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Effect of different nitrogen fertilizer treatments on the conversion of Miscanthus × giganteus to ethanol
Accepted Manuscript Effect of different nitrogen fertilizer treatments on the conversion of Miscanthus × giganteus to ethanol Bogdan Dubis, Katarzyna Bułkowska, Małgorzata Lewandowska, Władysław Szempliński, Krzysztof Józef Jankowski, Jakub Idźkowski, Natalia Kordala, Karolina Szymańska PII: DOI: Reference:
S0960-8524(17)31091-X http://dx.doi.org/10.1016/j.biortech.2017.07.005 BITE 18424
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
11 May 2017 30 June 2017 1 July 2017
Please cite this article as: Dubis, B., Bułkowska, K., Lewandowska, M., Szempliński, W., Jankowski, K.J., Idźkowski, J., Kordala, N., Szymańska, K., Effect of different nitrogen fertilizer treatments on the conversion of Miscanthus × giganteus to ethanol, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech. 2017.07.005
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1
Effect of different nitrogen fertilizer treatments on the conversion of Miscanthus ×
2
giganteus to ethanol
3
Bogdan Dubisa, Katarzyna Bułkowskab,*, Małgorzata Lewandowskac, Władysław Szemplińskia,
4
Krzysztof Józef Jankowskia, Jakub Idźkowskic, Natalia Kordalac, Karolina Szymańskac
5 6
a
7
University of Warmia and Mazury in Olsztyn, Oczapowskiego 8, 10-719 Olsztyn, Poland
8
b
9
Słoneczna 45G, 10-709 Olsztyn, Poland
Department of Agrotechnology, Agricultural Production Management and Agribusiness,
Department of Environmental Biotechnology, University of Warmia and Mazury in Olsztyn,
10
c
11
Olsztyn, Heweliusza 1, 10-718 Olsztyn, Poland
12
*Coresponding autor: PhD Katarzyna Bułkowska, Department of Environmental Biotechnology,
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University of Warmia and Mazury in Olsztyn, Słoneczna 45G, 10-709 Olsztyn, Poland, e-mail:
14
[email protected]
15
Keywords: ethanol fermentation, fertilization effect, sequential and simultaneous
16
hydrolysis and fermentation
Chair of Food Biotechnology, Faculty of Food Sciences, University of Warmia and Mazury in
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Abstract
19
Miscanthus × giganteus is a perennial rhizomatous grass which is used as a biofuel crop.
20
Due to its high yields, low production costs, resistance to low temperatures, low soil
21
requirements and, above all, high cellulose content, miscanthus can be a useful resource for
22
ethanol production. The aim of this study was the determine the effect of two fertilization
23
regimes (sewage sludge/mineral NPK) during miscanthus cultivation on the chemical
24
composition of biomass, the content of major lignocellulosic factions and the effectiveness
25
of miscanthus conversion to bioethanol. The results indicate that fertilization treatments
26
influenced biomass yield and the content of major lignocellulosic fractions. Bioethanol 1
1
production was higher when hydrolysis and fermentation processes were conducted
2
separately than when saccharification and fermentation were conducted simultaneously.
3
Ethanol production increased by 30% and 40% in response to sewage sludge and NPK
4
(equivalent nitrogen content = 160 kg N/ha) fertilization, respectively, in comparison with
5
unfertilized crops.
6 7
Introduction
8
In Europe, Miscanthus (Miscanthus × giganteus) is classified as an energy crop.
9
Miscanthus × giganteus grows to a height of 4 m, produces stems with a length of up to 3
10
m and a diameter of approx. 10 mm, and can be grown in one location for up to 15 - 20
11
years. Miscanthus is a C4 plant and a highly efficient crop which is characterized by high
12
yield, environmental benefits, the ability to grow without pesticides and fertilizers, long
13
life and high cellulose (CEL) content (40-48%). For these reasons, Miscanthus enjoys the
14
status of a popular energy crop in Europe. Miscanthus can be harvested twice a year in
15
autumn (October to November or early December) and spring (February to the end of
16
March). The biomass yield of Miscanthus varies depending on the date of harvest. Its dry
17
matter content is estimated at 35%-45% in the autumn harvest and 60%-70% in the spring
18
harvest. Miscanthus yields range from 1 to 3 t/ha in the first year of cultivation, 8 to 15 t/ha
19
in the second year, and 15 to 30 t/ha in the third and subsequent years. Due to a rapid
20
increase in yield, Miscanthus should not be harvested in the first years of cultivation. The
21
calorific value of Miscanthus is estimated at 18 MJ/ha (Greenhalf et al., 2013; D. J. M.
22
Hayes, 2013), and its theoretical ethanol yield (TEY) is around 0.45 L/kg of biomass.
23
Other lignocellulosic substrates, such as switchgrass, maize straw and bagasse, have
24
similar TEY values of approximately 0.40 L/kg. However, the biomass yield of
25
Miscanthus is 2-24 times higher in comparison with other substrates, therefore, its ethanol 2
1
yield per ha is high at 4600 to 12400 L/ha (Kim et al., 2015). It is estimated that by 2050,
2
Miscanthus could satisfy up to 12% of primary energy demand in Europe (HASTINGS et
3
al., 2009). Miscanthus is easy to cultivate because it has an extensive root system that
4
effectively absorbs and utilizes nutrients. Nutrient recovery is significant during transport
5
from the shoot to rhizomes and from falling leaves. Miscanthus roots are colonized by
6
nitrogen-fixing bacteria, which increases nitrogen inputs to the ecosystem (Cadoux et al.,
7
2012).
8
The most widely used raw materials in bioethanol production include sugar cane (Brazil)
9
and corn (United States). However, the popularity of lignocellulosic substrates is on the
10
rise due to their high availability, low cost and, above all, sustainability. These substrates
11
include agricultural wastes, grasses, wood and sawdust (Sarkar et al., 2012).
12
Lignocellulose is a polymer consisting of three main fractions: CEL, hemicellulose (HEM)
13
and lignin. The CEL content of substrates ranges from 35% to 50% of total dry weight, that
14
of HEM from 20 to 35%, and that of lignin from 10 to 25%. Hemicellulose is hydrolyzed
15
more easily than CEL due to the presence the numerous branches in the main chain. Lignin
16
has a highly complex structure, it and it imparts resistance to degradation to lignocellulosic
17
material. Lignin is strongly associated with CEL and HEM, and it creates an impermeable
18
barrier. The physicochemical properties of lignocellulose also offer protection against
19
microbial degradation (Balat, 2011; Mussatto and Teixeira, 2010).
20
During bioethanol production, pretreatment is the first step in reducing the recalcitrance of
21
lignocellulosic biomass to conversion into sugars, as it increases the availability of
22
enzymes to the polysaccharide fraction. There are many pretreatment methods, including
23
addition of hydrolysate (Li et al., 2016).
24
Pretreatment methods can be classified as biological, chemical or physical methods, or as
25
combinations of these methods (Zheng et al., 2009, Harmsen et al., 2010; Agbor et al., 3
1
2011). Biological methods utilize fungi and actinomycetes for reduction of the degree of
2
cellulose polymerization, and for partial degradation of hemicelluloses and lignin.
3
Chemical or physical methods affect biomass by increasing its solubility, decreasing its
4
degree of polymerization, partially or completely delignifying it, and partially or
5
completely hydrolyzing the hemicelluloses within the biomass. Examples of chemical
6
pretreatment methods include acid hydrolysis, alkaline hydrolysis, ozonolysis, oxidative
7
delignification, the organosolv process, and ionic liquid pretreatment. Under the heading of
8
physicochemical methods come such processes as steam explosion (autohydrolysis), liquid
9
hot water pretreatment, ammonia fiber explosion (AFEX), and CO2 explosion. Finally,
10
there is physical pretreatment, which is used to reduce particle size, or to increase the size
11
of pores and the accessible surface area of the biomass.
12
The aim of this study was to determine: 1) the effect of different fertilization regimes
13
during Miscanthus × gigantheus cultivation on the crop’s chemical composition and
14
content of lignocellulosic fractions, 2) the influence of the absence of hydrolyzate
15
supplementation during Miscanthus × giganteus cultivation on fermentation efficiency in
16
systems involving sequential or simultaneous hydrolysis and fermentation, and 3) the
17
effect of fertilization on Miscanthus × giganteus productivity.
18
Materials and Methods
19
Plant material
20
Miscanthus × giganteus (7th year of cultivation, 2014 year) was cultivated in the
21
Production and Experimental Station in Bałcyny, Poland, which belongs to the University
22
of Warmia and Mazury in Olsztyn. Field experiments were conducted in a split-plot system
23
with each fertilizer variant tested in triplicate on 120 m2 plots. Rhizomes were planted
24
manually with a spacing of 70 x 70 cm. Chemical weed control was applied (1x triticale)
25
with iodosulfuron methyl sodium and amidosulfuron mixture at a dose 0.125 L/ha. 4
1
Biomass was collected using a self-propelled chopper. The experiment was established on
2
Haplic Luvisol developed from boulder clay (IUSS, 2006). The arable layer (0-30 cm) was
3
slightly acidic (pH in 1 M KCl – 6.5), and had a high content of available phosphorus and
4
magnesium, and moderate content of potassium.
