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Waste Management 32 (2012) 1131–1137

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Enzymatic pretreatment of lignocellulosic wastes to improve biogas production

K. Ziemiński a,⇑, I. Romanowska b, M. Kowalska a
Institute of Fermentation Technology and Microbiology, Technical University of Lodz, Poland
Institute of Technical Biochemistry, Technical University of Lodz, Poland

a r t i c l e i n f o a b s t r a c t

Article history: The effect of enzymatic pretreatment of sugar beet pulp and spent hops prior to methane fermentation
Received 22 September 2011 was determined in this study. These industrial residues were subjected to enzymatic digestion before
Accepted 20 January 2012 anaerobic fermentation because of high fiber content (of 85.1% dry matter (DM) and 57.7% DM in sugar
Available online 18 February 2012
beet pulp and spent hops, respectively). Their 24 h hydrolysis with a mix of enzymatic preparations Celu-
star XL and Agropect pomace (3:1, v/v), with endoglucanase, xylanase and pectinase activities, was most
Keywords: effective. Reducing sugars concentrations in hydrolysates of sugar beet pulp and spent hops were by
Lignocellulosic waste
88.9% and 59.4% higher compared to undigested materials. The highest yield of biogas was obtained from
Sugar beet pulp
Spent hops
the enzymatic hydrolysate of sugar beet pulp (183.39 mL/d from 1 g COD at fermenter loading with
Enzymatic hydrolysis organic matter of 5.43 g COD/L  d). Fermentation of sugar beet pulp gave 19% less biogas. Methane fer-
Anaerobic fermentation mentation of spent hops hydrolysate yielded 121.47 mL/d biogas from 1 g COD (at 6.02 g COD/L  d, 13%
more than from spent hops). These results provide evidence that suitable enzymatic pretreatment of lig-
nocellulosic wastes improve biogas yield from anaerobic fermentation.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction generated annually (Altundogan et al., 2007). Traditionally, sugar

beet pulp was utilized as a feed for ruminants. Recently it has been
World economy is based on non-renewable, fossil fuels such as also used in production of microbial proteins, pectin (Levigne et al.,
petroleum and natural gas that results in rapid depletion of their 2002), citric acid (Adham, 2002), pectinolytic enzymes and ferulic
reserves. Furthermore, their exploitation, processing and combus- acid (Ferreira et al., 2007) and in paper making (Vaccari et al.,
tion pose a serious threat to natural environment. To meet rising 2005).
energy demands and solve problems caused by climate changes Spent hops is the residue from hops used in the brewing indus-
and fast economic growth the alternative, low-emission energy try. In 1997, 1,954,000 tons of brewers’ spent hops and spent malt
technologies have to be developed urgently. According to IEA were generated in Germany (Laufenberg et al., 2003). In Poland the
(International Energy Agency), the implementation of environmen- annual production of these two wastes is above 1000 tons. Spent
tally friendly energy production technologies should halve carbon hops are powder-like solids and their utilization causes serious
dioxide emissions till 2050. In EU countries at least 20% of the total problems. They are utilized only as a fertilizer. Their usage in feed
energy supplies in 2020 should be derived from the renewable re- formulations is limited due to the component bitter acids such as
sources (EurObserv’er Report, 2008). humulons (a-acids) and lupulons (b-acids). Alternative, rational
Lignocellulosic biomass is an attractive, renewable feedstock for applications of spent hops have not been proposed yet (Nikolic
sustainable production of diverse biofuels including bioethanol et al., 2005).
and biogas (Saxena et al., 2009). The concept of biofuels manufac- The increasing knowledge about anaerobic fermentation pro-
turing is based on the assumption that they may be derived only cesses gives rise to higher product yields from diverse feedstocks
from the biomass and organic wastes (Naik et al., 2010). Sugar beet including lignocellulosic residues. Studies on anaerobic plant bio-
pulp and spent hops rank among lignocellulosic residues that can mass biodegradation revealed the slow lignin decomposition
be used in biofuel, e.g. biogas, production. (Tuomela et al., 2000; Sun and Cheng, 2002) and beneficial impact
The first one is a by-product remaining after sucrose extraction of enzymatic pretreatment (Micard et al., 1997; Kristensen et al.,
from sugar beets yielding approximately 70 kg dry matter (DM) of 2009; Frigon et al., 2012). Traditional biomass pretreatments were
pulp from 1 ton DM of sugar beets (Spagnuolo et al., 1997). Only in based on acid or base hydrolysis (Wang et al., 2008; Fischer and
EU countries as much as 14 million ton DM of this by-product is Bipp, 2005). Resulting hydrolysates required troublesome process-
ing before application in fermentation processes. Degradation of
⇑ Corresponding author. lignocelluloses by microbial enzymes outperforms chemical hydro-
E-mail address: (K. Ziemiński). lysis because enzymes display the high substrate and reaction

