organic matter characterization during the anaerobic digestion of different biomasses by means of...
TRANSCRIPT
ww.sciencedirect.com
b i om a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 1 1 1e1 2 0
Available online at w
http: / /www.elsevier .com/locate/biombioe
Organic matter characterization during the anaerobicdigestion of different biomasses by means of CPMAS 13C NMRspectroscopy
Fulvia Tambone a, Fabrizio Adani a, Giovanni Gigliotti b, Daniela Volpe b, Claudio Fabbri c,Maria Rosaria Provenzano d,*aDipartimento di Scienze Agrarie e Ambientali, Via Caloria 2, 20133. Universita di Milano, Milano, ItalybDipartimento di Scienze Agrarie e Ambientali, Borgo XX Giugno 74, University of Perugia, 06121 Perugia, ItalycCentro Ricerche Produzioni Animali, C.R.P.A. S.p.A., Corso Garibaldi 42, Reggio Emilia, ItalydDipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Via Amendola 165/A, University of Bari, 70123 Bari, Italy
a r t i c l e i n f o
Article history:
Received 3 March 2011
Received in revised form
9 October 2012
Accepted 15 November 2012
Available online 23 December 2012
Keywords:
Anaerobic digestion
Fresh biomass
Digestates13CPMAS-NMR spectroscopy
* Corresponding author. Tel.: þ39 080 544292E-mail address: mariarosaria.provenzano
0961-9534/$ e see front matter ª 2012 Elsevhttp://dx.doi.org/10.1016/j.biombioe.2012.11.
a b s t r a c t
The aim of this work was to characterize ingestates and their corresponding digestates
obtained in two full-scale biogas production plants processing a) mixtures of organic
wastes in co-digestion, and b) pig slurry in order to assess the organic matter trans-
formation during anaerobic digestion by means of chemical analysis and 13CPMAS-NMR
spectroscopy. Results proved that digestates obtained by different organic substrates
exhibited significant chemical differences related to the different initial composition of
substrates. We proposed the use of the aliphaticity index in order to highlight the different
chemical nature of ingestates and their corresponding digestates. In order to verify
whether the AD process leads to stabilized final products regardless the initial composition
of biomass in view of a possible agronomical use of digestate, a comparison of CPMAS 13C
NMR data of a number of ingestates and digestates available in literature was carried out.
Results indicated that most of the aromatic structures present in the substrate tend to
degrade during the process and that anaerobic digestion proceeds through preferential
degradation of carbohydrates such as cellulose and hemicellulose and, as a consequence,
concentration of more chemically recalcitrant aliphatic molecules occurs.
ª 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Anaerobic digestion has been known for centuries.
However, interest in the economical recovery of fuel
methane gas from different types of organic wastes on
industrial scale has recently enormously increased. This is
mostly owed to the changing socio economical situation in
the world characterized by depletion of fossil fuels and
urgent need to move to alternative energy supplies with
9; fax: þ39 080 [email protected] (M.R. Provenzaier Ltd. All rights reserved006
emphasis on renewable sources. Anaerobic digestion tech-
nology has evolved quickly and, at present, can be
competitive with aerobic treatments, especially for treating
industrial wastewater and organic solid wastes at high
organic loading [1]. As a consequence, the number of
anaerobic digestion plants in Europe has remarkably
increased [2].
Biological degradation of organic matter (OM) under
anaerobic conditions originates different products, the most
no)..
b i om a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 1 1 1e1 2 0112
abundant of which are methane, used to produce energy
and heat, and carbon dioxide. The anaerobic degradation
occurs in three steps: hydrolytic, acidogenic and methano-
genic phases. During the first step organic polymers such as
proteins, lipids and carbohydrates undergo hydrolysis and
acidification producing amino acids, fatty acids and mono-
saccharides. In the acidogenic phase volatile fatty acids, CO2
and H2, which are substrates for methanogenic microor-
ganisms, are produced. Finally, during the methanogenic
phase, methane is originated, thus closing the anaerobic
trophic chain [3e5]. Co-digestion strategies, referring to the
combined treatment of several wastes with complementary
characteristics, represent one of the main advantages of the
anaerobic digestion and are widely applied in order to
enhance the methane production [1]. A great variety of
different substrates are easily available in huge quantities.
In this work we considered the following: a) Energy crop.
The concept of energy crop has been around for many years
and nowadays they represent a renewable biomass whose
cost is expected to decline as technology improves; b) Olive
oil production residues. For olive oil producing countries on
the Mediterranean area such as Italy, treatment and
disposal of olive mill effluent and residues represent one of
the major environmental problem also because a large
volume of this effluent is produced in a short period in
wintertime. In Italy, Law No. 574/96 fixes an application of
50e80 m3 olive mill wastewater per year to agricultural soil
located far from built-up areas and in areas where the
aquifers are deeper that 10 m. Difficulties regarding the
treatment of olive mill wastewaters are associated to the
presence of polyphenols and long fatty acid compounds
which are toxic for plant growth [6,7] and may inhibit bio-
logical treatment [8]. In addition, these effluents present
a high organic content due to fats and carbohydrates; c)
Animal wastes. These substrates represent a good source of
biomass since they contain an abundance of organic matter
and nutrients. Using animal wastes as biomass offers many
advantages for livestock operations by minimizing waste
disposal costs and also reducing odors and contaminants
[9]; d) agro-industrial residues.