5
Five fertilization treatments were applied before the growing season: A – no fertilizer
6
(control), B – sewage sludge (1.75 t DM/ha), C – sewage sludge (2.8 t DM/ha), D – NPK
7
fertilizer (100 kg N, 50 kg P, 80 kg K/ha; equivalent nitrogen content of 1.75 t DM/ha),
8
and E - NPK fertilizer (160 kg N, 50 kg P, 80 kg K/ha; equivalent nitrogen content of 2.8 t
9
DM/ha). The dry matter content of biomass was estimated at 93%. Biomass was chopped
10
into pieces ranging from 1 to 2 mm in diameter. The characteristics of Miscanthus are
11
shown in Table 1.
12 13
Sequential hydrolysis and fermentation (SHF) of Miscanthus × gigantheus
14
Miscanthus × giganteus biomass was subjected to alkaline pretreatment with NaOH under
15
the following conditions: temperature, 121 °C; time, 1 h; NaOH addition of 0.1 g ∙g-1 d.m.;
16
and a ratio of solid to liquid fraction of 1:9. The supernatant was separated, and the solid
17
fraction was detoxified by rinsing with distilled water and re-centrifugation (Lewandowska
18
et al., 2016; Świątek et al., 2014).
19
After alkaline pretreatment, the samples were centrifuged at RCF 4240 g at a temperature
20
of 5°C for 10 min. The supernatant was decanted, distilled water was added, and the
21
samples were centrifuged at RCF 4240 g and 5°C for 10 min. The procedure was
22
performed twice. After centrifugation, the liquid fraction was decanted, and the solid
23
fraction was combined with distilled water to obtain 10% of suspended solids in the
24
sample. After centrifugation, acetic acid (~99%) was used to adjust pH to 5.0 (measured
25
with the Hanna 211 pH-meter). Next, the medium was pasteurized at 90°C for 20 min. 5
1
Enzymatic hydrolysis was performed by adding the following enzymes: cellulase from
2
Trichoderma longibrachiatum (15 U/g DM of the substrate), xylanase from T.
3
longibrachiatum (15 FXU/g DM of the substrate) and cellobiase (Novozyme 188) (30
4
CBU/g DM of the substrate). Hydrolysis was conducted for 72 h at 42°C (Innova 40
5
incubator, New Brunswick Scientific). During the process, samples were shaken at 250
6
rpm/min. After hydrolysis, the concentration of reducing sugars was determined using 3,5-
7
dinitrosalycilic acid.
8
The hydrolyzate and the hydrolyzate supplemented with mineral sources of nitrogen and
9
phosphorus ((NH4)2SO4 and KH2PO4) were inoculated with S. cerevisiae 7 (5% v/v). The
10
concentration of reducing sugars after inoculation was calculated from the following
11
equation: concentration of sugars in the hydrolyzate × 0.2 / 0.21. Fermentation was carried
12
out at 30°C under anaerobic conditions for 72 h.
13 14
Simultaneous hydrolysis and fermentation (SSF) of Miscanthus × gigantheus
15
The medium was prepared according to the described protocol. Hydrolysis was conducted
16
with enzymatic preparations and was conducted for 24 h according to the described
17
protocol. To compare the efficiency of fermentation with and without supplementation,
18
mineral sources of nitrogen and phosphorus were added to selected samples after 24 h. The
19
temperature was lowered to 38°C, and 10 cm3 (5% v/v) of the S. cerevisiae AS4 inoculum
20
was added. Fermentation with hydrolysis was carried out for 96 hours (38°C, anaerobic
21
conditions) in 3 replicates.
22 23
Chemical analysis
24
Ash content was determined by mineralization in a muffle furnace. Protein content was
25
measured using the Kjeldahl method in the FOSS Kjeltec 8400 analyzer. Crude fat content 6
1
was determined in the Foss Tecator Soxtec 2043 fat extraction system. Sugar content was
2
measured with Epoll-2-Spekol. The fiber fraction was determined in the FibertecTM 1020
3
system. The neutral fiber content (NDL) was determined according to Van Soest et al.
4
(1991). The content of acid detergent fiber (ADF) and acid detergent lignin (ADL) was
5
determined according to (PN-EN ISO 13906, 2009). Cellulose concentration was
6
calculated as the difference between ADF and ADL, and HEM concentration was
7
determined as the difference between NDF and ADF. The effects of hydrolysis were
8
evaluated based on the quantity of enzymatically released reducing sugars determined in a
9
reaction with 3,5-dinitrosalicylic acid (Miller, 1959). After fermentation, alcohol content
10
was determined by distillation (AOAC, 1990). The heating value was determined
11
according to PN-ISO9831 (2002).
12
The fermentation yield (YF, L A100/kg) was calculated according to the following equation:
13
where VE is a volume of ethanol after fermentation (L A100), MS is a mass of wet biomass
14
for fermentation (kg), and EPre is the efficiency of alkali pretreatment (–).
15
The ethanol yield (YE, L A100/kg) was calculated with the following equation:
16
where YF is a fermentation yield (L A100/kg) and YB is a biomass yield (t/ha).
17 18
Statistical analysis
19
The Duncan multiple range test was used after ANOVA (STATISTICA 10, StatSoft Inc.)
20
to determine the significance of differences between alcohol content in series A-E (p<0.05
21
was considered significant).
22 7
1
Results and Discussion
2 3
The effect of fertilization on the chemical composition and content of lignocellulosic
4
fractions in Miscanthus × gigantheus biomass
5
The aim of this study was to determine the influence of different fertilization regimes on
6
the chemical composition, proportions of basic lignocellulosic fractions and the
7
bioconversion efficiency of biomass polysaccharides (CEL and HEM) to ethanol. The
8
proximate composition of Miscanthus × giganteus biomass grown with the application of
9
different fertilizers is presented in Table 1.
10
Fertilization had no significant influence on the proximate composition of Miscanthus
11
biomass in any of the examined treatments. Ash content was 3.50% in the control
12
treatment (A). Ash content was lower in the experimental treatments, excluding treatment
13
D (3.65%) with mineral fertilization. Fertilized crops were characterized by lower ash
14
content, which could be attributed to harvesting season (October) during which plant parts
15
that fell to the ground accumulated large amounts of minerals and nutrients (Lewandowski
16
and Kicherer, 1997). The ash content of biomass is determined by the growth stage, time of
17
year, and cultivation site (Hayes, 2013). Similar results were reported by Lewandowski
18
and Heinz (2003) who cultivated Miscanthus in three different locations and observed
19
differences in the ash content of plants subject to location and season. In all crops, ash
20
content varied across seasons, and was higher in December and lower in March in the
21
range of 2% to 4.5%. Jingping Qin et al. (2012) also demonstrated significant variations in
22
the ash content of different Miscanthus species (2.89% to 5.69%).
23
Sewage sludge and NPK fertilizers increased the protein content of Miscanthus biomass.
24
Fertilizers are highly abundant in nitrogen which can be accumulated in biomass, thus
25
increasing the protein content of plants. In our study, protein content increased in all 8
1
treatments regardless of the fertilization method, excluding treatment B (4.00%) where
2
sewage sludge was used (1.75 t DM/ha) and where protein content was identical to that
3
noted in the control treatment (4.01%). The nitrogen content of Miscanthus biomass
4
increased with a rise in fertilizer rate, and similar results were reported by other authors
5
(Kahle et al., 2001; Larsen et al., 2014; Schwarz et al., 1994). The mineral fertilizer (NPK)
6
induced a higher increase in protein content than sewage sludge because mineral nitrogen
7
is more readily absorbed by plants.
8
The content of sugars was highest in the non-fertilized treatment (3.40% DM). Fertilization
9
induced a decrease in sugar content in the range of 2.84% DM (treatment C) to 3.32% DM
10
(treatment B). A proximate analysis revealed that fertilization had no effect on crude fat
11
content which ranged from 0.93 to 0.97%. Crude fat content was somewhat lower (0.86%
12
DM) only in the treatment exposed to the highest rate of NPK fertilizer (treatment E).
13
An analysis of lignocellulosic fractions revealed the highest CEL content of 44.93% DM in
14
the non-fertilized control treatment (A). Sewage sludge lowered CEL content to 42.21%
15
DM in treatment B and 41.98% DM in treatment C. The lowest CEL content was
16
determined in the treatments fertilized with NPK, at 40.49% DM in treatment D and
17
41.27% DM in treatment E. An inverse relationship was noted for the HEM fraction.
