Escolar Documentos
Profissional Documentos
Cultura Documentos
PII: S0960-8524(16)30437-0
DOI: http://dx.doi.org/10.1016/j.biortech.2016.03.134
Reference: BITE 16324
Please cite this article as: Corneli, E., Dragoni, F., Adessi, A., De Philippis, R., Bonari, E., Ragaglini, G., Energy
conversion of biomass crops and agroindustrial residues by combined biohydrogen/biomethane system and
anaerobic digestion, Bioresource Technology (2016), doi: http://dx.doi.org/10.1016/j.biortech.2016.03.134
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title
Authors
Affiliations
a
Institute of Life Sciences, Scuola Superiore Sant’Anna, Piazza Martiri della Libertà
Florence, Italy
d
CRIBE – Centro di Ricerche Interuniversitario Biomasse da Energia, Via Vecchia
Abstract
Aim of this study was to evaluate the suitability of ensiled giant reed, ensiled maize,
ensiled olive pomace, wheat bran for combined systems (CS: dark fermentation +
were also analyzed under a single stage AD batch test, in order to investigate the effects
of DF on estimated energy recovery (ER) in combined systems. In CS, maize and wheat
bran exhibited the highest hydrogen potential (13.8 and 18.9 NL kgVS-1) and wheat
bran the highest methane potential (243.5 NL kgVS-1). In one-stage AD, giant reed,
maize and wheat bran showed the highest methane production (239.5, 267.3 and 260.0
with hydrogen production (r=0.92). Wheat bran proved to be a promising residue for CS
Keywords: maize, giant reed, olive pomace, wheat bran, dark fermentation.
Abbreviations: eGR: ensiled biomass of Arundo donax L.; eZM ensiled biomass of Zea
mays L.; eOP: ensiled olive pomace; WB: wheat bran; CS: combined system; AD:
anaerobic digestion; DF: dark fermentation; CS_DF: first stage of combined system;
CS_AD second stage of combined system; ER: energy recovery; Rmax: maximum daily
production rate; T50: time, expressed in days, when 50% of methane production was
reached; TS: total solids; VS: volatile solids; WSC: Water Soluble Carbohydrates;
VFAs: short chain volatile fatty acids; BBP: Biochemical Biogas Potential; BMP:
1 Introduction
alternative to fossil fuels (Weiland, 2010). In AD, biomethane is the main metabolite
produced and it can be used as biogas for power and heat production. Otherwise
upgraded biomethane can be used as gaseous vehicle fuel or injected into natural gas
grids, replacing natural gas (Weiland, 2010; Adessi et al., 2012). AD fermentation can
be divided into two main stages (Weiland, 2010; Adessi et al., 2012). In the first stage,
the hydrolysis of complex organic substrates and their acidogenic fermentation occur,
producing hydrogen, carbon dioxide and organic end products, like short chain volatile
fatty acids (VFAs), lactic acid and ethanol (Weiland, 2010; Adessi et al., 2012; Ghimire
2
et al., 2015a). Then, in the second stage, acetogenesis and methanation of organic
products take place and main final products are methane (50-70%) and carbon dioxide
the spatial separation into a two-stage system, i.e. Combined System (CS), composed of
a first stage, named dark fermentation (DF), where hydrogen is obtained, and a second
one that is mostly methanogenic (Argun and Kargi, 2011; Adessi et al., 2012; Ghimire
et al., 2015a). Considering the utilization of CS biogas, it can provide benefit in the
form of increased combustion efficiency of the mixed biofuel, hydrogen and methane,
CO2 emissions, as compared to sole CH4 combustion (Hallenbeck et al., 2009; Cavinato
heterotrophic bacteria is enhanced and assessed and the hydrolysis of biomass can be
improved compared to one-stage AD (Hawkes et al., 2007; Giordano et al., 2011), thus
In CS_DF, the hydrogenase enzyme can reduce protons to hydrogen, neutralizing the
electrons coming from the organic compounds oxidation (Ghimire et al., 2015a). Since
the main fermentation end products of CS_DF are acetic acid and butyric acid, with
butyric acid in excess of acetic acid, the process is also called acetate/butyrate-type
fermentation and these acids allow the highest theoretical conversion of glucose to H2
comparing with the other acids. In particular, the conversion of 1 mol of glucose to
acetic acid involves the production of 4 mol of H2 and the conversion to butyric acid of
2 mol of H2 (Hawkes et al., 2007; Abo-Hashesh and Hallenbeck, 2012; Ghimire et al.,
2015a). Moreover, since the inoculum is a mixed non-sterile culture, like biogas plant
3
recommended in order to reduce the activity of H2 consumer microorganisms, like
syntrophic association with H2 producers (Argun and Kargi, 2011; Ghimire et al.,
2015a). The use of non-sterile mixed cultures is generally preferred over a pure culture
since it is easily available, low cost, not requiring aseptic environment and it allows
Temperature and pH are important factors influencing DF efficiency (Guo et al., 2010;
higher H2 yields, since they facilitate the hydrolysis of lignocellulosic component and
speed up the process. The correction of the initial pH values to 5-7 would increase the
At plant scale, a combined system composed by a first stage of dark fermentation and
the second stage of anaerobic digestion could be feasible based on conventional two-
stage biogas plants (Schievano et al., 2014). However, with the view of developing rural
economies, farmers and biogas plant owners would take advantage of using simple
features such as thermophilic temperature and pH correction, which can advance the
process, could imply additional costs. The use of local and low cost substrates, suitable
this technology, provided that these substrates are actually suitable for conversion into
hydrogen and methane. In particular, it has been reported that substrates for dark
4
low lignin contents, since generally, the higher is the biodegradability of the substrate,
Biomass crops and residues of the agroindustry are promising substrates for
fermentation systems (Ghimire et al., 2015a). Giant reed (Arundo donax L.), maize (Zea
mays L.), olive pomace and wheat bran were selected as vegetable biomasses for the
present work, since they are still poorly studied in biohydrogen/biomethane CS. To the
best knowledge of the authors, this is the first time that ensiled giant reed and ensiled
olive pomace are tested in a two-stage batch CS. Giant reed is an interesting perennial
rhizomatous grass, which represents a promising feedstock for bioenergy supply chains,
like AD (Corno et al., 2014; Ragaglini et al., 2014; Dragoni et al., 2015). It is a low
input crop with high biomass production and great adaptability to marginal land, thus
potentially reducing the competition with food production (Corno et al., 2014). On the
other hand, another opportunity consists in the use of agro-industrial residues that are
Olive pomace and wheat bran were selected being widespread residues in the
Mediterranean area. They come from olive oil and wheat milling industry, respectively.
