Prevention of volatile fatty acids production and limitation of odours from winery wastewaters by denitrification

ARTICLE IN PRESS WAT E R R E S E A R C H

41 (2007) 2987 – 2995

Available at www.sciencedirect.com

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Prevention of volatile fatty acids production and limitation of odours from winery wastewaters by denitrification Andre´ Boriesa,, Jean-Michel Guillotb, Yannick Sirea, Marie Couderca, Sophie-Andre´a Lemairea, Virginie Kreimb, Jean-Claude Rouxb a

Institut National de la Recherche Agronomique, Unite´ Expe´rimentale de Pech Rouge, 11430 Gruissan, France Laboratoire de Ge´nie de l’Environnement Industriel, Ecole des Mines d’Ale`s, 30319 Ale`s Cedex, France

b

art i cle info

ab st rac t

Article history:

The effect of the addition of nitrate to winery wastewaters to control the formation of VFA

Received 21 December 2006

in order to prevent odours during storage and treatment was studied in batch bioreactors at

Received in revised form

different NO3/chemical oxygen demand (COD) ratios and at full scale in natural

16 February 2007

evaporation ponds (2  7000 m2) by measuring olfactory intensity. In the absence of nitrate,

Accepted 9 March 2007

butyric acid (2304 mg L1), acetic acid (1633 mg L1), propionic acid (1558 mg L1), caproic

Available online 30 April 2007

acid (499 mg L1) and valeric acid (298 mg L1) were produced from reconstituted winery

Keywords:

wastewater. For a ratio of NO3/COD ¼ 0.4 g g1, caproic and valeric acids were not formed.

Volatile fatty acids

The production of butyric and propionic acids was reduced by 93.3% and 72.5%,

Odours

respectively, at a ratio of NO3/COD ¼ 0.8, and by 97.4% and 100% at a ratio of NO3/

Nitrate

COD ¼ 1.2 g g1. Nitrate delayed and decreased butyric acid formation in relation to the

Denitrification

oxidoreduction potential. Studies in ponds showed that the addition of concentrated

Winery wastewaters

calcium nitrate (NITCALTM) to winery wastewaters (3526 m3) in a ratio of NO3/COD ¼ 0.8

Ponds

inhibited VFA production, with COD elimination (94%) and total nitrate degradation, and no final nitrite accumulation. On the contrary, in ponds not treated with nitrate, malodorous VFA (from propionic to heptanoı¨c acids) represented up to 60% of the COD. Olfactory intensity measurements in relation to the butanol scale of VFA solutions and the ponds revealed the pervasive role of VFA in the odour of the untreated pond as well as the clear decrease in the intensity and not unpleasant odour of the winery wastewater pond enriched in nitrates. The results obtained at full scale underscored the feasibility and safety of the calcium nitrate treatment as opposed to concentrated nitric acid. & 2007 Elsevier Ltd. All rights reserved.

1.

Introduction

The heavy biodegradable organic load that characterises food industry wastewaters has multiple impacts on treatments and on the environment: excessive sludge production, oxygen requirements and nutrient deficiency linked to aerobic processes, risk of acidification as a result of anaerobic digestion and long-term storage of seasonal wastewaters in order to prevent overload. The noxious odours during storage and treatment of food industry wastewaters are a major Corresponding author. Tel.: +33 4 68 49 44 00; fax: +33 4 68 49 44 02.

E-mail address: [email protected] (A. Bories). 0043-1354/$ - see front matter & 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2007.03.022

environmental problem today (ADEME, 2005). Winery wastewaters clearly fall within this framework (Guillot et al., 2000; Bories, 2006; Chrobak and Ryder, 2006). Worldwide wine production—a total of 26.5  109 Lyear1 (OIV, 2005), of which 69% takes place in Europe—consumes approximately 0.8 L water L1 wine and generates large volumes of wastewater on a seasonal basis (Rochard et al., 1996; Duarte et al., 1998; OIV, 1999; Picot and Cabanis, 1998; ITV, 2000; Rochard, 2005). Natural evaporation in ponds, a rustic and economical technique (Duarte et al., 1998; TV, 2000; Le Verge and Bories,

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41 (2007) 2987– 2995

2004), well adapted to discharge variations and seasonal production, is a widely used treatment method in the largest wine production region in France, the Languedoc-Roussillon (approximately 1.6  109 L of wine/year, 10% of the overall European production) where 179 evaporation ponds were identified (Lambert and Lecharpentier, 2006, personal communication). Volatile fatty acids form the major products resulting from the fermentation of carbon compounds in winery wastewaters (Bories et al., 2005; Bories, 2006), and are responsible for characteristic foul odours as a result of their low olfactory perception threshold (Le Cloirec et al., 1991). Other odorous compounds such as esters, mercaptans and aldehydes were also identified from winery wastewater treated in ponds (Guillot et al., 2000). The degradation of VFA by denitrifying microorganisms was studied and used to eliminate nitrate in wastewaters (Min et al., 2002; Elefsiniotis et al., 2004; Sponza and Atalay, 2004). More generally, the denitrification treatment of wastewaters has been investigated with various carbon sources, microbial systems and models (Bolzonella et al., 2001; Chiu and Chung, 2003; De Lucas et al., 2005; Sage et al., 2006). However, the outlook for curative treatment of VFA by means of denitrification in winery wastewater evaporation ponds appears to be compromised because of the massive quantities of VFA already accumulated and the emission of odours previous and/or subsequent to the curative treatment. On the other hand, the prevention of VFA formation by orienting the degradation of organic matter in wastewater into odourless products through anaerobic respiration with nitrate as the electron acceptor (denitrification) was studied in winery wastewaters to which nitrate had been added in the form of concentrated nitric acid (Bories et al., 2005). The stoichiometric requirement in nitrate depends on the degree of carbon reduction, according to the following Eq. (1): Cm Hn Op þ 0:4ð2m þ 0:5n  pÞNO3  ! mCO2 þ 0:2ð2m þ 0:5n  pÞN2 þ ½p þ 0:8ð2m þ 0:5n  pÞ  2mH2 O þ 0:4ð2m þ 0:5n  pÞOH :

