Qualitative Characterization and Differentiation of Digestates from Different Biowastes Using FTIR and Fluorescence Spectroscopies

doi:10.4236/jep.2011.21009

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Journal of Environmental Protection, 2011, 2, 83-89

doi:10.4236/jep.2011.21009 Published Online March 2011 (http://www.SciRP.org/journal/jep)

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.

Email: [email protected]

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

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

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

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

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

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.

REFERENCES

[1] J. M. S. Mata-Alvarez and P. Llabres, “Anaerobic Diges-

tion of Organic Wastes. An Overview of Research

Achievements and Perspective,” Bioresource Technology,

Vol. 74, No. 1, 2000, pp. 3-16.

doi:10.1016/S0960-8524(00)00023-7

[2] J. J. Lay, Y. Y. Li, T. Noike, J. Endo and S. Ishimoto

“Analysis of Environmental Factors Affecting Methane

Production from High Solids Organic Wastes,” Water

Science and Technology, Vol. 35, No. 6-7, 1997, pp.

493-500 doi:10.1016/S0273-1223(97)00560-X

[3] S. Filidei, C. Masciandaro and B. Ceccanti “Anaerobic

Digestion of Olive Mill Effluents: Evaluation of Waste-

water Organic Load and Phytotoxicity Reduction,” Water,

Air and Soil Pollution, Vol. 145, No. 1-4, 2003, pp. 79-94.

doi:10.1023/A:1023619927495

[4] M. Hamdi, “Toxicity and Biodegradability of Olive Mill

Wastewaters in Batch Anaerobic Digestion,” Applied

Biochemistry and Biotechnology, Vol. 37, No. 2, 1992, pp.

155-163. doi:10.1007/BF02921667

[5] M. D. Greca, P. Monaco, G. Pinto,A. Pollio, L. Previtera

and F. Temussi, 2001. “Phytoxicity of Low-Molecular

Weightphenols from Olive Mille Wastewaters,” Bulletin

of Environmental Contamination and Toxycology, Vol.

67, 2001, pp. 352-359. doi:10.1007/s001280132

[6] R. Borja, J. Alba and C. J. Banks, “Impact of the Main

Phenolic Compounds of Olive Mill (OMW) Wastewaters

on the Kinetics of Acetoclastic Methanogenesis,” Process

Biochemistry, Vol. 32, 1997, pp. 121-133.

doi:10.1016/S0032-9592(96)00055-6

[7] N. Senesi, T. M. Miano, M. R. Provenzano and G. Bru-

netti, “Spectroscopic and Compositional Comparative

Characterization of IHSS Reference and Standard Humic

Acids of Various Origin,” The Science of the Total Envi-

ronment, Vol. 81, No. 82, 1989, pp. 143-156.

doi:10.1016/0048-9697(89)90120-4

[8] N. Senesi, T. M. Miano, M. R. Provenzano and G. Bru-

netti, “Characterization, Differentiation and Classification

of Humic Substances by Fluorescence Spectroscopy,”

Soil Science, Vol. 152, No. 4, 1991, pp. 259-271.

doi:10.1097/00010694-199110000-00004

[9] N. Senesi, T. M. Miano and M. R. Provenzano, “Fluores-

cence Spectroscopy as a Mean to Distinguish Fulvic and

Humic Acids from Dissolved and Sedimentary Aquatic

and Terrestrial Sources,” Humic Substances in the

“Aquatic and Terrestrial Environment” Lecture Notes in

Earth Sciences, Vol. 33, Springer-Verlag, Berlin, 1991,

pp. 63-73.

[10] M. R. Provenzano, N. Senesi and G. Piccone, “Thermal

and Spectroscopic Characterization of Organic Matter

from Municipal Solid Wastes,” Compost Science &

Utilization, Vol. 6, 1998, pp. 67-73.

[11] M. R. Provenzano, S. C. de Olivera, M. R. S. Silva and N.

Senesi, “Assessment of Maturity Degree of Composts

from Domestic Solid Wastes by Fluorescence and Fourier

Infrared Spectroscopies,” Journal of Agricultural and

Food Chemistry, Vol. 49, 2001, pp. 885-892.

doi:10.1021/jf0106796

[12] X. Gomez, M. J. Cuetos, B. Tartarosky, M. F. Marti-

nez-Nunez and A. Moran, “A Comparison of Analytical

Techniques for Evaluating Food Waste Degradation by

Anaerobic Digestion,” Bioprocess and Biosystem Engi-

neering, Vol. 33, 2010, pp. 427-438

doi:10.1007/s00449-009-0343-8

[13] S. Piccinini, C. Fabbri and M. Soldano, “Monitoring and

Assessment of Three Biogas Plants in Italy,” Proceeding

of the International Conference “Biogas science”, Ger-

many, 9-11 December 2009.

[14] APHA “Standard Methods for the Examination of Water

and Wastewater,” 18th Ed., American Public Health

Qualitative Characterization and Differentiation of Digestates from Different Biowastes Using FTIR and

Fluorescence Spectroscopies

Association, Washington, DC, USA, 1992.

[15] IRSA CNR “Metodi Analitici per le Acque,” Quaderni,

Istituto Politecnico e Zecca dello Stato, Rome, Italy,

1994.

[16] T. M. Miano and N. Senesi, “Synchronous Excitation

Fluorescence Spectroscopy Applied to Soil Humic Sub-

stances Chemistry,” The Science of the Total Environ-

ment, Vol. 117, No. 118, 1992, pp. 41-51.

doi:10.1016/0048-9697(92)90071-Y

[17] X. Melamane, R. Tandlich and J. Burgess, “Anaerobic

Digestion of Fungally Pre-Treated Wine Distillery Waste-

water,” African Journal of Biotechnology, Vol. 6, No. 17,

2007, pp. 1990-1993

[18] W. Chen, P. Westerhoff, J. A. Leeheer and K. Booksh,

“Fluorescence Excitation-Emission Matrix Regional In-

tegration to Quantify Spectra for Dissolved Organic Mat-

ter,” Environmental Science & Technology, Vol. 37, 2003,

pp. 5701-5710. doi:10.1021/es034354c