Italy is the second biggest EU producer of olive oil and thus it is the second EU
producer of olive pomace, with an annual output of 2.5-3 million tons (Mantovi et al.,
2013). Olive pomace is a substrate rich in fiber components, lipids and proteins. In
order to use this substrate for biogas production, the concentration of lignin-rich parts,
mainly represented by pits, should be reduced and the pretreatment indicated is pitting
(Mantovi et al., 2013; Mateo and Maicas, 2015). Wheat bran is a matrix consisting of
structural carbohydrates, starch and proteins (Pan et al., 2008) and in Italy it has an
annual output of 1.1- 1.4 million tons (ISTAT, 2012), considering that wheat bran
accounts about 14-19% of the grains’ weight (Pan et al., 2008). In this study, maize was
5
considered as a widespread bioenergy crop, which is an annual food species widely
distributed, requiring relevant input and high fertile soils (Amon et al., 2007; Dragoni et
al., 2015). One of the most important issues representing a key challenge for the
bioenergy use of biomasses is the storage, and ensiling is a widespread technology for
storing wet feedstocks with total solid content ranging from 25 to 35% (Weiland, 2010;
The aim of the present work was to investigate the suitability of biomasses that are
relevant in the Mediterranean area (ensiled giant reed, ensiled maize, ensiled olive
pomace, wheat bran) in a batch dark fermentation system, in order to evaluate their
fermentation test was then followed by a batch second stage of anaerobic digestion,
production and the overall energy recovery of the combined system. The same
substrates were also analyzed under a single stage AD batch test. The two processes (CS
and one-stage AD) were then compared in terms of energy recovery from each different
biomass.
Four different substrates were chosen for assessing the production of H2 and CH4 in the
two-stage CS: ensiled biomass of A. donax (eGR), ensiled biomass of Z. mays (eZM),
ensiled olive pomace (eOP) and wheat bran (WB). The CS, composed by a first stage of
6
Three samples of aboveground biomass were harvested from giant reed and maize
grown in experimental fields located in San Piero a Grado, Pisa, Italy (43° 40’ 49.21’’
North, 10° 20’ 47.15’’ East; 1 m above mean sea level and 0% slope). Giant reed was
cut in July 2013, before crop senescence, since juvenile traits of the crop have been
2014). Maize was harvested in September 2013, at milky-wax ripeness. Entire plants of
giant reed and maize were chopped in an electric power shredder AL-KO H1600 (AL-
KO Maschinenfabrik GmbH, Obdach, Austria). Pitted olive pomace and wheat bran
were collected from a three-phase olive processing plant and from a wheat milling
industry, respectively. The biomass of giant reed, maize and olive pomace was
separately ensiled in triplicate at lab scale in plastic buckets (5 L), while wheat bran,
having a low moisture content, did not need to be ensiled for storage. The ensiled
material was treated with a commercial bacterial ensiling inoculant, Pioneer® 11CH4,
according to manufacturer guidelines (Pioneer Hi-Bred Italia Srl, Cremona, Italy). This
decreases the concentration of lactic acid and increases acetic acid (Driehuis et al.,
1999) in order to make the substrate more suitable for AD. Anaerobic conditions were
obtained by pressing the biomass into the buckets and using silicone to seal the
connection between the lid and the bucket. The dispersion of gas produced was
penetration of air into the silage. Buckets were stored at room temperature for 60 days.
2.2 Inocula
Inocula were collected from a two-stage mesophilic commercial biogas plant, fed with
energy crops, agroindustrial residues, cattle and poultry manure. The CS_DF inoculum
7
was collected from the first stage of the plant; it was sieved and heat-treated for 1 hour
producers and to inhibit microorganisms that consume H2 (Argun and Kargi, 2011). The
total solids (TS) content of CS_DF inoculum after pretreatment was 119.6 g kg-1 and the
volatile solids (VS) content was 91.1 g kg-1. Methanogenic inoculum used both for
CS_AD and for AD was collected from the second stage of the biogas plant. In this case
the inoculum was sieved and incubated at 37°C for five days long in order to deplete the
methanogenic inoculum had a TS content of 102.6 g kg-1 and a VS content of 75.8 g kg-
1
.
