aplus, Germany) with an initial working volume of 1.5 L. Based on the succinic acid yield and productivity obtained in anaerobic bottles, the liquid fraction after steam/acid pretreatment was considered superior over stillage in batch tests performed in 200 mL anaerobic bottles and selected for further investigation in a larger scale. The fermentation was conducted by mixing sterile liquid fraction (after 1.5% acid-based pretreatment) and experimental medium at 100:0 and 75:25 (v:v) ratio (Fig. 3). The reactors were operated at 37 °C and 150 rpm for 72 h. The start-up of the fermenters was previously described (Gunnarsson et al., 2015). All batch fermentations (200 mL bottles and 3-L bioreactors tests) were inoculated with 5% (v v-1) of exponentially (OD660 = 4.5) growing inoculum. In both sets of fermentation, 1 g of solid MgCO3 per 1 g of total sugar was supplied as an

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indirect CO2 source and pH buffer of fermentation medium. In case of tests in anaerobic bottles, sugars (glucose, xylose) and acids (succinic, acetic, formic) were analysed in the beginning of the process and after 48 h. During batch fermentation in the 3-L reactors, 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. 2.5. Analytical methods Volatile solids (VS), ash and protein content, pH, were determined according to standards methods (APHA, 2005). Total soluble phenolic compounds (TPC) were determined using standard colorimetric procedure (Folin-Ciocalteu reagent). Synthetic solutions of sugars and furans were prepared at concentration similar to those present in tested liquids in order to eliminate the interference with color. Optical density at 660 nm (OD660) was determined by a spectrophotometer (Jenway Buch and Holm A/S 64050UV/VIS) after insoluble MgCO3 removal by mixing the sample with 7% (w/v) HCl at the ratio of 1:1. OD660 of 1.0 corresponded with concentration of 0.626 g dry cell weight (DCW)/l (data no shown). The concentrations of sugar monomers (glucose, xylose, arabinose), organic acids (succinic-, lactic-, formic- and acetic acid), inhibitors (furfural and 5-hydroxymethyl-2furaldehyde HMF) were determined by using high performance liquid chromatography HPLC (Agilent 1100) equipped with a BioRad Aminex HPX-87 H column at 63 °C, refractive index (RI) detector (RID 1362A) and ultraviolet (UV) detector using 4 mM H2SO4 as eluent at 0.6 ml/min flow rate. A two-step acid hydrolysis was performed for quantitative analysis of polymer sugars in solid samples. The first step involved addition of 1.5 mL of H2SO4 (72%) at 30 °C for 60 min. After addition 42 mL of distilled water,

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the final concentration of H2SO4 was 4%. Next, the mixture was treated at 121 °C for 60 min in the second step of hydrolysis. The hydrolysate was filtered and the liquid fraction was analysed for sugars (HPLC), while the difference between the weight of dried filter cake and ash content represented Klason lignin. Chemicals used in this study were of analytical grade (Sigma Aldrich ApS). 2.6. Calculations 2.6.1. Effectiveness of the pretreatment The effectiveness of the pretreatment methods applied was based on FIS (fraction insoluble solids) recovery (Eq.1), and degrees of cellulose and hemicelluloses recovery. The degrees of cellulose and hemicelluloses recovery were calculated as the amount of cellulose/hemicelluloses recovered in solid or liquid fractions after pretreatment and expressed as percentage related to cellulose/hemicelluloses content in biomass before pretreatment. The increase/decrease (%) of analyzed biomass components after pretreatment was calculated in accordance with its initial content in untreated biomass. The amount of FIS (fraction insoluble solids) was calculated according to Eq. (1):
 (%) =

    

∙ 100

(1)

where: Solid fractionDry – dry weight of solids after pretreatment, washed and dried at 60 °C for 24 h. SlurryDry – dry weight of slurry used for pretreatment after drying at 60 °C for 24 h. 2.6.2. Effectiveness of the enzymatic hydrolysis Glucose (YGluc.) and xylose (YXyl.) yield during enzymatic hydrolysis were calculated according to Eq. (2) and (3):  . (%) =

  !"#.

$%&'()) ∙ (

*+, ) *-.

∙ 100

(2)

10

 /. (%) =

/  !"#.

*0,

/$%&'()) ∙ (*1.)

∙ 100 (3)

Total yields of glucose (Eq. 4), xylose (Eq. 5) and overall sugar yield (Eq. 6) after hydrolysis (pretreatment and enzymatic hydrolysis) were calculated as follows:   =>?. @   !"#. *+, $%&'()) ∙ ( )∙ AB

2345678 9:83; < (%) = C93678 9:83; < (%) = DE8FG33 9:83; (%) =

*-.

/  =>?. @ /  !"#. *0, )∙ AB

/$%&'()) ∙ (

*1.

∙ 100 (4)

∙ 100 (5)

  =>?H!"#.@/  =>?.H!"#. *+, *0, ($%&'()) ∙I J@ /$%&'()) ∙I J)∙ AB *-.

