high-rate aerobic treatment of winery wastewater using bioreactors with free and immobilized...

Vol. 90, No. 4, 381-386. 2000

High-Rate Aerobic Treatment of Winery Wastewater Using Bioreactors with Free and Immobilized Activated Sludge

MAURIZIO PETRUCCIOLI,‘* JOSe CARDOSO DUARTE,2 AND FEDERICO FEDERICI’ Dipartimento di Agrobiologia e Agrochimica, University of Tuscia, Via S. C. De Lellk, 01100 Viterbo, Italy’ and INETI,

Departamento de Biotecnologia, Azinhaga dos Lameiros a Estrada do Paqo do Lumiar, 1649-038 Lisboa, Portugalz

Received 17 March 2OOO/Accepted 26 June 2000

COD (chemical oxygen demand) removal rate and efficiency of winery wastewater (WW) aerobic treatments were evaluated in an air-bubble column hioreactor using self-adapted microbial populations either free or immobilized on polyurethane particles and in a packed-bed bioreactor immobilized on Raschig rings. The bioreactors were fed continuously for up to 12 months using WW of different origins and with different pollution loads (COD range, 0.8-11.0 kg.mw3): the maximum loading rate was approx. 8.8 kg-COD me3.d-‘. The highest COD removal rate (6.6 kg.m-3.d-1) was obtained with free activated sludge in the bubble column bioreactor; treatment efficiency and hydraulic retention time were >!Xl% and approx. 0.8 d, respectively. The microbial populations in the three reactors were characterized.

[Key words: winery wastewater, bioreactor, aerobic treatment, free activated sludge, immobilized activated sludge]

The world wine production rate is approximately 2.65 x lo8 hi/year of which about 63% comes from the European Union. France and Italy, alone, represent almost 70% of the entire European wine production (1). Winemaking operations yield an equivalent, or even larg- er, amount of wastewater, mainly originating from the washing of equipment and bottles and from cooling (2- 5). Due to the high organic load and the large volumes with a pronounced seasonal variability, the environmen- tal impact of this effluent is noticeable; the different wine-making technologies used may also account for the marked variability in the amount and the composition of this waste (3, 5, 6).

In the last two decades, a large number of small wineries have disappeared in favor of large, often co-opera- tive, wineries that process large amounts of grapes in short periods of time; as a consequence, the daily wastewater volumes have increased dramatically during the grape-harvesting and wine-making periods (1, 4, 5, 7). Thus, ever bigger conventional aerobic-treatment plants have been built to deal with the constantly increasing effluent volumes. Often, wineries do not have sufficient space available for the installation of such plants nor the financial resources necessary for such an investment (6, 8). To date, several interesting solutions, such as evaporation-condensation by for example ultrafiltration and reverse osmosis (9), anaerobic digestion in UASB reactors (10) and aerobic treatment with rotating biological contactors (11) have been proposed. Most promising, however, appears to be the use of verti- cal reactors characterized by good oxygen transfer and a high biological conversion capacity (12, 13).

The aim of this work was to assess the performance characteristics of aerobic winery wastewater treatments using an air bubble column reactor with activated sludge either free or immobilized on polyurethane particles and a packed-bed reactor with activated sludge immobilized on Raschig rings. The effects of some physico-chemical parameters, including pH, aeration rate and tempera-

* Corresponding author.

ture, on treatment performances were also investigated.

MATERIALS AND METHODS

Winery wastewater (WW) WW was obtained from two different wineries, Lungarotti (Torgiano, Perugia, Italy) and Coop Vitivinicola of Orvieto (Orvieto, Italy), and used as such with no addition of either phosphorous or nitrogen; the results of analytical characterization are presented in Table 1. Unless indicated otherwise, the pH was adjusted to 7.5.

Bioreactors Two experimental reactors (working volumes of 1.5 r) were used; schematic diagrams are shown in Figs. 1A and 1B. Reactor A was a glass column (Fig. IA) for use with free activated sludge (air bubble column bioreactor, ABB) or sludge immobilized on polyurethane particles (fluidized-bed bioreactor, FBB) which were pneumatically agitated. Reactor B (Fig. 1B) was a glass column used with the same activated sludge but immobilized on Raschig rings (packed-bed bioreactor, PBB).

