Effects of hydrothermal pretreatment of sugar beet ...

Bioresource Technology 166 (2014) 187–193

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Effects of hydrothermal pretreatment of sugar beet pulp for methane production K. Ziemin´ski a,⇑, I. Romanowska b, M. Kowalska-Wentel a, M. Cyran c a

Institute of Fermentation Technology and Microbiology, Lodz University of Technology, Poland Institute of Technical Biochemistry, Lodz University of Technology, Poland c The Plant Breeding and Acclimatization Institute (IHAR) – National Research Institute, Poland b

h i g h l i g h t s The effect of LHW pretreatment of sugar beet pulp on methane fermentation was checked. The optimum processing condition for hydrothermal pretreatment was determined. Products of degradation of SBP were analysed. LHW pretreatment enhancement of methane yield from anaerobic fermentation.

a r t i c l e

i n f o

Article history: Received 4 April 2014 Received in revised form 8 May 2014 Accepted 10 May 2014 Available online 17 May 2014 Keywords: Sugar beet pulp Pretreatment Liquid Hot Water Anaerobic digestion

a b s t r a c t The effect of Liquid Hot Water treatment conditions on the degree of sugar beet pulp (SBP) degradation was studied. The SBP was subjected to hydrothermal processing at temperatures ranging from 120 to 200 °C. The relationship between processing temperature and parameters of liquid and solid fractions of resulting hydrolysates as well as the efficiency of their methane fermentation was determined. The highest concentration of free glucose (3.29 mg ml1) was observed when the hydrolysis was conducted at 160 °C (it was 4-fold higher than that after processing at 120 °C). Total acids and aldehydes concentrations in the liquid fractions were increased from 0.005 mg ml1 for the untreated SBP to 1.61 mg ml1 after its processing at 200 °C. Parameters of the hydrolysates obtained by the LHW treatment decided of the efficiency of methane fermentation. The highest cumulative methane yield (502.50 L CH4 kg1 VS) was obtained from the sugar beet pulp hydrolysate produced at 160 °C. Ó 2014 Published by Elsevier Ltd.

1. Introduction Utilization of lignocellulosic wastes for biofuels production has become a priority in UE countries (Menon and Rao, 2012). In contrast to depleting fossil resources, lignocellulosic materials are renewable, abundant, inexpensive and easily available in the global scale. One of them is sugar beet pulp (SBP), which is a process residue from sucrose manufacturing. One third of world 0 sucrose production is derived from sugar beets (Ro’per, 2002). Only in EU countries, as much as 16.4 million tons of sugar was produced within 2012/13 campaign, resulting in generation of near 5 million tons of sugar beet pulp dry matter. Poland is the third sucrose producer in Europe. Its annual production reaches around 1.87 million ton that causes a need to utilize approximately 0.6 million ton of solids contained in the sugar beet pulp (www.stat.gov.pl). Sugar beet pulp contains 22–30% cellulose, ⇑ Corresponding author. Tel.: +48 42 631 34 85. E-mail address: [email protected] (K. Ziemin´ski). http://dx.doi.org/10.1016/j.biortech.2014.05.021 0960-8524/Ó 2014 Published by Elsevier Ltd.

22–30% hemicellulose (mainly arabinans), 24–32% pectin and 1–3% lignin on a dry weight (Zheng et al., 2013). It has been used as animal feed but due to the low lignin content may be also applied in papermaking (Gigac et al., 2008), production of galacturonic acid and arabinose (Leijdekkers et al., 2013) or composites (Liu et al., 2005). However, other options, enabling complete utilization of large amounts of sugar beet pulp generated each year, have been still sought-after. One of potential alternatives consists of its conversion to biofuels (Hutnan et al., 2000; Ziemin´ski et al., 2012; Zheng et al., 2012). Multiple problems encountered in plant biomass processing are caused by the complex and tough structure of lignocellulosic complex. Its efficient deconstruction is a prerequisite of increased biofuels production. The presence of lignin and crystalline cellulose, and the limited access of enzymes to the surface of cellulose fibers, considerably reduce degradation of lignocellulosic biomass under anaerobic conditions and decrease the rate and efficiency of fermentations (Frigon and Guiot, 2010). Prior to enzymatic hydrolysis, which is a key step of bioconversion procedures, plant biomass has to be pretreated to weaken the