18
Hemicellulose content was lowest in treatment A (24.25% DM). In treatments fertilized
19
with sewage sludge, HEM content was determined at 27.46% DM (B) and 28.57% DM
20
(C). The highest concentration of HEM was observed in the treatments fertilized with NPK
21
at 29.12% DM (D) and 28.90% DM (E). Hemicellulose is the predominant fraction in the
22
green parts of plants. Higher nutrient supply promotes leaf growth and increases the HEM
23
content of biomass. In lignified parts of plants, such as the stem, CEL and lignin are the
24
predominant fractions. The most important macronutrients for plant growth are nitrogen
25
and phosphorus which are not accumulated in the above-ground parts of plants, but are 9
1
stored in roots and rhizomes (Cadoux et al., 2012; Hodgson et al., 2011). The content of
2
CEL and lignin also decreased in response to fertilization. Plants that were not fertilized
3
with nitrogen were characterized by higher concentrations of all lignocellulosic fractions,
4
including HEM. The negative impact of nitrogen fertilization on the proportions of
5
lignocellulosic fractions was also found in other plant species, such as sorghum or millet
6
(Blümmel et al., 2003; Reddy et al., 2003). These results show that Miscanthus × giganteus
7
can be a good source of lignocellulosic biomass without fertilization, which makes it a
8
cost-effective crop. The total content of CEL and HEM did not differ significantly between
9
fertilized treatments. Cellulose and HEM are synthesized during sugar hydrolysis, and they
10
are used in ethanol production during fermentation. Crops grown for ethanol should be
11
characterized by the highest possible content of CEL and HEM. However, raw material
12
pretreatment can lead to HEM degradation to simpler compounds that inhibit the metabolic
13
activity of microorganisms responsible for fermentation (furfural, 5-
14
hydroxymethylfurfural). In the non-fertilized treatment (A), total CEL and HEM content
15
was 69.18%. In fertilized treatments, total CEL and HEM content ranged from 69.39% to
16
70.55%. Lignin is the third major lignocellulosic fraction. This insoluble polymer is
17
undesirable in ethanol production because it limits the availability of lignocellulosic
18
polysaccharides, and slows down hydrolysis and fermentation. The lignin content of the
19
evaluated biomass samples was influenced by fertilization. The highest lignin content in
20
excess of 9% was noted in treatment A. A significant reduction in lignin content was
21
observed in fertilized treatments. The application of sewage sludge decreased lignin
22
content to 7.56% (B) and 7.53% (C). An even greater reduction in lignin content was
23
observed in response to NPK fertilization at 6.90% (D) and 7.25% (E). The higher lignin
24
content (17.2%) in Miscanthus × giganteus was reported by Guragian et al. (2014). The
25
reduction in lignin content is a positive factor because substrate pre-treatment leads to the 10
1
release of phenolic compounds that inhibit microbial activity during fermentation. At the
2
same time, a reduction in CEL content is a negative factor because it lowers the availability
3
of polysaccharides for hydrolysis. Polysaccharides are a source of glucose, the main sugar
4
involved in fermentation.
5 6
The effect of fertilization on sequential hydrolysis and fermentation with Saccharomyces
7
cerevisiae 7
8
Miscanthus was used as a source of carbohydrates for alcohol fermentation. The effects of
9
different fertilization treatments on the fermentation of Miscanthus × giganteus biomass
10
hydrolyzates in a sequential system (SHF) with S. cerevisiae strain 7 are shown in Figure
11
1.
12
All fertilization treatments resulted in significantly higher ethanol content than in the
13
control treatment (p<0.05) (Fig.1). This result may be caused by an increase in the
14
concentration of hemicelluloses in the Miscanthus biomass after fertilization, and a
15
decrease in the concentration of lignin. Hemicelluloses are hydrolyzed more easily than
16
CEL and lignin, which is resistance to degradation and creates an impermeable barrier
17
around CEL and HEM. According to Lee and Kuan (2015), the lignin and hemicellulose
18
contents of Miscanthus are have an important influence on the efficiency of enzyme
19
hydrolysis of pretreated biomass. Miscanthus that has a high concentration of
20
hemicelluloses, a low cellulose crystallinity index and a high concentration of lignin is not
21
saccharificated well after pretreatment. Xu et al. (2012) also state that cellulose and lignin
22
have negative effects on biomass digestibility, in contrast to hemicelluloses, which have a
23
positive effect.
11
1
In the variants fertilized with sewage sludge, alcohol content was 2.36% (B) and 2.29%
2
(C). The use of NPK fertilizer also produced favorable results. In biomass from treatments
3
D and E, alcohol content was 2.24% and 2.40%, respectively.
4 5
The effect of the absence of hydrolyzate supplementation during Miscanthus × giganteus
6
cultivation on fermentation efficiency in the SHF system
7
Miscanthus × giganteus plants grown in treatments B (the lowest dose of sewage sludge)
8
and E (the highest dose of NPK) were selected for the subsequent stage of the analysis due
9
to their highest alcohol content after fermentation, highest fermentation efficiency and
10
highest sugar content. Fermentation was conducted sequentially after hydrolysis with the
11
use of the same yeast strain (S. cerevisiae 7). Process conditions were identical to those
12
described in the first stage of research (Fig. 2).
13
The results of the analysis confirm the usefulness of selected biomass types for
14
fermentation without an additional supply of minerals. Treatment B produced significantly
15
more alcohol than treatment E (2.47% vs. 2.31%, p<0.05). Fermentation took place in the
16
SHF system without the addition of mineral salt, which indicates that nitrogen and
17
phosphorus fertilizers (in particular sewage sludge) create a supportive environment for
18
substrate fermentation by yeast.
19
The absence of hydrolyzate supplementation with nitrogen and phosphorus did not
20
significantly influence the alcohol content of biomass fermented in the SHF system. The
21
alcohol content of distillate obtained after the fermentation of biomass from treatment B
22
was even higher than that noted under standard conditions. Alcohol content was highest in
23
treatment E where plants were supplied with nitrogen and phosphorus fertilizers.
24
12
1
The effect of the absence of hydrolyzates supplementation during Miscanthus × giganteus
2
cultivation on fermentation efficiency in the SSF system
3
The bioconversion of lignocellulosic substrates by simultaneous hydrolysis and
4
fermentation should take place at a temperature that is optimal for cellulolytic enzymes. In
5
the following stage of our experiment, Saccharomyces cerevisiae strain AS4 was used, and
6
the optimal temperature was 38°C. Based on the results obtained in the previous stage,
7
biomass was fermented without the addition of mineral salts to the SSF system (treatments
8
B and E).
9
After simultaneous hydrolysis and fermentation, the alcohol content of digestate from both
10
fertilized treatments (B and E) was lower than in the hydrolyzates from the SHF system
11
containing S. cerevisiae strain 7. The alcohol content of fermented hydrolyzates from
12
treatments B and E was determined at 2.21% and 1.90%, respectively (v/v) (Fig. 3).
13
In treatment B, the alcohol content was virtually the same after fermentation (2.20% v/v;
14
p<0.05), whereas the addition of a standard dose of mineral salts in treatment E increased
15
the alcohol content after fermentation to some extent (2.11% v/v), but this difference was
16
also not statistically significant (p<0.05). Lower ethanol production from Miscanthus ×
17
gigantheus was obtained by Boakye-Boaten et al. (2016) where after 72 h of simultaneous
18
saccharification and fermentation using S. cerevisiae and a cocktail of enzymes at 35°C,
19
the ethanol concentration varied among the samples and the highest concentration was
20
0.7% (w/v) for the biomass sample grown with swine manure, wet processed and
21
pretreated.
22
The absence of nitrogen and phosphorus supplementation had a minor effect on the alcohol
23
content of hydrolyzates fermented in the SSF system. In treatment B (fertilized with
24
sewage sludge), the addition of salt did not increase alcohol content which was almost
25
identical to that noted in the distillate obtained from non-supplemented biomass, and the 13
1
differences were statistically insignificant (p<0.05). However, in treatment E (NPK
2
fertilizer), the alcohol content of the hydrolyzate increased after fermentation when the
3
substrate was supplemented with nitrogen and phosphorus. Biomass should be pretreated
4
to increase ethanol production by simultaneous enzymatic hydrolysis and yeast
5
fermentation. Yeh et al. (2016) subjected Miscanthus floridulus biomass to pretreatment
6
with alkali at 90°C and acid-catalyzed steam explosion. The ethanol yields of M. floridulus
7
biomass fermented in the SSF system for 72 h were determined at 78.4 ± 1.0% and 69.0 ±
8
0.1% (w/w), respectively. Biomass pretreatment, feedstock processing and handling can
9
lower total cost by 39 to 43.9%, including enzyme production costs that account for 15.7%
10
of total cost, which can contribute to the rapid commercialization of bioethanol from
11
Miscanthus biomass (Boakye-Boaten et al., 2017).
12 13
The effect of fertilization on Miscanthus × gigantheus productivity
14
The main parameters evaluated in energy crops include yield, expressed in tons of biomass
15
per hectare, heating value (MJ/kg) and energy efficiency of crops. Fermentation efficiency
16
can be evaluated by determining the alcohol content of 1 kg of biomass and then
17
calculating the ethanol yield per ha. Crops with the highest values of the above parameters
18
are most suitable for bioethanol production. Moreover, the time of harvesting influence on
19
energy yield as higher heating values. Godin et al. (2013) stated that the Miscanthus
20
harvested in autumn offer the highest energy yield per unit area compared to the other
21
crops. Therefore, in our research Miscanthus was harvested in autumn.
22
The data in Table 2 clearly indicate that fertilization had a positive influence on the
23
biomass yield, energy yield and ethanol yield of Miscanthus × giganteus. In all fertilized
24
treatments, these parameters increased proportionally with a rise in the rate and quality of
25
fertilizers. The NPK fertilizer was more effective than sewage sludge, however, the 14
1
effectiveness of sewage sludge in comparison with a lower dose of NPK has not been fully
2
resolved.
3
In the non-fertilized treatment, biomass yield was determined at 17.34 t DM/ha.
4
Fertilization increased biomass yield proportionally to the fertilizer rate and quality. The
5
biomass yield of Miscanthus fertilized with sewage sludge reached 18.16 t DM/ha
6
(treatment B) and 18.85 t DM/ha (treatment C). Biomass yield was higher in response to
7
NPK fertilization at 18.54 t DM/ha (treatment D) and 19.02 t DM/ha (treatment E).
