Journal of Environmental Protection, 2011, 2, 83-89
doi:10.4236/jep.2011.21009 Published Online March 2011 (http://www.SciRP.org/journal/jep)
83
Qualitative Characterization and Differentiation of
Digestates from Different Biowastes Using FTIR
and Fluorescence Spectroscopies
Maria Rosaria Provenzano1, Giuseppina Iannuzzi1, Claudio Fabbri2, Nicola Senesi1
1Dipartimento di Biologia e Chimica Agroforestale ed Ambientale, University of Bari, Bari, Italy; 2C.R.P.A. S.p.A.Corso Garibaldi,
Reggio Emilia, Italy.
Received September 14th, 2010; revised November 1st, 2010; accepted December 23rd, 2010.
ABSTRACT
Anaerobic digestion of biomasses originates different products, the most abundant of which are methane and carbon
dioxide. During this process, a 60-70% organic matter reduction occurs and the final product, the digestate, is charac-
terized by high biological stability and high contents of recalcitrant organic molecules and nutrients. In the present
work digestates obtained by different mixture of biomasses in a full-scale co-digestion plant operating in Italy were
characterized as whole samples without any pre-treatment or extraction by means of Fourier transform infrared spec-
troscopy and fluorescence spectroscopy in the synchronous-scan mode and results were compared to those obtained on
the single fresh substrates. Biomasses considered were: beef cattle slurry, maize or sorghum silage, agro-industrial
residues, olive residues and olive mill wastewater. These substrates exhibited typical spectra related to their different
chemical composition. Results obtained on digestates provided evidence of distinctive characteristic of the final product
as a function of the different composition of the biomasses loaded into the digestion plant. We concluded that FTIR and
fluorescence spectra of digestates produced in a real co-digestion plant “inherit” the main spectroscopic features of the
organic wastes from which they are produced. Spectroscopic techniques used in this work succeeded in qualitatively
characterizing and differentiating digestates obtained from biomasses of different chemical composition.
Keywords: Organic Wastes, Anaerobic Digestion, Digestates, Fourier Transform Infrared Spectroscopy,
Synchronous-Scan Fluorescence Spectra
1. Introduction
Anaerobic digestion has been known for centuries but
interest in the economical recovery of fuel methane gas
from different types of organic wastes on industrial scale
has recently enormously increased owing to the changing
socio economical situation in the world. In fact, depletion
of fossil fuels has posed the urgent need to move to al-
ternative energy supplies with emphasis on renewable
sources and the number of anaerobic treatment plants in
Europe has remarkably increased in recent years.
Anaerobic digestion is a biological degradation of or-
ganic matter under anaerobic conditions which originates
different products, the most abundant of which are
methane and carbon dioxide. The decomposition takes
place in three stages: Hydrolysis whereby organic poly-
mers such as proteins, lipids and carbohydrates are bro-
ken down into soluble monomers; Acid formation during
which fermentative bacteria degrade these molecules to
volatile fatty acids and to ammonia; Methane formation
in which the acids are converted to biogas and a residue
called digestate. During the anaerobic digestion, a 60-
70% organic matter reduction occurs and the digestate is
characterized by high biological stability and high con-
tents of recalcitrant organic molecules and nutrients.
Co-digestion strategies, referring to the combined
treatment of several biomasses with complementary
characteristics, in most cases enhance the biogas produc-
tion due to positive synergisms established in the diges-
tion medium and the supply of missing nutrients by
co-substrates [1]. Agro-industrial residues are known to
have a high potential for methane production since they
can be digested rapidly making them a good source of
material for anaerobic co-digestion [2]. The concept of
energy crops has been known for many years but recently
they have risen to represent a renewable biomass whose

Qualitative Characterization and Differentiation of Digestates from Different Biowastes Using FTIR and 84
Fluorescence Spectroscopies
cost is expected to decline as technology improves. For
olive oil producing countries on the Mediterranean area
such as Italy, treatment and disposal of olive mill effluent
and residues represents one of the major environmental
problems also because a large volume of this effluent is
produced in a short period in wintertime. 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 [3-5].
Spectroscopic investigation based on Fourier trans-
form infrared spectroscopy (FTIR) and fluorescence
spectroscopy have been widely applied to the characteri-
zation of humic and fulvic acids of different origin and
nature [7-9] and whole composts from various organic
wastes sampled at different composting time [10,11].
However, as far as we are concerned, very few data [12]
are available on spectroscopic characterization of prod-
ucts deriving by anaerobic digestion.