´ ski et al. / Bioresource Technology 166 (2014) 187–193 K. Ziemin

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structure of the lignocellulosic complex, reduce the crystallinity and polymerization degree of cellulose, and increase the availability of the polysaccharides to depolymerizing enzymes (Taherzadeh and Karimi, 2008). Degradation of lignocellulosic biomass is carried out using acids or bases, specific enzymes or hydrothermal treatment (Alvira et al., 2010; Zheng et al., 2013). According to literature, the efficiency of enzymatic saccharification of lignocelluloses is affected by numerous factors (Zhang et al., 2010) and results of acidic hydrolysis are not satisfying (Larsson et al., 1999). Therefore, these approaches may be replaced by hydrothermal processing, mainly by Liquid Hot Water (LHW) treatment. In this method, plant biomass is pretreated at elevated pressure for a few to several dozen minutes in the aqueous system (at an average dry matter concentration of 1–8%) at temperatures ranging from 120 to 260 °C (Zhang and Shahbazi, 2011). At these temperatures the dissociation of water molecules is enhanced and it acts as an acidic catalyst. Therefore, other reagents are not necessary (Mood et al., 2013). The LHW treatment causes dissolving of 40–60% biomass, including almost all hemicelluloses, 30–60% lignin, and 4–22% cellulose (Hu et al., 2008). Principal advantages of this technique are low costs, compared to enzymatic and chemical methods, and the inexpensive and easily available solvent. The resulting hydrolysates are characterized by high concentrations of monosaccharides and low levels of the inhibitors mentioned above, that increases the efficiency of their further enzymatic digestion (Weil et al., 1998). Thus, the LHW method is a highly attractive and promising procedure of plant biomass depolymerization before the anaerobic fermentation. In this study, we attempted to determine (i) the impact of thermohydrolysis conditions on the tough structure of lignocellulosic fraction of the sugar beet pulp, and (ii) the effect of parameters of thermohydrolysis products on the efficiency of methane fermentation.

Fig. 1. Schematic presentation of the reactor used in hydrothermal processes (1 – reactor chamber, 2 – sample collection unit, 3 – pressure control, 4 – regulation of temperature).

(Fig. 1) equipped with a stirrer (250 rpm). Samples of sugar beet pulp (100 g TS) were suspended in distilled water (300 ml) in a chamber (1) and processed for 20 min at temperatures varying from 120 to 200 °C (under variable pressure). When the processing was completed, the temperature was decreased to 25 °C. Resulting hydrolysates were used as feedstocks in methane fermentation processes. Before chemical analyses, the hydrolysates were filtered. Soluble sugars and acids were quantified in the filtrates while the insoluble residues were washed with distilled water and analyzed for the contents of sugars, uronic acids and Klason lignin.

2. Methods

2.3. Methane fermentation of pretreated sugar beet pulp

2.1. Materials

The batch anaerobic fermentation processes were conducted with stirring (4 rpm) under mesophilic conditions (at the temperature of 37 °C) in identical glass chambers (working volume of 1 L). The volume of gas produced within 24 h was measured using a system consisting of an electronic AalborgÒ GFM17 flow-meter governed by a computer system, which was also used for the continuous temperature monitoring. The constant fermentation temperature was maintained using a thermostat connected to a water jacket of the fermentor. Before fermentation, the pH of batches of sugar beet pulp (pretreated at 120, 130, 140, 150, 160, 170 and 200 °C, as described above) was adjusted to 7.2 using Na2CO3. The reference material was the sugar beet pulp suspended in water (without the thermal pretreatment). To initiate the fermentation process, the inoculum (described above) was added to the reactors in the amount of 20 g TS L1.

Parameters of the fresh sugar beet pulp and anaerobic sludge, which was used in this study, are shown in Table 1. The seed anaerobic sludge harvested from an agricultural crude biogas plant (supplied with sugar beet pulp) was used as an inoculum in fermentation processes after concentrating by sedimentation (for 24 h) using an Imhoff funnel. 2.2. Liquid Hot Water treatment of sugar beet pulp Batches of sugar beet pulp were processed in a high-pressure, 600 ml thermostatic (equipped with a water jacket) reactor

Table 1 Main characteristics of the fresh sugar beet pulp and concentrated anaerobic sludge. Parameter TS VS Ash COD pH VFA TKN Phosphorus Cellulose Hemicellulose Pectin Lignin

Unit 1

g kg g kg1 g kg1 gO2 kg1 TS gCH3COOH kg1 S g kg1 TS g kg1 TS % TS % TS % TS % TS

n.d. – not determined.

