Proress Biorhrmirrry 27 (I 992) 231-237

Enhancement of the Anaerobic Digestion of Olive Mill Wastewater by the Removal of Phenolic Inhibitors

R. Borja,a A. Martin,0 R. Maestro,a J. Alba,” & J. A. Fiestasa ” Tnstituto de la Grass y sus Derivados (CSIC), Avda. Padre Garcia Tejero 4, 41012-Seville, Spain bDepartamento de Ingenieria Quimica, Facultad de Ciencias, Universidad de Ckdoba, Avda. San Albert0 Magno s/n, 14004-Cbrdoba, Spain

(Received 5 July 1991; revised version received and accepted 28 December 1991)

Olive mill wastewater f OMWj has a high organic pollutant load (45-130 g COD/Kitre) including various phenolic compounds, of which cafleic acid, thyrosol and hydroxythyrosol occur in the highest proportions. These two features make it impossible to purify OMW anaerobically unless a prior dilution is eflected. Partial removal of some of the organic matter and phenolic compounds, by aerobic pretreatment with CJeotrichum candidum is described. This provides a partially pur$ed efluent that, for the same COD level as that of the original OMW, is degraded anaerobically more rapidly than original OMW as a result of the increase in methanogenic activity by 39 ml CH, STP/g VSS day for a COD of 7 g/Zitre. Finally, the presence of a sepiolite support (Pansil) in the digestors is shown to increase the biodegradubility vf ihr OMWs and the yield coeflcient of the product

(Y,,.

NOTATION

G Volume of methane accumulated (ml)

G, Volume of methane accumulated at infinite retention time (ml)

K Kinetic constant as defined in eqn (1)

K?, Apparent kinetic constant (day-‘) S Substrate concentration, expressed as COD

(g O,/litre)

X. Initial substrate concentration, expressed as

1 Time (days) x Biomass concentration (g/litre)

Y, Cellular yield coefficient (g cell/g COD)

r, Yield coefficient of the product (ml CH,/g CnOD)

MA Methanogenic activity (g COD/g VSS/day) V Digestor volume (litres)

INTRODUCTION COD (g O,/litre)

Corresponding author: Dr R. Borja. Tclcphone: (95) 4611550; Among the polluting agents acting on the Fax : (95) 4616790 Mediterranean basin, olive mill wastewater (OMW)

231 process Biochemislry 0032-9592/92/$5.00 Q 1992 Elsevier Science Publishers Ltd, England

232 R. Borja, A. Marlin, R. Maestro, J. Alba, J.A. Fiestas

Table 1. Features of the OMW Related to its Polluting Power

Batch process

PH COD SOD ss TS

v”s”

4._5-5 4-7-5.2 12G130 4540 90-100 3548

1 9 120 60

1:: S

55 Fat 0.5-l 3-10

SS. suspcndcd solids: TS, total solids: MS, mineral sohds; VS, volatile solids. All concentrations expressed in g/litre.

is worth special attention on account of its environmental impact. This type of wastewater is composed of the vegetation water of olives plus washing and processing waters in addition to soft pulp tissue and oil in the form of a very stable emulsion. Its composition varies widely depending essentially on the type of process involved in obtaining the oil, while little or no water is used in batch systems, continuous systems employ about 1 litre of water per kg olives. Table 1 summarizes the features of OMW in relation to its polluting power.

In addition to its high polluting power, OMW usually possesses a high antibacterial activity exerted by various phenolic compounds.’ 5

Although the anaerobic digestion of this type of residue is feasible and quite appealing from an energetic point of view, the presence of the phenolic inhibitors decelerates the process, hinders removal of part of the COD and detracts from its economic viability. In this work the elimination of these phenolic compounds by aerobic fermentation with Grotrichum candidurn removes most of the phenolic compounds and yields an eflluenl that can be readily biodegraded anaerobically. This is of prac- tical interest in the design of purifying plants for this type of waste.