Start up and treatment conditions Activated sludge of different origins was used to inoculate 1OOml of WW in 500ml flasks which were incubated at 30°C on a rotary shaker (120 rpm, diameter of rotary motion 25 mm) for 4-6 d. The activated sludge was recovered by sedimentation of the mixed liquor and reused as an inoculum for lOOmI of WW, as above; after a period of 2 months the activated sludge was considered to be adapt-

TABLE 1. Chemical characteristics of the winery wastewater (WW)

Chemical oxygen demand (COD) Biochemical oxygen demand (BODS) Total suspended solids (TSS) Total phosphorous Phosphate (P-PO,) Nitrogen (N-NH3) Total polyphenols

Unit Range kg.m-3 0.8-11.0 kg.m-3 0.5-6.9 kg.mm3 0.2-l .3 g.m-3 4.0-35.0 g.m-] 0.3-30.0 g.mm3 0.001-2.0 g.rn-’ 5.8-33.6

382 PETRUCCIOLI ET AL.

A

B

FIG. 1. (A, B) Schematic diagram of the experimental column reactors used in this study. Reactor A was a glass column (Fig. 1A) for use with free activated sludge (air bubble column bioreactor, ABB) or immobilized on polyurethane particles (fluidized-bed bioreactor, FBB) pneumatically agitated. Reactor B (Fig. 1B) was a glass column used with activated sludge immobilized on Raschig rings (packed-bed bioreactor, PBB). 1, Warm or cold water inlet and outlet; 2, air inlet; 3, air outlet condenser; 4, feed inlet; 5, effluent outlet; 6, recycling of mixed liquor: 7, packed-bed; 8, glass beads; P, pump.

ed to the WW (Eusebio, A. et al., Abstr. 9th European Congress of Biotechnology, Brussels, Belgium, 1999) and was used as an inoculum for the bioreactors.

Immobilization of the activated sludge on polyure-

J . BIOSCI. BIOENG ,

thane sponge particles (approx. 5.0 mm cubed particles; pore size of 0.4-0.6mm; 8-10 particles per cm3 of sponge, approx. 20% of the bioreactor working volume) and on Raschig rings (approx. 6.0mm, in both height and internal diameter) was carried out in repeated-batch processes. At the end of each batch (duration, 2d), the free activated sludge was collected by sedimentation and reused in a subsequent batch for a total of 7 cycles; the indicator of immobilization was the browning of the carriers as a consequence of microbial biomass adhesion.

Effects of varying physico-chemical parameters such as pH, aeration rate and temperature, on the activated sludge performance were assessed using ABB. Standard conditions of fermentation were: aeration rate, 1.0 volume per volume per minute (vvm); WW pH, 7.5; temperature, 30°C; COD loading rate, 2.65 kg.m 3d ‘.

When PBB was used, the recycle flow through the ex- ternal loop was ca. 1 .O dm3h l.

Parameters investigated and methods of analysis During the study, samples of the influent, effluent and mixed liquor were taken daily from each reactor and the following parameters were determined: COD, phosphate (P-PO,) content, total suspended solids (TSS), mixed liquor suspended solids (MLSS), sludge volume index (SVI) and pH. In some cases, the biochemical oxygen demand (BODS), total polyphenols, total phosphorous and nitrogen (both N-NH4 and N-N03) contents were also determined.

All the analyses were carried out using standard methods for the analysis of water and wastewater (14).

Microbiological characterization Samples of mixed liquor and colonized carriers were taken from the bioreactors during the study period. Isolation of bacteria and fungi was carried out on Plate Count Agar and Rose- Bengal Chloramphenicol Agar Base (at 35°C). ldentifica- tion of the isolates, mostly at the genus level, was achieved by performing specific biochemical (catalase) and morphological (Gram staining and sporogenesys) tests as well as API tests (Bio-Merieux ltalia S.p.A.) such as API lOS, API 20NE, API 20E and API CO- RYNE for bacteria and API 20C AUX for yeasts and yeast-like fungi. Filamentous fungi were identified on a morphological basis only.

RESULTS AND DISCUSSION

Setting up of physico-chemical parameters The effects of aeration rate, pH of the WW and process temperature on the COD removal rate and efficiency and on the microbial biomass were studied in the air bubble column reactor (ABB) inoculated with activated sludge previously adapted to the wastewater and continuously fed with the same waste. Results are reported in Table 2.

Increasing the aeration rate from 0.5 to l.Ovvm re- sulted in increased dissolved oxygen (DO), which changed from less than 10% to approximately 25-30%. The microbial biomass (MLSS) increased from 4.0 to 5.8 kg. me3 and the COD removal rate and efficiency from 2.33 to 2.54 kg.mm3d-’ and from 88.1 to 96.0%, respectively. A further increase of the aeration rate to 1.5 vvm (DO around 35%) was not accompanied by proportional increases in either COD removal rate or efficiency.