0956-053X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
1132 K. Ziemiński et al. / Waste Management 32 (2012) 1131–1137

specificity, operate under mild conditions and do not generate meter AalborgÒ GFM17 (5) connected to the computer system.
by-products (Micard et al., 1996). These advantageous properties The constant temperature inside the fermentation chambers was
caused that enzymatic preparations have been increasingly applied kept by a thermostat connected to their water jackets (4 and 1).
in chemical, fuel, food and textile industries, in washing powder After 24 h enzymatic hydrolysis (carried out at 50oC) of 10% (w/v,
formulations and in paper making (Howard et al., 2003). DM) sugar beet pulp and spent hops, treated with the mixture of
Celustar XL and Agropect pomace preparations (3:1, v/v), at en-
2. Materials and methods zyme dose of 0.15 FPU/g DM, the hydrolysates (in aliquots of
50 g wet mass) were directed into the fermenter. They were
2.1. Sugar beet pulp and spent hops pumped twice a day by using peristaltic pumps (9) into the fer-
mentation chambers. Aqueous suspensions of the two residues
Chemical composition of the two residues used throughout the (without enzymatic pretreatment) were fermented as controls.
study such as sugar beet pulp and spent hops is shown in Table 1. Na2CO3 (10) was used to regulate pH of the sugar beet pulp hydro-
lysate during fermentation. An anaerobic sludge derived from an
2.2. Enzyme preparations agricultural biogas facility was used as an inoculum in a dose of
20 g DM/L. Microorganisms contained in this sludge were adapted
Processes of hydrolysis of sugar beet pulp and spent hops were to fermentation conditions for 4 months at retention time of
conducted by using enzymatic preparations characterized in Table 2. 30 days. After 4 months this time was reduced to 20 days.