(1)

Since the chemical oxygen demand (COD) is a function of the degree of carbon reduction according to the following Eq. (2): Cm Hn Op þ 0:5ð2m þ 0:5n  pÞO2 ! mCO2 þ 0:5nH2 O;

(2)

the nitrate requirement in relation to the COD is defined as a molar ratio NO3/O2 ¼ 0.8 mol mol1, resulting in a weight ratio of NO3/COD ¼ 1.55 g g1. In this study, the influence of the different NO3/COD ratios in relation to VFA formation from a winery wastewater was studied, and the effectiveness of the preventive treatment of a pond using calcium nitrate was assessed by measuring olfactory intensity in addition to physico-chemical analysis. The addition of nitrate in increasing quantities to reconstituted winery wastewater was studied on the basis of the evolution of the redox potential and VFA formation kinetics in order to underscore competition between acidogenic fermentation pathways and denitrification, and to determine the NO3/COD ratio required to inhibit VFA formation.

Since the general aim was to prevent odours, it was necessary to study and to compare winery wastewater evaporation ponds both treated and not treated with calcium nitrate under real conditions, with discharges over a long period of time and involving microbial systems.

2.

Materials and methods

2.1.

Winery wastewaters

For the study of the NO3/COD ratios, reconstituted winery wastewater was prepared with a mixture of red grape must and red wine, in equal proportions and then diluted 10-fold with distilled water. The composition is presented in Table 1. The pH of the reconstituted wastewater was then adjusted to pH 6.5 with sodium hydroxide. The reconstituted winery wastewater was divided between four 1-L closed glass bioreactors, equipped with a gas exhaust pipe (i.d.: 6 mm) and butyl septa for sampling and reagent addition, thermostatically controlled at 25 1C by water circulating through double-walled tubing, stirred at a speed of 250 rpm with a magnetic stirrer. The pH was measured with an Ingold electrode linked to an INGOLD 2300 pH metre, and adjusted to pH 6.5 by the addition of 10 N sodium hydroxide.

2.2.

Study of the NO3/COD ratio

Three bioreactors containing reconstituted wastewater were supplemented with nitrate in the form of soluble concentrated calcium nitrate (50% w/w), according to the quantities corresponding to the NO3/COD mass ratios (w/w): 0.4; 0.8; 1.2. The bioreactors supplemented with nitrate and a fourth without nitrate (control) were inoculated to a volume rate of 5% (vol/vol) with a suspension of sediments taken from evaporation ponds whose microbial activity was tested beforehand in the laboratory. The wastewaters were incubated for 21 days and samples (4 mL) were regularly taken with a syringe through a septum and maintained at 18 1C before being analysed.

Table 1 – Composition of the reconstituted winery wastewater

pH COD Ethanol Glucose Fructose Glycerol Tartaric acid a

Except pH.

Concentration (g L1)a

Theoretical COD (g O2 g1)

3.5 22.3 4.5 5.2 4.9 0.52 0.25

2.09 1.07 1.07 1.22 0.53

COD balance (%)

100 42.2 24.9 23.4 2.8 0.6

ARTICLE IN PRESS WAT E R R E S E A R C H

2.3. Winery wastewater treatment in ponds, with and without nitrate enrichment Experiments in evaporation ponds were carried out with wastewater from a winery that produces 180,000 hL wine year1 (mainly red). Wastewater was collected in a settling tank (40 m3), sieved and then evacuated to the evaporation ponds with a pump (20 m3 h1). Wastewaters were alternatively discharged into two 7000m2 evaporation ponds, whose impermeability was ensured by a layer of clay and bentonite. The volume of wastewater was measured by an integrating flowmetre. Soluble calcium nitrate (50% w/w), (NITCALs, YARA France, Paris, France), stored in 1-m3 tanks was injected into the wastewater discharge pipe, using a variable flow feed pump (100–200 L h1), coupled with the wastewater backflow pump. Liquid samples (250 mL) from the ponds were taken at three different locations and then mixed together to form the sample to be subjected to physico-chemical analyses (conserved at 18 1C).

2.4.

Chemical analyses

Nitrate and nitrite were measured using an ion HPLC: Waters IC Pak A column (50  4.6 mm) with eluent: borate/gluconate buffer, pH 8.5 and 10% acetonitrile (1.2 mL min1), Waters 717 autosampler, Waters 432 conductivity detector and EMPOWER software (Waters, Millipore, USA). Volatile fatty acids, sugars, ethanol, glycerol and organic acids were analysed by HPLC: mobile phase water/H2SO4 0.002 M (0.4 mL min1) with on-line degassing, Waters 717 autosampler, Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA), Waters RI 2410 refractive index detector and EMPOWER software (Waters, Millipore, USA). The COD was measured with an MN029 COD-1500 test kit (Macherey-Na¨gel, Du¨ren, Germany). The redox potential (ORP) was measured using a SenTix ORP (Ingold) electrode and a WTW millivoltmetre.