The initial pH of the WW (from 3.5 to 7.5) did not significantly affect the treatment performance, thus in- dicating that no neutralization of the incoming WW was

VOL. 90, 2000 HIGH-RATE AEROBIC TREATMENT OF WINERY WASTEWATER 383

TABLE 2. Effect of aeration rate, pH and temperature on the aerobic treatment of WW in an air-bubble column bioreactor

with free activated sludge

COD COD removal Final removal rate efficiency COD MLSS pH (kg. me3&l) (%I (kg.mM3) (kg.m-3)

Aeration rate (wm): 0.5 2.33t0.01 88.1t0.4 0.63kO.02 4.0ir0.4 8.5-tO.l 1.0 2.54t0.02 96.020.6 0.21+-0.03 5.8kO.2 8.7kO.O 1.5 2.5620.01 96.4kO.2 0.19IfIO.01 6.0f0.5 8.8kO.2

WW pH: 3.5 2.55kO.01 96.220.2 0.20+0.01 5.1t0.3 7.8kO.2 5.5 2.56t0.01 96.510.3 0.18&0.01 6.OkO.4 8.5tO.l 1.5 2.54t0.02 96.OkO.6 0.21+0.03 5.8f0.2 8.7kO.O

Temperature (“C) at pH 3.5: 30 2.55t0.01 96.250.2 0.20t0.01 5.1t0.3 7.8-t0.2 35 2.57kO.02 97.OkO.6 0.16+0.03 6.1kO.4 7.4kO.l 40 2.3lkO.02 87.3f0.6 0.67kO.03 6.0f0.3 6.9t0.2

Temperature (“C) at pH 5.5: 30 2.56+0.01 96.5kO.3 0.18t0.01 6.OkO.4 8.5kO.l 35 2.59+0.01 97.9t0.2 O.ll+O.Ol 5.8t0.2 8.4-tO.l 40 2.58t0.01 97.420.4 0.13+-0.02 5.8kO.5 8.2kO.l

a Standard conditions: aeration rate, 1.0 vvm; WW pH, 7.5; temperature, 30°C; initial WW COD, 5.30 kg.m-‘; COD loading rate, 2.65 kg.mm3&‘; hydraulic retention time, 2.0d.

Data are the means of three determinations t standard deviations.

necessary. Similarly, Ehlinger (Ehlinger, F., French patent application, FR 2689493-Al, 1993) did not observe any reduction in treatment efficiency upon lowering the initial pH of the wastewater to 3.5; only the microbial growth was consistently limited. On the contrary, the initial pH was found to influence the treatment performance when the process temperature was increased: at 4O”C, for example, the COD removal efficiency was 97.4% at pH 5.5 but it was only 87.3% at pH 3.5.

As a consequence, all further experiments were performed under the following conditions: aeration rate, l.Ovvm; initial WW pH, 5.5; treatment temperature, 35°C.

Treatment performances with different bioreactors In the last two decades, several attempts have been made to investigate the possibility of using cell immobilization in the technology of aerobic wastewater treatment (15, 16). The very early experiments, however, were restricted to the use of selected pure cultures immobilized in/on solid supports for the degradation of specific toxic compounds (17, 18). Later, immobilized consortia of two or more selected strains were employed (19, 20) but only recently has activated sludge been immobilized on different carriers and used for wastewater treatment (16, 21-23).

To investigate whether the COD removal rate and efficiency of WW aerobic treatment could be improved by the use of immobilized microbial cell systems, two experimental glass-column reactors were employed. The first (Fig. 1A) was pneumatically agitated and used with activated sludge either free (ABB) or immobilized on polyurethane particles (fluidized-bed bioreactor, FBB), while the second (Fig. 1B) was a glass-column reactor used with the same activated sludge but immobilized on Raschig rings (packed-bed bioreactor, PBB).

Figures 2, 3 and 4 show the effects of increased COD loading rates on the performances of the ABB, FBB and PBB, respectively. In all cases, the COD removal rates increased with increasing loads. However, in the case of the FBB and PBB, the maximum loading rates were only

2 Li 0.0 140 2 3 4 5 6 7 6 9

Loading rate (kg-COD m-3 d-‘)

FIG. 2. Effect of loading rate on COD removal rate (0), final COD ( w ) and efficiency (A) in WW treatment using an ABB with free activated sludge. Values represent means of three determinations; standard deviations are lower than 5%.

oh-, 7100 L 40 1.0 15 20 25 3.0

Loading rate (kg-COD m3 de’)

FIG. 3. Effect of loading rate on COD removal rate (0), final COD ( n ) and efficiency (A) in WW treatment using a FBB with activated sludge immobilized on polyurethane sponge particles. Values represent means of three determinations; standard deviations are lower than 5%.

