Structural characterization of olive mill waster-water after aerobic digestion using elemental analysis, FTIR and 13C NMR

Process Biochemistry 40 (2005) 2615–2622 www.elsevier.com/locate/procbio

Structural characterization of olive mill waster-water after aerobic digestion using elemental analysis, FTIR and 13C NMR Mohamed Hafidia,*, Soumia Amira, Jean-Claude Revelb a

Unite´ Sol et Environnement (Lab. Eco-Ve´ge´tale), Faculte´ des Sciences Semlalia, De´partement de Biologie, BP/2390 Marrakech, Maroc b Equipe Agronomie, Environnement et Ecotoxicologie (A2E), Ecole Nationale Supe´rieure Agronomique, Auzeville-Tolosane, BP/107 Toulouse, France Received 19 November 2003; accepted 19 June 2004

Abstract Aerobic digestion of olive mill waster-water (OMW) has been conducted under various medium conditions to determine the best treatment involving good stabilisation and maturity of these residues. Analysis by various chemical methods of elemental analysis, FTIR and 13C NMR spectroscopies show the high content of raw olive mill waster-water of the less condensed structures of phenols, organic acids, alcohol, fatty acids and simple sugars. Aerobic digestion involved an humification process through two mechanisms—biodegradation and polycondensation. These mechanisms were highly influenced by medium conditions. In the cases of natural acid pH, either in the presence of soil microflora or the yeast Saccharomyces cerevisiae, aerobic digestion has been less developed due to the antimicrobial effect of free phenol. Neutralization of pH enhances the development of microbial activity and the humification process seems greatly influenced by means of neutralization. In the case of neutralization by lime, the intense oxidation of organic compounds occurred and humification involved polyphenol condensation. While, in the case of neutralization by phosphate, more oxidation of sugars has been observed, and polycondensation in contrast developed through N-linkage. Accordingly, treatment of olive mill waste-waters by soil micro-flora with neutralization of pH by phosphate could be considered the best treatment that allows good stabilisation of organic matter and high preservation of nitrogen in humic form. This treatment corrects further the deficiency of the two elements phosphorus and nitrogen. # 2004 Elsevier Ltd. All rights reserved. Keywords: Olive mill waste-water; Aerobic digestion; Humification; Neutralization; Lime; Phosphate; Soil micro-flora; Yeasts

1. Introduction Morocco is a Mediterranean country in which a part of the economy is based on olive culture and the production of olive oil. It has been subjected to numerous difficulties in the disposal of olive mill waster-water OMW produced after trituration [1–3]. A ton of triturated olives produces about 500 to 1200 kg of effluents [4]. This waste causes large-scale environmental problems because of its high polluting power due to a high organic load and a high antimicrobial activity exerted mainly by various phenolic compounds [5,6]. The organic fraction includes sugars, tannins, polyphenols, polyalcohols, pectins and lipids. Some of these substrate * Corresponding author. E-mail address: [email protected] (M. Hafidi). 0032-9592/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2004.06.062

including sugars and polyalcohols can serve as carbon and energy sources for growth of microorganisms. However, its high content of monomeric and polymeric phenols involves high chemical and biochemical oxygen demands (COD and BOD) [7]. Monomeric phenols exhibit phytotoxic effects [8] and antimicrobial activity [9]. Polymeric phenols have a lignin-like structure as their most recalcitrant fractions and are mainly responsible for the typical colour of OMW [9]. The main difficulties recorded of treatment of olive mill effluents are related to: (a) seasonal operation; (b) high territorial scattering and high organic loading composed mainly of long-chain fatty acids and phenolic compounds which are difficult to biodegrade [10]. A preliminary treatment of these effluents and their valuation is recommended. A number of OMW treatment methods have been employed in recent years and these can be divided