Sugar beet pulp

Anaerobic sludge

234.82 ± 2.02 223.39 ± 1.99 10.87 ± 1.00 988.04 ± 18.24 5.25 ± 0.01 5.28 ± 0.43 98.00 ± 1.20 2.56 ± 0.04 30.0 ± 2.4 26.8 ± 1.82 24.2 ± 2.1 4.1 ± 1.6

99.40 ± 1.04 72.50 ± 1.00 26.90 ± 1.02 994.50 ± 20.65 6.05 ± 0.01 4.28 ± 0.08 40.42 ± 0.82 10.91 ± 0.46 n.d. n.d. n.d. n.d.

2.4. Analytical methods Total solids (TS), volatile solids (VS), ash, volatile fatty acids (VFA), total Kjeldahl nitrogen (TKN) and phosphorus were quantified according to relevant standard methods (APHA, 1998). Total chemical oxygen demand (COD) of the sugar beet pulp, before and after hydrothermal processing, was determined by a modified method described by Raposo et al. (2008). Cellulose was quantified by the acid-base method consisting of dissolving of lignin and hemicelluloses in nitric acid and sodium hydroxide, followed by the gravimetric determination of cellulose content on a dry weight (Druce and Willcox, 1949). Hemicelluloses were quantified by Ermakov method (Arasimovich and Ermakov,


1987) based on their hydrolysis to monosaccharides that were further assayed using the alkaline DNS reagent according to Miller (1959). Pectins were assayed as calcium pectate according to Nanji and Norman (1928). Lignin was quantified according to the NREL’s standard laboratory analytical procedure (LAP) for determination of structural carbohydrates in biomass (Sluiter et al., 2008). Soluble sugars released via the thermal treatment were quantified in filtrates of the sugar beet pulp hydrolysates by HPLC (HPAEC technique), using a DIONEX ICS-3000 chromatograph equipped with an electronic detector and a Carbo Pac PA1 column (4 250 mm). The rate of flow of the mobile phase (16 mM aqueous solution of NaOH) was 1.0 ml min1. The volume of samples was 25 ll and the resolution was carried out at 30 °C. Phenolic acids and aldehydes in the filtrates were assayed using a liquid KNAUER chromatograph, equipped with a UV–VIS detector and a LiChrospher–100, RP–18 column (5 lm, 250 4 mm). Fractions of phenolics were eluted using the gradient of solutions A (formic acid in HPLC water, 10:90 v/v) and B (HPLC water: acetonitrile: formic acid, 40:50:10 v/v/v). The gradient profile was as follows (solution A contents): 88–70% (0–26 min), 70–0% (26–40 min), 0% (40–43 min), 0–88% (43–48 min) and 88% (48–50 min). The rate of flow of the mobile phase was 1.0 ml min1, and the separated fractions of phenolics were detected at 3 wavelengths: 280 nm (hydroxybenzoic acids), 320 nm (hydroxycinnamic acids) and 360 nm. Contents of hydroxybenzoic and hydroxycinnamic acids were expressed as gallic acid concentration. The analysis of non-cellulosic polysaccharides contained in cell walls of sugar beet roots was performed according to Englyst and Cummings (1984) with some modifications. The samples were suspended (1: 25 w/v) in sodium acetate buffer (100 mM, pH 5.0) and incubated with a thermostable a-amylase (10 ll, 3000 U ml1, Megazyme) in a boiling water bath for 1 h. The samples were cooled to 45 °C, mixed with an amyloglucosidase (40 ll, 3300 U ml1, Megazyme) and incubated in a shaking water bath at 45 °C for 16 h. The suspension was mixed with 4 volumes of 96% ethanol, and kept at 6 °C for 4 h to precipitate water-extractable polysaccharides. The pellet containing both water-extractable and waterinsoluble polymers, was separated by centrifugation (20 min, 10,000g, room temperature) in a Sigma 4–15 centrifuge (Sigma Laborzentrifugen, Osterode, Germany) and dried at 45 °C for 6 h. The monosaccharide content and composition of total polysaccharide fraction in the pellet were analyzed by gas chromatography of alditol acetates obtained after hydrolysis with 1 M sulfuric acid (2 h, 100 °C) (Englyst and Cummings, 1984). Alditol acetates were separated on wide-bore capillary column (Rtx-225, 30 m, 5.53 mm i.d., Restek, Bellefonte, PA, USA) in a HP 5890 Series II Plus gas chromatograph (Hewlett–Packard, Waldbronn, Germany) equipped with a flame ionization detector. The column was heated at 190 °C for 2 min, then the temperature programme was 190–220 °C at 5 °C min1 and 220 °C for 5 min. Output signals were collected and integrated by ChemStation software (Hewlett–Packard) using meso-erythritol as an internal standard. Blanks were run to measure any contribution from the reagents. The Klason lignin was measured gravimetrically as the residue left after two-step acid hydrolysis [72% (w/w) H2SO4, 1 h 35 °C; 1 M H2SO4, 3 h, 100 °C] and corrected for ash (Theander et al., 1995). The content of uronic acid was determined according to Scott (1979). Methane and carbon dioxide concentrations in gaseous fermentation products were determined by GC, using an Agilent 7890A GC chromatograph equipped with a TCD detector and a 2D column system (connected by a pneumatic switch): molecular sieve 5A, 60/80 mesh 6 ft 1/8 in and Porapak Q 80/100 mesh, 6 ft 1/8 in.