2.3. Pretreatment of raw materials before enzymatic hydrolysis 2.5. Analytical methods

2.3.1. Mechanical comminution Contents of dry mass, organic substance, volatile fatty acids,
Before enzymatic hydrolysis the sugar beet pulp was milled by ammonium nitrogen and phosphate ions were determined accord-
using a Spruto Waldron 374 disc mill. The ultimate diameter of ing to standard methods (APHA, 1998). COD levels of suspensions
particles was 0.25 cm. Spent hops powder was not milled. and enzymatic hydrolysates of sugar beet pulp and spent hops
were estimated by a modified Raposo method (Raposo et al.,
2.3.2. Enzymatic hydrolysis of sugar beet pulp and spent hops 2008). Protein content was assayed by Kjeldahl method.
To initiate enzymatic hydrolysis, enzymatic preparations shown Concentrations of methane, carbon dioxide and hydrogen sulfide
in Table 2 were mixed with suspensions of sugar beet pulp and in the biogas obtained by anaerobic fermentation were determined
spent hops in tap water (10% w/v, DM). Enzyme doses varied be- by gas chromatography by using Perkin Elmer (USA) autosystem
tween 0.03 and 0.75 FPU/g DM and hydrolysis was conducted at equipped with PERMABONDÒ P-100 column (100 m  0.25 mm ID)
50 °C. Reducing sugars concentration and pH were determined at and flame ionization detector (FID). The flow rate of carrier gas
24 h intervals. To evaluate the synergy of action of Celustar XL (hydrogen) was 2.3 ml/min (200 kPa) and the injector temperature
and Agropect pomace, and Optimash VR and Agropect pomace was 250 °C. The preliminary analysis of the biogas composition was
preparations, these preparations were mixed in volume ratios of: conducted by using a Madur GA-21 plus biogas analyzer.
1:0, 1:1, 2:1 and 3:1, and added to the suspensions of sugar beet Reducing sugars concentration was estimated by Miller method
pulp and spent hops in analogous doses to the single preparations. with alkaline 30 ,50 -dinitrosalicylic acid (DNS) solution (Miller,
Reducing sugars concentration in enzymatic hydrolysates was 1959). Hemicelluloses were quantified by Ermakov method
compared with that in relevant controls containing only the resi- (Arasimovich and Ermakov, 1987) consisting in their saccharifica-
dues (without any enzyme). The most effective combination of tion to monosaccharides and their assay by Miller method.
enzymatic preparations was selected on this basis. In the next step, Percentage content of cellulose was determined gravimetrically
both the dose of this mix and hydrolysis duration were optimized. after solubilization of lignin and hemicelluloses in nitric acid and
The processes were conducted for 8 days at 50 °C. sodium hydroxide according to Druce and Willcox (Druce and
Willcox, 1949). Pectin was quantified as calcium pectate by the
2.4. Experimental set-up method of Carre and Haynes (Carre and Haynes, 1922) modified
by Nanji and Norman (Nanji and Norman, 1928). The content of lig-
Semi-batch anaerobic fermentation processes were conducted nin was estimated using the standard procedure established by the
under mesophilic conditions at 37 °C by using the set-up schemat- National Renewable Energy Laboratory (NREL). DM of the precipi-
ically shown in Fig. 1. Fermentations were run in two identical tated Klason lignin was determined gravimetrically (Lin and Dence,
glass fermentation chambers with working volume of 2 L (2 and 3). 1992).
Their contents were stirred at a rate of 4 rpm (9). The volume of Cellulolytic activity was determined according to Ghose (Ghose,
biogas produced in 24 h was measured by an electronic flow rate 1987) and expressed in filter paper units (FPU), which denote
1 lmol of reducing sugars released in 1 min from Whatman no 1
Table 1 filter paper under standard reaction conditions.
Chemical composition of sugar beet pulp and spent hops. Soluble sugars contained in enzymatic hydrolysates were quan-
tified by HPLC by using Dionex ICS-3000 chromatograph equipped
Biomass component Unit Sugar beet pulp Spent hops
with HPAEC detector and Carbo Pac PAI column (4  250 mm). The
DM [g/kg] 260.88 890.00
sugars were eluted isocratically with 16 mM NaOH at 30 °C and at
Ash [g/kg] 12.84 102.35
COD [g/kg DM] 988.04 1155.75
a flow rate of 1.0 ml/min. The volume of injected samples was
Ammonium nitrogen [g/kg DM] 14.8 23.47 25 ll.
Phosphorus PO3 [g/kg DM] 3.2 5.51
Protein [% DM] 10.3 20.0
Fiber, including: [% DM] 85.1 57.7 3. Results and discussion
Cellulose 30.0 19.6
Hemicellulose 26.8 12.5 Chemical composition of the 2 tested residues is shown in Table 1.
Pectin 24.2 4.0
The content of DM and organic substance in spent hops is by 70.6%
Lignin 4.1 21.0
and 68.5%, respectively, higher than in the sugar beet pulp. The
K. Ziemiński et al. / Waste Management 32 (2012) 1131–1137 1133

Table 2
Characterization of enzyme preparations.

