Professional Documents
Culture Documents
DOI 10.1007/s00449-007-0114-3
ORIGINAL PAPER
Received: 11 December 2006 / Accepted: 10 January 2007 / Published online: 15 February 2007
Springer-Verlag 2007
Abstract Selected biodegradable municipal solid cantly higher (49.39 and 47.70%, respectively), than
waste fractions were subjected to fifteen different pre- the contribution of temperature during steam treat-
hydrolysis treatments to obtain the highest glucose ment (0.13%) to the glucose yield.
yield for bio-ethanol production. Pre-hydrolysis treat-
ments consisted of dilute acid (H2SO4, HNO3 or HCl, 1 Keywords Bioethanol Glucose Lignocellulose
and 4%, 180 min, 60C), steam treatment (121 and Municipal solid waste
134C, 15 min), microwave treatment (700 W, 2 min)
or a combination of two of them. Enzymatic hydrolysis
was carried out with Trichoderma reesei and Tricho- Introduction
derma viride (10 and 60 FPU g–1 substrate). Glucose
yields were compared using a factorial experimental The use of biofuels for transport is becoming of
design. The highest glucose yield (72.80%) was ob- increasing importance for a number of reasons, such
tained with a pre-hydrolysis treatment consisting of as environmental concerns relating to climate change,
H2SO4 at 1% concentration, followed by steam treat- depletion of fossil fuel reserves, and reduction of
ment at 121C, and enzymatic hydrolysis with Tricho- reliance on imports [1, 2]. This is leading to inter-
derma viride at 60 FPU g–1 substrate. The contribution national, national and regional focus upon alternative
of enzyme loading and acid concentration was signifi- energy sources. In Europe, the European Commis-
sion has proposed indicative targets for biofuel sub-
stitution of 5.75% by 2010 [1]. A potential source for
low-cost biofuel (i.e., bio-ethanol) production is to
utilize lignocellulosic materials such as crop residues,
A. Li B. Antizar-Ladislao M. Khraisheh grasses, sawdust, wood chips, and solid waste. Addi-
Department of Civil and Environmental Engineering, tionally, European legislative pressures target for
University College London, Gower Street, minimising landfill use in European countries, and
London WC1E 6BT, UK
the amount of biodegradable municipal solid waste
A. Li (BMSW) going to landfill must be reduced by 25%
e-mail: aiduan.li@ucl.ac.uk
by 2010, 50% by 2013 and 65% by 2020 [1]. Thus, the
M. Khraisheh BMSW fraction may be considered an alternative
e-mail: m.khraisheh@ucl.ac.uk
sustainable source of bio-ethanol [3, 4]. In fact,
Present Address: London Boroughs (UK) collected 4.4 thousand ton-
B. Antizar-Ladislao (&) nes of total municipal solid waste (MSW) in 2004–
Department of Water and Environmental Science 2005 [5]. Although the recycling/composting rate in
and Technology, Universidad de Cantabria,
London has increased over the past 5 years from 8%
Bulevard Ronda Rufino Peón, 254,
39316 Torrelavega, Cantabria, Spain in 2000–2001 to 15% in 2004–2005, still 65% MSW is
e-mail: antizarb@unican.es disposed by landfill, of which 81.4% may be biode-
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190 Bioprocess Biosyst Eng (2007) 30:189–196
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Bioprocess Biosyst Eng (2007) 30:189–196 191
Table 1 Experimental conditions in pre-hydrolysis treatment of BMSW fractions selected in this study
Experiment Dilute acid Steam treatment Microwave treatment Enzymatic
hydrolysis
HNO3 Time, HCl Time H2SO4 Time T Time Power Time Cellulase
(%) min (%) (min) (%) (min) (C) (min) (W) (min) (FPU/g)
1 4 180 – – – – – – – – 60
2 – – 4 180 – – – – – – 60
3 – – – – 4 180 – – – – 60
4 4 180 – – – 121 15 – – 60
5 – – 4 180 – – 121 15 – – 60
6 – – – – 4 180 121 15 – – 60
7 – – – – 4 180 – – 700 2 60
8 – – – – 4 180 134 15 – – 60
9 – – – – 1 180 121 15 – – 60
10 – – – – 1 180 134 15 – – 60
11 – – – – 4 180 121 15 – – 10
12 – – – – 4 180 134 15 – – 10
13 – – – – 1 180 121 15 – – 10
14 – – – – 1 180 134 15 – – 10
15a 4 180 121 15 – – 60
a
Enzyme used is T. reesei
treatment, which lasted for 50 min. A few bubbles comparison and at 2, 5, 9, 24, 48, 72, and 96 h for ki-
started to appear on the surface of the material indi- netic studies.
cating the onset of boiling. Following microwave
treatment, the samples were washed with deionised Analytical methods
water until the pH of the filtrate remained constant
pH = 4.5. The pre-hydrolysis treated samples were The analytical methods were performed according to
drained and air-dried overnight to a final 45% moisture the following procedures. Ash content was determined
content. using the Standard Biomass Analytical Methods [15].