Batch assays were established in order to perform both the CS and the AD of the four
substrates (eGR, eZM, eOP, WB). The experimental biogas plant was composed by
connected to a PC, as described by Ragaglini et al. (2014). During the assays, three
In the CS_DF assay, each reactor was filled with biomass and 100 g of inoculum with a
substrate to inoculum ratio of 2:1 based on VS and ultrapure water was added up to a
volume of 650 mL. Thus, reactors were sealed and flushed with N2 in order to substitute
the air in the headspace, allowing to obtain anaerobic conditions and to reduce the
partial pressure of hydrogen (pH2) (Abo-Hashesh and Hallenbeck, 2012). CS_DF batch
assay was realized in triplicates for each biomass and was kept for four days, about 10
times shorter retention time compared to the CS_AD (Pakarinen et al., 2008; Schievano
8
et al., 2014). The assay was made without any pH control, allowing the pH to be
dependent on the process natural conditions. At the end, samples of CS_DF effluents
(200 g) were collected from each replicate for chemical analysis. Then, methanogenic
inoculum was added to each replicate up to the starting volume of 650 mL (Pakarinen et
al., 2008) and reactors were closed and flushed again with N2, to allow anaerobic
Conventional AD assay was carried out in triplicate for each biomass by adding in each
reactor the biomass and 200 g of inoculum with a substrate to inoculum ratio of 2:1
(VS/VS) and ultrapure water was added up to a volume of 650 mL. Thus, reactors were
sealed and flushed with N2 as in the combined system assay. The entire process had a
The Biochemical Biogas Potential (BBP) was calculated according to the Ideal Gas Law
and to the Molar Volume of Ideal Gases at Standard Temperature and Pressure
conditions (1 bar, 273.15 K). The composition of biogas was measured at discrete
Shangai, China): once at the end of CS_DF (the fourth day); four times for both the
CS_AD and the AD, more frequently at the beginning of the fermentation to better
assess the kinetics (at time intervals of 8, 15, 29 and 40 days after the beginning of the
assay). Both the pressure reduction due to biogas removal at each sampling time and the
biogas composition of the sampled gas were considered for estimating the cumulative
Then, the Biochemical Methane Potential (BMP) and the Biochemical Hydrogen
Potential (BHP) of the substrates, as well as the Methane Concentration (MC) and the
Hydrogen Concentration (HC) of the biogas, were determined. BBP, BMP, BHP of
9
inocula were subtracted from the biogas production of the biomasses tested, as reported
by Ragaglini et al. (2014). Moreover, the Energy Recovery (ER) of biomasses was
and hydrogen (12.74 MJ Nm-3), with the view to compare the two-stage combined
where: A represents the upper limit; M the initial relative growth rate; D the relative
growth rate at inflection; r the rapid initial phase; s the slow final phase.
Goodness of fit was assessed for each substrate, by means of R2adj and Root Mean
Square Error (RMSE). Then, the function was used to calculate the maximum daily
production rate (Rmax) through the analysis of model derivative, and the time, expressed
in days, when 50% of methane production was reached (T50). Curve fitting and model
parameterization were performed using the R software, version 3.0.1, and the nmle
package. Details of the model parameterization and the goodness of fit are reported in
Appendix.
Samples for chemical analyses were prepared by milling the biomass of different
substrates in a Retsch SM1 rotor mill (Retsch, Düsseldorf, Germany) equipped with a 1
(Ragaglini et al., 2014 and references therein). TS were estimated by drying the material
at 105°C to constant weight of the sample and VS were estimated by determining the
ash content of dry samples in a muffle furnace at 550°C. TS and VS values of silages
10
were corrected according to volatilization coefficients of lactic acid, total VFAs and
total alcohols (0.375, 0.892, 0.975, respectively) as reported by Porter and Murray
(2001). Elemental analysis was carried out with the LECO CHN Elemental TruSpec
total nitrogen and C/N ratio. Neutral Detergent Fiber (NDF), Acid Detergent Fiber
(ADF) and Acid Detergent Lignin (ADL) were determined with Van Soest method
using the FiberCap ™ 2021/2023 system (FOSS Analytical AB, Höganäs, Sweden),
Further analyses, specific for the three ensiled substrates, were made. pH value was
Concentration of ethanol, lactic acid and VFAs (acetic acid, propionic acid, butyric acid,
isovaleric acid and valeric acid) was determined according to Bianchi et al. (2010).
The pH value of CS_DF effluents and digestates was obtained with a CyberScan pH
110 pH meter (Eutech Instruments, Singapore). The concentration of ethanol, lactic acid
and VFAs was determined with HPLC chromatograph as described in Bianchi et al.
(2010) and the ammonium concentration was determined with Nessler method (Bianchi
et al., 2010). The molar ratio between butyrate and acetate (B/A) present in each
H2 production by anaerobic microflora during CS_DF (Kim et al., 2006; Hawkes et al.,
11
2.7 Statistical analysis
Biogas parameters (BBP, BMP, BHP, MC and HC) of different substrates were
analyzed one by one with one-way analyses of variance (ANOVA), considering the
substrate as independent variable. For each substrate, the ER of CS and AD and the
kinetic parameters (Rmax and T50) of CS_AD and AD were assessed one by one with
two-way ANOVA, considering the fermentation system and the substrate as factors.