In the present work a number of digestates obtained in
a full-scale co-digestion plant operating in Italy will be
analyzed by means of fluorescence spectroscopy in the
synchronous-scan mode and FTIR spectroscopy and re-
sults will be compared to those obtained on the fresh
substrates processed in the plant in order to qualitatively
characterize and differentiate digestates obtained from
different mixture of organic wastes.
2. Materials and Methods
The co-digestion plant is located in the city of Forlì, in
Emilia Romagna region, Northern Italy. In this plant, a
continuously stirred tank + 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 (maize or sorghum silage), beef cattle slurry, and
agro-industrial residues in about 50:20:30 percent vol-
ume ratio with an average of about 60 ton/day of bio-
masses processed. Loading of biomasses is totally com-
puterized and performed continuously with a 30 minutes
frequency. The organic loading rate is 3 kg·VS/m3/day
with a hydraulic retention time of 95 days. During the
olive oil production season, the beef cattle slurry is par-
tially substituted with olive mill wastewaters and agro-
industrial residues are mixed with a 10% of olive resi-
dues. The average biogas composition is quite constant
during the year due to the low variability of the composi-
tion of biomasses utilized. The methane production is
0.356 Nm3·kg·VS-1 while the electrical energy yield is
1.48 kW·h/kg·VS-1. The biogas, constituted by 53%vol
CH4, 47%vol CO2, traces of H2 and H2S, is totally deliv-
ered to a power generator to be converted into electricity
with an average daily production of about 18 MWh [13].
From this digestion plant, five samples of digestates
(D1-D5) were retrieved. D1 and D2 were obtained from
beef cattle slurry (BCS), maize or sorghum silage (SS),
agro-industrial residues (AIR). D3, D4 were obtained
from olive mill wastewater (OMW), maize or sorghum
silage (SS), agro-industrial residues (AIR), olive residues
(OR), D5 were obtained from olive mill wastewater
(OMW), maize or sorghum silage (SS), agro-industrial
residues (AIR).The fresh biomasses loaded into the plant
were sampled as well. For sampling, several sub-samples
were retrieved from the plant and thoroughly mixed and
homogenized to obtain a final sample of about 1 kg for
analysis. All samples were dried for 24 h at 105˚C,
shredded in a blender, sieved using a 2-mm mesh, and
stored in a refrigerator. Fresh matter (FM), total solids
(TS), and volatile solid (VS) were determined according
to standard procedure [14]. NH3-N and total nitrogen
(NTK) were determined according to the analytical
methods for wastewater sludge [15]. FTIR spectra were
generated on pellets obtained by pressing under vacuum
about 200 mg of the mixture obtained by crushing in a
agate mortar 400 mg KBr, spectrometry grade and 1 mg
of whole sample with precaution taken to avoid moisture
uptake. Spectra were recorded in the 4000 to 400 cm-1
wavelength range using a Nicolet 5P5 FTIR spectropho-
tometer, with a resolution of 2 cm-1 and a scan rate of 64
scans/min. Synchronous-scan fluorescence spectra were
obtained on the whole sample without any pre-treatment
or extraction using a Perkin-Elmer LS55 Luminescence
Spectrophotometer on water solution of samples at a
concentration of 100 mg/L after overnight agitation and
equilibration at room temperature, successsive filtration
with Whatman n˚ 1 paper and adjustment to pH 8 with
0.05 N NaOH for comparison with previous works [8].
Spectra were generated by changing simultaneously both
the excitation and the emission wavelengths over a scan
range of 300 - 550 nm while maintaining a constant
wavelength difference Δλ = λem – λex = 18 nm [16].
3. Results and Discussion
3.1. Chemical Analysis
Chemical analysis of each biomass and digestates are
reported respectively in Table 1 and Tab le 2. Fresh sub-
strates exhibit distinctive chemical characteristics related
to their different nature. High values of TS, VS and TOC
are shown by OR, whereas BCS and OMW are charac-
terized by high TKN content as opposite to lower values
shown by SS and OR. On the contrary, chemical data do
not differ significantly among digestates. All pH values
are slighly alkaline due to VFA degradation and ammo-
nia production [17]. TKN values are very similar whereas
NH3-N content decreases slightly in D5 likely due to the

Qualitative Characterization and Differentiation of Digestates from Different Biowastes Using FTIR and
Fluorescence Spectroscopies
85
Table 1. Chemical data of biomasses.