Briefly, triplicate 10 g samples were dried at 105C for
24 h (moisture content) and then transferred to a
Enzymatic hydrolysis muffle furnace at 575C for 24 h. The cellulose content
of the selected biodegradable MSW fractions was
Enzymatic hydrolysis of 0.5 g of pre-hydrolysed sub- analyzed by the method of Crampton and Maynard
strate was carried out at 50C in vials (50 ml) placed in [16]. Sugar concentrations were determined routinely
an orbital agitator at 68 rpm for 96 h. The liquor pH from centrifuged samples (13,000 rpm, 5 min) using a
was kept at pH = 4.8 using 0.1 M citrate acid-sodium glucose meter (MediSense Ltd, UK) after the method
citrate buffer. Two enzymes, T. viride and T. reesei was validated in the laboratory. Calibration graphs
(Cellulase from T. viride and cellulase from T. reesei were linear for the concentration range 0.5–5 g l–1
ATCC26921, respectively; Sigma, UK) were separately (R2 = 0.98).
used as received in combination with the enzyme b-
glucosidase (Sigma, UK). Possible differences in the Data analysis
cellulase activity of the enzymes [11] was measured by
the Filter Paper assay developed by Mandels et al. [12], All compositional analyses were done in triplicate, and
and the activity was expressed in terms of Filter Paper data were corrected to a 100% dry matter (DM) basis.
Units (FPU). The b-glucosidase activity was measured Glucose yield as percentage of the theoretical yield
by the PNPG assay [13], and reported as PNPG units [percentage digestibility, obtained from the equation
(PNPGU). All the experiments were performed at which involves the transfer of cellulose to sugar
enzyme to substrate ratios of 60 FPU g–1 substrate [14] (C6H10O5)n + nH2O fi (C6H12O6)n] was computed
and 64 PNPGU g–1 substrate, and 10 mg ml–1 puro- by using the formula given by the National Renewable
mycin for 96 h. Samples were withdrawn from the Energy Laboratory (Standard Biomass Analytical
reaction media after 24 h for pre-hydrolysis treatment Procedures) [17]:
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192 Bioprocess Biosyst Eng (2007) 30:189–196
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Bioprocess Biosyst Eng (2007) 30:189–196 193
to effectively maximize enzymatic hydrolysis was suspected that although microwave treatment may be
investigated. Subsequently, the best experimental more efficient in removing hemicellulose and lignin
conditions were compared with dilute acid treatment present in the selected BMSW fractions and therefore
followed by microwave treatment. enabling the accessibility of enzymes to the cellulosic
The effective pre-hydrolysis treatment for the se- chains, it also may have facilitated glucose production
lected BMSW fractions in this study occurred com- at concentrations, which may have been inhibitory
bining dilute H2SO4 (4%) followed by steam treatment during enzymatic hydrolysis. However, more research
(Fig. 2). Highest glucose yield after 24 h pre-hydrolysis is currently undertaken in order to provide with a
treatment was obtained from grass and carrot peelings rigorous explanation to this observation.
(62 and 60%, respectively), followed by potato peel- Pre-hydrolysis treatment methods investigated in
ings (32%). The glucose yield from waste paper was this study indicated that in general lower glucose yield
relatively lower, where the scrap paper and newspaper resulted from newspaper and scrap paper BMSW
resulted in 25 and 6% glucose yield, respectively. fractions than from kitchen waste BMSW fractions,
Previous studies have reported that softwood newspa- i.e., carrot peelings and potato peelings. Nguyen and
per is the most difficult component to digest in a lig- Saddler [14] reported a maximum glucose yield
nocellulosic MSW mixture, due to its high lignin approximately 60% after two-stage dilute-acid pre-
content [23]. In this study, no pre-treatment or com- treatment of softwood, which is the source for news-
bining dilute HCl with steam treatment resulted in the paper and scrap paper. A lower glucose yield obtained
highest glucose yields from scrap paper and newspaper from newspaper and scrap paper in this study than by
(32–46% scrap paper and 18% newspaper) as com- Nguyen and Saddler [14] may have occurred due to the
pared to other pre-hydrolysis treatments. Comparing dense structure and presence of chemicals, such as
the combined treatments H2SO4 with steam treatment fillers, ink and other additives in the newspaper and
and H2SO4 with microwave, generally higher glucose scrap paper (biosurfactants and antimicrobials to block
yields were obtained using the former treatment, but pulp degradation), which may have inhibited the
the glucose yield from newspaper was doubled using enzymatic hydrolysis [24].