Preparation Enzyme source Main activities* FPU [lmol/min]

Cellusoft Conc. L Trichoderma reesei Endoglucanase 17.96
Celustar XLb Trichoderma longibrachiatum Endoglucanase, xylanase 10.82
Optimash™VRc Penicillium funiculosum Xylanase, endoglucanase 16.94
Agropect pomaced Pectinase 7.05
Novozymes (Denmark).
Dyadic International, Inc. (Florida USA).
Genencor (San Francisco, USA).
Danisco Poland Ltd.
Activities given by producers.

Fig. 1. Experimental set-up (1). Thermostat, (2 and 3). Fermentation chambers (fermenters), (4). Temperature sensor, (5). Electronic gas flowmeter, (6). Peristaltic pumps, (7).
Tank for hydrolysates, (8). Tank for suspensions not subjected to enzymatic pretreatment (controls), (9). Stirrer, (10). pH adjustment.

high fiber content in the latter (85.1% DM) and in the first material and the level of reducing sugars was observed in hydrolysates of
(57.7% DM) can be reduced by their enzymatic pretreatment spent hops (Fig. 2B). At the dose of 0.15 FPU/g DM the concentra-
(Schwarz, 2001) yielding fermentable soluble sugars (Micard tion of reducing sugars was by 59.4% higher than in the control.
et al., 1997). An increase in enzymes dose up to 0.75 FPU/g DM resulted in only
18% rise in reducing sugars content.
3.1. Enzymatic hydrolysis of sugar beet pulp and spent hops HPLC analysis of resulting sugar beet pulp hydrolysates (di-
gested with the mix of Celustar XL and Agropect pomace) revealed
The interplay between the enzyme dose and reducing sugars con- mainly glucose (44.39%), mannose (33.46%) and xylose (19.53%).
centration after 24 h hydrolysis of sugar beet pulp and spent hops is Hydrolysates of spent hops obtained under the same conditions
presented in Fig. 2. The difference in the efficiency of action between contained mainly glucose (51.06%), rhamnose (30.71%) and galact-
the applied enzyme preparations and their combinations is appar- ose (14.45%) (Table 3). The difference in the composition of the
ent. The highest degree of sugar beet pulp and spent hops sacchari- hydrolysates stems from different composition of these lignocellu-
fication was achieved when these substrates were digested with a losic residues. The high xylose concentration in the first hydroly-
mix of Celustar XL and Agropect pomace preparations (volume ratio sate was caused by the high hemicellulose content in sugar beet
of 3:1, respectively). Among the 3 tested preparations only Agropect pulp (Table 1).
pomace displays pectinolytic activity. Pectinases play important role In the next step, duration of hydrolysis was optimized and
in degradation of sugar beet pulp and other lignocellulosic materials changes in pH during this process were determined. The substan-
(Spagnuolo et al., 1997; Considine et al., 1988) and improve an access tial increase in reducing sugars concentration was observed only
of endoglucanase to cellulose chains (Considine et al., 1988). This within the first 24 h of enzymatic digestion, by 88.9% and 59.4%
pectinolytic preparation contains also cellulases and therefore in hydrolysates of sugar beet pulp and spent hops, respectively
its activity in filter paper hydrolysis was also determined (Table 2).
For relatively small doses of the mix of Celustar XL and Agropect Table 3
Percentage content of monosaccharides in hydrolysates used for biogas production.
pomace (up to 0.15 FPU/g DM) the concentration of reducing sug-
ars in hydrolysates of sugar beet pulp increased with enzymes dose Sugar [%] Sugar beet pulp Spent hops
(Fig. 2A). At the dose of 0.15 FPU/g DM it was by 88.9% higher com- Xylosis 19.53 0.45
pared to the control. When the dose of enzymes was increased Glucose 44.39 51.06
from 0.15 FPU/g DM to 0.75 FPU/g DM, the concentration of reduc- Galactose 0.61 14.45
Mannose 30.46 2.42
ing sugars in sugar beet pulp hydrolysates was increased by only
Rhamnose - 30.71
24.5%. The similar relationship between the dose of the enzymes
1134 K. Ziemiński et al. / Waste Management 32 (2012) 1131–1137