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3. Results and discussion 3.1. Thermal hydrolysis of sugar beet pulp The dependence of chemical composition of sugar beet pulp hydrolysates on the temperature of LHW treatment is shown in Table 2. Presented data provide evidence that this processing caused a decrease in the total solids and volatile solids contents compared with the untreated pulp. This decrease was brought about by the catalytic action of water. Simultaneously, an apparent increase in volatile fatty acids concentration was observed. It grew proportionally to the temperature of LHW process and varied from 14.23 g CH3COOH kg1 TS (after the treatment at 120 °C) to 55.89 g CH3COOH kg1 TS (after the treatment at 200 °C). Values of COD were only slightly affected by the temperature of LHW treatment. Presented results are consistent with that reported by Budde et al. (2014). The pH of untreated sugar beet pulp was 5.25 (Table 1). The LHW processing caused a considerable decrease in pH, which was clearly proportional to the temperature (to 5.01 after processing at 120 °C and to 3.20 after processing at 200 °C). This phenomenon was caused by the presence of acids among products of hemicelluloses and lignin degradation (such as vanillic, hydroxybenzoic, coumaric, syringic and ferulic acids) (Table 4) and elevated concentrations of volatile fatty acids in the hydrolysates (Table 2). Temperature of sugar beet pulp hydrolysis strongly affected amounts and types of products generated by degradation of non-starch polysaccharides. The degree of their destruction was estimated based on concentrations of acids, aldehydes (Table 4) and monosaccharides (Fig. 2) that were released by this process into the liquid phase of the hydrolysates, and amounts of sugars, uronic acids and lignin, constituting the insoluble residues (Table 3). Dominating components of the solids, remaining after LHW processing of fresh sugar beet pulp over the temperature range 120–150 °C, were arabinose, galactose and uronic acids (Table 3). It is not surprising because the sugar beet pectin is rich in galacturonic acid (54.4–77.9%) and arabinose (1.8–12.5%). Like in pectin molecules from other plants, its backbone consists of a-1–4-linked galacturonic acid residues, either free or esterified. Around every tenth of these residues bears a-1–2-linked rhamnose residue. Some of the latter residues are substituted with a-arabinose units, attached in position 3 (Hutnan et al., 2000). The percentage content of arabinose in the insoluble fractions decreased with the rise in processing temperature (from 66% at 120 °C to 2% at 200 °C), that provides evidence of the weak thermal stability of pectin. The same tendency was observed for the contents of rhamnose, galactose and uronic acids, while amounts of xylose, mannose, fucose and glucose remained virtually constant, irrespective of the sugar beet pulp hydrolysis temperature. Concentrations of xylose and mannose were low and their appearance was caused by the weak thermal stability of hemicelluloses such as xylan and glucomannan, which undergo degradation at temperatures above 180 °C. The source of glucose, which was also present in small amounts, was thought to be residual sucrose, which remained in the sugar beet pulp. The similar results were reported by Kühnel et al. (2011) who subjected sugar beet pulp to the hydrothermal pretreatment at 120–170 °C, before its enzymatic saccharification yielding the feedstock for ethanol fermentation. However, in contrast to a decrease in neutral sugars concentration from 24.76% in the untreated sugar beet pulp to 2.52% in the insoluble residues after thermohydrolysis at 200 °C, the concentration of recalcitrant lignin was increased from 1.14% to 21.71%, respectively. Phenolic acids contained in plant biomass are usually components of esters and glycosides attached to lignin. Thermal