During the anaerobic digestion, a 60e80% organic matter
reduction occurs and the residual organic matrix called
digestate is characterized by high biological stability and
high contents of recalcitrant organic molecules and nutri-
ents such as nitrogen and phosphorus [10] and can be used
as nutrient fertilizer and/or organic amendment [11].
However, prior to selecting land application of the digestate,
the degree of stabilization of the treated waste should be
evaluated. During the stabilization process, organic matter
undergoes mineralization and conversion into humus-
related or humic substances, and as a result the energy
available for the metabolism of microorganisms is reduced
[12]. Analyzing anaerobic process stability of the end
product and assessment of organic matter of waste during
the process are necessary for obtaining the best information
about the stabilization process carried out [13]. In general,
amendment properties of biomass are related to the ability
of the contained organic matter to contribute in maintaining
the soil organic matter balance [14]. This ability depends by
the degradability of the organic matter [15]. An indirect
measurement of the degradability of a biomass is the
detection of the degree of the biological stability [16]. Bio-
logical stability is defined as the decomposition degree of
easily degradable OM in the substrate. High biological
stability is related to a minor environmental impact (e.g.
phytotoxicity for plant and odors emission).
Solid-state 13C cross-polarization magic angle spinning
nuclear magnetic resonance spectroscopy (13C CPMAS-NMR)
represents one of the most powerful tools for examining the
carbon composition of organic matter [17] providing the
distribution of organic carbons in a wide range of solid
matrices [18].
This technique has been recently proposed for moni-
toring the composting process of different mixtures of
organic substrates by comparing the carbon distributions of
fresh materials to those of final products [19]. Results
confirmed previous data available in literature [20e22]
showing that during the process the carbohydrate content
decreased and the aromatic and carboxyl groups contents
increased. However the percentage of increase/decrease of
the different form of carbon was related to the different
composition of the initial substrate providing evidence of
different OM evolution patterns as a function of the initial
substrates composition. Very few spectroscopic data are
available on the transformations suffered by the organic
matter present in different organic substrates when
submitted to anaerobic digestion [11,23]. In this work we
aimed at characterizing different ingestates and their cor-
responding digestates produced in two full-scale biogas
production plants located in Italy by means of 13C CPMAS-
NMR spectroscopy. This in order to verify possible chem-
ical differences among digestates obtained by different
organic substrates, to relate these differences to the initial
chemical composition of substrates and finally to ascertain
whether the AD process leads to stabilized final products
regardless the initial composition of biomass.
2. Material and methods
2.1. Anaerobic co-digestion plant
The co-digestion plant is located in the city of Forlı, Emilia
Romagna region, Northern Italy. In this plant, a continu-
ously stirred tank þ plug flow reactor is heated to
a temperature of 40 �C and the 6300 m3 digesters (5700 m3
net volume) are fed for most of the year with energy crops
(sorghum silage (SS), beef cattle slurry (BCS), and agro-
industrial residues (AIR)) with an average of about 60 Mg/
day of biomasses processed. The biomasses input is totally
computerized and performed continuously with a 30 min
frequency. The organic loading rate is 3 KgVS/m3/day with
a hydraulic retention time of 95 days. During the olive oil
production season, the beef cattle slurry is substituted with
olive mill wastewaters and agro-industrial residues are
mixed with a 10% of olive residues. The average biogas
composition is quite constant during the year due to the low
variability of the composition of biomasses utilized. The
methane production is 0.356 Nm3 kgVS�1 while the electrical
energy yield is 1.48 kWh/kgVS�1. The biogas, constituted by
b i om a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 1 1 1e1 2 0 113
530 ml l�1 CH4, 470 ml l�1 CO2, traces of H2 and H2S, is totally
delivered to a power generator to be converted into elec-
tricity with an average daily production of about 18 MWh
[24].
2.2. Pig slurry anaerobic digestion plant
The plant is located in Marsciano, Umbria region, Central
Italy, in an area characterized by outstanding livestock activ-
ities and productivity. The plant has been operating since 1987
and collects slurries from several nearby pig farms. During the
years, the plant has undergone relevant improvements and
currently it processes about 155,000 Mg y�1 of pig slurry (PS).
The loading is continuous and computerized and the
hydraulic retention time is about 25 days. The biogas
produced, constituted by about 600 ml l�1 CH4, 400 ml l�1 CO2
and traces of H2 and H2S, is delivered to a power co-generator
to be converted into electricity (3,800,000 kWh per year) and
heat energy. The anaerobic plant is integrated with an aerobic
treatment plant so that the digestate is centrifuged to obtain
two by-products: a liquid, directly used as a nitrogen fertilizer,
and a sludge delivered to the composting plant.
2.3. Sampling
From the two plants, single substrates, feeding materials
(ingestates) and their corresponding digestates were sampled.