The difference between each pair of means were determined by honest significant
difference test (Tukey’s test). Pearson correlation coefficient (r) was determined in
order to assess the relationship between two parameters, B/A and BHP in CS_DF.
digestion
Under the CS_DF process, WB showed the highest BBP (88.4 ± 20.6 NL kgVS-1),
comparable with eGR and eZM (62.5 ± 4.3 and 63.4 NL ± 1.8 kgVS-1, respectively) and
significantly higher than eOP (41.6 ± 1.1 NL kgVS-1) (P < 0.01). In general, eZM, WB
and eGR reached a plateau in biogas production, while eOP showed an early stage of
cumulative biogas production, not reaching the plateau in 4 days of CS_DF (Fig. 1).
Significant differences were observed for hydrogen production, in fact eZM and WB
showed higher values of BHP (13.8 and 18.9 NL kgVS-1 respectively), than eGR and
eOP (< 0.1 NL kgVS-1). HC in eZM and WB, 21.7 and 21.1% respectively, was
significantly higher than in eGR and eOP, where it was less than 0.2 %. On the
contrary, eGR and eOP showed the highest BMP, 19.8 and 20.7 NL kgVS-1
12
significantly higher than those of eZM and WB (3.0 and 2.3 %, respectively), that
Concerning the CS_AD, WB produced the highest value of BBP (387.7 NL kgVS-1) and
eOP produced the lowest value (204.1 NL kgVS-1), while eGR and eZM were
comparable, with BBP equal to 347.4 and 357.7 NL kgVS-1, respectively. WB showed
the highest BMP value (243.5 NL kgVS-1), while eZM, eGR and eOP showed lower
results (216.7, 195.7 and 136.6 NL kgVS-1 respectively). eOP had the highest MC (67.3
%), while eZM and WB had similar values, equal to 60.7 and 63.3 % respectively, and
In relation to the CS_DF process, the two better performing substrates in hydrogen
production, eZM and WB, had the lowest methane potential and they reached the
plateau for biogas cumulative production, showing that 4 days of dark fermentation
were enough to reach the maximum hydrogen potential of these biomasses in the
present experimental conditions. Regarding wheat bran, BHP of the present study was
in line with the results obtained by Noike and Mizuno (2000) in a DF batch assay, at
35°C in shaking conditions and using fermented soybean meal as inoculum (10-43 NL
kgVS-1). On the other hand, Pan et al. (2008) reported a maximum BHP value of 128.2
NL kgVS-1, using a pretreated wheat bran in mesophilic mixed batch test with activated
sludge of paper mill, as inoculum, and with initial correction of pH value at 5.0. Also
Giordano et al. (2011) assessed wheat bran performance in a lab scale CS, reporting
higher BHP for both common wheat and durum wheat (53 and 90 NL kgVS-1
mesophilic batch test with the initial correction of pH at 7. Schievano et al. (2014)
carried out the CS_DF of maize silage and olive pomace in semicontinuously operated
13
assessing nine different combinations of organic matter concentration and hydraulic
retention time: regarding maize silage, their BHP ranged from 12.5 to 120.9 NL kgVS-1,
hydrogen potential ranging from 0.1 to 13.0 NL kgVS-1. Another study, analyzing the
plant as inoculum and a retention time of 6 days, reported a higher BHP value, equal to
54.9 NL kgVS-1 (Ghimire et al., 2015b). Furthermore, concerning the retention time of
olive pomace in batch tests, Borja et al. (2005) analyzed the anaerobic acidogenic
fermentation of olive pomace at lab scale in mesophilic mixed batch, highlighting that a
retention time of 12 days is the optimum in the view of obtaining the maximum
production of acetate and butyrate. Thus, it could be hypothesized that a retention time
longer than 4 days would be necessary for olive pomace to achieve a higher hydrogen
production. Concerning eGR, Guo et al. (2014) reported the BHP of giant reed leaves
and stems separately, giving values of 25.1 and 34.2 NL kgTS-1 respectively, assessed
digester treating vinasses. However, the study of Guo et al. (2014) differs from the
present one, since the giant reed biomass was not ensiled and the harvest time was not
indicated.
After four days of DF, no CS_DF effluent had pH values lower than 5, which is the
minimum threshold that most heterotrophic bacteria can tolerate (Abo-Hashesh and
Hallenbeck, 2012). In general, it has been reported that substrates rich in carbohydrates
have higher hydrogen production, because they imply acidification of the medium,
sustaining optimal conditions for the hydrolysis of biomasses and for the inhibition of
hydrogen consumers (Ghimire et al., 2015b). This is in accordance with this study,
where the two substrates showing higher BHP, eZM and WB, had a Non-Structural
14
Carbohydrates concentration (NSCs = starch + WSCs) equal to 12.4 % and 8.6 %,
resulting pH values of the final CS_DF effluents equal to 5.12 and 5.66, respectively.