BCS SS AIR OMW OR
pH 6.98 ± 0.02 5.65 ± 0.01 5.44 ± 0.01 5.09 ± 0.03 5.41 ± 0.02
TS (g kgFM-1) 45 ± 7 305 ± 10 133 ± 4 49 ± 16 459 ± 4
VS (g kgTS-1) 688 ± 27 774 ± 10 784 ± 12 668 ± 6 972 ± 12
TOC (%TS) n.a. 38 ± 14 39 ± 8 48 ± 1 59 ± 1
TKN (mg kgTS-1) 50 ± 5 13 ± 5 19 ± 6 46 ± 11 9 ± 2
NH3-N (%TKN)) n.a. 6 ± 3 0.03 ± 12 11 ± 9 n.a.
Each value represents the mean of 3 determinations ±SE; n.a. = not available
Table 2. Chemical data of digestates.
D1 D2 D3 D4 D5
pH 7.87 ± 0.01 7.77 ± 0.01 7.65 ± 0.01 7.54 ± 0.01 7.74 ± 0.01
TS (g kgFM-1) 109 ± 2 107 ± 2 112 ± 8 106 ± 6 108 ± 8
VS (g kgTS-1) 604 ± 1 670 ± 3 610 ± 7 627 ± 4 627 ± 2
TKN (mg kgTS-1) 47 ± 2 45 ± 13 44 ± 10 45 ± 6 41 ± 8
NH3-N (%TKN)) 43 ± 12 44 ± 8 42 ± 12 42 ± 9 35 ± 9
Each value represents the mean of 3 determinations ±SE.
higher presence of polyphenols in the ingestates which
lower the pH of the substrates resulting in a lower am-
monia production.
3.2. FTIR Spectra
The assignment of FTIR bands is reported in Table 3.
FTIR spectra of all biomasses are illustrated in Figure 1.
By comparing results, it emerges that they feature char-
acteristic bands assigned to functional groups typical of
each substrate. OMW and OR show similar spectra
characterized by prominent peaks at 1560 and 1380 cm-1
due to the massive presence of proteins, aliphatic mole-
cules and phenolic groups in these substrates. A higher
relative intensity of peaks at 2920 cm-1 and 2850 cm-1
assigned to aliphatic structures of fatty acids is evident in
OMW spectrum with respect to OR. On the contrary,
BCS, SS and AR spectra feature a main peak at 1040
cm-1 and a number of peaks at 1630, 1420, 1240, 1380
cm-1 revealing the heterogeneous chemical nature of
these biomasses. BCS shows, in addition, a peak at 1560
cm-1 due to protinaceous materials of which this sludge is
rich.
A direct comparison between OMW and BSC spectra
highlights the notable different chemical nature of these
wastes (Figure 2) evidencing the predominant proteina-
ceous and polysaccharidic nature of BCS. When consid-
ering digestates, D1 and D2 produce overlapping spectra
and the same is found for D3 and D4 samples (Figure 4).
Table 3. Main absorbance bands in FTIR spectra and their
assignments.
Wavenumber (cm-1)Assignments
3400 -OH (phenols, alcohols and carboxylic groups)
2925 and 2854 C-H stretching of alkyl structures
1710-1740 C = O stretching in carboxyls, acids and ketones
1630-1650 Aromatic C = C, C = O in amides (I), ketone an
d
quinone groups
1660 Aromatic C = C, COO-, C = O
1520-1550 Amide II
1450-1460 C-H stretching in aliphatic structures
1400 OH of phenols, COO-, -CH3
1380 COO– antisymmetric stretching, C-H and bend-
ing of CH2 and CH3 groups
1200-1100 C-O stretching of aryl ethers and phenols, C-O
stretching of secondary alchols
1043-1034 C-O stretching of polysaccharides
The main differences emerging from the comparison of
these spectra are a higher relative intensity of the peak at
1380 cm-1 and a lower relative intensity of the peak at
1040 cm-1 shown by D3 and D4 as compared to D1 and
D2. These results are likely arising from the typical
bands observed on the single biomasses spectra and dis-
cussed above. The D5 spectrum is similar to those of D3

Qualitative Characterization and Differentiation of Digestates from Different Biowastes Using FTIR and 86
Fluorescence Spectroscopies
Figure 1. FTIR spectra of (from top to bottom): BCS;
OMW; OR; SS; AR.
Figure 2. FTIR spectra of: (a) OMW (top) and (b) BCS
(bottom).
Figure 3. FTIR spectra of (from top to bottom): D1, D2, D3,
D4 and D5.
(a)
(b)
(c)
(d)
(e)
Figure 4. Synchronous-scan spectra of: (a) Sorghum silage,
(b) Agro-industrial residues; (c) Beef cattle manure; (d)
Olive residues; (e) Olive mill wastewaters.

