Bioresource Technology 3 6 ( 19 91 ) 17 3-17 8

Effects of Agitation and Pretreatment on the Batch Anaerobic Digestion of Olive Mill Wastewater M. Hamdi*

Laboratoire de Microbiologie ORSTOM, Universit6 de Provence, 3 Place Victor-Hugo, F-13331 Marseille c6dex 03, France

(Received 5 January 1990; revised version received 4 August 1990; accepted 9 August 1990)

Abstract

The anaerobic digestion of ofive mill wastewaters (OMW) can be carried out only on a diluted sub- strate because aromatic compounds and lipids are toxic for methanogenic bacteria. Agitation decreases methane formation in anaerobic diges- tion of unmodified OMW. Acidified OMW is less toxic than is raw waste. Pretreatment of OMW by fermentation with Aspergillus niger decreases the toxicity for methanogenic bacteria and facilitates anaerobic digestion. Moreover, agitation did not affect gas production.

Key words: Olive mill wastewater, anaerobic digestion, phenolic compounds, methanogenic bacteria inhibition, pretreatment, agitation.

INTRODUCTION

The extraction of olive oil, which is carried out in small, seasonally-operating agroindustrial units located mainly in the Mediterranean region, results in the production of high-density olive mill wastewater (OMW) with a population equivalent in excess of 17 million people (Tsonis & Grigoro- poulos, 1988). The maximum BOD 5 and COD concentrations reach 100 and 200 g/litre respect- ively (Balice et al., 1982).

Anaerobic biological processes can be useful for dealing with this problem, since their well known advantages include savings in energy and chemicals and low sludge production. Moreover, the seasonal operation of olive oil mills is not a disadvantage for anaerobic processes because the observed methanogen decay rates are very low

*Present address: Centre de Biotechnologie, BP-W, 3038 Sfax, Tunisia.

Bioresource Technology 0960-8524/91/S03.50 © 1991 Great Britain

and a digester can be easily restarted after several months of shut-down (Lettinga et aL, 1980).

Several anaerobic processes such as anaerobic contact and Upflow Anaerobic Sludge Blanket (UASB) have been applied to diluted OMW treat- ment. However many problems concerning the high toxicity and the biodegradability of this efflu- ent, and the acidification of reactors have been encountered (Fiestas Ros de Ursinos et al., 1982; Boari et al., 1984; Andreoni et al., 1986). During start-up, acidifying microorganisms grow easily on the carbohydrates dissolved in the waste. The methanogenesis, which represents the limiting step in the anaerobic digestion of soluble com- pounds, is severely hindered by the combined inhibition caused by the high concentration of aromatic compounds and the build-up of volatile acids (Boari et al., 1984).

In the present study we investigated the effects of agitation, and the pretreatment of OMW by Aspergillus niger on the batch anaerobic digestion of OMW and examined the problems arising with processes involving the anaerobic digestion of OMW. We also describe a new method which gives more satisfactory results.

M E T H O D S

Culture methods The sludge used in all experiments was obtained from an anaerobic contact digester, treating diluted OMW at an average loading rate of 1 g/litre/day. Two litres of sludge were washed three times in Doffing (1985) solution under nitrogen to decrease the residual substrate of OMW. After each wash, the sludge was separated by sedi- mentation in a 12-h process. After the elimination of the supernatant solution the sludge was con-

173 Elsevier Science Publishers Ltd, England. Printed in

174 M. Hamdi

tinuously stirred and then distributed into serum bottles of 120 ml capacity. To each bottle was added 20 ml of sludge under strictly anaerobic conditions (02 free). The bottles were closed with black butyl rubber stoppers (Bellco Glass Inc., Vineland, N J, USA). The total solids and volatile solids in the sludge were 28.3 and 21.7 g/litre respectively. The maximum specific activity of this sludge measured at 20 mM acetate and formate (50:50) was about 8 mM methane/g VS d. The loaded serum bottles were stored at 4°C.