On the other hand, NSCs in eGR was 1.7 % while in eOP starch was not detected and
WSCs was 0.1 % (Table 1). eOP did not produce hydrogen, while it showed a
continuously increasing biogas production during the 4 days of the trial and a final pH
value of 7.62, above the inhibitory threshold of methanogenic archaea (Weiland, 2010).
Therefore, chemical characteristics of eOP, under the tested conditions, did not allow
occur, while the trend of biogas production and the final pH, equal to 5.35, suggested
that methanogenesis developed until it was inhibited by acidic conditions. Probably the
low performance of giant reed in CS_DF can also be related both to the early harvest
and to the ensiling process. In fact, the crop at early stages generally has lower NSCs
content than the mature crop (Ragaglini et al., 2014) and NSCs are metabolized to
VFAs during ensiling (Liu et al., 2015). Acetate concentration (3.37 g L-1) in CS_DF
effluents of eGR was higher than in the other substrates, even if this substrate had low
BHP. In fact, high concentrations of acetate do not imply a high H2 production if the
According to the present results, the higher is the butyrate to acetate ratio (B/A), the
higher is the BHP. This observation is in line with the results reported by Ghimire et al.
(2015b). In fact, eZM and WB showed the highest B/A, 3.4 and 3.5 respectively, while
eGR and eOP were less than 0.4 (Table 3). Kim et al. (2006) found that a 2.6 is a B/A
threshold value beyond which the production of H2 is enhanced. Actually, B/A and BHP
were significantly and positively correlated, with a Pearson’s r value equal to 0.92 (P <
0.001). Lactic acid and ethanol were not detected in any of CS_DF effluents.
15
Ammonium concentrations of CS_DF effluents varied from a minimum of 0.35 g L-1 in
value for the methanogenic activity in the following CS_AD stage (Chen et al., 2008).
By the literature, Schievano et al. (2014) reported BMP of maize silage in a CS_AD
stage ranging from 304 to 504 NL kgVS-1, which is higher than BMP of this study
(216.7 NL kgVS-1), while they did not obtain methane from olive pomace. Regarding
wheat bran, Giordano et al. (2011) reported BMP of CS_AD ranging from 230 to 289
NL kgVS-1, that is comparable to the value obtained in this study (243.5 NL kgVS-1).
To the best knowledge of the authors, the investigation in a combined system of ensiled
olive pomace and ensiled giant reed have not yet been assessed. At the end of CS_AD
batch tests, the total VFAs concentration in digestates (acetate, propionate and butyrate)
was minimum in eZM (0.08 g L-1) and higher in the other substrates, varying from 1.05
g L-1 in eGR to 1.42 g L-1 in eOP. Ammonium analyses showed concentrations from a
minimum of 1.08 g L-1 in eGR to a maximum of 1.32 g L-1 in WB, that were consistent
with not inhibitory values for methanogenic activity (Schievano et al., 2012; Corno et
al., 2015). pH was similar for all digestates, comprised in a range from 8.27 to 8.44, that
2010).
Biogas parameters of one-stage AD were analyzed. eGR, eZM and WB showed BBP
values (341.5, 394.4 and 377.4 NL kgVS-1, respectively) significantly higher than eOP
(206.7 NL kgVS-1). Similar results were obtained for BMP, since eGR, eZM and WB
showed significantly higher values (239.5, 267.3 and 260.0 NL kgVS-1, respectively)
than eOP (172.0 NL kgVS-1). eOP had MC (83.0 %) higher than the other substrates,
i.e. eGR, eZM and WB (70.2, 67.7 and 68.9 %, respectively) (Fig. 4). Compared with
16
the literature, MC values were quite high for all substrates. In eZM and WB, this could
be related to a relatively low content of starch (Table 1) (Amon et al., 2007; Pan et al.,
2008), while in eOP it could be related to the lipid content, typically higher than in the
other substrates (Weiland, 2010; Mateo and Maicas, 2015). By the literature, few
studies analyzed the BMP of giant reed harvested in summer. Ragaglini et al. (2014)
reported a BMP value of fresh giant reed harvested in July of 325.2 NL kgVS-1, while
Dragoni et al. (2015) reported a similar value (323.0 NL kgVS-1) for ensiled giant reed
cut at the same time. Both these values are higher than what observed in the present
study. However, the methane potential of giant reed of this study is higher than those
reported by Liu et al. (2015) and Corno et al. (2015), 160 and 185 NL kgVS-1
respectively. The latter result was obtained in a continually stirred tank reactor (CSTR)
fed with ensiled giant reed, while Liu et al. (2015) analyzed the BMP of ensiled giant
reed in a mesophilic batch test. In both cases the harvest time of the crop was not
indicated. Methane potential of maize silage of the present study is consistent with BMP
reported in the literature by Corno et al. (2015) (261 NL kgVS-1). Higher BMP values
are reported in the literature. Amon et al. (2007) obtained BMPs ranging from 312 to
365 NL kgVS-1, in a mesophilic batch test using four different ensiled maize varieties
harvested at milk ripeness, and Schievano et al. (2014) obtained values ranging from
pomace resulted unsuitable for AD. Concerning olive pomace, Fantozzi and Buratti
(2009) reported a BMP of 110 NL kgVS-1 using a mixed mesophilic batch test, which is
a lower value than the methane potential of the present study. Another study (Alagöz et
al., 2015) stated a BMP value of shredded olive pomace in mesophilic fed batch reactor,
equal to 180 NL kgVS-1, which is similar to the value obtained in this study. Finally,
Kolbl et al. (2014), comparing the BMP of wheat bran and maize silage under
17
mesophilic batch conditions, reported results consistent with those of the present study
butyrate, varied from 0.3 g L-1 in WB to 0.21 g L-1 in eOP. Ammonium analyses
L-1 in WB. pH was similar for all of digestates, comprised in a range from 8.32 to 8.50,
higher than the minimum threshold of 7.0, that inhibits the methanogenic activity
Energy recovery (ER, MJ kgVS-1) was calculated in order to compare the overall
bioenergy potential of the substrates (eGR, eZM, eOP, WB) in the two fermentation
systems (CS, AD). The interaction of the two factors (substrates and fermentation
system) was not significant. The effect of the fermentation system was significant, with
substrates was also significant: the highest ER values were obtained from WB (9.0 MJ
kgSV-1) and eZM (8.6 MJ kgSV-1). The latter was similar to eGR (8.0 MJ kgSV-1),
while the ER achieved from eOP was significantly the lowest (5.8 MJ kgSV-1) (Fig. 5).