Qualitative Characterization and Differentiation of Digestates from Different Biowastes Using FTIR and 87
Fluorescence Spectroscopies
and D4 but in addition it shows a significant peak at 1560
cm-1. This result may be ascribed to an accumulation
effect caused by the huge quantities of OR and OMW
loaded into the plant during the previous months. By
these results it can be concluded that FTIR spectra of
digestates show absorption bands associated to functional
groups reflecting the main chemical characteristics of
fresh biomasses from which they are originated.
3.3. Fluorescence Spectra
Synchronous-scan spectra of the organic substrates sub-
mitted to anaerobic digestion are illustrated in Figure 4.
Each biomass provides a typical spectrum regardless the
sampling month. The only common evidence to all spec-
tra is the peak at 390 nm whereas the general trend of
each spectrum is related to the different chemical com-
position of the organic substrate. SS (Figure 4(a)) shows
the maximum at lower wavelengths (340 nm), whereas
AIR (Figure 4(b)) and BCS (Figure 4(c)) feature
maxima at 390 nm and shoulders at 340 nm (more pro-
nounced for BCS) and at 440 nm. OR (Figure 4(d))
shows a major peak at 390 nm and a secondary peak at
340 nm. On the contrary, OMW spectrum (Figure 4(e))
is characterized by maxima shifted at much longer wave-
lengths (520 nm and 440 nm). Generally, peaks at low
wavelength values (<380 nm) are considered protein-
origin substances, whereas high emission wavelength
peaks (>380 nm) are considered humic-like substances
[17] (Chen et al. 2003). In fact, low wavelengths are as-
sociated to simple structural components, a low degree of
aromatic polycondensation and a low level of conjugated
chromophores. On the other hand, the shift towards
longer wavelengths is the result of an increased probabil-
ity of π-electron transitions between the singlet state and
ground state occurring in highly aromatic macromole-
cules [8]. The longer wavelengths values found in the
OMW spectrum are likely imputable to the massive
presence of polyphenols, whereas, as for SS, the silage
process performed by placing the entire green plant in a
silo after being chopped and packed and allowing it to
undergo anaerobic fermentation would degrade the
vegetal materials producing simpler organic compounds
that fluoresce at lower wavelengths. Very different re-
sults are evident when considering spectra of digestates.
D1 and D2 samples (Figure 5) show three peaks at 340,
390 (of highest intensity) and at 440 nm thus reflecting
the main peaks of AIR and BCS, whereas D3, D4 and D5
spectra (Figure 6) are dominated by maxima at 440 nm
and two shoulders at 390 and 520 thus reflecting the
main features of OMW spectrum. A comparison between
D1 and D4 spectra with their corresponding fresh bio-
masses as reported in Figure 7 and Figure 8 respectively
highlights these evidence and allows to conclude that,
similarly to FTIR results, synchronous-scan fluorescence
spectra of digestates obtained in a real co-digestion plant
Figure 5. Synchronous-scan spectra of D1(a) and D2 (b).
Figure 6. Synchronous-scan spectra of D3, D4 and D5.
Figure 7. Comparison between synchronous-scan spectra of
D1 and its corresponding biomasses (a) beef cattle manure;
(b) agro-industrial residues; (c) D1; (d) sor ghum silage.

Qualitative Characterization and Differentiation of Digestates from Different Biowastes Using FTIR and 88
Fluorescence Spectroscopies
Figure 8. Comparison between synchronous-scan spectra of
D4 and its corresponding biomasse: (a) Olive mill wastewa-
ters; (b) D4; (c) Sorghum silage; (d) Olive residues; (e)
Agro-industrial residues.
“inherit” the main fluorescence peaks and consequently
the main chemical characteristics of the organic wastes
from which they are produced.
4. Conclusions
FTIR and fluorescence spectroscopies are simple and
reliable tools which succeeded in qualitatively character-
izing and differentiating digestates obtained from bio-
masses of different chemical composition. Results pro-
vided evidence of peculiar characteristic related to the
chemical composition of biomasses from which they are
produced. Similar FTIR and fluorescence spectra were
found for digestates obtained loading into the digestion
plant organic mixtures with a quite constant composition.
During the olive oil season, digestates revealed the pres-
ence of characteristic features deriving from olive oil
production wastes. On the basis of our evidence we con-
cluded that digestates produced in a full-scale co-digestion
plant “inherit” the main chemical character of the organic
wastes from which they are produced.
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Fluorescence Spectroscopies
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