A Zeiss microscope equipped with epifluore- scence was used to detect any methanogenic bacteria, which exhibited a blue-green fluoresc- ence under UV illumination at 420 nm. Three substrates were studied (Table 1 ): OMW; acidified OMW resulting from batch anaerobic digestion of OMW at 80 g/litre of COD, then elimination of sludge by sedimentation; fermented OMW result- ing from A. niger growth for 72 h. The fungal strain used was Aspergillus niger strain A10 (Raimbault and Alazard, 1980). The mycelium was collected by filtration and the liquid digested.

In all experiments, the substrates were buffered with Na2CO3, keeping the 0.5 eq mole Na2CO3/g COD ratio constant and adjusting the pH to 7.25 by means of Ca(OH)2. At the time of use, 20 ml of substrates were outgassed with N 2 and added with a syringe. The pressure in all serum bottles was 1 atmosphere unless otherwise indicated. The bottle contents were not mixed unless noted. In each experiment, a bottle control was always carried out to subtract the methane or volatile fatty acids (VFA) produced by the sludge from the methane or VFA produced from sludge and substrate. Methane and VFA were analysed in separate bottles.

The study of the substrate toxicity was based on the methane production at increasing con- centrations of substrate. At each concentration, 1 control serum bottle and 1 serum bottle contain- ing substrate were used. For the study of the effect

Table 1. Characteristics of olive mill wastewaters

of agitation speed on the anaerobic digestion of OMW, four serum bottles were necessary to determine the methane and VFA production; two control serum bottles for methane and VFA pro- duction and two serum bottles containing sub- strates for methane and VFA production. The concentration of unmodified OMW and OMW fermented by A. niger used to study the effects of agitation was 20 g COD/litre. The agitation rate was varied by increasing the rotation speed of the shaker. All tests were done at 35°C.

Analytical methods Gas samples were taken with a syringe from the headspace of the serum bottle and analysed by gas chromatography with a Delsi instrument chromatograph (DELSI-Nermag, Argenteuil, France) equipped with a flame ionization detector. The flame ionization detector was fitted with a 80 cm stainless steel column packed with 4% H3PO 4 on Porapack Q (80-100 mesh). N 2 was used as carrier gas at 28 ml/min with H: and air flows of 25 and 30 ml/min, respectively. The oven, in- jector and detector temperatures were 200°C. The methane concentration was calculated with an ENICA 10 integrator (DELSI-Nermag). The same apparatus was used for VFA analysis. The sample was centrifuged at 4000 rpm for 10 min and acidified by 1% of H3PO 4 (50%). The total solids were determined after drying the sludge overnight at 105°C. The ash content was deter- mined after calcination of the dry sludge at 600°C for 1 h. The difference between total solids and ash content was defined as volatile solids (VS).

Glucose and reducing sugars were estimated using the glucose-oxidase method (Werner et al., 1977) and Somogyi micromethod (Nelson, 1944; Somogyi, 1945). The chemical oxygen demand (COD) was estimated according to Standard Methods (American Public Health Association, 1975). The method used for vegetable extracts (Peri & Pompei, 1971) was adapted to raw olive

Unmodified OM W Fermented OM W A cidified OM W

pH 5-4 5-6 5.05 Characteristics (g/litre) COD 165.4 72.8 64.3 TS 157.4 68.2 58.1 VS 144-7 61"2 48.6 VFA 1.27 0"38 13.2 Reducing sugars 37-5 2.6 6"7 Glucose 7.3 0.4 0 Oil 11-3 4"9 3"6 Total phenolics 16-8 7.2 5.8

Digestion of olive mill wastewater 175

mill effluent and fermented effluent with a few modifications (Balice et al., 1988). Total phenolic compounds were determined with phospho- molybdic-phosphotungstic reagent (Folin & Ciocalteau, 1927) and expressed as g pyrogallol/ litre.

RESULTS

The methanogenic bacterial genera observed in sludge used in these experiments were morpho- logically Methanobacterium, Methanococcus, Methanospirillum, Methanogenium and Methano- sarcina. Methane formation by this sludge was studied with three substrates.