For each sampling, several sub-samples of about 2 kg were
retrieved, thoroughly mixed and homogenized to obtain
a representative sample of about 1 kg. All samples were dried
for 24 h at 105 �C, shredded in a blender to pass through a 1-
mm mesh and stored in refrigerator.
2.4. Ingestates composition
Fresh biomasses were loaded into the Forlı co-digestion plant
as follows (% of dry matter). During the olive oil season: 70%
SS, 10% OMW, 10% OR and 10% AIR to obtain ingestate 1 (I1).
During the rest of the year: 70% SS, 10% BCS and 20% AIR to
obtain ingestate 2 (I2). PS is the only ingestate for the Mar-
sciano plant.
2.5. Digestates samples
D1 obtained from I1 and D2 obtained from I2 were retrieved
from the Forlı digestion plant. D3 and D3Cwere retrieved from
the Marsciano plant and were both obtained from PS: D3
represents the whole digestate before centrifugation, D3C is
the solid part of the digestate (sludge) after centrifugation, but
before composting.
2.6. Chemical analysis
For all samples, total organic carbon (TOC) was determined by
the wet dichromate oxidation method. Fresh samples were
used for total Kjeldahl Nitrogen determination (TKN) by
means of macro-Kjeldahl distillation methods. Moisture
content (data expressed as Total Solids e TS) of fresh samples
was determined as weight loss upon drying at 105 �C in an
oven for 24 h, while total volatile solids (VS) were determined
as weight loss (sample previously oven-dried at 105 �C) uponashing at 550 �C for 24 h in a muffle furnace.
All analyses were carried out in triplicate and standard
error (SE) was calculated.
2.7. 13C CPMAS-NMR analysis
The solid-state CPMAS 13C NMR spectra of the samples were
acquired at 10 kHz on a Bruker AMX 600 spectrometer (Bruker
BioSpin GmbH, Rheinstetten) using a 4 mm CP-MAS probe.
The pulse repetition rate was set at 0.5 s, the contact time at
1 ms and the number of scans was 3200.
A contact time of 1 ms was obtained after the VCT exper-
iments. The error in signal acquisition caused by the use of the
average contact timewas determined by comparing the signal
intensity in the absence of carbon relaxation e I0 and the
intensity of the signal Itcp measured at the optimal contact
time. In these conditions, it was shown that CPMAS 13C NMR
provides a quantitative representation of the C content in
humic substances [25]. The chemical shift scale of 13CPMAS-
NMR spectra was referred to tetramethylsilane (d ¼ 0 ppm).
Spectra were elaborated using TOPSPIN 1.3 software
(Bruker BioSpin GmbH, Rheinstetten, Germany).
2.8. Biological stability test
The biological stability was determined by the Specific Oxygen
Uptake Rate method (SOUR-test) [26]. The SOUR-test is a bio-
logical aerobic assay which measures the oxygen uptake rate
in an aqueous solution during microbial respiration of a sus-
pended solid matrix. The microbial respiration is measured
under standardized moisture conditions and maximized
oxygenation and bacteriaesubstrate interaction conditions,
amplifying the differences between different samples.
Dried and mechanically shredded samples (Ø < 1 mm)
were used in the SOUR-test. Briefly, 0.2 g of total solids were
set in a flask to which the following were added: 500 ml of
deionized water, 12 ml of phosphate buffer solution (KH2PO4
0.062 mol l�1, K2HPO4 0.125 mol l�1, Na2HPO4.7H2O
0.125 mol l�1; pH 7.2), and 5 ml of nutritive solution (CaCl20.25 mol l�1, FeCl3 0.9 mmol l�1 and MgSO4 0.09 mol l�1) made
up according to the standard BOD test procedures [27]. No
nitrogenwas added. During the test, standard conditionswere
maintained to ensure optimum microbial activity and reac-
tion rates. To allow oxygen diffusion, the sample was stirred
by using a magnetic stirrer and by performing intermittent
aeration every 15 min. Potential oxygen uptake wasmeasured
as the cumulative oxygen demand during the 20-h test (OD20,
mg O2 g TS�1 20 h�1).
3. Results and discussion
3.1. Chemical analysis
In Table 1 the chemical characteristics of samples from fresh
to final products are reported. During the anaerobic digestion,
VS and TOC contents decreased due to consumption of sugars,
proteins, amino acids and fatty acids which are all used by
microorganism as C source. The C loss was about 25% in both
Table 1 e Chemical characteristics of samples from fresh to final products during the anaerobic digestion.
I1 D1 I2 D2 PS D3 D3C
TOC (g kg TS�1) 465 � 7b 350 � 7a 436 � 9b 320 � 8a 345 � 4b 256 � 6a 342 � 10b
TS (g kg FM�1) 220 � 9b 122 � 6a 139 � 6b 112 � 1a 49 � 3a 60 � 2b 318 � 8c
VS (g kg TS�1) 837 � 6b 702 � 8a 741 � 6b 576 � 5a 771 � 21b 650 � 17a 750 � 15b
TNK (g kg TS�1) 43 � 2a 45 � 3a 33 � 1a 36 � 2a 44 � 1b 49 � 1c 29 � 1a
C/N ratio 10.81 � 0.7b 7.78 � 0.2a 13.21 � 0.5b 8.89 � 0.3a 7.8 � 0.3b 5.2 � 0.2a 11.8 � 1.0c
Means followed in the same raw (I1 vs D1; I2 vs D2; PS vs D3 vs D3C) by the same letter are not statistically different ( p < 0.05) according to
Tukey’s test. Each value represents the mean of 3 determinations �SE.