A Celustar XL+Agropect Cellusoft Celustar XL

0ptimash VR 0ptimash VR+Agropect

Reducing sugars [mg/mL]






0,15 0,30 0,45 0,60 0,75

B 18
Reducing sugars [mg/mL]

0,15 0,30 0,45 0,60 0,75

Enzyme dose [FPU/g DM]

Fig. 2. The dependence of reducing sugars concentration in enzymatic hydrolysates of: (A) sugar beet pulp, (B) spent hops, on enzyme dose and type.

Sugar beet pulp control Sugar beet pulp enzyme*

Spent hops control Spent hops enzyme*
Reducing sugars [mg/mL]

0 1 2 3 4 5 6 7 8
Time of hydrolysis [days]

Fig. 3. The effect of time on reducing sugars concentration in controls and in enzymatic hydrolysates of sugar beet pulp and spent hops. ⁄Enzyme preparation – a mixture of
enzymes Celustar XL and Agropect pomace (3:1, v/v) in a dose of 0.15 FPU/g DM.

(versus the relevant controls) (Fig. 3). Reducing sugars were re- 584 (Novo Nordisk, Denmark). In spent hops hydrolysis processes,
leased from sugar beet pulp during the first 4 days of hydrolysis the highest level of reducing sugars was observed after 24 h
but their concentration was increased by only 28% within the per- (Fig. 3). It was slightly decreased when the process was continued
iod between the 1st and 4th day. Further continuation of sugar till the 8th day. Therefore, enzymatic pretreatment of sugar beet
beet pulp hydrolysis caused a drop in reducing sugars concentra- pulp and spent hops before methane fermentation was conducted
tion (by 25% versus that on the 4th day), which was thought to re- only for 24 h.
sult from enzyme inactivation and microbial contamination. This Sugar beet pulp was more susceptible to enzymatic digestion
result was consistent with finding of Hutnan et al. (Hutnan et al., because of lower lignin content (4.1% DM) compared to spent hops
2000) who digested sugar beet pulp with enzyme preparation SP that are rich in lignin (21.0% DM). The latter incrusts cellulose and
K. Ziemiński et al. / Waste Management 32 (2012) 1131–1137 1135

Spent hops control Spent hops enzyme*

Sugar beet pulp control Sugar beet pulp enzyme*
0 1 2 3 4 5 6 7 8
Time [days]

Fig. 4. Changes in pH during enzymatic hydrolysis of spent hops and sugar beet pulp. ⁄Enzyme preparation – a mixture of enzymes Celustar XL and Agropect pomace (3:1, v/
v) in a dose of 0.15 FPU/g DM.

Table 4
Chemical composition of substrates subjected to anaerobic fermentation and anaerobic sludge.

Tested sample DM [g/kg] Organic matter [g/kg] COD [g/kg DM] Volatile acids [g/kg DM] N-NH4 [g/kg DM] PO3
4 [g/kg DM]

Sugar beet pulp Control 102.8 88.32 988.04 6.80 14.80 3.20
After enzymatic hydrolysis 104.0 90.82 1087.20 8.09 16.04 3.80
Spent hops Control 99.80 87.46 1155.75 4.20 23.47 5.51
After enzymatic hydrolysis 100.80 89.17 1204.02 4.45 25.28 6.02
Anaerobic sludge 80.54 67.10 862.33 3.19 22.31 36.00

HRT 30 days HRT 20 days

Volume og biogas [mL]

Time [days]
Sugar beet pulp control Sugar beet pulp after enzymatic hydrolysis
Spent hops pulp control Spent hops after enzymatic hydrolysis

Fig. 5. Biogas yield during anaerobic fermentation.