Batch anaerobic digestion of unmodified and acidified OMW Batch cultures containing proportions of OMW ranging from 10 to 30 g COD/litre were studied. Figure l a summarizes the cumulative methane production in these batch cultures over a 15-day incubation period. Since the pH of the media was buffered, the decrease in methane production observed when the COD concentration increased was due to the presence of toxic compounds in this effluent, such as tannins, phenolic compounds and oils. For example, similar results have been observed in anaerobic batch digestion of phenol and some alkyl phenolics (Fedorak & Hrudey, 1984).

The batch anaerobic digestion of unmodified OMW at various agitation speeds gave several levels of methane production (Fig. 2a). The cumu- lative methane production decreased as the agita- tion increased.

The VFA analysis in batch cultures at various agitation speeds is given in Fig. 3. This shows that agitation speeds of up to 50 rpm increase the VFA production rate during the first day. At an agita- tion speed of 200 rpm, the VFA microbial pro- duction was disrupted and accumulation of acetate began only after the first day. The kinetics of ethanol production and utilisation were modi- fied by agitation. With an agitation speed of 50 rpm, the ethanol was more quickly converted into acetate than with 0 and 200 rpm. When methano- genie bacteria are inhibited, the pressure of hydrogen increases and ethanol accumulates. Thauer et al. (1977) noted that it is only at low partial pressures of H2 that ethanol can be con- verted into acetate.

Acidified OMW was slightly less toxic than unmodified OMW, as shown in Fig. lb. In the case of anaerobic digestion of unmodified OMW, 10 g COD/litre was found to give more methane than 20 and 30 g COD/litre. In the case where OMW was acidified, 20 g COD/litre gave more methane than 10 g COD/litre, but 30 g COD/litre gave the same methane as a batch with 20 g COD/ litre. Thus the toxicity threshold is greater with acidified OMW than unmodified OMW. How- ever, 10 g unmodified OMW/litre gave 600 #M methane, while 10 g COD/litre of acidified OMW gave only 400/~M methane.

Batch anaerobic digestion of OMW fermented by A spetgillus niger At COD concentrations up to 30 g/litre the methane production was not inhibited. In fact, wastewater fermented by A. niger had enhanced methane production (Fig. lc). This shows that treatment by A. niger decreases the toxicity of OMW to methanogenic bacteria. In fact, the oil and phenolic compounds contained in fermented OMW were lower than in the unmodified OMW, as shown in Table 1. This fermented OMW can

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176 M. Hamdi

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therefore be easily treated by anaerobic digestion, with less toxicity and with less dilution.

Contrary to the case of unmodified OMW, agitation did not affect the extent of methane pro- duction in batch digestion of OMW fermented by A. niger (Fig. 2b). The rates of acetate production were faster than in the digestion of unmodified OMW, and ethanol was not detected (Fig. 4). In the digestion of OMW fermented by A. niger, acetate and butyrate accumulated but were immediately removed because the methanogenic bacteria were relatively uninhibited. The agitation also increased the rate of elimination of acetate (Fig. 4).

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DISCUSSION

Tannin concentrations range from 8-16 g/litre in OMW (Balice et al., 1982). The phenolic com- pound concentrations pass 10 g/litre (Fiestas Ros de Ursinos, 1981; Balice et al., 1988) and residual oil depends on the olive oil processing method and can reach 50 g/litre.

Results concerning the inhibition of methane production from unmodified OMW were expected since it was reported that 2 g tannins/ litre (Field & Lettinga, 1987), 1 g phenolic com- pounds/litre (Fedorak & Hrudey, 1984) and 5 mM oleic acid (Koster & Cramer, 1980) are toxic to methanogenic bacteria. OMW had to be diluted to 40 g/litre from 100 and 200 g COD/litre (Balice et al., 1982) in order to reduce the inhibi- tion effect of lipids and phenolic compounds (Table 2).