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Table 2 Characteristics of hydrothermally treated and untreated sugar beet pulp. Hydrolysis temperature (°C)

TS g kg1

VS g kg1

VFA gCH3COOH kg1 TS

COD gO2 kg1 TS

Untreated 120 130 140 150 160 170 200

100.22 ± 2.82 33.42 ± 0.74 33.02 ± 0.68 32.68 ± 0.65 32.02 ± 0.60 31.12 ± 0.59 31.08 ± 0.56 30.08 ± 0.42

87.12 ± 2.67 32.10 ± 0.72 31.75 ± 0.67 31.63 ± 0.65 30.82 ± 0.61 29.57 ± 0.60 28.98 ± 0.58 28.74 ± 0.45

6.30 ± 0.84 14.23 ± 1.02 18.26 ± 1.08 20.35 ± 1.20 26.04 ± 1.23 32.84 ± 1.25 40.67 ± 1.52 55.89 ± 2.00

988.04 ± 18.24 952.10 ± 16.54 940.78 ± 16.23 936.42 ± 15.98 930.63 ± 15.75 920.89 ± 16.05 912.72 ± 16.21 900.56 ± 16.78

Table 3 Contents of selected monosaccharides, uronic acids and Klason lignin in the fresh sugar beet pulp and insoluble residues after its hydrothermal processing. Hydrolysis temperature (°C)

Rhamnose

Fucose

Arabinose

Xylose

Mannose

Galactose

Glucose

Uronic acids

Klason lignin

4.21 ± 0.14 4.93 ± 0.15 4.82 ± 0.15 4.16 ± 0.07 3.90 ± 0.09 2.54 ± 0.07 2.06 ± 0.01 0.25 ± 0.02

0.81 ± 0.01 0.75 ± 0.05 0.82 ± 0.01 0.80 ± 0.03 0.89 ± 0.05 1.13 ± 0.04 1.01 ± 0.03 1.20 ± 0.04

15.02 ± 0.22 13.41 ± 0.35 9.47 ± 0.38 8.03 ± 0.12 6.05 ± 0.33 2.92 ± 0.01 1.51 ± 0.05 –

1.14 ± 0.01 2.51 ± 0.16 3.26 ± 0.19 3.21 ± 0.22 5.45 ± 0.20 8.13 ± 0.40 10.43 ± 0.25 21.71 ± 0.14

% TS Untreated 120 130 140 150 160 170 200

0.95 ± 0.06 1.04 ± 0.00 0.98 ± 0.01 0.85 ± 0.01 0.87 ± 0.01 0.44 ± 0.02 0.32 ± 0.00 –

0.11 ± 0.00 0.12 ± 0.00 0.13 ± 0.00 0.14 ± 0.01 0.14 ± 0.02 0.10 ± 0.01 0.05 ± 0.00 –

16.98 ± 0.16 16.57 ± 0.26 14.19 ± 0.35 9.97 ± 0.16 5.51 ± 0.20 2.28 ± 0.15 1.03 ± 0.01 0.06 ± 0.01

1.26 ± 0.08 1.40 ± 0.04 1.68 ± 0.16 1.63 ± 0.04 1.61 ± 0.09 1.81 ± 0.07 1.32 ± 0.02 0.68 ± 0.02

0.16 ± 0.01 0.26 ± 0.04 0.23 ± 0.03 0.12 ± 0.02 0.28 ± 0.04 0.25 ± 0.02 0.15 ± 0.04 0.34 ± 0.01