8
Fertilization of crops with sewage sludge increased the energy yield of biomass from
9
316.17 GJ/ha (treatment B) to 328.74 GJ/ha (treatment C). In treatments supplied with
10
NPK fertilizer, the increase in energy yield was somewhat higher than that noted for
11
sewage sludge at 323.52 GJ/ha (treatment D) and 333.23 GJ/ ha (treatment E). Similar
12
results were obtained by (Lisowski and Porwisiak, 2010) who evaluated the impact of
13
sewage sludge fertilization on Miscanthus × giganteus yield. In their study, fertilizers with
14
a higher content of mineral nitrogen (90 kg N/ ha) led to a higher increase in crop yield
15
than sludge fertilization alone. However, the application of a fertilizer with half the
16
nitrogen dose decreased yield. In contrast, Ociepa-Kubicka and Pachura (2013)
17
demonstrated that Miscanthus fertilization with sewage sludge promoted a higher increase
18
in biomass yield than mineral fertilizer containing up to 120 kg N/ha. These results
19
indicate that the composition of sewage sludge influences the biomass yield of fertilized
20
crops. The composition of sewage sludge is determined by the type and origin of treated
21
wastewater. Sewage sludge with a high content of heavy metals can inhibit plant growth.
22
Energy yield is also an important parameter of energy crops. In this study, no significant
23
differences in energy yield were observed between Miscanthus biomass from different
24
fertilization treatments, but plants fertilized with NPK were characterized by higher
25
heating value. In a study by Lewandowski et al. (2000), the heating value of Miscanthus 15
1
ranged from 17 to 19.2 MJ/kg, which is consistent with our results. Schwarz et al. (1994)
2
and Ercoli et al. (1999) demonstrated that fertilization does not contribute to an increase in
3
energy yield. In our study, however, energy efficiency was determined by both biomass
4
yield and its heating value.
5
The results of this study indicate that fertilization has a significant effect on the growth
6
performance of energy crops. Ethanol yield was highest (40% higher than in the control
7
treatment) when NPK fertilizer was applied at 160 kg N/ha. There are no published reports
8
describing the influence of fertilization on Miscanthus productivity and cellulosic ethanol
9
production. The impact of fertilization was analyzed in other energy crops. Sindelar et al.
10
(2012) demonstrated that maize fertilization with nitrogen increased the ethanol yield of
11
corn stover and corn cob substrate. In their study, nitrogen rates were similar to those
12
applied in our study, and they induced a similar increase in ethanol yield (30-50%).
13 14
Conclusions
15
The alcohol content of hydrolyzates of Miscanthus × giganteus from treatments fertilized
16
with sewage sludge at 1.75 DM/ha (treatment B) and NPK (160 kg N, 50 kg P, 80 kg K)
17
(equivalent nitrogen content of 2.8 t DM/ha) (treatment E) was determined at 2.47% and
18
2.31%, respectively. After fermentation in the SSF system, alcohol content reached 2.21%
19
in distillates from treatment B and 1.90% in distillates from treatment E. The type of
20
supplementation did not induce significant differences in alcohol content (p<0.05). The
21
alcohol content of hydrolyzates fermented in the SHF system was determined at 2.36% in
22
biomass from treatment B and 2.40% in biomass from treatment E. The SSF fermentation
23
system was less efficient, and the alcohol content of distillates was determined at 2.2%
24
(treatment B) and 2.11% (treatment E).
25 16
1
Acknowledgments
2
This study was financed by the University of Warmia and Mazury in Olsztyn as part of
3
statutory research grant No. 20.610.020-300.
4 5 6
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pretreatments in Miscanthus. Biotechnol. Biofuels 5, 1–12. doi:10.1186/1754-6834-5-58 40. Yeh, R.-H., Lin, Y.-S., Wang, T.-H., Kuan, W.-C., Lee, W.-C., 2016. Bioethanol
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18 19
21
1 2 3
Figure Captions
4
× giganteus biomass hydrolyzates in the SHF system with S. cerevisiae strain 7. Values
5
are given as means (n = 3, ± standard deviation). Means followed by the same letter are not
6
significantly different at p < 0.05, using the Duncan multiple range test.
Figure 1. The effect of different fertilization treatments on the fermentation of Miscanthus
7 8
Figure 2. The effect of the absence of hydrolyzate supplementation (treatments B and E)
9
on the fermentation of Miscanthus × giganteus hydrolyzates in the SHF system with S.
10
cerevisiae strain 7. Values are given as means (n = 3, ±standard deviation). Means
11
followed by the same letter are not significantly different at p < 0.05, using the Duncan
12
multiple range test.
13 14
Figure 3. The alcohol content of fermented hydrolyzates of Miscanthus × giganteus from
15
treatments B (fertilized with sewage sludge at 1.75 DM/ha) and E (fertilized with NPK,
16
equivalent nitrogen content of 2.8 t DM/ha) containing S. cerevisiae 7 or AS4, with and
17
without nitrogen and phosphorus supplementation, in SHF and SSF systems. Values are
18
given as means (n = 3, ±standard deviation). Means followed by the same letter are not
19
significantly different at p < 0.05, using the Duncan multiple range test.
20
22
1
Table 1. Proximate composition of Miscanthus × giganteus biomass from different
2
fertilization treatments (mean±standard deviation, n = 3). Parameter
Unit
Ash Protein Total sugar Crude fat NDF ADF Cellulose (CEL) Hemicellulose (HEM) Total CEL and HEM Lignin (ADL)
%DM %DM %DM %DM %DM %DM %DM %DM %DM %DM
A 3.5 (±0.08) 4.01(±0.08) 3.4 (±0.07) 0.96 (±0.05) 78.19(±0.46) 53.94(±2.43) 44.93 (±1.56) 24.25 (±1.90) 69.18 (±0.74) 9.01(±0.92)
Fertilization treatment B C D 3.42 (±0.10) 3.17 (±0.10) 3.65 (±0.04) 4.00 (±0.08) 4.25 (±0.04) 5.38 (±0.01) 3.32 (±0.01) 2.84 (±0.05) 2.77 (±0.11) 0.93 (±0.06) 0.97(±0.14) 0.95 (±0.04) 77.23(±0.64) 78.08(±0.38) 77.29(±0.20) 49.77(±1.70) 49.51(±0.30) 48.17(±0.81) 42.21 (±1.04) 41.98 (±0.20) 41.27 (±0.61) 27.46 (±1.58) 28.57 (±0.67) 29.12 (±0.75) 69.67 (±1.23) 70.55 (±0.43) 70.39 (±0.26) 7.56(±0.72) 7.53(±0.08) 6.90(±0.24)
E 3.36 (±0.08) 4.46 (±0.04) 3.11(±0.06) 0.86 (±0.03) 76.64(±0.41) 47.74(±0.20) 40.49 (±0.25) 28.9 (±0.21) 69.39 (±0.46) 7.25(±0.10)
3 4
23
1
Table 2. Energy efficiency and fermentation efficiency of Miscanthus × giganteus from
2
different fertilization treatments (mean±standard deviation, n = 3). Treatment
Heating value [MJ/kg]
A B C D E
17.17 (± 0.02) 17.41 (± 0.02) 17.44 (± 0.02) 17.45 (± 0.02) 17.52 (± 0.02)
Fermentation yield [L A100/kg] 0.105 (±0.05) 0.132 (±0.08) 0.128 (±0.02) 0.125 (±0.04) 0.134 (±0.02)
Biomass yield [t DM/ha]
Energy yield [GJ/ha]
17.34 (±0.47) 297.73 (±8.04) 18.16 (±0.48) 316.17 (±8.70) 18.85 (±0.56) 328.74 (±9.57) 18.54 (±1.77) 323.52 (±30.72) 19.02 (±1.31) 333.23 (±23.02)
Ethanol yield [L A100/ha] 1864 (±105) 2469 (±163) 2483 (±44) 2396 (±69) 2633 (±44)
3 4 5
24
3
2.36b
125
100
2.29b
2.40b 2.24b
1.87a
2.5
2
75
1.5
50
1
25
0.5
0
Alcohol content (% v/v)
Concentration of reducing sugars (g/L)
150
0 A hydrolyzate
B
C after fermentation
D
E alcohol content
1 2 3
Figure 1. The effect of different fertilization treatments on the fermentation of Miscanthus
4
× giganteus biomass hydrolyzates in the SHF system with S. cerevisiae strain 7. Values
5
are given as means (n = 3, ± standard deviation). Means followed by the same letter are not
6
significantly different at p < 0.05, using the Duncan multiple range test.
7 8
25
3
2.47a
100
2.31b
2.5
80
2
60
1.5
40
1
20
0.5
0
Alcohol content (% v/v)
Concentration of reducing sugars (g/L)
120
0 B hydrolyzate
E after fermentation
alcohol content
1 2
Figure 2. The effect of the absence of hydrolyzate supplementation (treatments B and E)
3
on the fermentation of Miscanthus × giganteus hydrolyzates in the SHF system with S.
4
cerevisiae strain 7. Values are given as means (n = 3, ±standard deviation). Means
5
followed by the same letter are not significantly different at p < 0.05, using the Duncan
6
multiple range test.
7 8
26
3
a Alcohol content (%v/v)
2.5
ab b
b
a' b' d' a' b'
c' d'
a' c' d'
2 1.5 1 0.5 0 B
E
SHF without supplemantation
SHF with supplementation
SSF without supplementation
SSF with supplementation
1 2
Figure 3. The alcohol content of fermented hydrolyzates of Miscanthus × giganteus from
3
treatments B (fertilized with sewage sludge at 1.75 DM/ha) and E (fertilized with NPK,
4
equivalent nitrogen content of 2.8 t DM/ha) containing S. cerevisiae 7 or AS4, with and
5
without nitrogen and phosphorus supplementation, in SHF and SSF systems. Values are
6
given as means (n = 3, ±standard deviation). Means followed by the same letter are not
7
significantly different at p < 0.05, using the Duncan multiple range test.