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into physico–chemical methods or biological methods. Physico–chemical methods including flocculation, ultrafiltration and thermal concentration, are generally very expensive and/ or unable to solve the problem completely of the need to dispose of a sludge derived from the process. Biological methods are based on production of proteins, activated carbons, poly-hydroxy-b-butyrates, exopolysaccharides, composting, anaerobic and aerobic digestion [7,9]. The latter methods have certain clear benefits due to the potential utilisation of their bioproducts. Most studies have been focused on bioremediation as a means of reducing the polluting effect of OMW. Removal of the monocyclic aromatic compounds from OMWs greatly reduces the toxicity of this waster-water. Aerobic treatment has a great efficiency in degrading polyphenolic compounds [11,12]. This great decrease has been explained either by microbial metabolism of phenols as an energetic source or their incorporation during humic substance neoformation [2,7,9,13,14]. Humification is widely recognised as a process allowing a turn-over of organic matter and has been suggested in numerous studies as a good index of stability and for assessing of the agronomic value of organic residues intended for agricultural manuring [15–17]. Accordingly, for the purpose of investigating the importance of humification as a detoxification process involving a transformation of polyphenols from their free forms to more stable humic structures, aerobic digestion of olive mill waster-water has been conducted. Different preliminary treatments have been performed to determine the best medium conditions for the humification process. Medeci et al. [18] has proposed neutralization of pH as a mean of avoiding the antimicrobial effect of polyphenols following their transformation to phenate form. Therefore, in this study, aerobic digestion has been carried out in the presence of soil micro-flora in natural acid pH or after neutralization by lime or natural phosphate, as well as after inoculation by yeasts (Saccharomyces cerevisiae) at natural acid pH. Various chemical techniques to follow humification process (elemental analysis, 13C NMR, FTIR) have been applied to olive mill waster-water at raw state RW and after 10 days of aerobic digestion of various trials.

Table 1 Principal physico–chemical characteristics of the raw olive mill wasterwater Parameters

Values

pH Electric conductivity (ms/cm) Dry matter (g/L) Ash (g/L) Chemical oxygen demand (g of O2/L) Organic carbon (% M.S.)

4.7 16.2 85.9 16.6 161  5 47  0.27

Total phenols (g/L)a Total Kjeldahl Nitrogen (mg/L) Ratio C/N

4  0.01 588  0.04 67.4

N–NH4+ (mg/L) N–NO3 (mg/L)

108 126

Total phosphorus (mg/L)

110

a

Phenols are expressed as catechin.

antimicrobial potential. A dilution of the raw olive mill waster-water with distilled water of 1/10 was applied [19]. To avoid any contribution of the natural flora of olive mill waster-water, sterilisation of the solution was carried out in an autoclave at 120 8C during 15 min was carried out. Two types of inoculation were applied: 1) Inoculation by soil micro-flora: 2 l of sterilized olive mill waster-water are mixed with 5 g soil, and a trial was conducted at natural acid pH (Trial 1: E1) or at pH neutralized by addition of lime CaCO3 (Trial 2: E2) or by addition of 50 g of natural phosphate taken from Khouribga city (Morocco) (Trial 3: E3). Microbiological characterization of soil inocula was achieved and results are expressed as colony forming units CFU/g fresh soil: fungus = 15.106 UFC/g fesh soil; yeasts = 11.106 UFC/g fesh soil; mesophilic flora = 24.106 UFC/g fesh soil. 2) Inoculation by baker’s yeasts (Saccharomyces cerevisiae): 1 g of yeast are dissolved in 2 l sterilized olive mill waster-water and the trial was carried out at natural acid pH (Trial 4: E4). 2.3. Aerobic digestion

2. Material and methods 2.1. Olive mill waster-water sampling The olive mill waster-water studied were taken from a semi-industrial unit of trituration of olive by pressure in Marrakech (Morocco), during the oliveculture season 2000/ 2001. Table 1 illustrates the various physico–chemical characteristics of these effluents [14]. 2.2. Preliminary preparation of olive mill waster-water Olive mill waster-water cannot be directly treated since it is high in organic matter, mainly in phenols with a high

