Improvement of the anaerobic biodegradation of olive mill wastewaters by prior ozonation pretreatment

Improvement of the anaerobic biodegradation of olive mill wastewatersby prior ozonation pretreatment

F.J. Benitez, J. Beltran-Heredia, J. Torregrosa, J.L. Acero

Abstract The purification of olive mill wastewaters(OMW) is investigated by a single anaerobic digestion in abatch reactor containing immobilized microorganisms,and by the combination of an ozonation pretreatmentfollowed by an anaerobic digestion. In the single anaerobicdigestion the removal of the COD is determined and themethane yield coefficient, which is the best measure of theextent of transformation of the biodegradable substrate, isalso obtained, its value being 194 ml CH4/g COD. A kineticstudy is performed by using the Monod model combinedwith the Levenspiel model, due to the presence of inhibi-tion effects. Both models lead to the determination of thekinetic parameters of this anaerobic treatment: kineticconstants, critical substrate concentration of inhibitionand inhibitory parameter. In the combined process, theozonation pretreatment of OMW achieves a great reduc-tion in the phenolic compounds, leading to a significantincrease in the methane yield coefficient in the followinganaerobic digestion, its value being 266 ml CH4/g COD.

List of symbolsAlk Alkalinity, g CaCO3/l.BOD Biological Oxygen Demand, g/l.COD Chemical Oxygen Demand, measurement of the

substrate concentration, g/l.CODnb Non-biodegradable concentration of substrate, g/l.CODm Average concentration of substrate in an experi-

ment, g/l.COD� Inhibition COD (substrate) concentration, g/lDMS Dissolved Mineral Solids concentration, g/l.DVS Dissolved Volatile Solids concentration, g/l.KS Monod Saturation constant, g/l.ko Constant defined by Eq. (10), days)1.k Rate constant without inhibitory effects, days)1.MS Total Mineral Solids concentration, g/l.MSS Mineral Suspended Solids concentration, g/l.

N Ammonia nitrogen, g N/l.n Inhibitory parameterP Phosphorus, g/l.RS Reducing sugars, g/l.)rCOD Biological reaction rate, g of COD/l.dayS Biodegradable substrate concentration, g/l.TKN Total Kjeldahl Nitrogen, g N/l.TOC Total Organic Carbon, g/l.TP Total phenolic content, g caffeic acid/l.VM Volume of methane produced, ml.VA Volatile acidity, g acetic acid/l.VS Total Volatile Solids concentration, g/l.VSS Volatile Suspended Solids concentration, g/l.XCOD COD removal yield, %.XTP Total phenolic compounds conversion, %.X Biomass concentration, g/l.YP/S Methane production yield coefficient, ml of CH4/g

of COD.YX/S Cellular yield coefficient, g cell mass/g of sub-

strate.l Specific growth rate of microorganisms, day)1.lmax Maximum specific growth rate of microorganisms,

day)1.

1IntroductionThe manufacture of olive oil is carried out in numeroussmall plants which operate seasonally. They produce anaqueous phase (around 50% of the total volume treated)arising from the water content of the fruit combined withthe water used to wash and process the original olives.This combination is named ‘‘olive mill wastewater’’(OMW), and its annual production in the Mediterraneancountries is estimated over 3 × 107 m3 (1).

Usually these effluents are disposed of into evaporationponds or through public sewers. As they contain a highload of organic matter, including sugars, tannins, poly-alcohols, polyphenols (2), they represent a large-scale en-vironmental problem, mainly owing to the bad smellcaused by fermentation processes and the possibility of thepollution of surface waters and underground aquifers.Therefore, several treatments have been investigated toreduce the polluting power, mainly biological processes(3, 4).