Table 2 e Biological stability of fresh substrates,ingestates and their corresponding digestates.
Samples OD20
(mg O2 g TS�1 20 h�1)
OMW 49.89 � 5.05
AIR 54.79 � 1.29
BCS 65.57 � 4.81
OR 71.61 � 7.66
SS 95.10 � 15.35
I1 77.58 � 3.62
I2 75.57 � 2.64
PS 140.08 � 19.88
D1 23.80 � 0.01
D2 34.51 � 3.27
D3 37.55 � 3.10
D3C 27.59 � 0.93
b i om a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 1 1 1e1 2 0114
Forlı andMarsciano plants.When expressing OM loss in terms
of VS balance, results were related to the composition of
ingestates. In the Forlı plant the OM loss was about 61% when
OMWandORwere loaded into the plant, whereas it was about
44% for the ingestate containing BCS. This result may be
attributed to the higher degradability of olive mill industry by-
products with respect to beef cattle slurry. When considering
the pig slurry, a minor OM loss was observed for D3 (26%)
whereas it was negligible for D3C in which the liquid fraction
was removed by centrifugation. An increase in TKN was
observed in all digestates as compared to their corresponding
ingestates (þ5%, þ9% and þ11% for D1-I1, D2-I2 and D3-PS,
respectively). This trend in TKN has been already observed
[10] and can be attributed to a concentration effect associated
to the degradation of organic compounds. In the Forlı plant,
the smaller increase in TKN observed for D1 is likely due to the
higher presence of cellulose, hemicelluloses and lignin in the
I1 with respect to I2. The mechanical separation operated in
the Marsciano plant caused the soluble N to remain in the
liquid part thus causing a smaller TKN value for D3C sample.
The changes observed in TOC and TKN concentrations
during the anaerobic digestion influenced the C/N ratios. The
loss in TOC and the relative increase in TKN resulted in
a decrease of the C/N ratios during the processes. Of course
the opposite was observed for D3C.
3.2. Biological stability
TheOD20 values for fresh substrates, ingestates and digestates
are reported in Table 2. High values of the respirometric index
associated to high oxygen consumption rates refer to insta-
bility of organic compounds supporting microbial respiration.
PS was characterized by the highest OD20 which indicates the
very low biological stability of this substrate. The silage
process accounts for the high value observed for SS.
The stabilization undertaken by the organic matter during
theprocess is indicatedbythe lowerOD20valuesmeasured inall
digestates includedD3. A furtherdecrease ofOD20wasobtained
for D3C in which the liquid part of the digestate containing the
most easily degradable organic molecules was separated by
centrifugation. Data obtained in this work for digestates are
comparable to recent bibliographical evidences [10,11] that
report values of OD20 between 30 and 100mgO2 g TS�1 20 h�1 as
a range todefine a goodbiological stability degree for digestates.
3.3. 13C CPMAS-NMR analysis
For a semi-quantitative approach, the overall chemical shift
range of 13C NMR spectra is subdivided in sub-regions. The
types of carbon that can be distinguished in the NMR spec-
trum are: aliphatic C (0e47 ppm); methoxyl C (OeCH3,
47e60 ppm); O-alkyl C of polysaccharides (60e115 ppm);
aromatic C and phenols (115e160 ppm); carbonyl, carboxyl C
and amide carbonyl (160e210 ppm). In Table 3 the CPMAS 13C
NMR integrated area of different carbon types of fresh
substrates, ingestates and digestates are reported.
3.4. CPMAS 13C NMR spectra of single biomasses andingestates
Each substrate showed different contents of C containing
groups (Fig. 1). The highest content of aliphatic C was found
for BCS and PS according to the following order:
BCS > PS > OMW >> AIR w OR w SS. Aliphatic C content was
associated to the highest carboxyl C content whose order was:
BCS > PS > OMW > AIR > SS > OR, attributable to fatty acids
and lipids, although this result could also reflect the presence
of proteins [28]. The highest content of polysaccharides was
found in SS, OR, and AIR (in the order
SS > OR w AIR > OMW > BCS). On the other hand, the OMW
and OR were characterized by the highest content in aromatic
C and phenols that are typical components of olive oil resi-
dues. In Fig. 2 the relative distribution of carbon in the
assigned chemical shift regions for I1, I2 and PS is illustrated.
PS showed the highest aliphatic and carboxyl C and the lowest
O-alkyl C and aromatics contents, whereas the presence of
olive oil production residues accounted for the higher
contents of methoxyls, O-alkyl C and aromatics in I1 as
compared to I2.