*1.

∙ 100 (6)

where: GlucosePret. and XylosePret. – the amount of glucose and xylose released to pretreated liquor during pretreatment, (g); GlucoseEnz.. and XyloseEnz. – the amount of glucose, xylose released during enzymatic hydrolysis respectively, (g) GlucanBiomass and XylanBiomass – the amount of glucan and xylan in the biomass, respectively, (g); 180/162 and 150/132 – stoichiometric conversion factors of glucan to glucose and xylan to xylose, respectively. 2.6.2. Effectiveness of ethanol and succinic acid fermentation 2.6.2.1. Ethanol production Ethanol yield (YEthanol) calculation was based on the following equitation (Eq. 7) and on the assumption that all glucose found in the feedstock could be converted by S. cerevisiae into ethanol, with a yield of 0.51 g EtOH/g of glucose. Ethanol production yield was also expressed as the amount of ethanol produced per unit of biomass treated (g-ethanol/100g biomass).

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YLMNO (%) =

LMNOPQRS. (T)

LMNOUVWRQWX. (T)

∙ 100 (7)

where: EtOHProd. – the highest ethanol amount obtained in fermentation process (g) EtOHTheoret. = theoretical ethanol production based on stoichiometric conversion of glucose to ethanol, i.e. 0.51 g-EtOH/g-glucose - assuming that all glucose found in the feedstock could be fermented by S. cerevisiae into ethanol. 2.6.2.2. Succinic acid production Succinic acid yield (YSuc.) was calculated as the amount of succinic acid obtained (g) per 1 g of sugars (glucose + xylose) consumed (Eq.8). The yield of succinic acid was expressed either as succinic acid amount (kg) per Mg of dry hemp or succinic acid (kg) per ha of hemp grown (Fig. 3). YYZ[.(%) =

Y\PQRS. (T)

YZT]^_R`abcWS (T)

∙ 100 (8)

where: SAProd. – the highest concentration of succinic acid produced (g L-1); Sugarconsumed – amount of glucose and xylose consumed during succinic acid fermentation (g L-1). 2.6.3. Statistical analysis Results are presented as average values (n =4) with standard deviations (±). The characteristics of untreated and pretreated hemp as well as data obtained for enzymatic hydrolysis, ethanol fermentation and succinic production 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. Normal

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distribution and homogeneity of variances were tested by Shapiro-Wilk and Levene test, respectively. Data significantly equivalent were indicated by the same letters. 3. Results and discussion 3.1. Biomass composition The main components of the industrial hemp (Fedora 17 strain) was glucan (46%), xylan (20%) and lignin (15%) (Table 1). Arabinan content was detected in amounts smaller than 1% of dry matter (data not shown). In total, the carbohydrates content amounted to 67% DM, which is a bit higher compared to previously analysed hemp strains, i.e. Uso (Gunnarsson et al., 2015) and Felina strain (Kuglarz et al., 2014). This makes this strain of industrial hemp a promising feedstock for biofuels and biochemicals production. Contents of various components vary significantly with maturity of the plant and it is also associated with the nature of particular strain (Godin et al., 2013). The differences in chemical composition between different hemp strains might have been a result of nutrients availability in various stages of hemp growth (Thomsen et al., 2005). 3.2. Cellulosic ethanol production 3.2.1. Biomass pretreatment All pretreatment methods used in this study had a significant influence on the carbohydrate content of hemp material. Recovery of FIS (fraction insoluble solids) ranged from 67-72%; depending on the conditions of pretreatment applied. As a principle, more severe conditions of the pretreatment led to lower recovery of insoluble fraction. The main components of the solid fraction after pretreatment were glucan, xylan and lignin (Table 1). Glucan content in solid fractions dry matter increased by 3240% (data equivalent statistically, p > 0.05) compared to its initial content. Regardless

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of conditions of pretreatment applied, more than 95% of glucan was retained in the solid fraction and there were insignificant concentration of glucose detected in liquid fractions (Table 1 and 2). Results obtained are in accordance with previous studies (Kuglarz et al. 2014; Gunnarsson et al. 2015). This is an advantage of the pretreatment conditions used. The analyzed pretreatment methods caused a partial hemicelluloses solubilization to its monomers, mostly xylose. The arabinose was observed in very low concentrations (< 0.5 g L-1) and only after alkaline H2O2 pretreatment (data not shown). Degree of hemicelluloses solubilization after steam/acid method was roughly 3 times higher than the one obtained with alkaline oxidative approach (Table 2). In contrary to the glucan content, applying harsher conditions of the pretreatment (higher H2SO4 concentration) played an important role in hemicelluloses solubilization (data statistically different, Table 2). Compared to cellulose, structure of hemicelluloses is more susceptible to being hydrolyzed into its constituents sugars, especially during dilute-acid pretreatment methods (Taherzadeh and Karimi, 2008). Table 1 Table 2 Besides carbohydrates, the second major component of biomass after pretreatment constituted lignin. The majority of lignin after steam/acid pretreatment (98-99%) was recovered in solid fraction; with no lignin detected in liquid fraction (Table 1). It is widely reported in the literature that steam treatment with addition of sulphuric acid is a method which does not cause a significant lignin solubilization (Silverstein et al., 2007). In this case, content of polymers, might also increase due to re-polymerization of dissolved lignin during polysaccharide degradation, which could generate pseudo-lignin and increase of the Klason lignin content (Barta et al., 2010). In contrast to acid-based