renders it less accessible to cellulolytic enzymes. Therefore, this 3.2. Methane fermentation of sugar beet pulp, spent hops and their
polymer cannot be degraded under anaerobic conditions. hydrolysates
Monitoring of pH revealed that it was maintained at the same
level (pH 6.0) during enzymatic hydrolysis of spent hops and in Sugar beet pulp, spent hops and their enzymatic hydrolysates
controls (Fig. 4). In contrast, pH was considerably decreased (from were subjected to methane fermentation and its results were com-
5.1 to 3.6 in 8 days) during enzymatic hydrolysis of sugar beet pulp pared. Although, the described above enzymatic pretreatment
because of the appearance of free organic acids such as galact- caused a significant increase in concentration of reducing sugars
uronic, ferulic, vanilic, coumaric and syringic, which were released (Fig. 3), values of the total content of organic matter and COD were
from hemicelluloses, lignin and pectin (Sun and Hughes, 1999). at the similar level in all the substrates subjected to methane fer-
Because of a decrease in pH to 4.17 within the first 24 h of sugar mentation (Table 4).
beet pulp hydrolysis, pH of this hydrolysate was adjusted to 6.6 Enzymatic hydrolysis of sugar beet pulp caused 18% rise in vol-
through addition of Na2CO3 (0.4 mg/g DM, this dose was deter- atile acids content (versus the control). Their level in spent hops
mined experimentally). To maintain pH of the control at 6.6 it and their hydrolysate was comparable and by 35% and 44% lower
was necessary to add sodium carbonate in a dose of 0.01 mg/g DM. compared to the sugar beet pulp hydrolysate, respectively (Table 4).
1136 K. Ziemiński et al. / Waste Management 32 (2012) 1131–1137

Table 5
Parameters and results of anaerobic fermentation of sugar beet pulp, spent hops and their hydrolysates.

Fermentation parameters and results Unit Sugar beet pulp Spent hops
Control After hydrolysis Control After hydrolysis
Hydraulic retention time [d] 20
Fermentation time [d] 220
Fermenter working volume [L] 2
Fermenter feeding [g/d] 100
Organic loading rate [g COD/L d] 4.94 5.43 5.77 6.02
Total volume [L] 177.28 219.08 139.8 160.88
Volume per day [mL/d] 805.82 995.84 635.45 731.27
Biogas 1 g COD supplied [mL/d] 163.12 183.39 110.129 121.47
Yield from: 1 L fermenter working volume [mL/d] 402.91 497.92 317.75 365.66
Methane content in biogas [%] 62 64 59 60

partial saccharification, positively affected outcomes of anaerobic

start of fermentation end of fermentation
fermentation. The observed increase in the yield of biogas produc-
1600 tion (by 19% and 13% from hydrolysates of sugar beet pulp and
spent hops, respectively, versus relevant controls) and the concom-
1400 itant, greater decrease in total organic matter content (by 23% and
1200 7% greater in anaerobically fermented hydrolysates of sugar beet
COD [g/kg DM]

pulp and spent hops, respectively, versus relevant controls) pro-

vide evidence that enzymatic pretreatment may be used to inten-
800 sify anaerobic organic biomass degradation processes in efficient
600 and environmentally friendly manner.