Table 4 The impact of processing temperature on concentrations of selected acids and aldehydes in filtrates of sugar beet pulp hydrolysates. Hydrolysis temperature (°C)

p-hydroxybenzoic acid

Vanillic acid

4-hydroxybenzoic aldehyde

Syringic acid

Vanillin

p-coumaric acid

Syringic aldehyde

Ferulic acid

0.04 ± 0.10 3.2 ± 0.80 3.6 ± 0.92 6.2 ± 1.20 10.0 ± 3.00 11.3 ± 2.50 22.0 ± 4.10 22.5 ± 3.80

– – – – 1.2 ± 0.80 3.2 ± 1.21 6.0 ± 2.10 7.5 ± 2.20

– – – – 2.6 ± 0.90 1.8 ± 0.62 3.4 ± 1.02 3.5 ± 0.92

– 1.2 ± 0.40 1.4 ± 0.80 1.9 ± 0.60 1.7 ± 0.50 2.0 ± 0.20 3.8 ± 0.60 4.0 ± 0.80

lg ml1 Untreated 120 130 140 150 160 170 200

4.6 ± 1.40 5.7 ± 0.02 18.3 ± 1.00 33.7 ± 3.00 104.9 ± 10.00 184.6 ± 12.00 617.1 ± 41.00 1481.4 ± 92.00

– 1.2 ± 0.10 2.9 ± 0.50 8.2 ± 2.00 21.3 ± 2.50 32.9 ± 3.00 48.9 ± 3.30 54.4 ± 4.80

– 0.4 ± 0.02 0.8 ± 0.04 1.3 ± 0.01 2.7 ± 0.20 4.2 ± 0.80 4.3 ± 0.90 4.5 ± 1.00

processing at acidic pH brings about the hydrolysis of ester and glycosidic bonds, leading to an increase in concentrations of free phenolic acids. Concentrations of phenolic acids and aldehydes in the liquid phase increased from 0.005 mg ml1 in the untreated sugar beet pulp to 1.61 mg ml1 after its processing at 200 °C. The dominating of these compounds was p-hydroxybenzoic acid, which constituted 96% of this fraction when the treatment was conducted at 200 °C (Table 5). At so high concentration, this acid may cause inhibition of biogas fermentation. Banks et al. (1996) studied the effect of phenolics contained in wastes from olive oil extraction on the course of methanogenesis and found that p-hydroxybenzoic acid in concentrations up to 700 mg l1 had no impact on methane production. However, at levels P1000 mg l1 it inhibited the process (Banks et al., 1996). Concentrations of the other phenolic compounds were low and ranged from 0.0004 to 0.0054 mg ml1. Concentrations of arabinose and galactose in the liquid phase of the hydrolysates apparently grew when the temperature of LHW processing was increased from 120 to 170 °C, from 0.07899 to 3.68 mg ml1, and from 0.0172 to 0.3099 mg ml1, respectively (Fig. 2). However, processing at 200 °C decreased their levels by 84% and 59%, respectively. Amounts of xylose and mannose, which probably originated from degradation of hemicelluloses contained in the sugar beet pulp, only slightly increased when the temperature of treatment was increased from 120 to 160 °C, from

0.05 ± 0.02 0.8 ± 0.10 0.7 ± 0.20 4.9 ± 0.60 12.4 ± 1.8 13.1 ± 1.60 20.2 ± 5.60 36.4 ± 4.01

1.3779 and 1.1903 mg ml1to 1.3902 and 1.5035 mg ml1, respectively. When the temperature of LHW processes was increased to 170 and 200 °C, concentrations of these monosaccharides were decreased by 77% and 81%, and by 41% and 75%, for xylose and mannose, respectively. The highest concentration of free glucose (3.2881 mg ml1) was observed when the thermal treatment of sugar beet pulp was carried out at 160 °C. It was 4-fold higher compared to that after processing at 120 °C and by 62% and 91% higher than after the treatment at 170 °C and 200 °C, respectively. The same tendency was reported by Kühnel et al. (2011). Also Martínez et al. (2010) observed the high solubility of arabinan and galacturonate from sugar beet pulp after its exposure to hydrothermal processing over the temperature range of 150–175 °C. 3.2. Methane fermentation of treated and untreated sugar beet pulp Results of methane fermentation of LHW treated and untreated sugar beet pulp batches are shown in Figs. 3 and 4 and in Table 5. Fig. 3 presents the cumulative amounts of methane derived from 1 kg VS of the sugar beet pulp. Processes of methane fermentation were conducted until termination of the biogas production after 28 days. The reference process consisted of fermentation of the untreated sugar beet pulp. Yields of methane synthesis were clearly dependent on the temperature of LHW treatment, deciding of the chemical composition of the hydrolysates. The analysis of