2.5.

2989

4 1 (200 7) 298 7 – 299 5

Determination of olfactory intensity

Odour intensities were determined by comparison with an nbutanol reference. Five solutions of 0.01, 0.05, 0.1, 0.5 and 1 g L1 were placed in glass flasks corresponding to five intensity levels (from 1 to 5), respectively, characterised as follows: level 1 (very low), level 2 (low), level 3 (medium), level 4 (slightly strong) and level 5 (strong). The odour intensity of each liquid effluent (pond with or without nitrate during two different periods) placed in a glass flask, was compared with n-butanol solutions by a jury of five selected panellists, and the average level was then calculated. Panellists were submitted to tests of sensitivity to n-butanol solutions and VFA solutions beforehand. The quantitative data obtained per intensity test were accompanied by a qualitative description of the smell. An experiment with 150 L of effluent without nitrate was carried out in a wind tunnel expressly designed for emission studies from areal sources (Leyris et al., 2000, 2005). The purpose of this experiment was to simulate the emission from the pond under controlled wind speed conditions (1 m s1). Air samples taken at 3 cm from the water surface

in Tedlar bags, were studied in both situations without nitrate: on site at the pond and in the tank of the wind tunnel. The odour intensity of these gas samples was quantified according to the levels as described above. A final experiment was carried out to determine the relationship between odour and VFA from the water. To do this, the most odorous VFA were selected and synthetic solutions of VFA were made into a buffer composed of Na2HPO4 (6 mM), NaH2PO4 (2 mM), Na2EDTA (1 mM) and NaCl (185 mM), as described by Comanici et al. (2006). The conditions of effluent without nitrate were reproduced, including VFA concentration and a pH value of 6.8. The concentrations (November period) were 513, 301 and 307 mg L1 for propionic, butyric and valeric acids, respectively. The concentrations (December period) for these acids were the same as those values given in Table 5 (without nitrate). The synthetic solutions were also evaluated by the jury in order to compare odour intensity on an n-butanol scale.

3.

Results and discussion

3.1. VFA

Influence of the NO3/COD ratio on the formation of

3.1.1.

VFA production from winery wastewater

Reconstituted winery wastewater has a COD of 22.3 g O2 L1, 91% of which consists of ethanol and sugars: glucose and fructose (Table 1). Its organic load and its composition correspond to that of winery wastewaters produced during the winery’s maximum activity period: grape harvest, vinification (Bories et al., 2005, 1998; Colin et al., 2005). The use of a reconstituted winery effluent (mixture of wine and must, diluted 10-fold) made it possible to carry out a laboratory study of the NO3/DCO ratio under pre-established and reproducible conditions, using a medium representative of winery effluents and easily available, with a pre-determined and stable composition. In this way, we were able to avoid the high degree of variability of the composition of winery effluents (Rochard et al., 1996; Duarte et al., 1998; Picot and Cabanis, 1998; ITV, 2000; Rochard, 2005). Composition in VFA produced during incubation of the reconstituted winery wastewater with evaporation pond microflora and at different NO3/COD ratios is given in Table 2. In the absence of nitrate, microflora produces a mixture of VFA: butyric acid (2304 mg L1), acetic acid (1633 mg L1),

Table 2 – VFA production from the reconstituted winery wastewater at various NO3/COD ratios NO3/COD (g g1) Acetic acid (mg L1) Propionic acid (mg L1) Butyric acid (mg L1) Valeric acid (mg L1) Caproic acid (mg L1)

0

0.4

0.8

1.2

1633 1558 2304 298 499

1369 1639 809 0 0

1841 428 155 0 0

0 0 59 0 0

ARTICLE IN PRESS 41 (2007) 2987– 2995

propionic acid (1558 mg L1), caproic acid (499 mg L1) and valeric acid (298 mg L1), through fermentation of the carbon compounds in the wastewater: sugars, ethanol, glycerol and organic acids (Table 1). VFA represent 45% of the initial COD of the reconstituted wastewater. Butyric, propionic, caproic, valeric and heptanoic acids form the major fraction of products resulting from the anaerobic degradation of the constituents of winery wastewaters by evaporation pond microflora. Their low perception threshold, their unpleasant odour, their high concentrations and large accumulated quantities all contribute to the typical characteristic noxious odour. Butyric and propionic acids are generally produced by clostridia and propionibacteria, respectively, from simple substrates such as sugars, lactic acid, glycerol, etc., according to well-known fermentation pathways (Barbirato et al., 1997; Colin et al., 2001). However, the formation of butyric, valeric, caproic and heptanoic acids from ethanol, a characteristic constituent of winery wastewaters (Colin et al., 2005), appears to be related to particular fermentation processes, given its reduction level (Cred ¼ 6). VFA accumulation subsequent to the overload of an anaerobic digester supplied with ethanol was demonstrated by Smith and McCarty (1988) and attributed to a reverse b-oxidation process. Butyric and caproic acid production by co-fermentation of ethanol and acetic acid was observed in Clostridium kluyveri (Gottschalk, 1986), and comparable metabolic properties were demonstrated in Eubacterium pyruvativorans (Wallace et al., 2004). Ethanol alone is not fermented and propionic acid can be used instead of acetic acid. On the basis of the energy production of this pathway 1 ATP/6 ethanol (Gottschalk, 1986), it is possible to project low microbial growth and a low VFA production rate from the anaerobic catabolism of the ethanol, which could explain the formation of VFA with long carbon chains during extended storage of winery wastewaters in evaporation ponds.