than the ER reported in this study for eZM in CS (7.9 MJ kgSV-1) and in AD (9.4 MJ
Nonetheless, in the present study the CS resulted to be less efficient than AD in terms of
ER. Probably, in a batch assay, the hydrogen potential can be assessed, while the
18
provides a constant feeding composition for methanogenic archaea and an overall
greater stability (Hawkes et al., 2007; Schievano et al., 2012, 2014). In general, even if
the ER in CS was lower than the ER in AD, the final biogas composition of CS was
more suitable for combustion than the one of AD, since the mixture of methane with
hydrogen, i.e. hythane, allows better engine performances and a lower release of
nitrogen oxide and CO2 into the atmosphere (Hallenbeck et al., 2009; Cavinato et al.,
2012). Considering the best performing substrates, i.e. WB and eZM, the obtained
biohythane would potentially have about 7 % of biohydrogen, complying with the best
(Cavinato et al., 2012). Evaluating the amount of CO2 released per each MJ produced
by biogas combustion, WB and eZM could reduce the CO2 production of -2.5 %
Considering the kinetics of CS_AD and of AD, the interaction of the two factors
(substrates and fermentation system) was significant for both T50 (P < 0.05) and Rmax (P
< 0.001) (Appendix). In CS_AD, the 50% of the methane production was reached in 2.9
days in eOP, which was not significantly different from the T50 value of WB (3.7 days),
that did not differ from eZM and eGR (4.4 and 4.6 days, respectively). In AD, all the
T50 values were similar one to the other, ranging from 4.7 days in WB to 5.5 days in
eGR, and were similar to eGR in CS_AD (Fig. 6C and 6D). Concerning the maximum
daily production rate, Rmax values of eZM, eOP and WB in CS_AD (32.4, 32.2 and 34.9
NL kgVS-1 day-1, respectively) were significantly higher than those of eGR in CS_AD
(24.9 NL kgVS-1 day-1) and of all substrates in AD: eGR, eZM, eOP and WB (26.8,
25.0, 22.9 and 23.5 NL kgVS-1 day-1, respectively) (Fig. 6A and 6B). Compared to AD,
the maximum rates in CS_AD were increased of 22.8, 28.9 and 32.8 % in eZM, eOP
and WB, respectively. On the contrary, in eGR a decrease in Rmax (-7.6 %) was
19
observed in CS_AD compared with AD. Kinetics of fermentations are related to
(Weiland, 2010; Ragaglini et al., 2014). In eZM and in WB the increase of Rmax in the
CS_AD compared with the AD system can be attributed to the high concentration of
VFAs which were obtained in DF (Table 3). The high Rmax and the low T50 of eOP in
CS_DA could be attributed to the highly biodegradable components remaining from the
CH4 production, that was still far from reaching the plateau. Conversely in eGR, Rmax of
CS_AD was significantly lower than in the other substrates, likely due to the fact that
readily fermentiscible components were more depleted during the CS_DF stage.
eGR and eOP resulted to be not so suitable for a combined fermentation system under
components like hemicellulose, cellulose and lignin, which are slowly degraded (Corno
et al., 2014). A possible enhancement of the process for lignocellulose matrix could be
the process and the production of inhibitory compounds, like furfural, can negatively
In general, it is worth stressing that in combined systems, DF has low hydrogen yields
owing to the inability of bacteria to overcome the thermodynamic barrier for a complete
supply is mandatory since they require light and electricity, respectively (Adessi et al.,
4 Conclusions
20
The obtained results showed that the substrate significantly influenced H2 and CH4
potentials of the fermentation systems. Indeed, the higher content of non- structural
carbohydrates made WB and eZM more suitable than eGR and eOP for biohythane
production in a two-stage process (i.e. sequential dark fermentation and second stage of
Conversely, eOP and eGR resulted unsuitable for a combined system performed under
5 Acknowledgements
The authors wish to thank Sergio Cattani, Fabio Taccini and Cristiano Tozzini for their
6 References
Adessi, A., De Philippis, R., & Hallenbeck, P. C. (2012). Combined systems for
digestion efficiency of wastewater sludge and olive waste: Synergistic effect of co-
188.