Attempts were supervised for 10 days in polyethylene mini-digestors with a capacity of 2 l (diameter: 11 cm; height: 24 cm), equipped permanently with air flow, which has been adapted after numerous trials to obtain optimal microbial activity. The experiment was conducted at ambient temperature of about 25  3 8C. Sampling of olive mill waster-water were carried out before the treatments (diluted and sterilized raw olive mill waster-water = RW) and after various attempts I, II, III, IV (end of treatments = E1; E2; E3; E4). The lyophylised samples of raw olive mill waster-water RW and of solutions obtained after various treatments E1; E2; E3; E4 were analysed by various chemical techniques:

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2.4. Elemental analysis Elemental analysis was performed for C, H, N in a Fison Carlo Erba EA 1110, and for O in a Carbo Erba 1106. 2.5. FTIR spectroscopy Infrared (FTIR) spectra were recorded on KBr pellets (250 mg of dried KBr and 2 mg of lyophilized samples pressed under vacuum as described by Cross [20] with a FTIR Perkin Elmer 1600 spectrophotometer over the 4000– 400 cm 1 range, at a rate of 16 nm/s. 2.6.

13

C NMR spectroscopy

13

C NMR spectra with 1H broadband decoupling were recorded at 75.469 MHz with a Bruker AM WB 300 MHz Spectrometer. A 100 mg of each crushed sample was dissolved in 3 ml of NaOD/D2O (0.5 M) and the solution obtained filtered. The spectra were obtained using inverse-gated-decoupling to suppress the nuclear Overhauser enhancement in order to obtain quantitative results [21]. Acquisition time was 0.98 sec, relaxation delay was 1.8 sec, pulse of 35 degrees, total acquisition time was 72 h. Free induction decays were processed by applying 50 Hz linebroading and baseline corrections. The spectral area integration (0–50 ppm, 50–110 ppm, 110–165 ppm, 165– 200 ppm) was carried out using a Program ‘Win NMR software, Bruker’.

3. Results and discussion The elemental composition of olive mill waste-waters at raw state (RW) and those treated by soil micro-flora at natural acid pH (E1), at pH neutralised by lime (E2) or at pH neutralised by natural phosphate (E3) and those treated by an inoculum of yeasts at natural acid pH (E4) are illustrated in Table 2. Raw olive mill waster-water in comparison to other trials (E1– 4) shows a high content of C and H, but a low content of

Table 2 Elemental composition of olive mill waster-water at raw state (RW) and after aerobic digestion at various conditions of medium (E1; E2; E3; E4) Trial

RW E1 E2 E3 E4

C

55.64 48.59 44.74 47.00 46.66

H

7.57 6.16 5.83 5.70 6.39

O

34.69 42.74 47.32 44.41 44.49

N

2.11 2.52 2.11 2.89 2.46

Atomic ratio O/C

C/N

C/H

0.47 0.66 0.79 0.71 0.72

30.81 22.50 24.73 18.98 22.18

0.61 0.66 0.64 0.69 0.61

RW: raw olive mill waster-water; E1: treated by soil micro-flora at natural acid pH; E2: treated by soil micro-flora at pH neutralized by lime; E3: treated by soil micro-flora at pH neutralized by natural phosphate; E4: treated by an inoculum of yeasts at natural acid pH.