2400

NO3 /COD = 0 NO3 /COD = 0.4 NO3 /COD = 0.8 NO3 /COD = 1.2

2000 Butyric acid (mg L-1)

WAT E R R E S E A R C H

1600 1200 800 400 0 0

100

200 300 Time (h)

400

500

Fig. 1 – Effect of various NO3/COD ratios (w/w): 0, 0.4, 0.8 and 1.2 on butyric acid production from reconstituted winery wastewater.

100 50 0 -50 ORP (mV)

2990

-100 -150 -200

NO3 /COD = 0 NO3 /COD = 0.4 NO3 /COD = 0.8 NO3 /COD = 1.2

-250 -300 -350 -400 -450 0

100

200

300

400

500

Time (h)

3.1.2.

Effect of COD/NO3 ratios

For a ratio of NO3/COD ¼ 0.4 g g1, caproic and valeric acids, whose carbon reduction degrees (Cred) are high (5.33 and 5.2, respectively), were not produced. Butyric acid (Cred ¼ 5) concentration was reduced by 64.9% and that of propionic and acetic acids (Cred ¼ 4.67 and 4, respectively) was not considerably modified. At a ratio of NO3/COD ¼ 0.8, butyric acid production was reduced by 93.3% in relation to the control, and that of propionic acid by 72.5%. The formation of acetic, propionic, caproic and valeric acids was null for the ratio of NO3/COD ¼ 1.2, and butyric acid production was very low (59 mg L1), a decrease of 97.4% in relation to the control without nitrate. Butyric acid production at different NO3/COD ratios is presented in Fig. 1. In the absence of nitrate, butyric acid formation was observed at the very beginning of wastewater incubation with microflora, linked to the consumption of sugars, glycerol and organic acids, and it occurred regularly throughout the 21 days of incubation, at an average rate of 4.6 mg L1 h1. The addition of nitrate delayed and decreased butyric acid production. At a ratio of NO3/COD ¼ 0.4, butyric acid production only began after 185 h of incubation and, as of the 330th hour, the production rate of butyric acid (4.1 mg L1 h1) was as high as the one observed in the

Fig. 2 – ORP behaviour from reconstituted winery wastes at various NO3/COD ratios (w/w): 0; 0.4; 0.8 and 1.2.

absence of nitrate. With a ratio of NO3/COD ¼ 0.8, the production rate of butyric acid remained low (0.7 mg L1 h1). For the ratio of NO3/COD ¼ 1.2, butyric acid production was only observed after the 330th hour and at a very low rate: 0.35 mg L1 h1. The behaviour of the redox potential of the reconstituted wastewater during incubation with microflora and with different NO3/COD ratios is illustrated in Fig. 2. This figure shows that in the absence of nitrate, the ORP is very low (o400 mV) during the most active butyric production phase and then remains within the value range of 77 and 185 mV. At a ratio of NO3/COD ¼ 0.4, ORP values ranged from +12 to 32 mV during the phase when butyric acid was not produced, and rapidly dropped to 186 mV when the production of butyric acid began. The ORP always remained negative when butyric acid production took place. For the ratio of NO3/ COD ¼ 0.8, the ORP remained positive until the 402nd hour and only slightly decreased (30 mV) at the end of the test when butyric acid production began. The ORP remained

ARTICLE IN PRESS

3.2. Full-scale study of winery wastewater ponds supplemented or not with nitrate A comparative study of the two winery wastewater evaporation ponds, treated or not with nitrate, was carried out in order to assess the benefits of nitrate for the prevention of VFA formation in actual winery wastewater, at full scale, over a period of time corresponding to industrial activity, and with the natural pond microflora. Table 3 presents the characteristics of the evaporation ponds (7000 m2 each) and of preventive wastewater treatment through the addition of calcium nitrate. The evaporation ponds were supplied with

2

Area (m ) Wastewater intake (m3) Nitrate supply Nitrate (g NO3 L1) NO3/COD (g g1)

With nitrate

Without nitrate

7000 3521 Calcium nitrate 13.3 0.8

7000 2236 None 0 0

Table 4 – Composition of the winery wastewater discharged into ponds Concentrationa (g L1)b

COD balance (%)

4.19 16.62 3.91 1.96 2.86 0.40 0.33

100 49.2 12.6 18.4 2.9 1.1

pH COD Ethanol Glucose Fructose Glycerol Tartaric acid a b

Except pH. Mean on eight samples.

1000 900

Butyric acid with NO3

800

Valeric acid with NO3

700

Butyric acid without NO3

600

Valeric acid without NO3

500 400 300 200 100 /0 9 06 /05 /1 0 13 /05 /1 0 20 /05 /1 0 27 /05 /1 0 03 /05 /1 1 10 /05 /1 1 17 /05 /1 1 24 /05 /1 1 01 /05 /1 2 08 /05 /1 2 15 /05 /1 2/ 05