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method, a significant part of lignin (35-41%) was solubilized during H2O2 pretreatment and longer reaction time led to higher lignin solubilization (data different statistically, Table 2). Pretreatment of lignocellulosic biomass may produce degradation products, such as: furfural, hydroxymethylfurfural (HMF), acetic acid, lactic acid, formic acid, phenolic compounds etc., which are generally considered inhibitory for fermentation processes. Their concentration depend mostly on the type of biomass treated as well as pretreatment conditions. All pretreatments tested generated carboxylic acids and soluble phenolic compounds (Table 2), which have been widely reported in liquid fractions after pretreatments (Palmqvist and Hahn-Hägerdal, 2000). In this study, acetic acid was observed as the main degradation product (Table 2) and its occurrence is mostly associated with hydrolysis of acetylated hemicelluloses and lignin linkages. After alkaline oxidative method, besides acetic and formic acid, soluble phenolic compounds were generated, most probably due to partial breakdown of lignin, which is usually observed in this type of biomass pretreatment (Palmqvist and Hahn-Hägerdal, 2000). Increasing the reaction time of H2O2 treatment resulted in higher concentrations of inhibitors, especially acetic acid and soluble phenolic compounds (Table 2). Results obtained in this study clearly showed that acid-based pretreatment of hemp biomass was superior in comparison to alkaline oxidative method due to generation of significantly lower amounts of degradation products. 3.2.2. Hydrolysis (saccharification) Influence of the pretreatment methods applied on the effectiveness of enzymatic hydrolysis, using the commercial celluclast and ß-glucosidase mixtures, was analysed. The glucose yield of untreated feedstock amounted to 30%, which is in middle of the

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range previously reported (Pakarinen et al., 2011; Kuglarz et al., 2014; Gunnarsson et al., 2015). This indicates that untreated hemp is relatively difficult to hydrolyze, which is thought to be connected with specific plant cell-wall structure and more than double higher crystallinity index compared to other types of biomass, e.g. sorghum (Kamireddy et al., 2013). Both acid- and alkaline oxidative-based method applied in this study influenced the enzymatic hydrolysis in a positive way. Based on the solid fraction saccharification, the highest glucose and xylose yields were reached when the hemp was previously pretreated with H2O2 for 2h and the values were 8-13% (glucose yield) and 11-27% (xylose yield) higher compared to yields recorded after other pretreatment conditions applied (Table 3). Alkaline pretreatment turned out to be also superior to acid-based method in respect to the rate of enzymatic hydrolysis. Enzymatic hydrolysis of H2O2 pretreated biomass resulted in 60-70% higher glucose rate (3.1-3.2 g L-1 h-1, between 0 and 12 h) compared to the saccharification rate of acid treated feedstock (Fig. 1). This result is most likely due to lignin solubilization achieved during H2O2 pretreatment (Chang and Holtzapple, 2000). Significantly lower values of xylose yield during enzymatic hydrolysis of biomass pretreated by steam/acid can be explained by the fact that most of the xylose was released during pretreatment (Table 2), and thus the proportion of more resistant and less accessible hemicelluloses increased (Barta et al., 2010). Considering the sugar losses and effectiveness of pretreatment and enzymatic hydrolysis in total, both pretreatment methods (acid-based, alkaline H2O2) allowed to reach a comparable overall sugar yields (data equivalent statistically, Table 3). However, based on sugar yields from both solid and liquid fractions obtained after enzymatic hydrolysis and pretreatment respectively, 9-21% higher yields of total xylose

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were achieved after steam method with acid, compared to alkaline H2O2 method. This can be an advantage of this method, taking into account succinic acid production from liquid fractions after pretreatment (rich in pentoses) (Table 2). Table 3 3.2.3. Ethanol fermentation In all tested batches, ethanol production started immediately without any lag until the glucose was completely consumed. The xylose concentration did not change its concentration significantly during the fermentation (Fig. 1), which showed that native S. cerevisiae (non-engineered) was not able to use xylose (Talebnia et al., 2010; Kuglarz et al., 2014). The xylose concentration continued to slightly increase during ethanol fermentation due to the presence of enzymes used during enzymatic hydrolysis (Fig. 1). The ethanol yield amounted to 81-89% (0.41-0.45 g-ethanol/g glucose released during enzymatic hydrolysis) of theoretical yield (Table 3). The ethanol yield of “pure glucose” was also analysed and the values amounted to 0.50-0.52 g EtOH/g-glucose (data not shown). The slight decrease in the ethanol concentration between 36 h and 48 h of the process was observed (Fig 1). The possible explanation include simultaneous consumption of glucose and accumulated ethanol by Saccharomyces cerevisiae, which was previously reported (Ramon-Portugal et al., 2004) or catabolic ethanol oxidation at low sugar concentration and/or ethanol evaporation. More than double ethanol production was achieved after biomass pretreatment compared to fermentation of hemp without pretreatment, which was connected with higher availability of glucose in pretreated biomass (Table 3). In total, 7-17% higher ethanol production was achieved after alkaline oxidative methods compared to the values after acid-based method. Whilst, higher severity of pretreatment (higher acid concentration or longer reaction