Preferently, the anaerobic digestion of the material isconducted because of its advantages: low costs, it demandslittle energy and very low amounts of nutrients, and theanaerobic bacteria are capable of transforming most of the

Bioprocess Engineering 17 (1997) 169–175 Springer-Verlag 1997

169

Received: 11 November 1996

F.J. Benitez, J. Beltran-Heredia, J. Torregrosa, J.L. AceroDepartamento de Ingenieria Quimica y EnergeticaUniversidad de Extremadura. 06071 Badajoz. SpainFax number: 34-24-271304

Correspondence to: F.J. Benitez

This research has been granted by the ‘‘Comision Interministerialde Ciencia y Tecnologia’’ (CICYT) of Spain, under Project AMB96-0815.

organic substances present in the waste into methane.However, some organic compounds like phenols, are toxicfor methanogenic bacteria and inhibit the anaerobic di-gestion. So, several pretreatments are often proposed, likechemical ozonations (5) or biological processes (6), whichremove phenolic compounds, decrease the toxicity formethanogenic bacteria and facilitate the anaerobic diges-tion.

In this work, the single anaerobic degradation of OMWis studied in a first step with the aim of providing results ofthe removal of the COD obtained and the methane pro-duced by determining the yield coefficient YP=S. And akinetic study, which takes into account the inhibition ef-fects that appeared in the process, is conducted to reportseveral kinetic parameters of practical interest in the de-sign of purifying plants for this type of wastes. In a secondstep, an ozonation pretreatment is performed, in order toverify if this chemical oxidation improves the biodegrad-ability of the OMW by removing the phenolic inhibitors ina great extent and enhances the yield of the followinganaerobic degradation. In that case, the production ofmethane per mass unit of introduced COD must be higherthan that obtained in the single anaerobic digestion.

2Material and methods

2.1Olive mill wastewatersThe original wastewaters used as substrate were obtainedfrom an olive oil production plant (‘‘Cooperativa SantaMarta Virgen’’, Extremadura Community, south west ofSpain), which uses the traditional mill and pressing pro-cess. The main chemical characteristics and compositionsof these OMW were evaluated and are summarized inTable 1. Those analyses were performed according to theprocedures described in the Standard Methods (7), whilethe total phenolic content was determined by the Folin-Ciocalteau method (8). Prior to each experiment, theOMW were centrifuged and filtered to remove suspendedsolids.

2.2Fermentation system and inoculumThe anaerobic experiments were conducted in a magnet-ically stirred batch anaerobic digestion unit. It consisted ofa 1000 ml cylindrical Pyrex glass vessel, provided with acover containing inlets for loading feedstocks and bub-bling the inert gas (required for unloading), and outlets forremoving effluents and venting biogas. The reactor wassubmerged in a thermostatic bath with the necessary ele-ments to maintain the temperature constant at 35 °Cwithin ± 0.2 °C. The microorganisms effecting the processwere supported on sepiolite, a micronized fibrous silicate(Mg4Si6O15(OH)2.6H2O) that retained methanogenic bac-teria preferentially. The volume of methane produced wasmeasured by using an 1 l Boyle-Mariotte reservoir whichwas connected to the reactor. The biogas produced waspassed through a NaOH solution to retain CO2 and thevolume of methane yielded was determined from theamount of water displaced by the gas.

The reactor was inoculated with a sludge taken from anOMW anaerobic treatment plant. The digester was initiallycharged with 850 ml of distilled water and 150 ml of theinoculum in order to obtain a biomass concentrationaround 14 g/l of VSS, and 15 g of support. A previous stagewas necessary to acclimatize the bacterial flora to this sub-strate over a 5 months period, by incrementing the volumeof OMW loaded to the reactor in order to reach increasinginitial substrate concentrations, measured as COD, from 1 to5 g/l. During this period, every new fresh feed was addedeach time when the COD concentration remained almostconstant and the methane production had stopped. Thebiomass acclimatization was finally achieved when afterthree experiments conducted with the higher COD initialconcentration, a similar removal of COD was obtained.