0

29

positive (4+40 mV) throughout the test made with the ratio of NO3/COD ¼ 1.2. The effect of increasing nitrate concentrations was revealed by the suppression of VFA production according to decreasing order of the carbon reduction degree: caproic and valeric acids first, butyric acid followed by propionic acid and, finally, acetic acid. Competition between catabolic pathways favoured the oxidative pathway through anaerobic respiration, as opposed to fermentation processes. The prevention of malodorous VFA formation (propionic to caproic acids) obtained for NO3/COD ratios ranging from 0.8 to 1.2 g g1, less than the stoichiometric ratio (NO3/COD ¼ 1.55 g g1), suggests that acidogenic substrates of winery wastewaters (sugars, ethanol) were preferentially used by anaerobic respiration with nitrate whose redox potential conditions inhibited anaerobic catabolic pathways. When nitrate is no longer available, carbon is the final electron acceptor and acidogenic fermentation pathways appear. Sobieszuck and Szewczyk (2006) demonstrated that for carbon substrates with a reduction degree lower that 4.67, the critical denitrification ratio, COD/N, is equal to 7.6 g O2 g1 N, corresponding to a NO3/COD ratio of 0.58 g g1. The prevention of H2S and mercaptan formation with nitrate was studied and applied to urban wastewater (Bentzen et al., 1995; Hobson and Yang, 2000). Garcı´a de Lomas et al. (2005) showed that the mechanism for preventing H2S is due to the development of Thiomicrospira denitrificans, bacteria that reduce nitrate by oxidising sulphide. The strategy and the mechanisms involved in preventing VFA formation by nitrate from wastewaters rich in carbon studied here, based on the oxidation of carbon substrates by denitrification, differ from the H2S prevention treatment with nitrate that does not prevent H2S production by sulphate reduction bacteria but that biologically oxidises H2S by reducing the nitrate.

Table 3 – Characteristics of the winery wastewater ponds supplemented or not with nitrate

2991

4 1 (200 7) 298 7 – 299 5

Butyric, valeric acid (mg L-1)

WAT E R R E S E A R C H

Fig. 3 – Behaviour of butyric and valeric acids in winery wastewater ponds supplemented or not with nitrate.

wastewaters produced during the winery’s maximum activity period: grape harvest/vinification (29 September–14 December). The average composition of wastewaters discharged into the ponds (Table 4) is characterised by a COD (16.6 g O2 L1) and a proportion of ethanol (49% of the COD) and sugars (31% of the COD) close to those of the reconstituted winery wastewaters (Table 2), confirming the representativeness of the study carried out with reconstituted wastewater. One pond was supplied with winery wastewaters enriched with nitrate (3521 m3), with an average concentration of 13.3 g NO3 L1, or a ratio of NO3/COD ¼ 0.8. The other pond was supplied with winery wastewaters (2236 m3), without the addition of nitrate (control). As can be seen in Fig. 3, no accumulation of butyric and valeric acids, nor any other VFA (results not shown) were observed in the pond supplied with wastewaters enriched in nitrate during the wastewater discharge period (212 months). On the other hand, the accumulation of butyric acid (143–997 mg L1) and valeric acid (99–343 mg L1) was observed throughout the study period in the pond supplied with wastewater without nitrate. Table 5 provides the VFA composition in the two ponds at the end of the maximum discharge period (14 December). In addition to the VFA observed in reconstituted winery wastewaters (acetic, propionic, butyric, valeric and caproic acids), the formation of

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41 (2007) 2987– 2995

Table 5 – VFA composition of the winery wastewater ponds supplemented or not with nitrate Without nitrate

n.d.a n.d. n.d. n.d. n.d. n.d.

822 211 354 178 83 69

Acetic acid (mg L1) Propionic acid (mg L1) Butyric acid (mg L1) Valeric acid (mg L1) Caproic acid (mg L1) Heptanoic acid (mg L1)

Nitrite Nitrate raw COD

3000

7 6 5 4

2000 1000

3 2 1

0

n.d.—not detected.

70

With NO3

60 VFACOD/COD (%)

4000

8

0

29 /0 06 9/05 /1 13 0/05 /1 20 0/05 /1 27 0/05 /1 03 0/05 /1 1 10 /05 /1 1 17 /05 /1 1 24 /05 /1 1 01 /05 /1 08 2/05 /1 15 2/05 /1 2/ 05

a

Nitrate, nitrite (mg L-1)

With nitrate

5000

COD (gO2 L-1)

2992

Without NO3

Fig. 5 – Behaviour of COD, nitrate and nitrite in winery wastewater pond supplemented by nitrate.

50 40 30 20 10

/0 5 /1 1 /0 1/ 5 12 /0 5 8/ 12 /0 5 15 /1 2/ 05

05

24

17

1/ /1

10

/1 1

5

5

/0

/0 3/

11

05 27

/1 0

05

0/

20

/1

5

/1 0/

13

/0

10 6/

29

/9

/0

5

0

Fig. 4 – Part of the VFA (propionic to heptanoic) in the COD of winery wastewater ponds supplemented or not by nitrate (each VFA is expressed as equivalent COD, and the sum of VFA-COD is expressed as a percent of the COD measured).