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time of hydrogen peroxide) did not have an influence on total ethanol production. The highest values of ethanol production obtained in this study (17.5 g/100g of biomass) (Table 3) were superior compared to the values reported for other lignocellulosic materials after the same type of fermentation applied (SSH), e.g. rapeseed straw (14-15 g/100g of biomass) (Luo et al., 2011), which demonstrates that the hemp pretreated by the methods presented in this study is a promising feedstock for cellulosic bioethanol fermentation. Fig. 1. 3.4. Succinic acid fermentation More than 80% of xylose, the main sugar component of analyzed substrates, was consumed as long as the fermentation mixture contained at least 25% of medium (nutrients + nitrogen) (Table 4). Glucose present in small concentrations in liquid fractions after steam/acid pretreatment was completely consumed in case of feedstock with and without nutrients addition (medium). In both types of feedstock (liquid fraction or stillage), the succinic yields above 64% were obtained; providing that the liquid fraction was supplemented with at least 25% of medium (75:25 ratio of feedstock to medium). Fermentation of raw liquid fraction (without nutrient addition) after pretreatment resulted in 4-6 times higher residual xylose levels compared to processes with 25-50% content of medium (Table 4). This indicates that nutrients (nitrogen, minerals) availability could be a limiting factor in succinic acid production from liquid fractions, leading to lower sugar utilization (Du et al., 2007; Gunnarsson et al., 2015). Whilst, stillage used as feedstock for succinic acid production did not require nutrients supply, which can be explained the fact that this substrate, is a complex feedstock containing not only nutrients (derived from nitrogen and nutrients added in ethanol

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fermentation step), but also yeast biomass structural biopolymers (e.g. starch, proteins, lipids), VFA (mainly acetate) from yeast metabolism and some leftover xylose (Kaparaju et al., 2009). Slightly higher yields (7-10%) obtained from fermentation of liquid fractions after acid-based pretreatment compared to stillage can be explained with presence of glucose, which is normally associated with higher succinic yields compared to xylose (Chen et al., 2011). In total, the highest succinic acid production (g/100g of biomass) was achieved for liquid fractions pretreated with 1.5% and 1.0% acid, which were 38-48% and 20-31% higher from the succinic production of stillage respectively (Table 4). This indicates that acid-based pretreatment of hemp, resulting in generation of rich in pentoses liquid fractions, can be considered as superior to alkaline oxidative method. However, stillage can be fermented without additional medium with nutrients, which is considered as one of the main factor influencing the cost of bio-succinic acid production (Chen et al., 2011). Depending on fermentation conditions, other metabolites (acetic, formic, lactic acid or ethanol) can be produced in different amounts during fermentation (McKinlay et al., 2005). In a present study, acetic- and formic acid were generated as main by-products (Table 4). However, their concentrations were below the levels impacting the succinic acid process performance (Corona-González et al., 2008). Table 4 Experiments at controlled conditions (bioreactors) were performed to optimize utilization of xylose and succinic acids yields as well as to verify the influence of medium addition (nutrients) on the succinic acid production. Moreover, the effect of pH control on succinic acid fermentation was tested. Results obtained showed that cell density (shown as DCW in Fig. 3 A-B) reached maximum in about 25% shorter time when fermenting mixture (liquid fraction + nutrients) compared to substrate without

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nutrients addition. This could be associated with positive effect of nutrients presented in the medium, on the cell growth (Chen et al., 2011). As a result of fermentation in bioreactors, which allowed to maintain optimal pH at 6.8 during the process, succinic acid yield amounted to 79-82%, which is 6-9% higher compared with processes in bottles and was mostly associated with improved non-fermentable (xylose) consumption (Table 4, Fig. 2). These values are only slightly lower compared to succinic production using fermentable sugars from hemp (Gunnarsson et al. 2015). Fig. 2. 3.5. Output of the combined ethanol and succinic acid production During pretreatment of one ton of hemp, 70% of the DM was transferred to WIS fraction, whilst 20% was transferred to liquid fraction. In both cases of pretreatment, more than 95% of cellulose was retained in solid fractions after pretreatment. Using ordinary baker’s yeast, Saccharomyces cerevisiae, 149 kg EtOH/Mg of dry hemp (1923 kg EtOH/ha) after acid-based (1.5% H2SO4) pretreatment and even 11-17% (Table 3) more after alkaline oxidative method was produced. In case of combined generation of ethanol and succinic acid, the biomass pretreatment with 1.5% of acid allowed to generate 115 kg of succinic acid/Mg of dry hemp (1484 kg/ha), which is about 80% higher compared to production of succinic acid from stillage (Fig. 3, Table 4). Succinic acid produced has been recognized as one of the twelve most promising building block chemicals and it is precursor for the production of wide spectrum of commodities used in food, chemical, and pharmaceutical industries (McKinlay et al., 2007; Bechthold et al., 2008). Taking into account the recent progress in succinic acid downstream processing (Kurzrock and Weuster-Botz, 2010; Cheng et al. 2012a), production of succinic acid from lignocellulosic biomass during ethanol production would result in