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O and N elements. The ratios O/C and C/H exhibit low values and the C/N ratio is much higher. Basing on FTIR spectra of (RW) and the others trials (E1– 4) (Fig. 1) and the assignments of the main recorded bands reported in numerous studies [22–24] (Table 5), raw olive mill waster-water (RW) is characterized by a high content of phenols, alcohol and organic acids demonstrated by high absorbance in range of 4000–3600 cm 1 and a band around 3400 cm 1. Aliphatic compounds are also highly predominant in raw olive mill waste-water and presented by a strong peak at 2922 and at 2855 cm 1 corresponding mainly to a long lipidic chain. The high peak around 1711 cm 1 could be attributed also to free carboxyl groups of fatty acid or simple sugars. According to the signals in 13C NMR spectra of (RW) and of the others trials (E1– 4) (Fig. 2), and their main chemical groups attribute reported in numerous studies [22–24] (Table 4) and area integration of spectra (Table 3), raw olive mill waster-water (RW) show a similar carbon distribution between long polymethylene chains, alkyl carbon substituted by oxygen attributed mainly to polysaccharides and aromatic carbon mainly benzenic and phenolic compounds. Martinez and Garrido [25] reported that the organic matter of olive mill waste-water composed primarily of simple sugars (13–55%), oil (1–10%), proteines (8–16%), organic acids (3–10%), polyalcohols (3–10%) and polyphenols (2–15%). Therefore, results suggest the high content of raw olive mill waster-water of less condensed structures as organic acids, phenols, alcohol, simple sugars and fatty acids and their lower content in nitrogenic structures compared to other organic wastes [26,27]. This could explain the acid pH of medium (4.71) and agree with the high content of these effluents on polyphenols in the order of 4.02 g/l (Table 1). After 10 days of aerobic digestion, the various trials (E1– 4) showed a less content of C and H elements, but a high content of O and N (Table 2). The FTIR spectra of all trials (Fig. 1) showed a high decrease of absorbance at 2922 and 2855 cm 1 assigned to aliphatic structures and also a disappearance of absorbance around 4000–3600 cm 1 and at 1711 cm 1 correspond to less condensed subunits with free –OH and –COOH groups. In parallel, an intense absorbance of the bands centred at 3400 and around 1620–1634 cm 1 exhibit the intensity of more condensed aromatic structures and N-containing moities. As well as, an arise of structures around 1040 cm 1 corresponds to highly polymerized aliphatic structures as polysaccharides. The 13C NMR spectra of different treatments (E1– 4) show a high decrease of C-alkyl structure 0–50 ppm in parallel with the relative increase of intensity of O-alkyl 50–100 ppm, aromatic 140–160 and carboxylic structures 168–190 ppm (Fig. 2, Table 3). These changes during aerobic digestion could be explained by a strong decomposition of less condensed structures as readily biodegradable compounds, and by that more resistant and polycondensed structures are preserved or newformed [15,28–30]. The microbial oxidation of side

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Fig. 1. FTIR spectra of olive mill waster-water at raw state (RW); after aerobic digestion in presence of soil micro-flora at natural acid pH (E1), at pH neutralized by lime (E2) or neutralized by natural phosphate (E3); after aerobic digestion in presence of inoculum of yeasts at natural acid pH (E4).

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Fig. 2. 13C NMR spectra of olive mill waster-water at raw state (RW); after aerobic digestion in presence of soil micro-flora at natural acid pH (E1), at pH neutralized by lime (E2) or neutralized by natural phosphate (E3); after aerobic digestion in presence of inoculum of yeasts at natural acid pH (E4).

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Table 3 Structural carbon distribution in olive mill waster-water at raw state (RW) and after aerobic digestion at various condition of medium (E1; E2; E3; E4) Trials

C-alkyla

O-alkyl/N-alkyla

Aromatica

Carboxylica

RW E1 E2 E3 E4

28.04 9.42 25.95 8.35 9.52

25.21 30.10 29.61 23.14 27.51

31.20 37.87 28.43 44.03 37.61

15.55 22.62 16.01 24.48 25.36

RW: raw olive mill waster-water; E1: treated by soil micro-flora at natural acid pH; E2: treated by soil micro-flora at pH neutralized by lime; E3: treated by soil micro-flora at pH neutralized by natural phosphate; E4: treated by an inoculum of yeasts at natural acid pH. a Values are expressed as percentages of the whole spectrum area.