heptanoic acid was observed in untreated pond of winery wastewaters. None of these VFAs were detected in the wastewater pond enriched with nitrate (Table 5). VFA concentrations in the untreated pond were lower than those obtained in batch tests with reconstituted wastewaters. These differences can be explained by dilution due to autumn rains (Mediterranean climate) and the eventual degradation by the pond’s microflora. Fig. 4 illustrates the evolution of the portion of VFA (propionic to heptanoic acids) in the COD of the ponds. It shows that 30–60% of the COD of the untreated pond consisted of VFA higher than acetic acid, revealing the preponderance of malodorous compounds and acidogenic fermentation. On the contrary, the portion of VFA in the residual COD of wastewaters treated with nitrate was negligible (Fig. 4). Fig. 5 shows the evolution of the raw COD, of the nitrate and nitrite in the pond supplied with wastewaters enriched in nitrate. The COD of the pond never exceeded 5 g L1 from the beginning of the discharge period, decreased to 1 g L1 during the first month and remained close to this value throughout the discharge period. In relation to the average COD of discharged winery wastewaters (16.6 g L1), the apparent COD degradation rate in the pond treated with nitrate almost reached 94%, revealing that the organic load of the wastewater enriched in nitrate was oxidised and eliminated as carbon dioxide through anaerobic respiration. Moreover, the

complementary analysis of the effluent in the treated pond revealed a suspended matter content of 0.5 g L1, and a dissolved COD and BOD of less than 300 and 50 mg L1, respectively (data not shown). These levels of dissolved COD and BOD are consistent with effluent standards in the receiving environment and correspond to the characteristics of winery effluents treated biologically. Therefore, discharge in a receiving environment of an effluent treated with nitrate in the pond could be considered after clarification. Related research and experiments are being carried out at this time by the laboratory. The nitrate concentration in the treated pond developed parallel to the COD. The nitrate totally disappeared by the end of the first month and did not accumulate thereafter. The transitory formation of nitrite in the pond was observed at the beginning of the discharge period of wastewater enriched in nitrate (first month) and occasionally thereafter when nitrate input was greater. Under these conditions of strong denitrifying activity, the degradation rate of nitrite into nitrogen was less than the degradation rate of nitrate into nitrite. However, the nitrite totally disappeared during the subsequent periods and, at the end of the discharge of wastewaters enriched with nitrate, no nitrate or nitrite accumulation could be observed in the evaporation pond. The evolution of the ammoniacal nitrogen concentration in the pond, ranging from 0 to 14 mg NH+4 L1 (results not shown), did not reveal an accumulation during nitrate degradation, confirming that the nitrate was totally transformed into N2. The phenomenon of the reductive dissimilation of nitrate into ammonia (Payne, 1973; Samuelsson, 1985; Vigneron et al., 2005) did not take place in the pond. The absence of VFA accumulation for a ratio of NO3/ COD ¼ 0.8 and at full scale (3526 m3 of wastewater) confirmed that anoxic respiration process (denitrification) were in competition with the acidogenic pathways, although the NO3/COD ratio was clearly lower than the stoichiometry. Compared to previous studies of preventive treatments based on concentrated nitric acid (a less expensive source of nitrate), the implementation of the calcium nitrate treatment (a non-corrosive product) proved itself to be technically feasible both for determining the quantity of nitrate to be

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used (concentrated calcium nitrate solution) and in relation to human safety (neutral product) and facility maintenance (no corrosion, ease of storage), matters of particular importance because wineries do not always have the adequate structures or the means to enable them to use large quantities of concentrated acids.

3.3. Contribution of VFA to the odour of winery wastewater and effect of nitrate enrichment Among the VFA produced from winery wastewaters, acetic acid is the one that has the least pronounced foul odour. All the others, from propionic to heptanoic acid, have unpleasant odours and low perception thresholds of only several dozen mg m3 (Le Cloirec et al., 1991). The fraction of malodorous VFA (higher than acetic acid) represents 74% and 52% (w/w) of the VFA produced from reconstituted wastewaters and in winery wastewater ponds, respectively. In order to complete chemical measurements of VFA and to assess the odours of the VFA and the winery wastewater ponds, olfactory intensity measurements were made on model VFA solutions and on liquid samples taken from the ponds at two different times during the discharge period (middle and end).

3.4.

Relationship between odour intensity and VFA

The odour intensity of liquid samples, given in Fig. 6, clearly shows that nitrate addition decreases the intensity level. If treated wastewater is not odourless, the decrease from strong to medium odour impression is significant even if the RSD of these experiments was calculated as 0.5. The decrease is also linked to a major transformation of odour quality. The odour is very unpleasant without nitrate, whereas it is very acceptable with nitrate. The unpleasant odour from liquid samples, subsequently confirmed by gas samples (3/11/2005) from the site and from the experiment in the wind tunnel, reveal average levels of 3 and 2.75, respectively. This small decrease may be due to a slight evolution of the liquid (1 day between sampling at the site and the experiment in the wind tunnel) and mainly to the volatilisation of some odorous compounds during operations to transfer the effluent into the tank of the wind tunnel. Because the procedure of smelling air from a Tedlar bag is different than the procedure involving direct sniffing of a flask, levels estimated on gas samples are

Real effluent

Levels of odour Intensity

Without nitrate

5 4 3

VFA solutions

5 4 3 2 1 nov-05

dec-05

Fig. 7 – Comparison of odour intensity of wastewater without nitrate and a solution with the major odorous VFA at the same concentration.

lower than those obtained directly on liquid samples that contain many volatile odorous compounds. All these results indicate that the decrease of odour is due to VFA decrease obtained by the addition of nitrate to wastewater. To confirm this close relationship between odour intensity and VFA, synthetic solutions were made with the same selected VFA concentrations as those in the pond without nitrate for both periods (November and December). The VFA selected were propionic, butyric and valeric acids. The intensity levels given in Fig. 7 show the high degree of correspondence between odours from real wastewater without nitrate and, therefore, with VFA solutions. These three acids are very odorous and present low perception thresholds (around 80, range 4–50 and around 5 mg m3 for C3–C5 acids, respectively, according to data from INERIS, a research institute under the supervision of the French Ministry of the Environment and Sustainable Development). These three VFA mainly contribute to overall odour. When acetic acid, the most abundant but potentially less odorous with a perception threshold at 900 mg m3 (INERIS data), was added to these synthetic solutions, it is more difficult to evaluate because of the irritant aspect of this acid. This acid (C2) gives a sour qualitative feeling without increasing the odour intensity. Acetic acid is less responsible for odour intensity than others, so the mixture of the three acids (C3–C5) is an easy way to simulate the global intensity level of winery wastewaters.