Table 3 e 13CPMAS-NMR integrated area of different carbon type of fresh substrates, ingestates and their correspondingdigestates.
C type
Samples Aliphatic C bondedto other aliphatic
chain or to H
MethoxylC OeCH3
OeCH3 or N-alkyl O-alkyl C di-O-alkyl C
Aromatic C phenolor phenyl ether C
Carboxyl Cketo C
0e47 47e60 60e115 115e160 160e210
Band d range (ppm)
OMW 32.8 7.7 36.8 13.0 9.7
AIR 16.1 5.7 63.4 7.9 6.9
BCS 40.4 9.1 30.7 9.1 10.7
SS 11.5 4.7 69.5 8.2 6.1
OR 12.8 7.8 64.1 10.3 5.0
I1 16.2 6.6 61.9 9.1 6.2
I2 18.4 6.1 59.5 8.2 7.8
PS 38.5 6.2 38.2 7.0 10.1
D1 21.2 8.7 48.6 12.7 8.8
D2 21.3 9.0 51.4 10.9 7.4
D3 29.5 8.1 30.4 6.9 25.1
D3C 16.2 6.9 64.2 7.4 5.3
b i om a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 1 1 1e1 2 0 115
3.5. CPMAS 13C NMR spectra of digestates
Results are illustrates in Fig. 3. A similar distribution of C
containing groups is evident for D1 and D2. However, D2
showed the highest methoxyl C content whereas D1 was
characterized by the highest aromatic and phenols contents.
D3 exhibited the highest alkyl C and carboxyl C and the lowest
O-alkyl C and aromatics contents whereas D3C showed lower
0
10
20
30
40
50
OMW BCS AIR OR SS PS
0
20
40
60
80
OMW BCS AIR OR SS PS
024681012
OMW BCS AIR OR SS PS
a
c
b
d
e
Fig. 1 e Relative distribution in the assigned chemical shift regi
alkyl C; (d) aromatic C and phenols; (e) carboxyl C.
alkyl C, methoxyl C and carboxyl C with respect to D3 and the
highest content of O-alkyl C. The relative percentage of the
increase or decrease of each typical resonance area during the
anaerobic digestion for each process was calculated as: (final
value in the digestate e initial value in the ingestate/initial
value in the ingestate) � 100, and results obtained are illus-
trated graphically in Fig. 4. A very different trend of percent
variations of C types are found in D3 and D3C as compared to
0
2
4
6
8
10
OMW BCS AIR OR SS PS
024791214
OMW AIR BCS SS OR PS
ons for fresh substrates of: (a) alkyl C; (b) methoxyl C; (c) O-
0
10
20
30
40
50
I1 I2 PS5,8
6
6,2
6,4
6,6
6,8
I1 I2 PS
010203040506070
I1 I2 PS0
2
4
7
9
I1 I2 PS
024681012
I1 I2 PS
a
c
b
d
e
Fig. 2 e Relative distribution in the assigned chemical shift regions for ingestates of: (a) alkyl C; (b) methoxyl C; (c) O-alkyl C;
(d) aromatic C and phenols; (e) carboxyl C.
05101520253035
D1 D2 D3 D3C0
2
4
6
8
10
D1 D2 D3 D3C
010203040506070
D1 D2 D3 D3C02468101214
D1 D2 D3 D3C
05
101520
2530
D1 D2 D3 D3C
a
c
b
d
e
Fig. 3 e Relative distribution in the assigned chemical shift regions for digestates of: (a) alkyl C; (b) methoxyl C; (c) O-alkyl C;
(d) aromatic C and phenols; (e) carboxyl C.
b i om a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 1 1 1e1 2 0116
b i om a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 1 1 1e1 2 0 117
D1 and D2. In details: a) alkyl C: a dramatic decrease was
evident in D3C (�58%) and D3 (�23%), whereas an increase
was observed in D1 (þ31%) and D2 (þ16%); b) methoxyl C: D2
underwent the major relative increase (þ47%), followed by D1
(þ32%) very similar to D3 (30%) whereas a smaller increase
was observed for D3C (11%); c) O-alkyl C: a relative decrease
was evident for D1 (�21%), D2 (�14%) and D3 (�20%), whereas
a notable þ68% was found for D3C indicating the accumula-
tion in this substrate of highly aliphatic biopolymers resistant
to decomposition [17]; d) aromatic C and phenols: a negligible
variation in D3 (�1%) and a very small increase in D3C (þ6%)
were found whereas marked relative increases were obtained
for D1 (þ39%) and D2 (þ33%). The increase in aromatic C and
phenols might be related to the degradation of non-aromatic
cell wall compounds, which leads to a relative enrichment
in aromatics. In general, the increase in aromatic and phenolic
C indicates the preference of microorganisms for easily
degradable C components. In other words, the higher
decomposition of carbohydrateswould result in accumulation
of recalcitrant molecules; e) carboxyl C: a �47% was observed
for D3C associated to the centrifugation of the whole digestate
which concentrates carbohydrates in the sludge, whereas
a remarkable þ148% was found for D3, þ42% for D1, and �5%
for D2.