20

generation of valuable bio-chemical and would influence positively the level of biomass utilization (sugars C5 and C6). Another possibility of increasing the economic feasibility of a hemp biorefinery is the production of heat or heat and power generation from lignin fraction. Lignin is generally not degraded during bioethanol or succinic acid fermentation and is retained in solid fraction (acid-based pretreatment) or partially solubilized into liquid fraction (alkaline oxidative method, Table 2). Dry lignin can be pelletized and considered as high quality solid biofuel (Larsen et al., 2012). As CO2 is consumed during succinic acid production, the CO2 generated during ethanol production can be integrated in this process. Another option would be to use CO2 from biogas to produce succinic acid as has previously been demonstrated (Gunnarsson et al., 2014). It should be taken into account that the present study is a simplified analysis, which is focused on products. The energy requirements for biofuels and biochemical processing (pretreatment, production, fuels recovery and purification, etc.) were not considered. However, the results obtained can be used as an important input for environmental and economic analyses to document sustainability of the hemp based biorefinery. Fig. 3. 4. Conclussions The results obtained clearly showed that industrial hemp can be used for cellulosic bioethanol and succinic acid production in a biorefinery concept. The highest ethanol production was achieved after hemp pretreatment by alkaline oxidative method. However, acid-based pretreatment of hemp was superior to alkaline oxidative method with respect to the combined ethanol and succinic acid production (149 kg EtOH/Mg of hemp + 115 kg of succinic acid/Mg of hemp). Optimal conditions of hemp

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bioprocessing identified in this study can pave the way towards sustainable hemp utilization for production of biofuels and bioproducts. Acknowledgements This study is the result of work done in the Biorefinery Øresund project. We would like to thank Interreg IVA for the financial support given. This work was also partially supported by the University of Bielsko-Biala (Poland) within internal funds for development of young scientists as well as EU project POKL04.01.02-00-196/09-00. We would like to thank Sven-Erik Svensson from Swedish University of Agricultural Sciences (SE-230 53 Alnarp, Sweden) for providing us with industrial hemp (Cannabis sativa L.). References 1.

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Cheng, K.K., Zhao, X.B., Zeng, J., Wu, R.C., Xu, Y.Z., Liu, D.H., Zhang, J.A., 2012a. Downstream processing of biotechnological produced succinic acid. Appl. Microbiol. Biotechnol. 95, 841-850.

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Corona-González, R.I., Bories, A., González-Álvarez, V., Pelayo-Ortiz, C., 2008. Kinetic study of succinic acid production by Actinobacillus succinogenes ZT-130. Process Biochem. 43, 1047-1053.

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Du, C., Lin, S.K.C., Koutinas, A., Wang, R., Webb, C., 2007. Succinic acid production from wheat using a biorefining strategy. Appl. Microbiol. Biotechnol. 76, 1263-1270.

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Upgrading and Biosuccinic Acid Production. Environ. Sci. Technol. 48, 1246412468. 14. Gunnarsson, I.B., Kuglarz, M., Karakashev, D., Angelidaki, I., 2015. Thermochemical pretreatments for enhancing succinic acid production from industrial hemp (Cannabis sativa L.). Bioresour. Technol. 182, 58-66. 15. Huang, H.J., Ramaswamy, S., Tschirner, U.W., Ramarao, B.V., 2008. A review of separation technologies in current and future biorefineries. Sep. Purif. Technol. 62, 1-21. 16. IEA. IEA bioenergy Task 42 on biorefineries: co-production of fuels, chemicals, power and materials from biomass. In: Minutes of the third Task meeting. Copenhagen, Denmark, 25-2 March 2007, 2008. 17. Kamireddy, S.R., Li, J.B., Abbina, S., Berti, M., Tucker, M., Ji, Y., 2013. Converting forage sorghum and sunn hemp into biofuels through dilute acid pretreatment. Ind. Crop Prod. 49, 598-609. 18. Kaparaju, P., Serrano, M., Thomsen, A.B., Kongjan, P., Angelidaki, I. 2009. Bioethanol, biohydrogen and biogas production from wheat straw in a biorefinery concept. Bioresour. Technol. 100, 2562-2568. 19. Kreuger, E., Sipos, B., Zacchi, G., Svensson, S-E., Björnsson, L., 2011. Bioconversion of industrial hemp to ethanol and methane: The benefits of steam pretreatment and co-production. Bioresour. Technol. 102, 3457-3465. 20. Kricka, W., Fitzpatrick, J., Bond, U., 2015. Challenges for the production of bioethanol from biomass using recombinant yeasts. Adv. Appl. Microbial. 92, 89125.