8 9 10
27
1 2
Highlights Fertilization influences biomass yield and content of lignocellulosic fractions
3
Bioethanol production was highest with hydrolysis and fermentation done separately
4
Sewage sludge or NPK fertilizer increased ethanol production 30 or 40%, respectively
5 6 7 8 9
28
S0960-8524(17)31091-X http://dx.doi.org/10.1016/j.biortech.2017.07.005 BITE 18424
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
11 May 2017 30 June 2017 1 July 2017
Please cite this article as: Dubis, B., Bułkowska, K., Lewandowska, M., Szempliński, W., Jankowski, K.J., Idźkowski, J., Kordala, N., Szymańska, K., Effect of different nitrogen fertilizer treatments on the conversion of Miscanthus × giganteus to ethanol, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech. 2017.07.005
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1
Effect of different nitrogen fertilizer treatments on the conversion of Miscanthus ×
2
giganteus to ethanol
3
Bogdan Dubisa, Katarzyna Bułkowskab,*, Małgorzata Lewandowskac, Władysław Szemplińskia,
4
Krzysztof Józef Jankowskia, Jakub Idźkowskic, Natalia Kordalac, Karolina Szymańskac
5 6
a
7
University of Warmia and Mazury in Olsztyn, Oczapowskiego 8, 10-719 Olsztyn, Poland
8
b
9
Słoneczna 45G, 10-709 Olsztyn, Poland
Department of Agrotechnology, Agricultural Production Management and Agribusiness,
Department of Environmental Biotechnology, University of Warmia and Mazury in Olsztyn,
10
c
11
Olsztyn, Heweliusza 1, 10-718 Olsztyn, Poland
12
*Coresponding autor: PhD Katarzyna Bułkowska, Department of Environmental Biotechnology,
13
University of Warmia and Mazury in Olsztyn, Słoneczna 45G, 10-709 Olsztyn, Poland, e-mail:
14
[email protected]
15
Keywords: ethanol fermentation, fertilization effect, sequential and simultaneous
16
hydrolysis and fermentation
Chair of Food Biotechnology, Faculty of Food Sciences, University of Warmia and Mazury in
17 18
Abstract
19
Miscanthus × giganteus is a perennial rhizomatous grass which is used as a biofuel crop.
20
Due to its high yields, low production costs, resistance to low temperatures, low soil
21
requirements and, above all, high cellulose content, miscanthus can be a useful resource for
22
ethanol production. The aim of this study was the determine the effect of two fertilization
23
regimes (sewage sludge/mineral NPK) during miscanthus cultivation on the chemical
24
composition of biomass, the content of major lignocellulosic factions and the effectiveness
25
of miscanthus conversion to bioethanol. The results indicate that fertilization treatments
26
influenced biomass yield and the content of major lignocellulosic fractions. Bioethanol 1
1
production was higher when hydrolysis and fermentation processes were conducted
2
separately than when saccharification and fermentation were conducted simultaneously.
3
Ethanol production increased by 30% and 40% in response to sewage sludge and NPK
4
(equivalent nitrogen content = 160 kg N/ha) fertilization, respectively, in comparison with
5
unfertilized crops.
6 7
Introduction
8
In Europe, Miscanthus (Miscanthus × giganteus) is classified as an energy crop.
9
Miscanthus × giganteus grows to a height of 4 m, produces stems with a length of up to 3
10
m and a diameter of approx. 10 mm, and can be grown in one location for up to 15 - 20
11
years. Miscanthus is a C4 plant and a highly efficient crop which is characterized by high
12
yield, environmental benefits, the ability to grow without pesticides and fertilizers, long
13
life and high cellulose (CEL) content (40-48%). For these reasons, Miscanthus enjoys the
14
status of a popular energy crop in Europe. Miscanthus can be harvested twice a year in
15
autumn (October to November or early December) and spring (February to the end of
16
March). The biomass yield of Miscanthus varies depending on the date of harvest. Its dry
17
matter content is estimated at 35%-45% in the autumn harvest and 60%-70% in the spring
18
harvest. Miscanthus yields range from 1 to 3 t/ha in the first year of cultivation, 8 to 15 t/ha
19
in the second year, and 15 to 30 t/ha in the third and subsequent years. Due to a rapid
20
increase in yield, Miscanthus should not be harvested in the first years of cultivation. The
21
calorific value of Miscanthus is estimated at 18 MJ/ha (Greenhalf et al., 2013; D. J. M.
22
Hayes, 2013), and its theoretical ethanol yield (TEY) is around 0.45 L/kg of biomass.
23
Other lignocellulosic substrates, such as switchgrass, maize straw and bagasse, have
24
similar TEY values of approximately 0.40 L/kg. However, the biomass yield of
25
Miscanthus is 2-24 times higher in comparison with other substrates, therefore, its ethanol 2
1
yield per ha is high at 4600 to 12400 L/ha (Kim et al., 2015). It is estimated that by 2050,
2
Miscanthus could satisfy up to 12% of primary energy demand in Europe (HASTINGS et
3
al., 2009). Miscanthus is easy to cultivate because it has an extensive root system that
4
effectively absorbs and utilizes nutrients. Nutrient recovery is significant during transport
5
from the shoot to rhizomes and from falling leaves. Miscanthus roots are colonized by
6
nitrogen-fixing bacteria, which increases nitrogen inputs to the ecosystem (Cadoux et al.,
7
2012).
8
The most widely used raw materials in bioethanol production include sugar cane (Brazil)
9
and corn (United States). However, the popularity of lignocellulosic substrates is on the
10
rise due to their high availability, low cost and, above all, sustainability. These substrates
11
include agricultural wastes, grasses, wood and sawdust (Sarkar et al., 2012).
12
Lignocellulose is a polymer consisting of three main fractions: CEL, hemicellulose (HEM)
13
and lignin. The CEL content of substrates ranges from 35% to 50% of total dry weight, that
14
of HEM from 20 to 35%, and that of lignin from 10 to 25%. Hemicellulose is hydrolyzed
15
more easily than CEL due to the presence the numerous branches in the main chain. Lignin
16
has a highly complex structure, it and it imparts resistance to degradation to lignocellulosic
17
material. Lignin is strongly associated with CEL and HEM, and it creates an impermeable
18
barrier. The physicochemical properties of lignocellulose also offer protection against
19
microbial degradation (Balat, 2011; Mussatto and Teixeira, 2010).
20
During bioethanol production, pretreatment is the first step in reducing the recalcitrance of
21
lignocellulosic biomass to conversion into sugars, as it increases the availability of
22
enzymes to the polysaccharide fraction. There are many pretreatment methods, including
23
addition of hydrolysate (Li et al., 2016).
24
Pretreatment methods can be classified as biological, chemical or physical methods, or as
25
combinations of these methods (Zheng et al., 2009, Harmsen et al., 2010; Agbor et al., 3
1
2011). Biological methods utilize fungi and actinomycetes for reduction of the degree of
2
cellulose polymerization, and for partial degradation of hemicelluloses and lignin.
3
Chemical or physical methods affect biomass by increasing its solubility, decreasing its
4
degree of polymerization, partially or completely delignifying it, and partially or
5
completely hydrolyzing the hemicelluloses within the biomass. Examples of chemical
6
pretreatment methods include acid hydrolysis, alkaline hydrolysis, ozonolysis, oxidative
7
delignification, the organosolv process, and ionic liquid pretreatment. Under the heading of
8
physicochemical methods come such processes as steam explosion (autohydrolysis), liquid
9
hot water pretreatment, ammonia fiber explosion (AFEX), and CO2 explosion. Finally,
10
there is physical pretreatment, which is used to reduce particle size, or to increase the size
11
of pores and the accessible surface area of the biomass.
12
The aim of this study was to determine: 1) the effect of different fertilization regimes
13
during Miscanthus × gigantheus cultivation on the crop’s chemical composition and
14
content of lignocellulosic fractions, 2) the influence of the absence of hydrolyzate
15
supplementation during Miscanthus × giganteus cultivation on fermentation efficiency in
16
systems involving sequential or simultaneous hydrolysis and fermentation, and 3) the
17
effect of fertilization on Miscanthus × giganteus productivity.
18
Materials and Methods
19
Plant material
20
Miscanthus × giganteus (7th year of cultivation, 2014 year) was cultivated in the
21
Production and Experimental Station in Bałcyny, Poland, which belongs to the University
22
of Warmia and Mazury in Olsztyn. Field experiments were conducted in a split-plot system
23
with each fertilizer variant tested in triplicate on 120 m2 plots. Rhizomes were planted
24
manually with a spacing of 70 x 70 cm. Chemical weed control was applied (1x triticale)
25
with iodosulfuron methyl sodium and amidosulfuron mixture at a dose 0.125 L/ha. 4
1
Biomass was collected using a self-propelled chopper. The experiment was established on
2
Haplic Luvisol developed from boulder clay (IUSS, 2006). The arable layer (0-30 cm) was
3
slightly acidic (pH in 1 M KCl – 6.5), and had a high content of available phosphorus and
4
magnesium, and moderate content of potassium.
5
Five fertilization treatments were applied before the growing season: A – no fertilizer
6
(control), B – sewage sludge (1.75 t DM/ha), C – sewage sludge (2.8 t DM/ha), D – NPK
7
fertilizer (100 kg N, 50 kg P, 80 kg K/ha; equivalent nitrogen content of 1.75 t DM/ha),
8
and E - NPK fertilizer (160 kg N, 50 kg P, 80 kg K/ha; equivalent nitrogen content of 2.8 t
9
DM/ha). The dry matter content of biomass was estimated at 93%. Biomass was chopped
10
into pieces ranging from 1 to 2 mm in diameter. The characteristics of Miscanthus are
11
shown in Table 1.