The aromaticity values, expressed as the ratio of aromatic
C and aliphatic C þ aromatic C [29], is known to provide an
-80
-60
-40
-20
0
20
40
D1 D2 D3 D3C
-40
-20
0
20
40
60
80
D1 D2 D3 D3C
-100
-50
0
50
100
150
200
D1 D2 D3 D3C
a
c
b
d
e
Fig. 4 e Percent increase/decrease in digestates of: (a) alkyl C; (b
carboxyl, during the anaerobic digestion.
evaluation of the evolution of humification during composting
[30]. Here we propose to use the aliphaticity index expressed
as the ratio of aliphatic C and aliphatic Cþ aromatic C. Results
obtained are illustrated in Fig. 5. PS and D3 exhibited the
highest aliphaticity indexes. A stronger reduction was evident
in D3C as compared to PS, whereas a slight decrease was
observed in D1 and in D2. This index highlights the aliphatic
nature of pig slurry as compared to other organicmatrices and
the distinctive aliphatic nature of their corresponding diges-
tates. The smaller aliphaticity index found for D3C with
respect to D3 is due to the separation of the liquid phase in
which most of the fatty acids are left.
3.6. Comparison with previous data
As stated in the Introduction, one of the aim of the present
paper was to ascertain whether the AD process leads to
stabilized final products regardless the initial composition of
biomass. This point is of major importance considering the
possible agronomical use of digestates. In order to accomplish
this task we took into consideration CPMAS 13C NMR data of
a number of ingestates and digestates available in literature
[11,23]. Results of this comparison are reported in Table 4.
The main results emerging from the analysis of data are
the following:
0
10
20
30
40
50
D1 D2 D3 D3C
-10
0
10
20
30
40
50
D1 D2 D3 D3C
) methoxyl; (c) O-alkyl C; (d) aromatic C and phenols (e)
0
0,2
0,4
0,6
0,8
1
D1 D2 D3 D3CI1-D1 I2-D2 PS-D3 PS-D3C
Fig. 5 e Aliphaticity index of ingestates (gray) and their
corresponding digestates (black).
b i om a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 1 1 1e1 2 0118
a) The increase in aromatic C is not statistically significant.
This result indicates that most of the aromatic structures
present in the substrate tends to degrade during the
process.
b) Alkyl C and O-alkyl C contents in digestates are positively
correlated with those in ingestates (r ¼ 0.88, p < 0.05 for
alkyl C and r ¼ 0.68, <0.05 for O-alkyl C), i.e., final Alkyl C
contents depend on the concentration of pre-existing
molecules that were most recalcitrant to the degrada-
tion. Both in ingestates and in digestates alkyl C and O-
alkyl C contents are inversely correlated (r¼�0.93, p< 0.05
Table 4 e 13CPMAS-NMR integrated area of a number ofingestates and digestates available in literature (Ref. [11]and [23]).
Band d range (ppm)
0e47 47e113 113e160 160e210
Ingestates
21.93 60.74 6.64 10.69
29.8 56.94 5.13 8.13
25.98 59.56 3.71 10.75
18.82 67.2 6.78 7.2
18.37 66.65 7.14 7.84
19.18 67.81 7.68 5.33
19.72 64.76 8.71 6.81
20.65 64.87 7.63 6.85
21.77 65.5 4.81 7.92
16.2 68.5 9.1 6.2
18.4 65.6 8.2 7.8
Mean 20.98a 64.37b 6.86a 7.77a
STD 3.87 3.69 1.69 1.67
Digestates
42.82 37.51 8.47 11.2
46.71 32.68 7.52 13.09
44.01 36.05 7.72 12.22
25.58 59.36 7.92 7.14
28.15 55.28 8.14 8.43
30.32 54.42 8.03 7.23
38.54 44.14 7.72 9.6
28.77 55.2 8.58 7.45
39.08 42.01 9.08 9.84
29.9 38.5 6.9 25.1
21.2 57.3 12.7 8.8
Mean 34.1b 46.58a 8.43a 10.91a
STD 8.4 9.83 1.52 5.11
for ingestates and r ¼ �0.88, p < 0.05 for digestates), i.e.
alkyl C concentration is due to degradation of O-Alkyl C
(cellulose, hemicelluloses).
c) Ingestates degradation results in reduction of O-alkyl C
and increase of alkyl C. The average increase of alkyl C is
68.4% whereas the average decrease for O-alkyl C is
�27.6%. Conversely, in our work a strong decrease of alkyl
C was observed for D3 and D3C. As reported in par. 3.4, pig
slurry is characterized by the highest aliphatic and
carboxyl C contents associated to fatty acids and lipids as
well as protein which all appear to be more easily
degradable in this biowaste as compared to other
substrates.