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21. Kuglarz, M., Gunnarsson, I.B., Svensson, S.E., Prade, T., Johansson, E., Angelidaki, I., 2014. Ethanol production from industrial hemp: Effect of combined dilute acid/ steam pretreatment and economic aspects. Bioresour. Technol. 163, 236-243. 22. Kurzrock, T., Weuster-Botz, D., 2010. Recovery of succinic acid from fermentation broth. Biotechnol. Lett. 32, 331-339. 23. Larsen, J., Haven, M.Ø., Thirup, L., 2012. Inbicon makes lignocellulosic ethanol a commercial reality. Biomass Bioenergy, 46, 36-45. 24. Luo, G., Talebnia, F., Karakashev, D., Xie, L., Zhou, Q., Angelidaki, I., 2011. Enhanced bioenergy recovery from rapeseed plant in a biorefinery concept. Bioresour. Technol. 102, 1433-1439. 25. McKinlay, J., Vieille, C., Zeikus, J.G., 2007. Prospects for a bio-based succinate industry. Appl. Microbiol. Biotechnol. 76, 727-740. 26. McKinlay, J.B., Zeikus, J.G., Vieille, C., 2005. Insights into Actinobacillus succinogenes fermentative metabolism in a chemically defined grownth medium. Appl. Environ. Microbiol. 71, 6651-6656. 27. Pakarinen, A., Maijala, P., Stoddard, F.L., Santanen, A., Tuomainen, P., Kymäläinen, M., Viikari, L., 2011. Evaluation of annual bioenergy crops in the boreal zone for biogas and ethanol production. Biomass Bioenergy 35, 3071-3078. 28. Palmqvist, E., Hahn-Hägerdal, B., 2000. Fermentation of lignocellulosic hydrolysates II: inhibitors and mechanisms of inhibition. Bioresour. Technol., 74, 25-33.

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29. Prade T., 2011. Industrial Hemp (Cannabis sativa L.) - A High Yielding Energy Crop. Swedish University of Agricultural Sciences. Doctoral Thesis, Alnarp, Sweden. 30. Ramon-Portugal, F., Pingaud, H., Strehaiano, P., 2004. Metabolic transition step from ethanol consumption to sugar/ethanol consumption by Saccharomyces cerevisiae. Biotechnol. Lett. 26, 1671-1674. 31. Rehman, M.S.U., Rashid, N., Saif, A., Mahmood, T., Han, J.I., 2013. Potential of bioenergy production from industrial hemp (Cannabis sativa): Pakistan perspective. Renew. Sustain. Energy Rev. 18, 154-164. 32. Silverstein, R.A., Chen, Y., Sharma-Shivappa, R.R., Boyette, M.D., Osborne, J., 2007. A comparison of chemical pretreatment methods for improving saccharification of cotton stalks. Bioresour. Technol. 98, 3000-3011. 33. Taherzadeh, M.J., Karimi, K., 2008. Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: A review. Int. J. Mol. Sci. 9, 1621-1651. 34. Talebnia, F., Karakashev, D., Angelidaki, I., 2010. Production of bioethanol from wheat straw: An overview on pretreatment, hydrolysis and fermentation. Bioresour. Technol. 101, 4744-4753. 35. Thomsen, A.B., Rasmussen, S., Bohn, V., Nielsen, K.V., Thygesen, A. Hemp raw materials: The effect ofcultivar, growth conditions and pretreatment on the chemical composition of the fibres. Risø-R-1507(EN), Risø National Laboratory, Roskilde, Denmark; 2005.

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Figure captions Fig. 1. Enzymatic hydrolysis and ethanol fermentation course (a - hemp pretreated by steam with 1.0% H2SO4; b - hemp pretreated by steam with 1.5% H2SO4; c – hemp pretreated by H2O2 for 1h; d – hemp pretreated by H2O2 for 2h; filled triangles represent glucose; filled squares represent xylose; filled circles represent ethanol) Fig. 2. Time course of succinic acid fermentation in 3-L batch bioreactors (A - liquid fraction after hemp pretreatment with 1.5% H2SO4 and fermented without medium; B liquid fraction after hemp pretreatment with 1.5% H2SO4 and mixed with medium at 75:25 ratio) Fig. 3. Mass balance of the hemp biorefinery concept, aimed at maximizing the ethanol and succinic acid production (a – hemp pretreated by 1.5% H2SO4; b – ethanol production in Pyrex flasks; c – succinic acid production in bioreactors at 75:25 ratio of feedstock to medium; d – calculations based on hemp dry matter of 930 kg Mg-1 and yield of 12 Mg of dry matter/ha; SA. – succinic acid; AcA – acetic acid; FA – formic acid; EtOH – ethanol)