200 References
0 Adham, N.Z., 2002. Attempts at improving citric acid fermentation by Aspergillus
Sugar beet Sugar beet Spent hops Spent hops niger in beet-molasses medium. Bioresour. Technol. 84 (1), 97–100.
pulp control pulp after control after Altundogan, H.S., Bahar, N., Mujde, B., Tumen, F., 2007. The use of sulphuric acid-
hydrolysis hydrolysis carbonization products of sugar beet pulp in Cr (VI) removal. J. Hazard. Mater.
144, 255–264.
APHA (American Public Health Association) 1998. Standard methods for the
Fig. 6. COD concentration after anaerobic fermentation of sugar beet pulp, spent
examination of water and wastewater, twentieth edn., APHA, Washington,
hops and their hydrolysates.
Arasimovich, V.V., Ermakov, A.I., 1987. Measurement of the total content of
These results are consistent with changes in pH observed during hemicelluloses. In: Ermakov, A.I. (Ed.), Methods for biochemical studies of
plants. Agropromizdat, Leningrad, pp. 164–165.
enzymatic hydrolysis. Carre, M.H., Haynes, D., 1922. The estimation of pectin as calcium pectate and the
Parameters and results of the anaerobic fermentation of sugar application of this method to the determination of the soluble pectin in apples.
beet pulp, spent hops and their enzymatic hydrolysates are shown Biochem. J. 16 (1), 60–69.
Considine, P.J., O’Rorke, A., Hackett, T.J., Coughlan, M.P., 1988. Hydrolysis of beet
in Fig. 5 and Table 5. The total yield of biogas production was in- pulp polisaccharides by extracts of solid-state cultures of Penicillium
creased by enzymatic treatment of sugar beet pulp and spent hops capsulatum. Biotechnol. Bioenerg. 31, 433–438.
by 19% and 13%, respectively (versus relevant controls). The high- Druce, E., Willcox, J.S., 1949. The determination of cellulose in nutritional studies:
Part II. A comparison of the applicability of three methods for the determination
est biogas production was achieved from sugar beet pulp hydroly- of cellulose. J. Agric. Sci. 39, 153–155.
sate (183.39 mL/d from 1 g COD at organic matter loading of the EurObserv’er Report, 2008. The state of renewable energies in Europe, 47–51.
fermenter of 5.43 g COD/L  d). COD was concomitantly reduced Frigon, J.-C., Mehta, P., Guiot, S.R., 2012. Impact of mechanical, chemical and
enzymatic pretreatments on the methane yield from the anaerobic digestion of
by 65% (Fig. 6). For comparison, fermentation of sugar beet pulp switchgrass. Biomass Bioenerg. 36, 1–11.
which was conducted at lower (by 9.9%) organic matter loading Ferreira, P., Diez, N., Faulds, C.B., Soliveri, J., Copa-Patińo, J.L., 2007. Release of ferulic
yielded 19% less biogas and COD was reduced by 52%. According acid and feruloylated oligosaccharides from sugar beet pulp by Streptomyces
tendae. Bioresour. Technol. 98, 1522–1528.
to Schimpf and Valbuena (Schimpf and Valbuena, 2009) the pre-
Fischer, K., Bipp, H.-P., 2005. Generation of organic acids and monosaccharide’s by
treatment of plant biomass with enzymes degrading structural hydrolytic and oxidative transformation of food processing residues. Bioresour.
polysaccharides may cause up to 20% increase in the biogas yield. Technol. 96, 831–842.
Methane fermentation of spent hops hydrolysate was con- Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257–
ducted at organic matter loading of 6.02 g COD/L  d (the highest Howard, R.L., Abotsi, E., Jansen van Rensburg, E.L., Howard, S., 2003. Lignocellulose
in this series of experiments). As much as 121.47 mL/d biogas biotechnology: Issues of bioconversion and enzyme production. Afr. J.
was produced from 1 g COD, it means by 34% less than from undi- Biotechnol. 2 (12), 602–619.
Hutnan, M., Drtil, M., Mrafkova, L., 2000. Anaerobic biodegradation of sugar beet
gested sugar beet pulp. Methane fermentation brought about a de- pulp. Biodegradation 11, 203–211.
crease in COD levels – by 59% and 55% in spent hops hydrolysate Kristensen, J.B., Felby, C., Jørgensen, H., 2009. Determining yields in high solids
and its control, respectively. Presented results cannot be compared enzymatic hydrolysis of biomass. Appl. Biochem. Biotechnol. 156, 557–562.
Laufenberg, G., Kunz, B., Nystroem, M., 2003. Transformation of vegetable waste
with other findings because no references concerning anaerobic into value added products: (A) the upgrading concept; (B) practical
fermentation of spent hops have been found. implementations. Bioresour. Technol. 87, 167–198.
Levigne, S., Ralet, M.-C., Thibault, J.-F., 2002. Characterisation of pectins extracted
from fresh sugar beet under different conditions using an experimental design.
4. Conclusions Carbohydr. Polym. 49, 14–153.
Lin, S.Y., Dence, C.W. (Eds.), 1992. Methods in lignin chemistry. Springer-Verlag,
It was found that enzymatic pretreatment of two lignocellulosic Micard, V., Renard, C.M.G.C., Thibault, J.-F., 1996. Enzymatic saccharification of
residues such as sugar beet pulp and spent hops, causing their sugar beet pulp. Enzyme Microb. Technol. 19, 162–170.
K. Ziemiński et al. / Waste Management 32 (2012) 1131–1137 1137