1 2 3 4 5

Loading rate (kg-GOD m” de’)

1.1 lco

10 I 0.9 90 g

06 8 6

o,7 E 180 .g 0'

0.6 Y ii o-70;

058

04- E g-60 a,

0.3 'L L

FIG. 4. Effect of loading rate on COD removal rate (C), final COD ( n ) and efficiency (A) in WW treatment using a PBB with activated sludge immobilized on Raschig rings. Values represent means of three determinations; standard deviations are lower than 5%.

3.30 and 5.77 kg-COD rne3.d-‘, respectively; at these feeding rates the final COD values were rather high, being 1.35 and 1.02 kg.mV3, respectively. With the ABB, however, at a loading rate as high as 8.24 kg of COD

384 PETRUCCIOLI ET AL. 1. BIOSCI. BIOEM,.

TABLE 3. WW treatment processes using the air bubble column bioreactor (ABB), the fluidized-bed bioreactor

(FBB) and the packed-bed bioreactor (PBB)

Parameters ABB FBB PBB

COD removal rate (kg .m ?,d- r) 6.61 1.79 3.22 Final COD (kg. m 3, 0.45 0.50 0.40 COD removal efficiency (%) 92.2 88.7 91.1 Hydraulic retention time (d) 0.8 2.2 1.2 MLSS (kg.m 3, 5.8 0.6 0.08 SVI (ml.g-r) 120.0 n.da n.d. P-PO, removal efficiency (%) 78.0 71.0 85.0

a nd., Not detectable. Data are the means of three determinations; values are given when

the COD removal rate was maximum and, simultaneously, the final CODs0.5 kg.mm3.

m m3. d- l, the COD of the resulting effluent was 0.9 kg. rnp3 and the treatment efficiency was still 86.3%.

Table 3 lists the optimal values of treatment parameters at the highest COD removal rates and when the final CODS were 10.5 kg.m-3. With the ABB, the COD removal rate was 6.6 kg+m m3. d l and the hydraulic retention time and MLSS were 0.8 d and 5.8 kg.mp3, respectively. With the FBB, however, the highest COD removal rate and the retention time were only 1.79 kg. m m3. d.-’ and 2.2 d, respectively, probably due to the formation of a layer of insoluble salts around the immobilized microbial biomass (micrographs not shown), thus limiting oxygen and nutrient transfer. The efficiency of the treatment process using the PBB was interesting: the COD removal rate and efficiency, and the hydraulic retention time were 3.22kg.mm3.d-l and 91.1%, and 1.2 d, respectively. The repeated passages through the packed bed led to a clearer final effluent in which the suspended solids were only 0.08 kg.mp3, a MLSS value similar to that (0.065 kg.mp3) reported by Hashimoto and Sumino (22) using activated sludge entrapped in polyethylene glycol prepolymer.

The sludge volume index (SVI) for the ABB (120 ml. g I) was well within the range (75-150 ml. g-t) generally recognized as acceptable (24). In the case of the FBB and PBB, the SVIs were not detected due to the very limited amount of MLSS (0.6 and 0.08 kg.mmm3, respectively) .

The initial content of phosphorus (as phosphate, P- POJ of the WW used in this study was low (530 g. mm3) and did not have any significant negative influence on the COD removal efficiency (results not shown). This result is somewhat surprising since the phosphorus content is considered to be of great importance in the wastewater biological treatment and the addition of this element has been suggested to improve treatment performances (23, 25).

From the above results, it can be concluded that no advantage of using immobilized cell systems was observed in the present study: the ABB, in fact, performed better than either the FBB or PBB.

Prolonged WW treatment with the ABB The ABB was fed continuously for 280 d; the WW was from different wineries (COD range, 2.7-6.6 kg.m m3) and the maximum loading rate was 8.8 kg of COD mm3.d-‘. Results are shown in Fig. 5.

As a rule, the COD of the final effluent ranged be- tween 0.11 and 0.3 kg.mp3 with a COD removal efficiency routinely higher than 90%. The critical loading rate

8 a

q- 7 E6

85

;;4

8" 2 1 0

0 50 100 150 200 250

Time (d)

FIG. 5. Time course of the performance of the WW treatment process using the ABB with free activated sludge. Line & symbols: -, loading rate; q , removal rate; A, MLSS; A, pH; 0, initial COD; 0, final COD; + , efficiency.

was 8.24 kg of COD mm 3 .d ‘, above which it was observed that the COD of the final effluent began to increase to reach a peak of 1.2 kg.m- 3 at the loading rate of 8.8 kg of COD m-m3.dm I. However, at this loading rate the removal efficiency was still high, being around 80%.