In general, it can be concluded that anaerobic digestion
proceeds through preferential degradation of carbohydrates
such as cellulose and hemicellulose and, as a consequence,
concentration of more chemically recalcitrant aliphatic
molecules occurs. On the other hand, it is well known that
biochemically recalcitrant soil organic matter (SOM) fractions
are enriched with alkyl carbon structures and resist decom-
position due to intrinsic molecular properties. Precursors of
these recalcitrant bio(macro)molecules such as glycerides,
waxes, and terpenoids occur in plants, microorganisms and
animals [31]. Several other studies have shown that, as
organic matter decomposition proceeds, aliphatic moieties
(rather than or together with aromatic moieties) tend to
accumulate in soils [21,32e35]. Lorenz et al. reported that “The
intrinsic biochemical stability of naturally occurring recalci-
trant aliphatic biomacromolecules may enhance the terres-
trial storage of atmospheric CO2” [31]. In this scenario,
anaerobic digestion may act as the perfect recycling cycle,
producing energy from biowaste and contributing to carbon
sink through the addition of digestate to soil.
Further research is needed in order to compare amend-
ment properties of digestate rich in alkyl C with those of
compost rich in aromatic C since they present this different
ascertained molecular composition.
4. Conclusions
Anaerobic digestionmodifies the relative concentration of the
different forms of carbon of the ingestate. Digestates obtained
by different organic substrates show significant differences
related to the different chemical composition of the input
materials. In particular, the co-digestion process based on the
use of energy crops, agro-industrial residues and beef cattle
slurry proceeds mainly through the degradation of
carbohydrates-like molecules which accumulates recalcitrant
aromatics and phenols. When beef cattle slurry is substituted
with olive oil production by-products, more aromatic C,
phenols and carboxyl groups are accumulated. The anaerobic
digestion of pig slurry occurs mainly through the degradation
of fatty acids. The sludge obtained after centrifugation of the
whole digestate appears most suitable for the composting
process to which it is afterwards subjected being character-
ized by a notable relative increase in polysaccharides and at
a minor extent in aromatic with respect to the whole diges-
tate. The qualitative/quantitative chemical modifications
b i om a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 1 1 1e1 2 0 119
occurring during the process are confirmed by the increasing
of the biological stability of the final products.
A comparison of CPMAS 13C NMR data of a number of
ingestates and digestates available in literature indicated that
most of the aromatic structures present in the substrate tend
to degrade during the process and that anaerobic digestion
proceeds through preferential degradation of carbohydrates
such as cellulose and hemicellulose and, as a consequence,
concentration of more chemically recalcitrant aliphatic
molecules occurs.
r e f e r e n c e s
[1] Fernandez A, Sanchez A, Font X. Anaerobic co-digestion ofa simulated organic fraction of municipal solid wastes andfats of animal and vegetable origin. Biochem Eng J 2005;26:22e8.
[2] Tani M, Sakamoto N, Kishimoto T, Umetsu K. Utilization ofanerobically digested slurry combined with other wastefollowing application to agricultural land. Int Congr Ser 2006;1293:331e4.
[3] Chynoweth DP, Owens JM, Legand R. Renewable methanefrom anaerobic digestion of biomass. Renew Energ 2008;22:1e8.
[4] Mata Alvarez J, Mace S, Llabres P. Anaerobic digestion oforganic solid wastes. An overview of research achievementsand perspectives. Biores Technol 2000;74:3e16.
[5] Nishio N, Nakashimada Y. Recent development of anaerobicdigestion processes for energy recovery from wastes. J BiosciBioeng 2007;103(2):105e12.
[6] Hamdi M. Toxicity and biodegradability of olive millwastewaters in batch anaerobic digestion. Appl BiochemBiotech 1992;37:155e63.
[7] Della Greca M, Monaco P, Pinto G, Pollio A, Previtera L,Temussi F. Phytoxicity of low-molecular weight phenolsfrom olive mill wastewaters. B Environ Contam Tox 2001;67:352e9.
[8] Borja R, Alba J, Banks CJ. Impact of the main phenoliccompounds of olive mill (OMW) wastewaters on the kineticsof acetoclastic methanogenesis. Process Biochem 1997;32:121.
[9] Borole AP, Klasson KT, Ridenour W, Holland J, Karim K, Al-Dahham MH. Methane production in a 100-L upflowbioreactor by anaerobic digestion of farm waste. ApplBiochem Biotech 2006;129/132:887e96.
[10] Schievano A, Pognani M, D’Imporzano G, Adani F.Predicting anaerobic biogasification potential of ingestatesand digestates of a full-scale biogas plant using chemicaland biological parameters. Bioresour Technol 2008;99:8112e7.
[11] Tambone F, Genevini P, D’Imporzano G, Adani F. Assessingamendment properties of digestate by studying the organicmatter composition and the degree of biological stabilityduring the anaerobic digestion of the organic fraction ofMSW. Bioresour Technol 2009;100:3140e2.
[12] Gomez X, Cuetos MJ, Tartakovsky B, Martinez-Nunez MF,Moran A. A comparison of analytical techniques forevaluating food waste degradation by anaerobic digestion.Bioproc Biosyst Eng 2010;33:427e38.
[13] Cuetos MJ, Gomez X, Otero M, Moran A. Anaerobic digestionof solid slaughterhouse waste: study of biologicalstabilization by Fourier Transform infrared spectroscopy andthermogravimetry combined with mass spectrometry.Biodegradation 2010;21:543e56.