Xylose

Glucose

Mannose

Galactose

3 2.5 2 1.5 1 0.5 0 120

140

160

180

200

Hydrolisis temperature [oC] Fig. 2. The effect of temperature on concentrations of selected sugars in the liquid phase of sugar beet pulp hydrolysates.

400

300

200

100

untreated

120

130

140

150

160

170

200

0 0

5

10

15

20

25

Time [day] Fig. 3. Cumulative methane yields from batch anaerobic digestion tests of untreated and treated sugar beet pulp.

CH4

CO2

CH4/CO2 ratio

80

2.5

70 60

2

50 40

1.5

30 20

1

10

CH4/CO2 ratio

curves depicting the dynamics of methane production (Fig. 3) provides evidence that amounts of methane that were synthesized in successive days of anaerobic fermentation depended on LHW processing temperature, which decided of concentrations of acids and aldehydes in the sugar beet pulp hydrolysates (Table 4). The highest total methane volume (502.50 L CH4 kg1 VS) was achieved from the sugar beet pulp hydrolysate produced at 160 °C (Table 5). For the first 4 days of this hydrolysate fermentation, methane was synthesized at the rate of 0.1 L d1 and as much as 79.68% of the cumulative methane yield was obtained. The efficient methane synthesis from this feedstock was thought to result from the relatively high concentration of soluble sugars and low levels of phenolic acids that act as fermentation inhibitors (Table 4). Furthermore, this fermentation process caused the largest decrease in COD of the hydrolysate (by 86%), which was 53.5% higher compared to the COD reduction observed when the untreated sugar beet pulp was fermented. For the first 2 days of anaerobic fermentation, the productivities of methane from the hydrolysates obtained by LHW treatments at 160 and 200 °C were comparable. The rate of methane synthesis from the feedstock produced at 200 °C was 0.08 L d1, and for the first 4 days as much as 75.47% of the cumulative methane yield was obtained. However, the further progress of this hydrolysate fermentation showed explicitly that the high concentrations of p-hydroxy-benzoic acid – 1481.4 lg ml1 and other phenolic compounds (Table 4) that were released at this temperature negatively affected methane yield. In consequence, the cumulative methane volume for this hydrolysate was only 424.52 L CH4 kg1 VS. It was lower compared with the other fermentation results obtained in this study. It means that too high processing temperatures (200 °C and above) should be avoided. Pretreatment under harsh conditions leads to formation of hemicellulose and lignin degradation products, like phenolic and heterocyclic compounds, e.g., vanillin, vanillic alcohol, furfural and hydroxymethylfurfural (HMF). Many of them are toxic to microorganisms and inhibit their growth (Barakat et al. 2012). The same observations were reported by Banks et al., (1996). Also Budde et al. (2014) reported that the most suitable feedstock for methane fermentation was obtained by LHW treatment at 160 °C while processing at temperatures 200–220 °C caused elevated

191

500

CH4 and CO2 contents [% (v/v)]

Sugars [mg ml-1]

3.5

Arabinose

Cumulative methane production [L CH4 kg-1 VS]


0.5

0

Temperature of hydrolisis [oC] Fig. 4. The impact of LHW treatment temperature on mean concentrations of methane and carbon dioxide as well as CH4/CO2 ratio in the biogas derived by methane fermentation of the sugar beet pulp.