TABLE 4. Characterization of the microbial populations present in the mixed liquor (ML) and on the immobilizing carriers (CA) after

5-6 months of WW treatment using the ABB, the FBB and the PBB

Species ABB FBB PBB

ML ML CA ML CA

Bacteria Acinetobacter sp. + (3)a + (2) t (2) - -~ Aeromonas sp. 4. (6) - ~- Alcaligenes faecalis f (3) - - - Bacillus sp f (4) -t (4) -t (6) + (2) * (5) Flavobacterium sp . + (2) + (2) + (1) + (2) +- (2) F. meningosepticum -~ -- +m Pseudomonas sp. -t (15) - (10)-t (4) v (8) - (4) P. paucimobilis + (2) -~ -~ - P. vescicularis - * (5) + (3) - Sphingobacterium multivorum + (1) ~-. - - Filamentous bacteria t (1) tt (5) ~ 1 (2) Not identified + (5) + (4) + (5) + (2) 4 (3)

Fungi Aspergillus niger t (2) ~ Candida sp . - + (1) i- (3) + (1) * (2) C. lambica - t (1) ti (2) - C. lypolitica - + (2) + (2) Fusarium sp . .- + (1) t- (2) Penicillium sp. -. i (2) P. roquefortii - t- (1) Saccharomyces cerevisiae r- (6) + (2) + (2) t (2) + (1) Trieoderma koningii - + (1) + (2) - a Number of isolates apparently different. Legend: + , presence; +t, high frequency; - , absence.

VOL. 90, 2000 HIGH-RATE AEROBIC TREATMENT OF WINERY WASTEWATER 385

The COD removal rate and efficiency were higher than those obtained by Ehlinger (Ehlinger, F., French patent application, FR 2689493-Al, 1993) with yeasts but similar to those obtained by Fumi et al. (6) in a multi-step plant; in the latter case, however, the hydraulic retention times were up to ten times higher than those recorded in the study reported in the present paper using the ABB. Also Miiller (11) obtained COD removal efficiencies of 80-95X with a rotating biological contactor but the loading rate was routinely less than 4.3 kg of COD me3.d-*.

Microbiological characterization After 5-6 months of operation, the microbial populations (either free or immobilized) that developed in the bioreactors were isolated and characterized; results are summarized in Table 4.

The bacterial species isolated from the reactor liquid belonged for the most part to the genus Pseudomonas, well known for the ability of several of its members to degrade aromatic compounds (26, 27) and to produce exo-polysaccharides that play an important role in floe formation (24, 28). On the contrary, most isolates from the colonized carriers belonged to species of the genus Bacillus. In the case of colonized polyurethane particles, several gram-positive filamentous bacteria, the iden- tification of which was not possible based on the API test, were also isolated. Their presence, together with that of some filamentous fungi, is considered of great importance in the development of immobilized microbial populations because of their adhesion ability (15, 29).

As was expected, Saccharomyces cerevisiae was isolated from all bioreactors; this yeast is always present in winery wastewaters, particularly during vintage and racking, and might play an important role in the WW degradation (Ehlinger, F., French patent application, FR 2689493-Al, 1993).

No strains belonging to species of the genus Spirillum were observed and/or isolated, a clear indication of ade- quate aeration (24).

Finally, Protozoa, such as, Opercularia sp. and Ar- celfa sp., and Rotifera were present both in the free activated sludge floes and on the colonized carriers.

In conclusions, from the above results, it can be concluded that the aerobic treatment of winery wastewaters using aerated column reactors with free activated sludge, as compared to the use of more conventional technologies, appears to be promising in view of the impact that it could have on the reduction of the space required for the realization of winery wastewater treatment plants. It has been shown to yield optimal efficiency and conversion rates. On the contrary, the immobilization of the activated sludge did not lead to any improvement in treatment performance, probably due to the development of opportunistic populations that lowered the concentration of the effective biomass. However, in particular situa- tions in which an effluent is required with very low MLSS, thus avoiding (or reducing) the need for further treatment, the use of immobilized systems might be an acceptable compromise.

ACKNOWLEDGMENT

This work was developed under a project funded by the Euro- pean Commission, DG Environment, Program LIFE (Project 96ENV/P/00602/INDW).

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