Micard, V., Renard, C.M.G.C., Thibault, J.-F., 1997. Influence of pretreatments on Schwarz, W.H., 2001. The cellulosome and cellulose degradation by anaerobic
enzymic degradation of a cellulose-rich residue from sugar-beet pulp. Lebensm. bacteria. Appl. Microbiol. Biotechnol. 56, 634–649.
Wiss. Technol. 30, 284–291. Spagnuolo, M., Crecchio, C., Pizzigallo, M.D.R., Ruggiero, P., 1997. Synergistic effects
Miller, G.L., 1959. Use of dinitrosalicyl acid reagent for determination of reducing of cellulolytic and pectinolytic enzymes in degrading sugar beet pulp. Bioresour.
sugar. Anal. Chem. 31, 426–428. Technol. 60, 215–222.
Naik, S.N., Goud, Vaibhav.V., Rout, Prasant.K., Dalai, Ajay.K., 2010. Production of first Sun, Y., Cheng, J.Y., 2002. Hydrolysis of lignocellulosic materials for ethanol
and second generation biofuels: A comprehensive review. Renew. Sust. Energ. production: a review. Bioresour. Technol. 83, 1–11.
Rev. 14, 578–597. Sun, R.C., Hughes, S., 1999. Fractional isolation and physico-chemical
Nanji, H., Norman, R., 1928. Studies on pectin. Part II. The estimation of the characterization of alkali-soluble polysaccharides from sugar-beet pulp.
individual pectic substances in nature. Biochem. J. 22, 596–604. Carbohydr. Polym. 38, 273–281.
Nikolic, D., Li, Y., Chadwick, L.R., Pauli, G.F., Breemen, R.B., 2005. Metabolism of Tuomela, M., Vikman, M., Hatakka, A., Itävaara, M., 2000. Biodegradation of lignin in
xanthohumol and isoxanthohumol, prenylated flavonoids from hops (Humulus a compost environment: a review. Bioresour. Technol. 72, 169–183.
lupulus L.) by human liver microsomes. J. Mass Spectrom. 40, 289–299. Wang, S., Zhuang, X., Luo, Z., Cen, K., 2008. Experimental study and product analysis
Raposo, F., de la Rubia, M.A., Borja, R., Alaiz, M., 2008. Assesment of a modiefied and of lignocellulosic biomass hydrolysis under extremely low acids. Front. Energy
optimised method for determining chemical oxygen demand of solid substrates Power Eng. China 2 (3), 268–272.
and solutions with high suspended solid content. Talanta 76, 448–453. Vaccari, G., Tamburini, E., Sgualdino, G., Urbaniec, K., Klemes, J., 2005. Overview of
Saxena, R.C., Adhikari, D.K., Goyal, H.B., 2009. Biomass-based energy fuel through the environmental problems in beet sugar processing: possible solutions. J.
biochemical routes: A review. Renew. Sust. Energ. Rev. 13, 167–178. Clean. Prod. 13, 499–507.
Schimpf, U., Valbuena, R., 2009. Increase in efficiency of biomethanation by enzyme
application. Bornimer Agrartechnische Berichte 68, 44–56.