12 13
Sequential hydrolysis and fermentation (SHF) of Miscanthus × gigantheus
14
Miscanthus × giganteus biomass was subjected to alkaline pretreatment with NaOH under
15
the following conditions: temperature, 121 °C; time, 1 h; NaOH addition of 0.1 g ∙g-1 d.m.;
16
and a ratio of solid to liquid fraction of 1:9. The supernatant was separated, and the solid
17
fraction was detoxified by rinsing with distilled water and re-centrifugation (Lewandowska
18
et al., 2016; Świątek et al., 2014).
19
After alkaline pretreatment, the samples were centrifuged at RCF 4240 g at a temperature
20
of 5°C for 10 min. The supernatant was decanted, distilled water was added, and the
21
samples were centrifuged at RCF 4240 g and 5°C for 10 min. The procedure was
22
performed twice. After centrifugation, the liquid fraction was decanted, and the solid
23
fraction was combined with distilled water to obtain 10% of suspended solids in the
24
sample. After centrifugation, acetic acid (~99%) was used to adjust pH to 5.0 (measured
25
with the Hanna 211 pH-meter). Next, the medium was pasteurized at 90°C for 20 min. 5
1
Enzymatic hydrolysis was performed by adding the following enzymes: cellulase from
2
Trichoderma longibrachiatum (15 U/g DM of the substrate), xylanase from T.
3
longibrachiatum (15 FXU/g DM of the substrate) and cellobiase (Novozyme 188) (30
4
CBU/g DM of the substrate). Hydrolysis was conducted for 72 h at 42°C (Innova 40
5
incubator, New Brunswick Scientific). During the process, samples were shaken at 250
6
rpm/min. After hydrolysis, the concentration of reducing sugars was determined using 3,5-
7
dinitrosalycilic acid.
8
The hydrolyzate and the hydrolyzate supplemented with mineral sources of nitrogen and
9
phosphorus ((NH4)2SO4 and KH2PO4) were inoculated with S. cerevisiae 7 (5% v/v). The
10
concentration of reducing sugars after inoculation was calculated from the following
11
equation: concentration of sugars in the hydrolyzate × 0.2 / 0.21. Fermentation was carried
12
out at 30°C under anaerobic conditions for 72 h.
13 14
Simultaneous hydrolysis and fermentation (SSF) of Miscanthus × gigantheus
15
The medium was prepared according to the described protocol. Hydrolysis was conducted
16
with enzymatic preparations and was conducted for 24 h according to the described
17
protocol. To compare the efficiency of fermentation with and without supplementation,
18
mineral sources of nitrogen and phosphorus were added to selected samples after 24 h. The
19
temperature was lowered to 38°C, and 10 cm3 (5% v/v) of the S. cerevisiae AS4 inoculum
20
was added. Fermentation with hydrolysis was carried out for 96 hours (38°C, anaerobic
21
conditions) in 3 replicates.
22 23
Chemical analysis
24
Ash content was determined by mineralization in a muffle furnace. Protein content was
25
measured using the Kjeldahl method in the FOSS Kjeltec 8400 analyzer. Crude fat content 6
1
was determined in the Foss Tecator Soxtec 2043 fat extraction system. Sugar content was
2
measured with Epoll-2-Spekol. The fiber fraction was determined in the FibertecTM 1020
3
system. The neutral fiber content (NDL) was determined according to Van Soest et al.
4
(1991). The content of acid detergent fiber (ADF) and acid detergent lignin (ADL) was
5
determined according to (PN-EN ISO 13906, 2009). Cellulose concentration was
6
calculated as the difference between ADF and ADL, and HEM concentration was
7
determined as the difference between NDF and ADF. The effects of hydrolysis were
8
evaluated based on the quantity of enzymatically released reducing sugars determined in a
9
reaction with 3,5-dinitrosalicylic acid (Miller, 1959). After fermentation, alcohol content
10
was determined by distillation (AOAC, 1990). The heating value was determined
11
according to PN-ISO9831 (2002).
12
The fermentation yield (YF, L A100/kg) was calculated according to the following equation:
13
where VE is a volume of ethanol after fermentation (L A100), MS is a mass of wet biomass
14
for fermentation (kg), and EPre is the efficiency of alkali pretreatment (–).
15
The ethanol yield (YE, L A100/kg) was calculated with the following equation:
16
where YF is a fermentation yield (L A100/kg) and YB is a biomass yield (t/ha).
17 18
Statistical analysis
19
The Duncan multiple range test was used after ANOVA (STATISTICA 10, StatSoft Inc.)
20
to determine the significance of differences between alcohol content in series A-E (p<0.05
21
was considered significant).
22 7
1
Results and Discussion
2 3
The effect of fertilization on the chemical composition and content of lignocellulosic
4
fractions in Miscanthus × gigantheus biomass
5
The aim of this study was to determine the influence of different fertilization regimes on
6
the chemical composition, proportions of basic lignocellulosic fractions and the
7
bioconversion efficiency of biomass polysaccharides (CEL and HEM) to ethanol. The
8
proximate composition of Miscanthus × giganteus biomass grown with the application of
9
different fertilizers is presented in Table 1.
10
Fertilization had no significant influence on the proximate composition of Miscanthus
11
biomass in any of the examined treatments. Ash content was 3.50% in the control
12
treatment (A). Ash content was lower in the experimental treatments, excluding treatment
13
D (3.65%) with mineral fertilization. Fertilized crops were characterized by lower ash
14
content, which could be attributed to harvesting season (October) during which plant parts
15
that fell to the ground accumulated large amounts of minerals and nutrients (Lewandowski
16
and Kicherer, 1997). The ash content of biomass is determined by the growth stage, time of
17
year, and cultivation site (Hayes, 2013). Similar results were reported by Lewandowski
18
and Heinz (2003) who cultivated Miscanthus in three different locations and observed
19
differences in the ash content of plants subject to location and season. In all crops, ash
20
content varied across seasons, and was higher in December and lower in March in the
21
range of 2% to 4.5%. Jingping Qin et al. (2012) also demonstrated significant variations in
22
the ash content of different Miscanthus species (2.89% to 5.69%).
23
Sewage sludge and NPK fertilizers increased the protein content of Miscanthus biomass.
24
Fertilizers are highly abundant in nitrogen which can be accumulated in biomass, thus
25
increasing the protein content of plants. In our study, protein content increased in all 8
1
treatments regardless of the fertilization method, excluding treatment B (4.00%) where
2
sewage sludge was used (1.75 t DM/ha) and where protein content was identical to that
3
noted in the control treatment (4.01%). The nitrogen content of Miscanthus biomass
4
increased with a rise in fertilizer rate, and similar results were reported by other authors
5
(Kahle et al., 2001; Larsen et al., 2014; Schwarz et al., 1994). The mineral fertilizer (NPK)
6
induced a higher increase in protein content than sewage sludge because mineral nitrogen
7
is more readily absorbed by plants.
8
The content of sugars was highest in the non-fertilized treatment (3.40% DM). Fertilization
9
induced a decrease in sugar content in the range of 2.84% DM (treatment C) to 3.32% DM
10
(treatment B). A proximate analysis revealed that fertilization had no effect on crude fat
11
content which ranged from 0.93 to 0.97%. Crude fat content was somewhat lower (0.86%
12
DM) only in the treatment exposed to the highest rate of NPK fertilizer (treatment E).
13
An analysis of lignocellulosic fractions revealed the highest CEL content of 44.93% DM in
14
the non-fertilized control treatment (A). Sewage sludge lowered CEL content to 42.21%
15
DM in treatment B and 41.98% DM in treatment C. The lowest CEL content was
16
determined in the treatments fertilized with NPK, at 40.49% DM in treatment D and
17
41.27% DM in treatment E. An inverse relationship was noted for the HEM fraction.
18
Hemicellulose content was lowest in treatment A (24.25% DM). In treatments fertilized
19
with sewage sludge, HEM content was determined at 27.46% DM (B) and 28.57% DM
20
(C). The highest concentration of HEM was observed in the treatments fertilized with NPK
21
at 29.12% DM (D) and 28.90% DM (E). Hemicellulose is the predominant fraction in the
22
green parts of plants. Higher nutrient supply promotes leaf growth and increases the HEM
23
content of biomass. In lignified parts of plants, such as the stem, CEL and lignin are the
24
predominant fractions. The most important macronutrients for plant growth are nitrogen
25
and phosphorus which are not accumulated in the above-ground parts of plants, but are 9
1
stored in roots and rhizomes (Cadoux et al., 2012; Hodgson et al., 2011). The content of
2
CEL and lignin also decreased in response to fertilization. Plants that were not fertilized
3
with nitrogen were characterized by higher concentrations of all lignocellulosic fractions,
4
including HEM. The negative impact of nitrogen fertilization on the proportions of
5
lignocellulosic fractions was also found in other plant species, such as sorghum or millet
6
(Blümmel et al., 2003; Reddy et al., 2003). These results show that Miscanthus × giganteus
7
can be a good source of lignocellulosic biomass without fertilization, which makes it a
8
cost-effective crop. The total content of CEL and HEM did not differ significantly between
9
fertilized treatments. Cellulose and HEM are synthesized during sugar hydrolysis, and they
10
are used in ethanol production during fermentation. Crops grown for ethanol should be
11
characterized by the highest possible content of CEL and HEM. However, raw material
12
pretreatment can lead to HEM degradation to simpler compounds that inhibit the metabolic
13
activity of microorganisms responsible for fermentation (furfural, 5-
14
hydroxymethylfurfural). In the non-fertilized treatment (A), total CEL and HEM content
15
was 69.18%. In fertilized treatments, total CEL and HEM content ranged from 69.39% to
16
70.55%. Lignin is the third major lignocellulosic fraction. This insoluble polymer is
17
undesirable in ethanol production because it limits the availability of lignocellulosic
18
polysaccharides, and slows down hydrolysis and fermentation. The lignin content of the
19
evaluated biomass samples was influenced by fertilization. The highest lignin content in
20
excess of 9% was noted in treatment A. A significant reduction in lignin content was
21
observed in fertilized treatments. The application of sewage sludge decreased lignin
22
content to 7.56% (B) and 7.53% (C). An even greater reduction in lignin content was
23
observed in response to NPK fertilization at 6.90% (D) and 7.25% (E). The higher lignin
24
content (17.2%) in Miscanthus × giganteus was reported by Guragian et al. (2014). The
25
reduction in lignin content is a positive factor because substrate pre-treatment leads to the 10
1
release of phenolic compounds that inhibit microbial activity during fermentation. At the
2
same time, a reduction in CEL content is a negative factor because it lowers the availability
3
of polysaccharides for hydrolysis. Polysaccharides are a source of glucose, the main sugar
4
involved in fermentation.