Amon, T., Amon, B., Kryvoruchko, V., Zollitsch, W., Mayer, K., & Gruber, L. (2007).
Biogas production from maize and dairy cattle manure influence of biomass
173-182.
21
Argun, H., & Kargi, F. (2011). Bio-hydrogen production by different operational modes
Bianchi, L., Mannelli, F., Viti, C., Adessi, A., & De Philippis, R. (2010). Hydrogen-
producing purple non-sulfur bacteria isolated from the trophic lake Averno (Naples,
Borja, R., Sánchez, E., Rincón, B., Raposo, F., Martın, M. A., & Martın, A. (2005).
Cavinato, C., Giuliano, A., Bolzonella, D., Pavan, P., & Cecchi, F. (2012). Bio-hythane
production from food waste by dark fermentation coupled with anaerobic digestion
37(15), 11549-11555.
Chen, Y., Cheng, J. J., & Creamer, K. S. (2008). Inhibition of anaerobic digestion
Corno, L., Pilu, R., & Adani, F. (2014). Arundo donax L.: A non-food crop for
Corno, L., Pilu, R., Tambone, F., Scaglia, B., & Adani, F. (2015). New energy crop
giant cane (Arundo donax L.) can substitute traditional energy crops increasing biogas
Dragoni, F., Ragaglini, G., Corneli, E., o Di Nasso, N. N., Tozzini, C., Cattani, S., &
Bonari, E. (2015). Giant reed (Arundo donax L.) for biogas production: land use saving
and nitrogen utilisation efficiency compared with arable crops. Italian Journal of
22
Driehuis, F., Elferink, S. J. W. H., & Spoelstra, S. F. (1999). Anaerobic lactic acid
degradation during ensilage of whole crop maize inoculated with Lactobacillus buchneri
inhibits yeast growth and improves aerobic stability. Journal of Applied Microbiology,
87(4), 583-594.
Fantozzi, F., & Buratti, C. (2009). Biogas production from different substrates in an
Ghimire, A., Frunzo, L., Pirozzi, F., Trably, E., Escudie, R., Lens, P. N., & Esposito, G.
Ghimire, A., Frunzo, L., Pontoni, L., d'Antonio, G., Lens, P. N., Esposito, G., & Pirozzi,
152, 43-48.
Giordano, A., Cantù, C., & Spagni, A. (2011). Monitoring the biochemical hydrogen
Guo, X. M., Trably, E., Latrille, E., Carrère, H., & Steyer, J. P. (2010). Hydrogen
Guo, X. M., Trably, E., Latrille, E., Carrere, H., & Steyer, J. P. (2014). Predictive and
explicative models of fermentative hydrogen production from solid organic waste: role
7476-7485.
23
Hallenbeck, P. C., Ghosh, D., Skonieczny, M. T., & Yargeau, V. (2009).
Hawkes, F. R., Hussy, I., Kyazze, G., Dinsdale, R., & Hawkes, D. L. (2007).
ISTAT (2012)
http://agri.istat.it/sag_is_pdwout/jsp/dawinci.jsp?q=plC010000010000012000&an=201
Kim, S. H., Han, S. K., & Shin, H. S. (2006). Effect of substrate concentration on
Kolbl, S., Paloczi, A., Panjan, J., & Stres, B. (2014). Addressing case specific biogas
plant tasks: Industry oriented methane yields derived from 5L Automatic Methane
substrate particle sizes and organic loading. Bioresource technology, 153, 180-188.
Liu, S., Ge, X., Liew, L. N., Liu, Z., & Li, Y. (2015). Effect of urea addition on giant
Mantovi, P., Fabbri, C., & Soldano, M., (2013). Si ottimizza la filiera del biogas se la
Mateo, J. J., & Maicas, S. (2015). Valorization of winery and oil mill wastes by
Noike, T., & Mizuno, O. (2000). Hydrogen fermentation of organic municipal wastes.
24
Pakarinen, O., Lehtomäki, A., & Rintala, J. (2008). Batch dark fermentative hydrogen
production from grass silage: the effect of inoculum, pH, temperature and VS ratio.
Pan, C., Fan, Y., & Hou, H. (2008). Fermentative production of hydrogen from wheat
47(16), 5812-5818.
Porter, M. G., & Murray, R. S. (2001). The volatility of components of grass silage on
oven drying and the inter‐relationship between dry‐matter content estimated by different
Ragaglini, G., Dragoni, F., Simone, M., & Bonari, E. (2014). Suitability of giant reed
(Arundo donax L.) for anaerobic digestion: effect of harvest time and frequency on the
Schievano, A., Tenca, A., Scaglia, B., Merlino, G., Rizzi, A., Daffonchio, D., Oberti,
Schievano, A., Tenca, A., Lonati, S., Manzini, E., & Adani, F. (2014). Can two-stage
instead of one-stage anaerobic digestion really increase energy recovery from biomass?.
25
Figure captions
(BBP), of ensiled giant reed (eGR), ensiled Zea mays (eZM), ensiled olive pomace
(eOP) and wheat bran (WB) in the first stage of dark fermentation (CS_DF).