2.3Ozonation systemThe ozonation experiment was conducted in a completelymixed batch reactor where the wastewaters, diluted withdistilled water up to 50% due to their high COD content,was loaded. It consisted of a 1200 cm3 cylindrical Pyrexglass vessel, provided with a cover containing inlets forbubbling the gas feed and stirring, and outlets for sam-pling and venting. The reactor was also submerged in athermostatic bath with the necessary elements to maintainthe temperature constant within ± 0.2 °C. For the ozonegeneration, oxygen taken from a commercial cylinder wasdried with silica gel traps and introduced into an ozonegenerator (Yemar, model HPA).

2.4Experimental procedureIn the single anaerobic digestion process, once the accli-matization period was finished, the experiments werecarried out by loading several volumes of the originalOMW leading to the different substrate initial concentra-tions CODo given in Table 2. Amounts of K2HPO4 and ureawere also added as nutrients to these OMW loads in orderto maintain the ratio COD:N:P around 250:5:1, this ratiobeing adequate to keep satisfactory microbial activitiesinto the inoculum culture (9).

Table 1. Chemical characteristics and contents of the OMW

Parameter Value Units

pH 4.84BOD 52 g/lCOD 112 g/lTOC 16.01 g/lN 0.042 g N/lTKN 0.126 g N/lP 1.61 g/lRS 14.4 g/lTP 2.20 g caffeic acid/lVA 0.27 g acetic acid/lAlk. 3.40 g CaCO3/lMS 31.14 g/lVS 59.83 g/lDMS 30.65 g/lDVS 55.18 g/lMSS 0.49 g/lVSS 4.65 g/l

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Bioprocess Engineering 17 (1997)

Prior to each new volume of material to be degraded beingintroduced into the reactor, an equal volume was removedfrom it, after a settlement period of one night in order toavoid biomass losses. Each experiment was run for the timenecessary for complete biomethanation of every load, thatis, when the methane production and the COD removalceased. At regular intervals through out an experiment, thesubstrate concentration COD and the methane released VMwere determined. Table 3 shows as an example the valuesobtained in an experiment (BN-2 of Table 2).

In the ozonation experiment of the combined process,when the temperature, pH (by adding orthophosphoricacid and sodium hydroxide) and ozone partial pressureswere adjusted to the desired values of 20 °C, 4.85 and1.30 kPa respectively, the ozone-oxygen gas stream wasfed to the the reacting medium through a bubble gassparger with a constant flow rate of 40 l/h at room con-ditions. Once the process started, the experiment lastedaround 8 hours, and several samples were taken periodi-cally to analyze the substrate concentration and the totalphenolic content present in the reacting mass.

Later, different volumes of this pretreated OMW wereloaded into the bioreactor, and anaerobic experimentswith different initial concentrations of substrate wereperformed in the same experimental procedure as hasbeen described for the single anaerobic digestion.

3Results and discussion

3.1Single anaerobic digestionThe anaerobic digestion of OMW has been conducted byseveral experimental series where the initial concentrationof substrate to be degraded was modified between 3.69 and11.79 g of COD/l.

3.1.1COD removal and product (methane) formationAs shown in Fig. 1, the COD concentration decreasedcontinuously with degradation time in all experimentsuntil a constant value was reached: this is the corre-sponding value to the non-biodegradable substrate con-centration represented by CODnb and it was estimated by agraphical procedure involving extrapolation of the CODconcentration at an infinite time of bioreaction. As severalauthors pointed out, this residual non-biodegradable or-ganic matter is due to the presence of compounds likephenols and polyphenols which are very difficult for theflora to break down.