levels of potential fermentation inhibitors. The rate of methane synthesis for the first 4 days of fermentation of the sugar beet pulp processed at 170 °C was 0.07 L d1, resulting in production of 57.14% of the cumulative methane volume during this period. Although this initial rate was lower compared to the rates observed for the batches of sugar beet pulp that were processed at 160 °C and 200 °C, the cumulative methane volume after 28 days of the anaerobic process was only 2.49% lower than that achieved by fermentation of the feedstock produced at 160 °C. This result shows that the microflora involved in the methane fermentation tolerated the high concentration of p-hydroxybenzoic acid (617.1 lg ml1) generated by LHW treatment at 170 °C, which also gave rise to the high concentration of soluble sugars (Fig. 2). The

Table 5 Methane yield and COD removal in anaerobic digestion of hydrothermally treated and untreated sugar beet pulp. Untreated

CH4 yield L CH4 kg1 VS COD removal %

285.42 56

Hydrolysis temperature (°C) 120

130

140

150

160

170

200

432.84 71

436.69 72

445.40 75

476.50 78

502.50 86

490.00 84

424.52 68

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latter parameter positively affected methane synthesis. The reduction of COD in this process was 84%. Yields of methane from 28 day processes of anaerobic fermentation of the sugar beet pulp hydrolysates produced at 120, 130, and 140 °C were on average 12% lower compared to the maximum yield, obtained from the hydrolysate prepared at 160 °C. The lowest methane production (285.42 L CH4 kg1 VS, 76% lower compared to the maximum yield) was observed in the process of anaerobic fermentation of the untreated sugar beet pulp, which also caused the lowest decrease in COD (by 56%) of this feedstock. The dependence of volumetric concentrations of methane and carbon dioxide as well as CH4/CO2 ratio in the biogas derived by methane fermentation of the sugar beet pulp on the temperature of LHW pretreatment is shown in Fig. 4. The lowest mean methane concentration (55%) and highest mean carbon dioxide concentration (40.5%) were observed in the biogas derived from the sugar beet pulp, which was not subjected to the LHW pretreatment prior to methane fermentation. For this biogas, the CH4/CO2 ratio was 1.35. When the pretreatment temperature was increased from 120 to 160 °C, the methane content grew from 59.5% to 68%, respectively. In consequence, the CH4/CO2 ratio was increased from 1.61 to 2.29, respectively. Further rise in LHW processing temperature from 160 °C to 170 °C and 200 °C caused a decrease in the CH4/CO2 ratio by 2.6% and 7.4%, respectively, which means that the temperature of 160 °C was most suitable for the LHW pretreatment of the sugar beet pulp. LHW pretreatment has been extensively carried out to improve methane yield from lignocellulosic biomass, including sunflower stalks, sugarcane bagasse, rice straw, wheat straw, Miscanthus (species of giganteus and sacchariflorus) and grass (Pennisetum hybrid), paper tube residuals, municipal solid waste, and microalgae (Zheng et al., 2014). Processing of various wastes under suitable conditions of temperature, pressure and time enables to increase methane yields by 7–222% compared with fermentation of untreated wastes (Zheng et al., 2014). The effectiveness of LHW varied for different biomass materials, depending on the chemical compositions and structural properties, and the optimal pretreatment conditions were highly biomass related. Findings of Chandra et al. (2012a,b) who produced methane from wheat straw (Chandra et al. 2012a) and rice straw (Chandra et al. 2012b), and used LHW for their deconstruction, clearly demonstrate that this technique is a promising and efficient approach to the pretreatment of lignocellulosic residues. Methane yields from pretreated wheat and rice straws were increased by 20% and 222% respectively, compared with the untreated straws. LHW hydrolysis of lignocellulosic residues may lead to intensified production of second generation fuels. This technique is competitive to other biomass hydrolysis methods. Furthermore, hydrothermal pretreatment process requires much lower need of chemicals for neutralization of the produced hydrolyzate and produces lower amounts of neutralization residues compared to many chemical processes. 4. Conclusions Results of this study explicitly demonstrate that the temperature of LHW treatment of SBP decides of amounts and types of products released from non-starch polysaccharides. The highest concentration of free glucose was achieved by processing at 160 °C (it was 4-fold higher compared to the treatment at 120 °C). Yields of methane derived from sugar beet pulp hydrolysates produced at 200 °C and 160 °C were by 48% and 76% higher, respectively, compared with yields from the untreated SBP. The significant enhancement of methane production provides evidence that LHW pretreatment is an efficient and environmentally friendly method of improvement of SBP utilization.

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