Methane Concentration (MC) and Hydrogen Concentration (HC) of the first stage of
dark fermentation (CS_DF) of ensiled giant reed (eGR), ensiled Zea mays (eZM),
ensiled olive pomace (eOP) and wheat bran (WB). Significance levels of ANOVA are
Fig.3. Biochemical Biogas Potential (BBP), Biochemical Methane Potential (BMP) and
ensiled giant reed (eGR), ensiled Zea mays (eZM), ensiled olive pomace (eOP) and
wheat bran (WB). Significance levels of ANOVA are shown: * (p < 0.05), ** (p <
Fig.4. Biochemical Biogas Potential (BBP), Biochemical Methane Potential (BMP) and
Methane Concentration (MC) of the anaerobic digestion (AD) of ensiled giant reed
(eGR), ensiled Zea mays (eZM), ensiled olive pomace (eOP) and wheat bran (WB).
Significance levels of ANOVA are shown: * (p < 0.05), ** (p < 0.01), *** (p < 0.001).
Fig.5. (A) Energy recovery (ER) from ensiled giant reed (eGR), ensiled Zea mays
(eZM), ensiled olive pomace (eOP) and wheat bran (WB) considering the mean
between the combined system (CS) and the anaerobic digestion (AD). (B) Energy
recovery from the combined system (CS) and the anaerobic digestion (AD) considering
the mean of the considered substrates. Significance levels of ANOVA are shown: * (p
26
Fig.6. Kinetics of fermentation of ensiled giant reed (eGR), ensiled Zea mays (eZM),
ensiled olive pomace (eOP) and wheat bran (WB). Rmax and daily methane production
rate estimated as the first derivative of cumulate production curves of CS_AD (A) and
of AD (B); cumulative methane production along 40 days and T50 of CS_AD (C) and of
AD (D).
Table captions
Table 1 Total solids (TS), volatile solids (VS), total nitrogen (N), carbon to nitrogen
ratio (C/N), Neutral Detergent Fiber (NDF), Acid Detergent Fiber (ADF), Acid
Detergent Lignin (ADL), starch and Water Soluble Carbohydrates (WSCs) of ensiled
giant reed (eGR), ensiled Zea mays (eZM), ensiled olive pomace (eOP) and wheat bran
(WB).
Table 2 pH, ethanol, lactic acid and volatile fatty acids (acetic acid, propionic acid,
butyric acid, isovalerian acid and valerian acid) of ensiled substrates (ensiled giant reed
Table 3 pH, ammonium, acetic acid, propionic acid, butyric acid, volatile fatty acids
(VFAs, as the sum of acetic acid, propionic acid and butyric acid) and butyrate to
acetate molar ratio (B/A) of dark fermentation (CS_DF) effluents of ensiled giant reed
(eGR), ensiled Zea mays (eZM), ensiled olive pomace (eOP) and wheat bran (WB).
Table 4 pH, ammonium, acetic acid, propionic acid, butyric acid and volatile fatty acids
(VFAs, as the sum of acetic acid, propionic acid and butyric acid) of the second stage of
anaerobic digestion (CS_AD) digestates of ensiled giant reed (eGR), ensiled Zea mays
Table 5 pH, ammonium, acetic acid, propionic acid, butyric acid and volatile fatty acids
(VFAs, as the sum of acetic acid, propionic acid and butyric acid) of anaerobic digestion
27
(AD) digestates of ensiled giant reed (eGR), ensiled Zea mays (eZM), ensiled olive
28
100
90
eGR
80 WB
70 eZM
eOP
60
BBP (NL kgVS-1)
50
40
30
20
10
0
1 10 20 30 40 50 60 70 80 90
Time (h)
29
30
31
32
33
Table 1 Total solids (TS), volatile solids (VS), total nitrogen (N), carbon to nitrogen
ratio (C/N), Neutral Detergent Fiber (NDF), Acid Detergent Fiber (ADF), Acid
Detergent Lignin (ADL), starch and Water Soluble Carbohydrates (WSCs) of ensiled
giant reed (eGR), ensiled Zea mays (eZM), ensiled olive pomace (eOP) and wheat bran
(WB).
butyric acid, isovalerian acid and valerian acid) of ensiled substrates (ensiled giant reed
2
Table 3 pH, ammonium, acetic acid, propionic acid, butyric acid, volatile fatty acids
(VFAs, as the sum of acetic acid, propionic acid and butyric acid) and butyrate to
acetate molar ratio (B/A) of dark fermentation (CS_DF) effluents of ensiled giant reed
(eGR), ensiled Zea mays (eZM), ensiled olive pomace (eOP) and wheat bran (WB).
3
Table 4 pH, ammonium, acetic acid, propionic acid, butyric acid and volatile fatty acids
(VFAs, as the sum of acetic acid, propionic acid and butyric acid) of the second stage of
anaerobic digestion (CS_AD) digestates of ensiled giant reed (eGR), ensiled Zea mays
4
Table 5 pH, ammonium, acetic acid, propionic acid, butyric acid and volatile fatty acids
(VFAs, as the sum of acetic acid, propionic acid and butyric acid) of anaerobic digestion
(AD) digestates of ensiled giant reed (eGR), ensiled Zea mays (eZM), ensiled olive
5
Highlights
system
• Wheat bran showed the highest H2/CH4 potential and energy recovery
• Olive pomace and giant reed were unsuitable for combined H2/CH4 production
39