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a.) 50

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Hydrolysate DM (kg): 728.1 Glucose: 343.7 Xylose: 28.5 Cel.: 119.7 Hem.: 25.0 Lignin: 141.9 Ash: 7.9 Others: 61.4

Solid fraction DM (kg): 660.0 Cel.: 429.0 Hem.: 50.1 Lig.: 143.9 Ash: 8.1 Others: 28.9

b

Ferm. Broth (kg) EtOH: 148.7 Glucose: 0.0 Xylose: 32.8

Hemp (DM): 930 kg

Cel: Hem: Lignin: Ash: Others:

431.5 186.9 139.5 22.13 149.9

Final output: EtOH: 149 kg/Mg (1923 kg/ha)d SA: 115 kg/Mg (1484 kg/ha)d Succinic acid c

fermentation

Liquid fraction DM (kg): 188.0 Glucose.: 1.7 Xylose: 118.6 Lignin: 0.0 Ash: 4.5 Others: 63.2

Fig. 3.

Ferm. Broth (kg) SA: 114.8 AcA: 38.6 FA: 21.5 Glucose: 0.0 Xylose: 12.9

Table 1. Characteristics of untreated biomass and solid fractions after pretreatment (recov. = recovery, average values n = 4, ± standard deviations, the same letters represent data equivalent statistically p > 0.05, Tukey’s HSD test used for statistical analysis) Pretreatment Untreated* 1% H2SO4-10min. 1.5% H2SO4-10min. 3% H2O2 – 1h. 3% H2O2 – 2h.

Glucan % DM % recov. 46.4±1.7b 61.0±1.8a 95.1±0.5a 65.0±1.7a 95.2±0.4a 62.5±1.2a 96.1±0.7a 65.1±1.5a 95.3±2.1a

Xylan % DM % recov. 20.1±0.9a 9.91±0.7b 35.8±2.2b 7.59±0.6c 25.8±2.8c 18.8±0.9a 67.0±3.2a 17.9±1.0a 60.9±4.0a

* - biomass not subjected to acid/steam or alkaline oxidative pretreatment

Lignin % DM % recov. 15.0±0.8b 20.3±0.7a 98.2±5.8a 21.8±1.0a 99.3±5.9a 12.3±0.8c 58.9±3.3b 9.93±0.8d 45.3±3.9c

Ash % DM 2.38±0.1a 1.84±0.1b 1.23±0.1b 1.02±0.1b 1.00±0.1c

WIS % 72±2a 68±2b 71±2ab 67±2ab

Table 2. Characteristics of liquid fraction after biomass pretreatment (recov. = recovery, b.d.l. – blow detection limit; TPC – total soluble phenolic compounds, average values n = 4, ± standard deviations, the same letters represent data equivalent statistically p > 0.05, n.d - not detected Pretreatment

Glucose

Xylose

Lignin

g L-1 % recov. g L-1 % recov. % recov. 2.32±0.3b 3.29±0.6a 49.8±3.5b 15.5±1.4b 1% H2SO4 -10min. 2.88±0.2a 3.72±0.2a 59.0±1.1a 20.1±0.7a 1.5% H2SO4 -10min. 0.27±0.1c 0.89±0.2b 17.1±1.7c 34.8±1.7 2.27±0.2c 3% H2O2 - 1h. 0.31±0.1c 0.96±0.2b 17.0±3.5c 41.1±2.9 2.45±0.5c 3% H2O2 - 2h. a - degree of acetylation, calculated as percentage of xylan content in biomass dry matter

Furfural

HMF

g L-1 0.15±0.1 0.21±0.1 b.d.l. b.d.l.

g L-1 0.25±0.1 0.37±0.1 b.d.l. b.d.l.

Acetic acid g L-1 2.3±0.3b 2.7±0.3b 2.6±0.3b 6.5±0.6a

%a 6.0±0.4b 6.5±0.8b 7.3±1.1b 16.7±2.6a

Formic acid TPC g L-1 1.2±0.2b 1.5±0.2b 1.9±0.2b 2.6±0.2a

g L-1 1.1±0.1c 1.4±0.3c 3.0±0.4b 4.6±0.3a

Table 3. Overall sugar yields after pretreatment and enzymatic hydrolysis as well as ethanol yield during fermentation (± standard deviation, n=4, the same letters represent data equivalent statistically p > 0.05).

Pretreatment Untreated* 1% H2SO4 -10min. 1.5% H2SO4-10min. 3% H2O2 – 1h. 3% H2O2 – 2h.