H2SO4 followed by microwave pre-hydrolysis treat-
ment. Enzymatic hydrolysis of MSW fractions
Glucose yields following the combined treatment of
dilute acid with microwave and the glucose yield fol- Efficient hydrolysis of cellulose requires the synergistic
lowing the same dilute acid treatment alone were activities of three types of enzymes. Endo-b–1,4-glu-
compared (Figs. 1, 2). Thus the effect of microwave canases hydrolyze accessible regions on cellulose
treatment following dilute acid treatment could be chains to provide new sites for attack by exo-acting
revealed. It was observed that the glucose yield was cellobiohydrolases, which removes successive cellobi-
generally lower after microwave treatment. It was ose units from newly created chains ends. Finally, b-
glucosidase hydrolyzes cellobiose, and smaller amounts
of higher cellooligomers, to glucose. Two cellulose-
SP digesting enzymes used in this study T. reesei and T.
viride contain endogenous b-glucosidase activity,
NP H2SO4+MW however, the activities of this enzyme are generally
H2SO4+SE insufficient to prevent the accumulation of cellobiose,
G HNO3+SE resulting in product inhibition of endoglucanases and
HCl+SE cellobiohydrolases [25]. Consequently, b-glucosidase
no pretreatment was supplemented during the enzymatic hydrolysis of
PP
MSW fractions.
The substrates included single substrate from the
CP selected MSW fractions (carrot peeling, potato peel-
ings, grass, newspaper and scrap paper) and multi-
0 20 40 60 80 100 substrates by combining different single substrates. All
Glucose yield, % substrates were pre-hydrolysis treated combining di-
lute acid (H2SO4, 4%) followed by steam treatment, as
Fig. 2 Effects of combined pre-hydrolysis treatments of selected
BMSW fractions in glucose yield after 24 h hydrolysis (dilute
this was shown the most efficient pre-hydrolysis treat-
acid concentration 4%, T.viride 60 FPU g–1). CP carrot peelings, ment for MSW fractions (Fig. 3). This study indicated
PP potato peelings, G grass, NP newspaper, SP scrap paper that using T. viride resulted in higher glucose yields
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194 Bioprocess Biosyst Eng (2007) 30:189–196
G Process optimization
PP
CP
Mathematical modelling is a valuable tool for the
Avicel
process optimization, and thus various studies have
developed mathematical models considering several
0 20 40 60 80 100
factors. For example, Nguyen and Saddler [14] used
Glucose yield, % and exponential function, R0 ¼ t eðTr Tb Þ =14:75, to
Fig. 3 Effects of cellulase (T. viride, T. reesei, 60 FPU g–1)
optimize the pre-treatment process including two fac-
during enzymatic hydrolysis of selected BMSW fractions in tors (temperature and residence time) and the process
glucose yield (55C, 96 h). Pre-hydrolysis treatment: H2SO4 (4%, was optimized with different severities by changing the
180 min) and steam treatment (121C, 15 min). CP carrot temperature. Where, R0 is the severity factor combin-
peelings, PP potato peelings, G grass, NP newspaper, SP scrap
paper
ing the effects of time and temperature, t is the resi-
dence time (min), Tr is the reaction temperature (C),
and Tb is a reference temperature (100C). Esteghla-
than using T. reesei, which corroborates previous lian et al. [20] modelled the hydrolysis of hemicellulose
studies indicating an activity (units glucose produced to its monomeric constituents and possible degradation
per mg enzyme) of T. viride and T. reesei of 3–10 and of these monomers by a series of first-order kinetic
1.0, respectively [26]. functions. The kinetic parameters for predicting the
Once pre-hydrolysis treatment (H2SO4 4% and xylose yield were determined using the actual acid
steam treatment) and cellulose-digesting enzymes (T. concentration in the reactor after accounting for the
viride) were optimized for maximum glucose yield neutralization effect of the substrates. Yañez et al. [18]
from selected BMSW fractions, a set of experiments of used a pseudo-homogeneous first order kinetic func-
enzymatic hydrolysis was carried out during 96 h, and tion to calculate the hemicellulose (H) and non-poly-
samples were withdrawn at intervals (Fig. 4). This ki- saccharide (NPF) fractions and an overall material
netic study indicated that the highest glucose yield was balance to estimate the cellulose content (Cl),
obtained using potato peeling (89%), followed by Cl = 100 – H – NPF. These studies have facilitated
carrot peelings (73%) or combination of carrot peel- process optimization, however, current technology
ings and potato peelings (70%). Newspaper produced development for the production of bio-ethanol focus
the lowest glucose yield (14%) following 96 h enzy- towards the utilisation of residual lignocellulosic
matic hydrolysis. The glucose yield produced by a materials at lower production costs. Thus, important
combination of five selected BMSW fractions (20% factors that need to be optimized in order to reduce the
each) increased from 41 to 52% after 24 and 96 h of cost of the production of bio-ethanol include acid
enzymatic hydrolysis, respectively. The presence of concentration, temperature and cellulase concentra-
newspaper was suspected to be the main responsible tion.