The COD removal yield is defined in the form:

XCOD �

CODo ÿ CODf

CODal� 100 ; �1�

where CODo and CODf are respectively the initial and finalCOD of an experiment; and CODal is the COD contained inthe initial volume of OMW loaded to the reactor prior toeach experiment. The values obtained for XCOD are shownin Table 2: the decrease in this yield can be attributed tothe toxic and inhibitor characteristics of the phenoliccompounds present in a greater extent when the concen-tration of organic matter is higher. Those compounds in-

Table 2. COD Removal yield and methane production in the single anaerobic digestion

Expt. CODo CODf CODal XCOD )rCOD VMF YP/S

BN-1 3.69 2.53 1.50 77.3 0.42 225 194BN-2 4.17 2.69 1.95 75.8 0.57 305 205BN-3 5.68 3.18 3.30 75.7 0.96 475 196BN-4 8.36 4.39 6.00 66.2 1.22 750 188BN-5 11.79 6.05 8.90 64.5 1.58 1060 185

g/l g/l g/l % g COD/day · l ml ml CH4/g COD

Table 3. Evolution of COD and methane procuded in experimentBN-2

Time COD VM

0 4.17 00.5 3.42 1651 3.21 2201.5 3.07 2572 2.92 2853 2.84 2933.5 2.75 3004.5 2.69 305

day g/l ml Fig. 1. Evolution of the COD concentration with reaction time forexperiments where COD0 was varied

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F. J. Benitez et al.: Improvement of the anaerobic biodegradation of olive mill wastewaters by prior ozonation pretreatment

hibit the methanogenic step, and subsequently, the wholeanaerobic process.

The biological reaction rate )rCOD (g of COD/l.day) wasalso determined in every experiment performed, calculat-ing (dCOD/dt) by fitting the experimental data to a poly-nomic expression by least squares regression analysis.Table 2 provides the values obtained at the first day ofreaction: it can be seen that this rate increases when theinitial substrate concentration was also increased, as canbe expected.

On the other hand, the volume of methane produced VMwas measured through each experiment. Figure 2 plots theVM values versus time: it can be observed that the highestproduction rates at initial times of reaction is obtained inexperiments where the CODo was highest. The final valuesof methane formed in every experiment VMF are given inTable 2.

The methane production yield coefficient YP/S (ml ofCH4/g of COD) is determined by the definition equation:

YP=S �VMF

CODo ÿ CODf; �2�

The results obtained are also shown in Table 2, and fromthese it can be assumed an average value of 194 ml CH4/gCOD. This value is in the range of those reported by sev-eral authors for similar processes: 150 ml CH4/g COD byHamdi et al. (3); 234 ml CH4/g COD by Cail and Barford(10) and 260 ml CH4/g COD by Martin et al. (11).

Finally, at the end of each experiment, the pH, the vol-atile acidity and the alkalinity were also measured. The pHvaried between 7.5 and 7.6, and therefore, it remainedthroughout the experiments within the optimal workingrange (6.4–7.8) for anaerobic digesters (9). The volatileacidity increased when the organic matter loaded to thereactor also increased, from 64 mg of HAc/l for experi-

ment BN-1 to 201 mg of HAc/l for experiment BN-5. In allcases, they were lower than 500 mg of HAc/l, the limitvalue reported by Cail and Barford (12) for the anaerobicreactors. The alkalinity remained virtually constantaround 4500 mg of CaCO3/l, a higher value than 2500 mgof CaCO3/l, minimum amount needed to assure the de-velopment of the process in optimum conditions (13).

3.1.2Kinetic studyAn anaerobic digester can be considered as a bioreactorwhere the biodegradable substrate S reacts with a con-centration of microbial sludge X. Both parameters arerelated by the cellular yield coefficient YX=S (g cell mass/gof substrate):

YX=S � ÿ

dXdS

; �3�

In such a process, the rate of production of biomass is welldescribed by a first order kinetic equation:

dXdt

� l X ; �4�

where l is the specific growth rate.The introduction of Eq. (3) into Eq. (4) leads to an ex-

pression for the rate of substrate decomposition:

ÿ

dSdt

�

l XYX=S

; �5�

For the determination of the specific growth rate l, theMonod equation can be used:

l �

lm SKs � S

: �6�

At low substrate concentration, where KS>>>S (14),Eq. (6) simplifies to:

l �

lm SKS

; �7�

which introduced into Eq. (5) leads to:

ÿ

dSdt

�

lm XYX=S Ks

S � K X S : �8�

Because of the low cellular yield coefficient YX=S inan anaerobic system (0.02–0.06 g cells/g of substrate)(15, 16), and taking into account the low amounts of theCOD consumed through these experiments (between 1 and6 g, see Table 2), it can be assumed that X remains almostconstant, and Eq. (8) is transformed into:

ÿ

dSdt

� koS ; �9�

with ko being equal to:

ko �lmX

KSYX=S; �10�

This aspect was confirmed by the experimental results:the measured biomass concentration was very similar in allthe experiments, with an average value of 13.8 g of VSS/l.

Fig. 2. Evolution of the methane production with reaction time forexperiments where COD0 was varied

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According to Eq. (9), the removal of organic matterfollows a first order kinetics, this aspect being previouslypointed out by several authors (17, 18). Therefore, its in-tegration with the condition S � So for t � 0, leads to:

lnSo

S� kot : �11�

It must be remarked that S represents the biodegradablesubstrate concentration, which in this research was de-termined by subtracting the non-biodegradable concen-tration of substrate CODnb from the substrateconcentration COD at any time. This CODnb was, estimatedas was previously described, and the values obtained foreach experiment are given in Table 4 together with theresulting initial So values.

According to Eq. (11), a plot of ln(So/S) versus time mustlead to straight lines. Figure 3 shows this plot for the ex-periments conducted: the straight lines obtained confirmthe validity of the proposed model. After least square re-gression analysis, the constants ko shown in Table 4 arededuced. It can be observed that those constants decreasewhen So increases, and specially when its value of So ishigher (experiments BN-4 and BN-5): it does mean thatsome inhibition effects by medium constituents such assubstrate or products takes place.

For the evaluation of this inhibition process, theLevenspiel model (19) is used. In this model the constantko is related to the concentration of substrate by means ofthe expression:

ko � k 1 ÿCODm

COD�

� �n

�12�

where k is the rate constant when no inhibitory substanceis present; n is an inhibitory parameter; CODm representsthe average concentration of substrate in the experiment(the mean value in this case, being reported in Table 4);and COD� is the substrate inhibition concentration, criticalvalue when the cells cease their activity because of theinhibition process.

Eq. (12) can be linearized in the form:

ln ko � ln k � n ln 1 ÿCODm

COD�

� ��13�

As COD� it is not previously known, this equation isresolved by non-linear regression analysis, which leads tothe following values: k � 0:96 daysÿ1, n � 1:50 andCOD�

� 20 g/l. Therefore, it can be proposed the fol-lowing expression for the constant ko of the Levenspielmodel:

ko � 0:96 1 ÿCODm

20

� �1:50

�daysÿ1� : �14�

In order to validate the robustness of the model usedand the proposed equations, the theoretical values of thebiodegradable substrate concentration S are calculatedfrom Eqs. (11) and (14), and plotted as continuous lines inFigure 4 with the experimental results obtained in theexperiments. As can be seen, there is a general agreementbetween both values, with discrepancies within ± 8%,which confirm the proposed model.

Table 4. Values of CODnb, So, ko constants, and CODm in thesingle anaerobic digestion

Expt. CODnb So ko CODm

BN-1 2.48 1.21 0.77 3.11BN-2 2.63 1.54 0.70 3.43BN-3 3.10 2.58 0.65 4.47BN-4 4.28 4.08 0.58 6.37BN-5 5.80 5.99 0.37 8.92

g/l g/l day)1 g/l

Fig. 3. Kinetic study: Determination of the ko constants

Fig. 4. Comparison between theoretical values (continuous lines)and experimental results for the biodegradable substrate concentra-tion

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3.2Combined process of ozonation and anaerobic digestion

3.2.1Ozonation pretreatment stageIn this experiment, it was observed that the substrateconcentration decreased continuously with time. Table 5shows the initial and final COD values obtained, and theconversion reached: it can be seen that the COD reductionis only of 6.2 %. This low conversion can be attributed tothe fact that, although the organic compounds are veryreactive towards ozone and generate in the ozonation re-actions numerous intermediates constituted by smallermolecules with less pollutant character in many cases,these intermediates still demand oxygen to reach their fi-nal stages of oxidation, and therefore, the reduction ofCOD is low.