alkyl chain to form more oxidized subunits may occurred [15,28–30]. The possibility of polymerization of phenols and tannins into condensed structures of high molecular weight was also previously observed occurring during humification process [7,9,15,29]. The polymerization of phenols has been proposed in a previous study [14] to explain the difference between the results of a quantitative experiment by a Folin–Cioccalteu method and the qualitative HPLC technique for analysis of phenols [2,14]. The qualitative method HPLC revealed the disappearence of phenols and the presence only of tracks in the case of all trials (except E1), however, the quantitative method demonstrated that the decrease of phenol was not more than 63%. As well as, the increase of pH from 5–7 to 7.7–9.0 occurring after aerobic digestion that has been explained by numerous authors as due to biodegradation of COOH and OH groups of organic compounds or also through release of NH4+ after degradation of proteins [2,9,14], could also be due to integration of these groups through polycondensation during neoformation of humic structures. Esra et al. [31] showed basing on HPLC that mainly substances which contain both phenolic and carboxyl groups were partially or even totally adsorbed during OMW treatment in contrast to those compounds that have only one phenolic or carboxyl group such as tyrosol and veratric acid. Casa et al. [32] explained the reduction in total phenols and ortho-diphenols respectively by 65 and 86% in OMW treated with laccase, by the phenolic polymerization as revealed by size-extaction chromatography. However, the changes occurred in various trials (E1–4) during aerobic digestion exhibit some differences. Elemental composition of the trial performed by soil micro-flora at acid pH (E1) show less changes compared to other treatments (Table 2). In fact, numerous authors suggested acid pH of olive mill waste-water, resulting from their high content of polyphenols, as antimicrobial factors [5,6,33]. The acid pH of medium avoids the development of microbial activity and organic matter decomposition. 13C NMR analysis of (E1) show a high content of O-alkyl structures around 70 ppm and a resonance in the carboxyl area around 164 ppm attributed to phenolate and particulary intense resonance around 174 ppm arised from esters. The aromatic

carbon shows an intense resonance around of 143 ppm corresponding more to benzenic structures. Therefore, the biodegradation of phenols and sugars appear less developed and polycondensation has occurred with ester linkage supported by a resonance at 78 and at 174 ppm compared to other treatments (Fig. 2). A low N-linkage has been shown by less intense resonance around 160 and 180 ppm, in spite of an increase of N content. In fact, a previous study show a high content of mineral nitrogen compared to organic nitrogen, in the case of the trial conducted with soil micro-flora at natural acid pH in contrast to other treatments [14]. This could be supported in FTIR spectra by a high absorbance around 1395 cm 1 attributed to nitrate. Therefore, aerobic digestion seems affected by acid pH of medium where a low microbial activity occurred. The trial with pH neutralized by lime (E2) exhibits a much increase of O content and O/C ratio and a high decrease of C and H elements, but a low increase of C/H ratio (Table 2). The C/N ratio shows a decrease, without significant change in N content (Table 2). The high increase of O content involved a decrease of relative intensity of other elements. FTIR spectra of E2 show an intense absorbance centred around 1609 cm 1 instead of 1634 cm 1 (Fig. 1). This may indicate a high intensity of aromatic structures and a lower presence of N-containing moities. These variations could support the strong microbial oxidation or mineralisation of organic structures and the strong oxidative coupling of biodegradation products in this case (E2) compared to other treatments. The protein structures have been subjected to high mineralisation than the incorporation to neoformed humic structures. Esra et al. [31] consider the removal of nutrient substances such as nitrogenous compounds 50–70% after lime pretreatment as a disadvantage of this process in a case using treated OMW as a fertilizer for agricultural purpose. The 13C NMR analysis of E2 shows a low decrease of alkyl carbon and aromatic carbon, but the significant increase of intensity of O-alkyl carbon (Table 3). The high intensity of O-alkyl carbon support the presence of more oxidized products. While, the high content of alkyl carbon in the end of aerobic digestion in this trial compared to other treatments could be explained by ring cleavage of oxidized aromatic products b-keto-adipate pathway [34]. However, a high resonance around 164 ppm (Fig. 2) corresponding to phenolate suggests a high degree of polyphenolic polycondensation rather than ring cleavage of polyphenolic entities [13,34]. Therefore, incorporation of microbially derived units into the humic macromolecules could be suggested to explain this high content of alkyl carbon [35,36]. The trial conducted at pH neutralized by natural phosphate (E3) shows a greater increase of N element and C/H ratio and a greater decrease of C/N ratio compared to other treatments (Table 2). The O content and O/C ratio also exhibit an important increase. The high values of O/C and C/ H ratios may be attributed to the predominence of aromatic structures [15]. The low C/N ratio and a high N content may indicate a greater incorporation of nitrogen-containing