4. With nitrate

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Levels of odour intensity

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Conclusion

This study revealed, both in the laboratory and at full scale, the effect of the addition of an electron acceptor, nitrate in this case, on the degradation of the components of winery wastewaters through anaerobic respiration, to the detriment of acidogenic and odorogenic fermentations, as well as its effect on odour prevention. The following conclusions can be drawn from this study:

2

 VFA and particularly those acids ranging from propionic to

1 nov-05

dec-05

Fig. 6 – Comparison of odour intensity of wastewater with or without nitrate during two different periods.

heptanoic that are very odorous and have low perception levels are produced by the degradation of the organic components of winery wastewaters by microflora. Butyric acid is preferentially produced by fermentation of sugars,

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glycerol and organic acids, whereas ethanol, the major component of winery wastewaters, leads to the formation of propionic, butyric, valeric, caproic and heptanoic acids. Increasing NO3/COD ratios (0.4–1.2) successively affect VFA production, going from the most highly reduced first (caproic, valeric) to the least reduced (butyric followed by propionic). The combination of the degradation of organic compounds and nitrate, and the increase of the redox potential shows that microflora activity is focused on an anaerobic respiration process (denitrification), in competition with acidogenic fermentation pathways. Full-scale experimentation (evaporation pond) with the addition of calcium nitrate at a ratio of NO3/COD ¼ 0.8 in winery wastewaters (3526 m3) revealed the absence of the formation of VFA and the complete degradation of the nitrate into nitrogen gas, with the considerable decrease of the COD. Olfactometric measurements underscored the relationship between the VFA concentration in the wastewaters and the odour, and confirmed the role of nitrate in relation to odour prevention.

Acknowledgements This study received financial support from the French Agency for the Environment and Energy Management (ADEME) within the framework of a request for proposals, ‘‘Odours and Industries’’ (Contract No. 0374C0036). We thank Cave Anne de Joyeuse in Limoux, France, for granting us access to its wastewater treatment facilities and for its contribution to test procedures. We are also grateful to the Socie´te´ YARA France for its assistance with calcium nitrate treatment. R E F E R E N C E S

ADEME, 2005. Pollutions Olfactives. Agence de l’Environnement et de la Maıˆtrise de l’Energie, Dunod, Paris, 388pp. Barbirato, F., Chedaille, D., Bories, A., 1997. Propionic acid fermentation from glycerol: comparison to conventional substrate. Appl. Microbiol. Biotechnol. 47, 441–446. Bentzen, G., Smith, A.T., Bennett, D., Webster, N.J., Reinholt, F., Sletholt, E., Hobson, J., 1995. Controlled dosing of nitrate for prevention of H2S in a sewer network and the effects on the subsequent treatment process. Water Sci. Technol. 31 (7), 293–302. Bolzonella, D., Innocenti, L., Pavan, P., Cecchi, F., 2001. Denitrification potential enhancement by addition of anaerobic fermentation products from the organic fraction of municipal solid waste. Water Sci. Technol. 44 (1), 187–194. Bories, A., 2006. Pre´vention et traitement des odeurs des effluents vinicoles. In: Techniques de l’Inge´nieur, Paris, G 1 960, pp. 1–12. Bories, A., Sire, Y., Colin, T., 2005. Odorous compounds treatment of winery and distillery effluents during natural evaporation in ponds. Water Sci. Technol. 51 (1), 129–136. Bories, A., Conesa, F., Boutolleau, A., Peureux, J-L., Tharrault, P., 1998. Nouvelle approche et nouveau proce´de´ de traitement des effluents vinicoles par fractionnement des constituants et thermo-concentration. Revue Franc- aise d’Oenologie 171, 26–29.