On the other hand, Table 5 also depicts the initial andfinal values of the total phenolic compounds present TPand the conversion XTP obtained in the experiment. As canbe observed for this parameter, an important removal of94.3% is reached after 8 hours of ozonation. As waspointed out in the Introduction, these phenolic com-pounds present in OMW are the main limiting factor forusing anaerobic digestion for these residues, since they aretoxic to methanogenic bacteria. Their elimination thusshould reduce the toxicity for a later anaerobic treatment.

3.2.2Anaerobic digestion stageAs was explained in the Experimental Procedure, with theOMW from the ozonation pretreatment, several anaerobicdigestion experiments were performed with the initialsubstrate concentrations CODo given in Table 6. In asimilar way as in Table 2 for the single anaerobic digestionprocess, Table 6 also shows the final substrate concentra-tion CODf at the end of the experiment, the containedCODal in the volume loaded to the reactor, the COD re-moval yield XCOD, the biological reaction rate ÿrCOD, the

final volume of methane produced VMF, the methaneproduction yield coefficient YP=S and the constant ko.

For these parameters, similar trends can be observedthan in the single digestion: thus, when CODo increases,XCOD and ko decrease, while ÿrCOD and VMF also increase.However, the most significant aspect is related with YP=S:in this case, again it remains almost constant, but now it isobtained an average value of 266 ml of CH4/g of COD,when the single fermentation produced only 194 ml ofCH4/g of COD. This means that the ozonation pretreat-ment, which eliminates most of the phenolic compounds,reduces the toxicity and facilitates the anaerobic digestionof OMW as it was expected.

4ConclusionsThe results show that olive mill wastewaters can be effec-tively treated by an anaerobic digestion, which resulted in agas production rate averaging around 196 ml of CH4/g ofCOD and COD removal efficiencies around 70% dependingon the initial COD of the waste loaded to the digester. Theapplication of the Monod and Levenspiel models providethe values of the kinetic constants of the substrate degra-dation rate, which are correlated as a function of the av-erage substrate concentration in the experiments.

The pretreatment of the OMW with ozone reducesconsiderably the total phenolic compounds concentrationwhich is the main cause of the toxicity to methanogenicbacteria of these wastes, and subsequently removes such atoxicity. This fact is demonstrated by a significant incre-ment of the methane yield coefficient in the pretreatedwater in relation to that obtained in the anaerobic diges-tion of untreated OMW. Therefore, as a general conclu-sion, it can be established that successive steps are verysuitable for removing the high content of COD and thephenolic compounds present in the OMW in order to meetdischarge norms, ozone being an excellent oxidizing agentin the specific destruction of phenolic compounds.

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Table 5. Ozonation pretreatment

CODo (g/l) 59.90CODf (g/l) 56.20XCOD (%) 6.2TPo (g/l) 1.120TPf (g/l) 0.064XTP (%) 94.3

Table 6. COD Removal yield and methane production in theanaerobic digestion of pretreated OMW

Expt. CODo CODf CODal XCOD )rCOD VMF YP/S ko

BON-1 3.88 2.81 1.37 77.8 0.435 308 288 0.88BON-2 4.82 3.13 2.25 76.9 0.642 441 262 0.70BON-3 5.85 3.26 3.40 74.1 0.937 653 252 0.66BON-4 9.58 4.93 6.40 72.7 1.560 1225 263 0.65

g/l g/l g/l % g/day · l ml mlCH4/gCOD

day)1

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Improvement of the anaerobic biodegradation of olive mill wastewaters by prior ozonation pretreatment