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moities during neoformation of humic substances suggested previously by other authors [37]. FTIR spectra of (E3) show a very weak absorbance around 1040 cm 1 and 1267 cm 1 attributed to the low presence of polysacharidic structures (Fig. 1). The intense absorbance around 1634–1505 cm 1 has arisen from aromatic structures or amides. In 13C NMR spectra in the case of E3, alkyl carbon substituted by oxygen decreased in contrast to other treatments, but a high increase of substituted aromatic carbon 120–160 and amide carbon around 180 ppm occurred (Fig. 2). Neutralization by natural phosphate enhances microbial attack on carbohydrates, mainly sugars compounds. A high resonance of N-containing structures could be explained by microbial metabolism having had a limited need for protein compounds. In addition, the strong degradation of O-alkyl compounds could contribute to the relative intensity of N-containing structures. Therefore, a polycondensation has occurred mainly through nitrogen linkage. The trial conducted with an inoculum of yeasts at natural acid pH (E4) shows also an increase of O and O/C ratio but a weak increase of C/H and low decrease of C/N ratios (Table 2). In this trial, a high intensity of peptidic links is demonstrated by intense resonance around 140–160 and particularly around 180 ppm (Fig. 2). These N-containing structures could be attributed to derived tissue entities of yeasts incorporated to the humic structures [9]. Hamdi and Ellouz [9] has observed during the use of yeast for production of protein of unicellular micro-organisms, that the polyphenolic compounds had been adsorbed to yeast. They proposed the high affinity of polyphenolic compounds to complexation with protein or nitrogen containing compounds. Accordingly, the trials of aerobic digestion conducted after pH neutralization (E2 and E3) exhibited a particular tendency compared to those carried out at natural acid pH (E1 and E4). Medeci et al. [18] showed that at neutralized or slightly alkaline pH (7.4–7.6), the phenols moities pass to phenates and loose their antimicrobial character. This allowed the development of an intense microbial activity. Thus, neutralization of pH of medium before aerobic digestion enhances the development of microbial activity, but the mean of neutralization has a great influence on structures selected for microbial degradation and on compounds incorporated during humic substance network. In the case of neutralization with lime, A more oxidative coupling through significant phenol oxidation resulting Table 4 Resonance signals in

13

C NMR spectra and attributed chemical groups

Signal (ppm)

Attributed chemical groups

0–50 50–110 110–130 130–145 145–160 160–200

Paraffinic C in alkyl chains Aliphatic carbons substituted by oxygen and nitrogen Unsubstituted aromatic C Carbon-substituted aromatic carbons Oxygen or nitrogen substituted aromatic carbons Carboxylic carbons, ester or amide

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Table 5 The main absorbance bands in FTIR spectra and their assignments Bands and peaks (cm 1) Assignments 3600 cm 3300–3400 cm

1

2920–2930 cm 1725 cm 1630–1650 cm

1

1600 cm 1520–1550 cm 1400 cm 1200–1100 cm

1

1

1 1

1 1 1

Free –OH of phenols or carbohydrates H-bonded OH groups of alcohols, phenols and organic acids, as well as H-bonded N–H groups C–H stretching of alkyl structures C=O of free COOH and aldehydes Aromatic C=C, C=O in amide (I), ketone and quinone groups Aromatic C=C, COO , C=O Amide (II) OH of phenols, COO , -CH3 Amide III, –C–O–C of carbohydrates, aromatic ethers, Si-O-C groups

highly reactive free-radicals that promote polymerisation of phenolic substrates [38]. The polymerised structures display humic like proprieties and a lower bioavailability than the free phenols or monocyclic aromatic compounds [39]. In contrast, neutralization of pH by phosphate allows further oxidation of carbohydrates and enhances polycondensation through peptidic linkage. The nitrogen and phophate are often lacking in olive mill waste-water (Table 1). Thus, neutralization using phosphate is better to correct also the deficiency of the two elements [40,41]. This allows us to suggest that the mean of pre-treatment exhibited a high influence on both mechanisms biodegradation and polycondensation nature or humification.

Acknowledgements This work was supported JER 6013 associated to the AUPELF-UREF and a Scholarship Program of Research Training from the French-Speaking University Agency.

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