5 6
The effect of fertilization on sequential hydrolysis and fermentation with Saccharomyces
7
cerevisiae 7
8
Miscanthus was used as a source of carbohydrates for alcohol fermentation. The effects of
9
different fertilization treatments on the fermentation of Miscanthus × giganteus biomass
10
hydrolyzates in a sequential system (SHF) with S. cerevisiae strain 7 are shown in Figure
11
1.
12
All fertilization treatments resulted in significantly higher ethanol content than in the
13
control treatment (p<0.05) (Fig.1). This result may be caused by an increase in the
14
concentration of hemicelluloses in the Miscanthus biomass after fertilization, and a
15
decrease in the concentration of lignin. Hemicelluloses are hydrolyzed more easily than
16
CEL and lignin, which is resistance to degradation and creates an impermeable barrier
17
around CEL and HEM. According to Lee and Kuan (2015), the lignin and hemicellulose
18
contents of Miscanthus are have an important influence on the efficiency of enzyme
19
hydrolysis of pretreated biomass. Miscanthus that has a high concentration of
20
hemicelluloses, a low cellulose crystallinity index and a high concentration of lignin is not
21
saccharificated well after pretreatment. Xu et al. (2012) also state that cellulose and lignin
22
have negative effects on biomass digestibility, in contrast to hemicelluloses, which have a
23
positive effect.
11
1
In the variants fertilized with sewage sludge, alcohol content was 2.36% (B) and 2.29%
2
(C). The use of NPK fertilizer also produced favorable results. In biomass from treatments
3
D and E, alcohol content was 2.24% and 2.40%, respectively.
4 5
The effect of the absence of hydrolyzate supplementation during Miscanthus × giganteus
6
cultivation on fermentation efficiency in the SHF system
7
Miscanthus × giganteus plants grown in treatments B (the lowest dose of sewage sludge)
8
and E (the highest dose of NPK) were selected for the subsequent stage of the analysis due
9
to their highest alcohol content after fermentation, highest fermentation efficiency and
10
highest sugar content. Fermentation was conducted sequentially after hydrolysis with the
11
use of the same yeast strain (S. cerevisiae 7). Process conditions were identical to those
12
described in the first stage of research (Fig. 2).
13
The results of the analysis confirm the usefulness of selected biomass types for
14
fermentation without an additional supply of minerals. Treatment B produced significantly
15
more alcohol than treatment E (2.47% vs. 2.31%, p<0.05). Fermentation took place in the
16
SHF system without the addition of mineral salt, which indicates that nitrogen and
17
phosphorus fertilizers (in particular sewage sludge) create a supportive environment for
18
substrate fermentation by yeast.
19
The absence of hydrolyzate supplementation with nitrogen and phosphorus did not
20
significantly influence the alcohol content of biomass fermented in the SHF system. The
21
alcohol content of distillate obtained after the fermentation of biomass from treatment B
22
was even higher than that noted under standard conditions. Alcohol content was highest in
23
treatment E where plants were supplied with nitrogen and phosphorus fertilizers.
24
12
1
The effect of the absence of hydrolyzates supplementation during Miscanthus × giganteus
2
cultivation on fermentation efficiency in the SSF system
3
The bioconversion of lignocellulosic substrates by simultaneous hydrolysis and
4
fermentation should take place at a temperature that is optimal for cellulolytic enzymes. In
5
the following stage of our experiment, Saccharomyces cerevisiae strain AS4 was used, and
6
the optimal temperature was 38°C. Based on the results obtained in the previous stage,
7
biomass was fermented without the addition of mineral salts to the SSF system (treatments
8
B and E).
9
After simultaneous hydrolysis and fermentation, the alcohol content of digestate from both
10
fertilized treatments (B and E) was lower than in the hydrolyzates from the SHF system
11
containing S. cerevisiae strain 7. The alcohol content of fermented hydrolyzates from
12
treatments B and E was determined at 2.21% and 1.90%, respectively (v/v) (Fig. 3).
13
In treatment B, the alcohol content was virtually the same after fermentation (2.20% v/v;
14
p<0.05), whereas the addition of a standard dose of mineral salts in treatment E increased
15
the alcohol content after fermentation to some extent (2.11% v/v), but this difference was
16
also not statistically significant (p<0.05). Lower ethanol production from Miscanthus ×
17
gigantheus was obtained by Boakye-Boaten et al. (2016) where after 72 h of simultaneous
18
saccharification and fermentation using S. cerevisiae and a cocktail of enzymes at 35°C,
19
the ethanol concentration varied among the samples and the highest concentration was
20
0.7% (w/v) for the biomass sample grown with swine manure, wet processed and
21
pretreated.
22
The absence of nitrogen and phosphorus supplementation had a minor effect on the alcohol
23
content of hydrolyzates fermented in the SSF system. In treatment B (fertilized with
24
sewage sludge), the addition of salt did not increase alcohol content which was almost
25
identical to that noted in the distillate obtained from non-supplemented biomass, and the 13
1
differences were statistically insignificant (p<0.05). However, in treatment E (NPK
2
fertilizer), the alcohol content of the hydrolyzate increased after fermentation when the
3
substrate was supplemented with nitrogen and phosphorus. Biomass should be pretreated
4
to increase ethanol production by simultaneous enzymatic hydrolysis and yeast
5
fermentation. Yeh et al. (2016) subjected Miscanthus floridulus biomass to pretreatment
6
with alkali at 90°C and acid-catalyzed steam explosion. The ethanol yields of M. floridulus
7
biomass fermented in the SSF system for 72 h were determined at 78.4 ± 1.0% and 69.0 ±
8
0.1% (w/w), respectively. Biomass pretreatment, feedstock processing and handling can
9
lower total cost by 39 to 43.9%, including enzyme production costs that account for 15.7%
10
of total cost, which can contribute to the rapid commercialization of bioethanol from
11
Miscanthus biomass (Boakye-Boaten et al., 2017).
12 13
The effect of fertilization on Miscanthus × gigantheus productivity
14
The main parameters evaluated in energy crops include yield, expressed in tons of biomass
15
per hectare, heating value (MJ/kg) and energy efficiency of crops. Fermentation efficiency
16
can be evaluated by determining the alcohol content of 1 kg of biomass and then
17
calculating the ethanol yield per ha. Crops with the highest values of the above parameters
18
are most suitable for bioethanol production. Moreover, the time of harvesting influence on
19
energy yield as higher heating values. Godin et al. (2013) stated that the Miscanthus
20
harvested in autumn offer the highest energy yield per unit area compared to the other
21
crops. Therefore, in our research Miscanthus was harvested in autumn.
22
The data in Table 2 clearly indicate that fertilization had a positive influence on the
23
biomass yield, energy yield and ethanol yield of Miscanthus × giganteus. In all fertilized
24
treatments, these parameters increased proportionally with a rise in the rate and quality of
25
fertilizers. The NPK fertilizer was more effective than sewage sludge, however, the 14
1
effectiveness of sewage sludge in comparison with a lower dose of NPK has not been fully
2
resolved.
3
In the non-fertilized treatment, biomass yield was determined at 17.34 t DM/ha.
4
Fertilization increased biomass yield proportionally to the fertilizer rate and quality. The
5
biomass yield of Miscanthus fertilized with sewage sludge reached 18.16 t DM/ha
6
(treatment B) and 18.85 t DM/ha (treatment C). Biomass yield was higher in response to
7
NPK fertilization at 18.54 t DM/ha (treatment D) and 19.02 t DM/ha (treatment E).
8
Fertilization of crops with sewage sludge increased the energy yield of biomass from
9
316.17 GJ/ha (treatment B) to 328.74 GJ/ha (treatment C). In treatments supplied with
10
NPK fertilizer, the increase in energy yield was somewhat higher than that noted for
11
sewage sludge at 323.52 GJ/ha (treatment D) and 333.23 GJ/ ha (treatment E). Similar
12
results were obtained by (Lisowski and Porwisiak, 2010) who evaluated the impact of
13
sewage sludge fertilization on Miscanthus × giganteus yield. In their study, fertilizers with
14
a higher content of mineral nitrogen (90 kg N/ ha) led to a higher increase in crop yield
15
than sludge fertilization alone. However, the application of a fertilizer with half the
16
nitrogen dose decreased yield. In contrast, Ociepa-Kubicka and Pachura (2013)
17
demonstrated that Miscanthus fertilization with sewage sludge promoted a higher increase
18
in biomass yield than mineral fertilizer containing up to 120 kg N/ha. These results
19
indicate that the composition of sewage sludge influences the biomass yield of fertilized
20
crops. The composition of sewage sludge is determined by the type and origin of treated
21
wastewater. Sewage sludge with a high content of heavy metals can inhibit plant growth.