Boari et al. (1984) reported that performance of anaerobic contact digesters was not satisfactory because of excessive mechanical mixing. Indeed, Fig. 2a shows that the methane production decreases when the agitation rate increases. Better anaerobic digestions of OMW were obtained with UASB and the anaerobic filter, where mixing is lower than in anaerobic contact digesters.

The inhibition obtained with unmodified OMW can be reduced during the acidification step (Fig. lc). This result is in conformity with the high gas

Digestion o f ofive mil l wastewater

Table 2. Performances of anaerobic processes fed with olive mill wastewaters

177

Process Volume Load Efficiency References ( l~ h (g COD/I n . day) (% COD) i

Contact 2 600 1-55 70 Antonacci et al. ( 1981 ) 70 000 2"55 80 Fiestas et al. (1982)

30 6"00 75 Balice et al. (1988)

UASB 15 16-00 70 Boari et al. (1984) 5 000 16"00 70 Boari et al. (1984)

20 15"40 70 Balice et al. ( 1988 )

HAUSBUFF 19"7 190"00 40 Tsonis and Grigoropoulos ( 1988)

Fixed bed 2 a 2-70 65 Hamdi (1987) 2 h 4.40 75 Hamdi (1987)

21 ' 2"80 83 Rigoni-Stern et al. (1988) 300 ' 8"00 87 Rigoni-Stern et al. (1988)

10 d 2"50 60 Rozzi et al. (1989) 10 e 2"50 55 Rozzi et al. (1989) 11Y 3"00 65 Rozzi et al. (1989) 11 ~ 3"00 60 Rozzi et al. (1989)

Media used. °Plastic, bClay, 'Polyurethane, dprisms, eCubes, JCyl. plugs T30, UCyl. plugs TR30. hlR: Litre reactor volume. i% COD = COD removal.

production rates (up to 30 m 3 of gas 78.5% C H 4 /

m3/day) obtained with a hybrid anaerobic upflow sludge blanket-upflow fixed filter (HAUSBUFF) treating acidified OMW from an open holding basin and stored for 5 to 7 months (Tsonis & Griopoulos, 1988). The acidification may be car- fled out in a stirred tank reactor since Fig. 3 shows that the kinetics of VFA production were improved by agitation.

The pretreatment of OMW is significant since the production of methane per gram of intro- duced COD was twice that obtained with un- modified OMW (Fig. 1). Moreover, the accumulated acetate was removed during the anaerobic digestion when OMW was pretreated by A. niger, while it remained when OMW was not modified (Figs 3 and 4). This means that the pretreatment of OMW by A. niger improves its biodegradability and reduces its inhibition effect. It may be considered as a main step in the OMW digestion.

These results show that besides dilution, acidi- fication or pretreatment can be a main means of carrying out the anaerobic digestion of OMW with less inhibition of methanogenic bacteria.

REFERENCES

American Public Health Association (1975). Standard Methods for the Examination of Waste and Wastewater 14th edn. pp. 550-4.

Andreoni, V., Ferrari, A., Ranali, G. & Sorlini, C. (1986). The influence of some phenolic acids present in oil mill waters on microbial groups for the methanogenesis. In Proc of Inter. Symp. on Olive Byproducts Valorization. FAO, 5-7 March, Espana.

Antonacci, R., Brunetti, A., Rozzi, A. & Santori, M. (1981 ). Trattamento anaerobico di acque di vegetazione di frantoio. 1. Risultati preliminari, lngegneria Sanitara, 6, 257-62.

Balice, V., Boari, G., Cera, O. & Abbaticchio, P. (1982). Indagine analitica sulle acque di vetetazione. Nota 1. Inquinamento, no. 7- 8, 49-53.

Balice, V., Carrieri, C., Cera, O. & Rindone, B. (1988). The fate of tannin-like compounds from olive mill effluents in biological treatments. In Proc. of Fifth Int. Symp. on Anaerobic Digestion. Bologna, Italy, 22-26 May, ed. E. R. Hall & E N. Hobson. Academic, pp. 275-9.

