organic matter characterization during the anaerobic digestion of different biomasses by means of...

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Organic matter characterization during the anaerobic digestion of different biomasses by means of CPMAS 13 C NMR spectroscopy Fulvia Tambone a , Fabrizio Adani a , Giovanni Gigliotti b , Daniela Volpe b , Claudio Fabbri c , Maria Rosaria Provenzano d, * a Dipartimento di Scienze Agrarie e Ambientali, Via Caloria 2, 20133. Universita ` di Milano, Milano, Italy b Dipartimento di Scienze Agrarie e Ambientali, Borgo XX Giugno 74, University of Perugia, 06121 Perugia, Italy c Centro Ricerche Produzioni Animali, C.R.P.A. S.p.A., Corso Garibaldi 42, Reggio Emilia, Italy d Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Via Amendola 165/A, University of Bari, 70123 Bari, Italy article info 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 Digestates 13 CPMAS-NMR spectroscopy abstract 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 13 CPMAS-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 13 C 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 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 * Corresponding author. Tel.: þ39 080 5442929; fax: þ39 080 5442813. E-mail address: [email protected] (M.R. Provenzano). Available online at www.sciencedirect.com http://www.elsevier.com/locate/biombioe biomass and bioenergy 48 (2013) 111 e120 0961-9534/$ e see front matter ª 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biombioe.2012.11.006

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Page 1: Organic matter characterization during the anaerobic digestion of different biomasses by means of CPMAS 13C NMR spectroscopy

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)..

Page 2: Organic matter characterization during the anaerobic digestion of different biomasses by means of CPMAS 13C NMR spectroscopy

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

Page 3: Organic matter characterization during the anaerobic digestion of different biomasses by means of CPMAS 13C NMR spectroscopy

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

Page 4: Organic matter characterization during the anaerobic digestion of different biomasses by means of CPMAS 13C NMR spectroscopy

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.

Page 5: Organic matter characterization during the anaerobic digestion of different biomasses by means of CPMAS 13C NMR spectroscopy

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-

Page 6: Organic matter characterization during the anaerobic digestion of different biomasses by means of CPMAS 13C NMR spectroscopy

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

Page 7: Organic matter characterization during the anaerobic digestion of different biomasses by means of CPMAS 13C NMR spectroscopy

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)

Page 8: Organic matter characterization during the anaerobic digestion of different biomasses by means of CPMAS 13C NMR spectroscopy

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

Page 9: Organic matter characterization during the anaerobic digestion of different biomasses by means of CPMAS 13C NMR spectroscopy

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.

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