Chiu, Y.C., Chung, M.S., 2003. Determination of optimal COD/ nitrate ratio for biological denitrification. Int. Biodeterior. Biodegrad. 51, 43–49. Chrobak, R.S., Ryder, R.A., 2006. Odors and control methods in winery wastewater treatment. In: IV International Specialized Conference on Sustainable Viticulture: Winery Wastes and Ecological Impact Management, 5–8 November, Vin˜a del Mare, Chile. Colin, T., Bories, A., Lavigne, C., Moulin, G., 2001. Effects of acetate and butyrate during glycerol fermentation by Clostridium butyricum. Curr. Microbiol. 43, 238–243. Colin, T., Bories, A., Sire, Y., Perrin, R., 2005. Treatment and valorisation of winery wastewater by a new bio-physical process. Water Sci. Technol. 51 (1), 99–106. Comanici, R., Gabel, B., Gustavsson, T., Markovitsi, D., Cornaggia, C., Pommeret, S., Rusu, C., Kryschi, C., 2006. Femtosecond spectroscopic study of carminic acid-DNA interactions. Chem. Phys. 325 (2), 509–518. De Lucas, A., Rodrigues, L., Villasen˜or, J., Fernandez, F.J., 2005. Denitrification potential of industrial wastewaters. Water Res. 39 (15), 3715–3726. Duarte, E., Martins, M.B., Carbalho, E.C., Spranger, I., Costa, S., 1998. An integrated approach for assessing the environmental impacts of wineries in Portugal. In: Second International Specialized Conference on Winery Wastewaters, Bordeaux, 5–7 May, CEMAGREF ed., pp. 61–69. Elefsiniotis, P., Wareham, D.G., Smith, M.O., 2004. Use of volatile fatty acids from an acid-phase digester for denitrification. J. Biotechnol. 114 (3), 289–297. Garcı´a de Lomas, J., Corzo, A., Gonzalez, J.M., Andrades, J.A., Iglesias, E., Montero, M.J., 2005. Nitrate promotes biological oxidation of sulfide in wastewaters: experiments at plantscale. Biotehnol. Bioeng. 93 (4), 801–811. Gottschalk, G., 1986. Bacterial Metabolism, second ed. Springer, pp. 234–236. Guillot, J.M., Desauziers, V., Avezac, M., Roux, J.C., 2000. Characterization and treatment of olfactory pollution emitted by wastewater in winery Mediterranean region. Fresen. Environ. 9, 243–250. Hobson, J., Yang, G., 2000. The ability of selected chemicals for suppressing odour development in rising mains. Water Sci. Technol. 41 (6), 165–173. ITV, 2000. Les filie`res d’e´puration des effluents vinicoles. Nouvelle e´dition. ITV, France. Lambert, J., Lecharpentier, D., 2006. Fe´de´rations des Caves Coope´ratives de l’Aude et du Gard, Narbonne France. Personal Communication. Le Cloirec, P., Fanlo, J.L., Degorce-Dumas, J.R., 1991. Traitement des odeurs et de´sodorisation. Innovation 128, Paris. Le Verge, S., Bories, A., 2004. Les bassins d’e´vaporation naturelle des margines. Le Nouvel Olivier, 41, Septembre–Octobre, pp. 5–10. Leyris, C., Maupetit, F., Guillot, J.M., Pourtier, L., Fano, J.L., 2000. Laboratory study of odour emissions from areal sources: evaluation of a sampling system. Analusis 28, 199–206. Leyris, C., Guillot, J.M., Pourtier, L., Fanlo, J.L., 2005. Comparison and development of dynamic flux chambers for the determination of odorous compound emission rate from area source. Chemosphere 59, 415–421. Min, K.S., Park, K.S., Khan, J.Y.J., Kim, Y.J., 2002. Acidogenic fermentation: utilization of wasted sludge as a carbon source in the denitrification process. Environ. Technol. 23 (3), 293–302. OIV, 1999. Gestion des effluents de cave et de distillerie. Cahier scientifique et technique, 78pp. OIV, 2005. World statistics, Third General Assembly of the OIV, June, Paris. Payne, J.W., 1973. Reduction of nitrogenous oxides by microorganisms. Bacteriol. Rev. 37, 409–452. Picot, B., Cabanis, J.C., 1998. Caracte´risation des effluents vinicoles: e´volution des charges polluantes de deux caves

ARTICLE IN PRESS WAT E R R E S E A R C H

4 1 (200 7) 298 7 – 299 5

vinicoles du sud de la France sur deux cycles annuels. In: Second International Specialized Conference on Winery Wastewaters, Bordeaux, 5–7 May, CEMAGREF ed., pp. 312–317. Rochard, J., 2005. Traite´ de Viticulture Et D’œnologie Durables. Oenoplurime´dia, Chaintre´, Fr., 310pp. Rochard, J., Kerner, S., Finazzer, E., 1996. Re´glementations relatives aux effluents vinicoles dans les principaux pays producteurs de vin. 76e`me Assemble´e Ge´ne´rale de l’OIV, Novembre. Sage, M., Dauphin, G., Ge´san-Guiziou, G., 2006. Denitrification potential and rates of complex carbon source from dairy effluents in activated sludge system. Water Res. 40 (14), 2747–2755. Samuelsson, M-O., 1985. Dissimilatory nitrate reduction to nitrate, nitrous oxide, and ammonium by Pseudomonas putrefaciens. Appl. Environ. Microbiol. 50 (4), 812–815. Smith, D.P., McCarty, P.L., 1988. Hydrogen partial pressure: effect on methanogenesis of ethanol and propionate in a perturbed

2995

CSTR. In: Tilche, A., Rozzi, A. (Eds.), Fifth International Symposium on Anaerobic Digestion. Monduzzi Editore, Bologna, pp. 75–80. Sobieszuck, P., Szewczyk, K.W., 2006. Estimation of (C/N) ratio for microbial dentrification. Environ. Technol. 25 (1), 103–106. Sponza, D.T., Atalay, H., 2004. Influence of nitrate and COD on phosphorus, nitrogen and dinitrotoluene (DNT) removals under batch anaerobic and anoxic conditions. Anaerobe 10 (5), 287–293. Vigneron, V., Bouchez, T., Bureau, C., Mailly, N., Mazeas, L., Duquennoi, C., Audic, J.M., He´be´, I., Bernet, N., 2005. Leachate pre-treatment strategies before recirculation in landfill bioreactors. Water Sci. Technol. 52 (12), 289–297. Wallace, R.J., Chaudhary, L.C., Miyagawa, E., McKain, N., Walker, N.D., 2004. Metabolic properties of Eubacterium pyruvativorans, a ruminal ‘hyper-ammonia-producing’ anaerobe with metabolic properties analogous to those of Clostridium kluyveri. Microbiology 150, 2921–2930.