Boari, G., Brunetti, A., Passino, R. & Rozzi, A. (1984). Anaerobic digestion of olive oil mill wastewaters. Agricul- tural Wastes, 10, 161-75.

Dolfing, J. (1985). Kinetics of methane formation by granular sludge at low substrates concentrations. Appl. Microbiol. Biotechnol., 22, 77-81.

Fedorak, P. M. & Hrudey, S. E. (1984). The effects of phenol and some alkyl phenolics on batch anaerobic methano- genesis. Water Res., 18,357-61.

Field, J. A. & Lettinga, G. (1987). The methanogenic toxicity and anaerobic degradability of a hydrolysable tannin. Wat. Res., 20, 367-74.

Fiestas Ros de Ursinos, J. A. (1981). In Proc: Diff6rentes utilisations des margines. In Proc. of S~minaire inter- national sur la valorisation des sous produits de l'olivier. Organisation des Nations Unies pour l'Alimentation et l'Agriculture. Tunisie. D~cembre. pp. 93-110.

Fiestas Ros de Ursinos, J. A., Navarro Gamero, R., Leon Gabello, R., Garcia Buendia, A. J. & Mastrojuan Saez de Jauragui, G. M. (1982). Depuracion anaerobia del apechin como fuente de energia. Grasas y Aceites., 33,265-70.

Folin, O. & Ciocalteau, V. (1927). On tyrosine and trypto- phan determinations in protein. J. Biol. Chem., 73, 627-50.

178 M. Hamdi

Hamdi, M. (1987). Digestion ana6robie des margines par filtres ana6robies. Dipl6me d'ing6nieur., Universit6 de Provence, Marseille.

Koster, I. W. & Cramer, A. (1980). Inhibition of methano- genesis from acetate in granular sludge by long-chain fatty acids. Appl. Environ. Microbiol., 53,403-9.

Lettinga, G., Van Velson, A. E M., lobma, S. W., de Zeeuw, W. & Klapwijk, A. (1980). Use of the upflow sludge blanket (USB) reactor concept for biological wastewater treatment, especially for anaerobic treatment. Biotech. & Bioeng., 222,699-734.

Nelson, N. (1944). Photometric adaptation of somogyl method for determination of glucose. J. Biol. Chem., 153, 375-80.

Peri, C. & Pompei, C. (1971). Estimation of different phenolic groups in vegetable extracts. Phytochem., 10, 2187-9.

Raimbault, M. & Alazard, D. (1980). Culture method to study fungal growth in solid fermentation. Eur. J. Appl. Microbiol. BiotechnoL, 9, 199-9.

Rigoni-Stern, S., Rismondo, R., Szpyrkowicz, L. & Zilio grandi, F. (1988). Anaerobic digestion of vegetation water

from olive oil mills on a fixed biological bed with a biogas production. In Proc. of Fifth Int. Symp. on Anaerobic Digestion. Bologna, Italy, 22-26 May, ed. A. Tilche & A. Rozzi. Moduzzi Editore SPA, pp. 561-5.

Rozzi, A., Passino, R. & Limoni, M. (1989). Anaerobic treat- ment of olive mill effluents in polyurethane foam bed reactors. Process Biochemistry, April, 68-74.

Somogyi, M. (1945). Determination of blood sugar. J. Biol. Chem., 160, 61-8.

Thauer, R. K., Jungermann, K. & Decker, K. (1977). Energy conservation in chemotrophic anaerobic bacteria. Bact. Rev., 41,100-80.

Tsonis Stelios, P. & Grigoropoulos Sotirios, G. (1988). High rate anaerobic treatment of olive oil mill wastewater. In Proc. of Fifth Int. Symp. on Anaerobic Digestion. Bologna, Italy, 22-26 May, ed. E. R. Hall & P. N. Hobson. Academic, pp. 115-24.

Werner, W., Rey, H. G. & Wielinger, R. H. (1977). Uber die eigenschoften eines neuen chromogens fur die Blutzucker nach der GOD/POD methode. Z. Anal. Biochem., 252, 224-8.