CP
15 min) and enzymatic
60 G
hydrolysis with T. viride
(60 FPU g–1). CP carrot SP
peelings, PP potato peelings, 40
G grass, NP newspaper, SP NP
scrap paper 20 CP+PP
NP+SP
0
0 20 40 60 80 100 C+P+G+NP+SP
Time, h
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Bioprocess Biosyst Eng (2007) 30:189–196 195
Thus, once the most effective pre-hydrolysis and tration (1%). An optimal ratio of cellulase to cellulose
enzymatic hydrolysis for the selected BMSW fractions 0.8 FPU g–1 substrate has been previously reported
were identified in this study, the hydrolysis process was using Avicel pH 105 and T. reesei [28], while a ratio
optimized by using a factorial experimental design 0.2 FPU g–1 substrate was reported to be inhibitory [29].
considering the aforementioned factors. Glucose yields This study indicated that BMSW fractions required
using pre-hydrolysis treatment of carrot peelings and higher cellulase to cellulose ratios to obtain high glucose
potato peelings (50% each), adding dilute H2SO4 acid yields, which will increase the cost of the process.
(1 and 4%; 180 min), steam treatment (T, 121 and
134C; 15 min), and T. viride (10 and 60 FPU g–1
substrate) following hydrolysis at 50C for 72 h were Conclusions
computed using the software package Design Ease
6.0.6 to find the optimal treatment conditions (Fig. 5). Results from the presented experimental work indi-
The highest glucose yield (72.80%) was obtained at cated that various BMSW fractions have a vast po-
dilute acid concentration (1%), low temperature tential for the production of sugars that eventually can
(121C) and high enzyme loading (60 FPU g–1 sub- be used for producing bio-ethanol. Dilute acid hydro-
strate). The contribution of enzyme loading and acid lysis followed by steam treatment is the most efficient
concentration to the glucose yield was significantly pre-hydrolysis method on the selected waste among all
higher (49.39 and 47.70%, respectively), than the con- the pre-hydrolysis methods used in this study. High
tribution of temperature during steam treatment glucose yields were obtained from both, single sub-
(0.13%). Additionally, the results obtained by the strates (70–89%) and multi-substrates (41–52%).
model presented a satisfactory correlation to the actual Newspaper produced the lowest glucose yield (14%)
values (R2 = 0.90). following 96 h enzymatic hydrolysis. The contribution
The use of dilute H2SO4 acid concentration is an of enzyme loading and acid concentration was signifi-
advantage, in particular since its cost is usually higher cantly higher (49.39 and 47.70%, respectively), than
than the cost of using other physico-chemical pre- the contribution of temperature during steam treat-
hydrolysis treatments [8, 9]. Additionally, this study ment (0.13%) to the glucose yield.
indicated that lower temperature (121C) and long Conversion of MSW to bio-ethanol can be an
residence time (96 h) during steam treatment produced alternative sustainable approach to disposal of waste
the highest glucose yields, which additionally has been and reduction of the biodegradable fraction of MSW to
recognized as one of the most cost-effective pre- landfill. Technology that has been applied over the past
hydrolysis treatment process for agricultural residues [9, years to other lignocellulosic materials may be applied
27]. Neutralization of pH is necessary for the down- to BMSW-to-bio-ethanol production; however, further
stream enzymatic hydrolysis or fermentation process, laboratory studies are needed to optimize the biocon-
which may be avoided using low H2SO4 acid concen- version of BMSW-to-bio-ethanol production.
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196 Bioprocess Biosyst Eng (2007) 30:189–196
Acknowledgments This research was undertaken at the 14. Nguyen QA, Saddler JN (1991) An integrated model for the
Department of Civil and Environmental Engineering, Labora- technical and economic-evaluation of an enzymatic biomass
tory of Environmental Engineering, Chadwick Building, Uni- conversion process. Bioresour Technol 35:275–282
versity College London, UK, AL acknowledges the financial 15. NREL (1994) Chemical Analysis and Testing Task Labora-
support provided by the Dorothy Hodgkin Postgraduate Award, tory Analytical Procedure: Standard Method for Ash in
and the technical support received at Wolfson Institute and Biomass. National Renewable Energy Laboratory. http://
Department of Biochemical Engineering at the University Col- www.devafdc.nrel.gov/pdfs/9338.pdf
lege London. 16. Crampton EW, Maynard LA (1938) The relation of cellulose
and lignin content to the nutritive value of animal feeds. J
Nutr 15:383–395
17. NREL (1995) SSF Experimental protocols: Lignocellulosic
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