Enzymatic hydrolysis after 48 h Glucose, Glucose Xylose, g L-1 yield, (Eq.2) g L-1 11.7±0.8d 30.3±1.9c 2.12±0.4b 35.0±0.9c 68.9±1.7b 3.73±0.3b 39.1±1.7b 72.2±3.1b 3.80±0.2b 39.0±1.0b 74.8±1.9b 11.5±0.5a 44.7±1.6a 82.4±3.0a 10.9±0.6a

Xylose yield, (Eq.3) 12.1±1.5d 44.3±3.3c 50.1±2.4c 61.2±2.7b 71.0±3.4a

Pretreatment + Enzymatic hydrolysis Glucose(T), Xylose(T), Overall (Eq.4) (Eq.5) yield(T), (Eq.6) 30.3±2.3c 12.1±1.5d 24.7±1.1b 69.0±3.4b 65.7±2.5b 67.9±1.5a 72.4±1.0ab 74.1±1.8a 72.9±2.2a 72.0±4.2ab 56.8±3.0c 67.3±3.3a 80.0±3.5a 53.2±1.8c 71.2±4.0a

* - biomass not subjected to acid/steam or alkaline oxidative pretreatment; a – g of ethanol obtained per 100 g of initial biomass

Ethanol fermentation YEtOH, Yg/100ga (Eq.7)

88.9±3.0a 83.0±1.5a 81.1±2.0a 86.6±3.2a 83.6±3.0a

7.17±0.3c 15.5±0.5b 14.9±0.5b 16.6±0.3a 17.5±1.0a

Table 4. Succinic acid production from by-products generated during hemp processing into ethanol, in sealed anaerobic bottles (average values n =4, ± represent standard deviations, the same letter represent data statistically equivalent p > 0.05) Initial 0h After 48 h Glucose, Xylose, Glucose, Xylose, Sugar Succinic Succinic Succinic Acetic Formic -1 -1 -1 -1 a -1 b -1 c gL gL gL gL utiliz. , % acid, g L yield ,% prod., g/100) acid, g L acid, g L-1 Liquid fraction after stem pretreatment/acid addition (1.0%) and enzymatic hydrolysis 0:100 2.25±0.2b 15.5±1.4b b.d.l. 9.14±0.3b 48.3±2.8b 4.42±0.2e 51.8±4.4d 3.22±0.2e 3.22±0.2a 1.87±0.1bc 25:75 1.74±0.2cd 11.6±0.7c b.d.l. 2.29±0.3d 82.7±3.1a 7.89±0.3b 71.8±2.9ab 7.68±0.5c 3.51±0.2a 1.94±0.1ab 50:50 1.15±0.1e 7.78±0.5d b.d.l. 1.42±0.2e 84.1±1.4a 5.44±0.3d 72.7±2.3ab 7.95±0.5bc 2.58±0.1b 1.52±0.1c Liquid fraction after stem pretreatment/acid addition (1.5%) and enzymatic hydrolysis 0:100 2.88±0.1a 19.7±0.3a b.d.l. 12.9±0.4a 42.8±1.4b 5.22±0.3d 54.7±5.0d 3.46±0.2e 3.41±0.2a 2.20±0.2a 25:75 2.16±0.1bc 14.8±0.2b b.d.l. 3.32±0.3c 80.4±1.9a 9.92±0.2a 72.9±1.5a 8.81±0.3ab 3.50±0.1a 2.02±0.2ab 50:50 1.44±0.1de 9.85±0.1c b.d.l. 2.01±0.2de 82.4±1.5a 6.76±0.3c 72.7±2.6ab 9.01±0.2a 2.48±0.2b 1.65±0.2bc Stillage after alkaline 3% H2O2 treatment (1 h) and enzymatic hydrolysis 0:100 b.d.l. 15.2±0.7b b.d.l. 2.10±0.4de 86.1±1.6a 8.38±0.1b 64.2±3.0bc 6.38±0.2d 2.48±0.1b 1.10±0.1d 25:75 b.d.l. 11.4±0.5c b.d.l. 1.69±0.2de 85.1±2.0a 6.15±0.2c 63.7±2.5c 6.24±0.4d 1.59±0.2c 0.96±0.1d 50:50 b.d.l. 7.60±0.3d b.d.l. 1.45±0.1e 80.8±1.9a 4.01±0.1e 65.0±2.9bc 6.06±0.3d 1.24±0.2c 0.76±0.1d a - sugar utiliz.(%) = [(initial sugar content - sugar content after succinic acid production)/initial sugar content)] ∙100, b - calculated as g succinic acid produced/g sugar consumed according to Eq. (8), c – calculated as g succinic acid produced/100 g of initial biomass, b.d.l. - below detection limit Medium to feedstock ratio

•

Ethanol and succinic acid production from hemp in a biorefinery concept

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Succinic acid from liquid after H2SO4 pretreatment (7.7-9.0g/100g) and stillage (6.1-6.4g/100g)

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3% H2O2 optimal for biomass conversion to ethanol (166-175 kg/Mg of hemp)

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1.5% H2SO4 method ensured the highest combined ethanol/succinic acid output

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