22
Energy yield is also an important parameter of energy crops. In this study, no significant
23
differences in energy yield were observed between Miscanthus biomass from different
24
fertilization treatments, but plants fertilized with NPK were characterized by higher
25
heating value. In a study by Lewandowski et al. (2000), the heating value of Miscanthus 15
1
ranged from 17 to 19.2 MJ/kg, which is consistent with our results. Schwarz et al. (1994)
2
and Ercoli et al. (1999) demonstrated that fertilization does not contribute to an increase in
3
energy yield. In our study, however, energy efficiency was determined by both biomass
4
yield and its heating value.
5
The results of this study indicate that fertilization has a significant effect on the growth
6
performance of energy crops. Ethanol yield was highest (40% higher than in the control
7
treatment) when NPK fertilizer was applied at 160 kg N/ha. There are no published reports
8
describing the influence of fertilization on Miscanthus productivity and cellulosic ethanol
9
production. The impact of fertilization was analyzed in other energy crops. Sindelar et al.
10
(2012) demonstrated that maize fertilization with nitrogen increased the ethanol yield of
11
corn stover and corn cob substrate. In their study, nitrogen rates were similar to those
12
applied in our study, and they induced a similar increase in ethanol yield (30-50%).
13 14
Conclusions
15
The alcohol content of hydrolyzates of Miscanthus × giganteus from treatments fertilized
16
with sewage sludge at 1.75 DM/ha (treatment B) and NPK (160 kg N, 50 kg P, 80 kg K)
17
(equivalent nitrogen content of 2.8 t DM/ha) (treatment E) was determined at 2.47% and
18
2.31%, respectively. After fermentation in the SSF system, alcohol content reached 2.21%
19
in distillates from treatment B and 1.90% in distillates from treatment E. The type of
20
supplementation did not induce significant differences in alcohol content (p<0.05). The
21
alcohol content of hydrolyzates fermented in the SHF system was determined at 2.36% in
22
biomass from treatment B and 2.40% in biomass from treatment E. The SSF fermentation
23
system was less efficient, and the alcohol content of distillates was determined at 2.2%
24
(treatment B) and 2.11% (treatment E).
25 16
1
Acknowledgments
2
This study was financed by the University of Warmia and Mazury in Olsztyn as part of
3
statutory research grant No. 20.610.020-300.
4 5 6
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doi:10.1016/j.biotechadv.2011.05.005
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4. Blümmel, M., Zerbini, E., Reddy, B.V.., Hash, C.., Bidinger, F., Ravi, D., 2003. Improving
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21
1 2 3
Figure Captions
4
× giganteus biomass hydrolyzates in the SHF system with S. cerevisiae strain 7. Values
5
are given as means (n = 3, ± standard deviation). Means followed by the same letter are not
6
significantly different at p < 0.05, using the Duncan multiple range test.
Figure 1. The effect of different fertilization treatments on the fermentation of Miscanthus
7 8
Figure 2. The effect of the absence of hydrolyzate supplementation (treatments B and E)
9
on the fermentation of Miscanthus × giganteus hydrolyzates in the SHF system with S.
10
cerevisiae strain 7. Values are given as means (n = 3, ±standard deviation). Means
11
followed by the same letter are not significantly different at p < 0.05, using the Duncan
12
multiple range test.
13 14
Figure 3. The alcohol content of fermented hydrolyzates of Miscanthus × giganteus from
15
treatments B (fertilized with sewage sludge at 1.75 DM/ha) and E (fertilized with NPK,
16
equivalent nitrogen content of 2.8 t DM/ha) containing S. cerevisiae 7 or AS4, with and
17
without nitrogen and phosphorus supplementation, in SHF and SSF systems. Values are
18
given as means (n = 3, ±standard deviation). Means followed by the same letter are not
19
significantly different at p < 0.05, using the Duncan multiple range test.
20
22
1
Table 1. Proximate composition of Miscanthus × giganteus biomass from different
2
fertilization treatments (mean±standard deviation, n = 3). Parameter
Unit
Ash Protein Total sugar Crude fat NDF ADF Cellulose (CEL) Hemicellulose (HEM) Total CEL and HEM Lignin (ADL)
%DM %DM %DM %DM %DM %DM %DM %DM %DM %DM
A 3.5 (±0.08) 4.01(±0.08) 3.4 (±0.07) 0.96 (±0.05) 78.19(±0.46) 53.94(±2.43) 44.93 (±1.56) 24.25 (±1.90) 69.18 (±0.74) 9.01(±0.92)
Fertilization treatment B C D 3.42 (±0.10) 3.17 (±0.10) 3.65 (±0.04) 4.00 (±0.08) 4.25 (±0.04) 5.38 (±0.01) 3.32 (±0.01) 2.84 (±0.05) 2.77 (±0.11) 0.93 (±0.06) 0.97(±0.14) 0.95 (±0.04) 77.23(±0.64) 78.08(±0.38) 77.29(±0.20) 49.77(±1.70) 49.51(±0.30) 48.17(±0.81) 42.21 (±1.04) 41.98 (±0.20) 41.27 (±0.61) 27.46 (±1.58) 28.57 (±0.67) 29.12 (±0.75) 69.67 (±1.23) 70.55 (±0.43) 70.39 (±0.26) 7.56(±0.72) 7.53(±0.08) 6.90(±0.24)
E 3.36 (±0.08) 4.46 (±0.04) 3.11(±0.06) 0.86 (±0.03) 76.64(±0.41) 47.74(±0.20) 40.49 (±0.25) 28.9 (±0.21) 69.39 (±0.46) 7.25(±0.10)
3 4
23
1
Table 2. Energy efficiency and fermentation efficiency of Miscanthus × giganteus from
2
different fertilization treatments (mean±standard deviation, n = 3). Treatment
Heating value [MJ/kg]
A B C D E
17.17 (± 0.02) 17.41 (± 0.02) 17.44 (± 0.02) 17.45 (± 0.02) 17.52 (± 0.02)
Fermentation yield [L A100/kg] 0.105 (±0.05) 0.132 (±0.08) 0.128 (±0.02) 0.125 (±0.04) 0.134 (±0.02)
Biomass yield [t DM/ha]
Energy yield [GJ/ha]
17.34 (±0.47) 297.73 (±8.04) 18.16 (±0.48) 316.17 (±8.70) 18.85 (±0.56) 328.74 (±9.57) 18.54 (±1.77) 323.52 (±30.72) 19.02 (±1.31) 333.23 (±23.02)
Ethanol yield [L A100/ha] 1864 (±105) 2469 (±163) 2483 (±44) 2396 (±69) 2633 (±44)
3 4 5
24
3
2.36b
125
100
2.29b
2.40b 2.24b
1.87a
2.5
2
75
1.5
50
1
25
0.5
0
Alcohol content (% v/v)
Concentration of reducing sugars (g/L)
150
0 A hydrolyzate
B
C after fermentation
D
E alcohol content
1 2 3
Figure 1. The effect of different fertilization treatments on the fermentation of Miscanthus
4
× giganteus biomass hydrolyzates in the SHF system with S. cerevisiae strain 7. Values
5
are given as means (n = 3, ± standard deviation). Means followed by the same letter are not
6
significantly different at p < 0.05, using the Duncan multiple range test.
7 8
25
3
2.47a
100
2.31b
2.5
80
2
60
1.5
40
1
20
0.5
0
Alcohol content (% v/v)
Concentration of reducing sugars (g/L)
120
0 B hydrolyzate
E after fermentation
alcohol content
1 2
Figure 2. The effect of the absence of hydrolyzate supplementation (treatments B and E)
3
on the fermentation of Miscanthus × giganteus hydrolyzates in the SHF system with S.
4
cerevisiae strain 7. Values are given as means (n = 3, ±standard deviation). Means
5
followed by the same letter are not significantly different at p < 0.05, using the Duncan
6
multiple range test.
7 8
26
3
a Alcohol content (%v/v)
2.5
ab b
b
a' b' d' a' b'
c' d'
a' c' d'
2 1.5 1 0.5 0 B
E
SHF without supplemantation
SHF with supplementation
SSF without supplementation
SSF with supplementation
1 2
Figure 3. The alcohol content of fermented hydrolyzates of Miscanthus × giganteus from
3
treatments B (fertilized with sewage sludge at 1.75 DM/ha) and E (fertilized with NPK,
4
equivalent nitrogen content of 2.8 t DM/ha) containing S. cerevisiae 7 or AS4, with and
5
without nitrogen and phosphorus supplementation, in SHF and SSF systems. Values are
6
given as means (n = 3, ±standard deviation). Means followed by the same letter are not
7
significantly different at p < 0.05, using the Duncan multiple range test.
8 9 10
27
1 2
Highlights Fertilization influences biomass yield and content of lignocellulosic fractions
3
Bioethanol production was highest with hydrolysis and fermentation done separately
4
Sewage sludge or NPK fertilizer increased ethanol production 30 or 40%, respectively
5 6 7 8 9
28