[14] Magdoff F, Weil RR. Soil organic matter managementstrategies. In: Magdoff F, Weil RR, editors. Soil organic matterin sustainable agriculture. New York: CRC Press; 2004. p.45e65.
[15] Kogel-Knabner I. The macromolecular organic compositionof plant and microbial residues as inputs to soil organicmatter. Soil Biol Biochem 2002;34:139e62.
[16] Lasaridi K, Stendiford EI. A simple respirometric technique forassessing compost stability. Water Res 1998;31(129):3717e23.
[17] Chen Y. Nuclear magnetic resonance, infra-red andpyrolysis: application of spectroscopic methodologies tomaturity determination of composts. Compost Sci Util 2003;11(2):152e68.
[18] Spaccini R, Piccolo A. Molecular characterization of compostat increasing stages of maturity. 2. Thermochemolysis-GC-MS and 13C-CPMAS-NMR spectroscopy. J Agric Food Chem2007;55:2303e11.
[19] Caricasole P, Provenzano MR, Hatcher PG, Senesi N.Evolution of organic matter during composting of differentorganic wastes assessed by CPMAS 13C-NMR spectroscopy.Waste Manag 2011;31:411e5.
[20] Chen Y, Inbar Y, Hadar Y, Malcolm RL. Chemical propertiesand solid-state CPMAS 13C-NMR of composted organicmatter. Sci Total Environ 1989;81/82:201e8.
[21] Baldock JA, Oades JM, Nelson PN, Skene TM, Golchin A,Clarke P. Assessing the extent of decomposition of naturalorganic materials using solid-state C-13 NMR spectroscopy.Aust J Soil Res 1997;35:1061e83.
[22] Tang J, Maie N, Tada Y, Katayama A. Characterization of thematuring process of cattle manure compost. ProcessBiochem 2006;41:380e9.
[23] Tambone F, Scaglia B, D’Imporzano G, Schievano A, Orzi V,Salati S, et al. Assessing amendment and fertilizingproperties of digestates from anaerobic digestion througha comparative study with digested sludge and compost.Chemosphere 2010;81:577e83.
[24] Piccinini S, Fabbri C, Soldano M. Monitoring and assessmentof three biogas plants in Italy. In: Proceeding of theInternational Conference “Biogas science”, 9e11 December2009, Erding (Germany).
[25] Conte P, Piccolo, Van Lagen B, Buurman P, Hemminga MA.Elemental quantitation of natural matter by CPMAS 13C-NMRspectroscopy. Solid State Nucl Mag 2002;21(3e4):158e70.
[26] D’Imporzano G, Adani F. The contribution of water solubleand water insoluble organic fraction to oxygen uptake rateduring high rate composting. Biodegradation 2007;18:103e13.
[27] APHA. Standard methods for the examination of water andwastewater. 18th ed. Washington, DC USA: American PublicHealth Association; 1992.
[28] Dignac MF, Derenne S, Ginestet P, Bruchet A, Kniker H,Largeau C. Determination of structure and origin ofrefractory organic matter in bio-depurated wastewater viaspectroscopic methods. Comparison of conventional andozonation treatment. Environ Sci Technol 2000;34:3389e94.
[29] Hatcher PG, Breger IA, Dennis LW, Maciel GE. Solid-state 13C-NMR of sedimentary humic substances: new revelations ontheir chemical composition. In: Christman RF, Gjessing ET,editors. Aquatic and terrestrial humic materials. Ann Arbor,MI: Ann Arbor Science; 1983. p. 37e81.
[30] Albrecht R, Ziarelli F, Alarcon-Gutierrez E, Le Petit J,Terrom G, Perissol C. 13C solid-state NMR assessment ofdecomposition pattern during co-composting of sewagesludge and green wastes. Eur J Soil Sci 2008;59:445e52.
[31] LorenzK,LalR,PrestonC.Strengtheningthesoilorganiccarbonpool by increasing contributions from recalcitrant aliphaticbio(macro)molecules. Geoderma 2007;142(1e2):1e10.
[32] Zech W, Senesi N, Guggenberger G, Kaiser K, Lehmann J,Miano TM, et al. Factors controlling humification and
b i om a s s a n d b i o e n e r g y 4 8 ( 2 0 1 3 ) 1 1 1e1 2 0120
mineralization of soil organic matter in the tropics.Geoderma 1997;79:117e61.
[33] Hu W, Mao J, Xing B, Schmidt-Rohr K. Poly(methylene)crystallites in humic substances detected by nuclearmagnetic resonance. Environ Sci Technol 2000;34(3):530e4.
[34] Stimler K, Xing B, Chefetz B. Transformation of plant cuticlesin soil: effect on their sorptive capabilities. Soil Sci Soc Am J2006;70(4):1101e9.
[35] Chefetz B, Tarchitzky J, DeshmukhAP,Hatcher PG, ChenY.Structuralcharacterizationofhumicsubstancesinparticle-sizefractionof anagricultural soil. Soil Sci SocAm J 2002;66:129e41.