MATERIALS AND METHODS

Fermentation systems Magnetically stirred batch anaerobic digestion units (ADU) (1 litre) were immersed in a water bath at 37 “C. The biogas produced was passed through an NaOH solution to retain CO, and the volume of methane yielded determined indirectly from the amount of water displaced by the gas.

In order to develop a continuous fluidized bed

Table 2. Features of the OMW Used in the Direct Anaerobic Digestion

OMW Fermented OMW

PH COD TS MS vs TSS MSS vss Volatile acidity (HAcO) Alkalinity (CaCO,) N-NH, Polyphenols (caffeic acid) o-diphenols (caffeic acid)

5.2 5.4 60.0 220 97.2 46.4 13.4 10.0 83.8 344 49.5 14.1

3.9 64 10.4 77 o-34 0.16 1.46 1.24 0.06 @04 0.300 O-103 0.023 0.003

All concentrations expressed in g/litre.

wastewater treatment process and avoid hydro- dynamic sweeping of the biomass, the influence of the support on the kinetics of the process was studied. The support material in the fluidized bed was a sepiolite (Pansil), a fibrous silicate of micronized size onto which methanogenic bacteria attach readily; since these bacteria are those featuring the lowest specific growth rate, they are retained preferentially. 6.7 The features of the sup- port were described in detail in a previous paper.*

Olive mill wastewater (OMW) and fermented OMW The OMW used stems from a continuous process factory. The features of the OMW and OMW fermented aerobically with Geotrichum candidum used are summarized in Table 2. The OMW is supplemented with NH, to obtain a C : N: P ratio of 100:0~5:0~1.

The aerobic fermentation of the OMW was carried out at 2.5 “C using 1 litre air/h/litrc of OMW with Geotrichum candidum which was iso- lated from OMW (according to Meyers’) and deposited in the IJFM, CIB-CSIC (Spain) culture collections as IJFM A 534, and UAMH collection as UAMH 6257 in the University of Alberta, Edmonton (Canada).

Inoculum The anaerobic digestion process was started by using previously diluted and neutralized biomass from an OMW storage and evaporation pond as inoculum. The composition of the inoculum is given

in Table 3.

Enhuncement of the anaerobic digestion of olive mill wastewater 233

Table 3. Composition of the Biomass Used as Inocuh~m

PH TS V.Y MS TSS VSS MSS

7.2 16.3 12.9 3.4 13.0 1 o-4 2.6

Abbreviations as in Table 1.

Experimental procedure Each anaerobic digestion unit was supplemented with 750 ml of distilled water, 250 ml of the inoculum and 10 g of support (if required). While larger amounts of support allowed an increase of biomass they could also increase the apparent viscosity of the medium and hence hinder mass transfer and decelerate the process of biodegrada- tion. In order to condition the biomass, experiments were preceded by daily feedings of S-2.5 ml OMW. The volume of OMW added was adjusted according to the rate of methane production.

Two sets of experiments were carried out; in one, OMW loads of 10, 20, 40 and 80 ml were added in the digestors (see Table 2); in the other, 60 ml and 320 ml loads of the effluent obtained by aerobic fermentation of OMW (see Table 2) with Geotrichum candidurn were processed. All loads were added to the bioreactor after drawing a volume of liquid equal to that to be subsequently treated. This drawing operation was only effected after allowing the system to stand for 2 h in order to avoid biomass losses. The loads used were virtually the same for every experiment and digestor, and ranged between 5.0 and 5.1 g VSS/litre. In every instance, the volume of methane released as a function of time and the initial and final COD were determined. The experiment duration was the time required for the complete biomethanation of each load.

Analyses Analyses were carried out in accordance with the

Srandurd Methods.for the Examination of Water and Wastewater.’

Phenolic compounds were isolated and identified by two-dimensional thin-layer chromatography on cellulose plates developed with 1: 4: 5 HAcO/ BuOH/H,O and 2 :98 HAcO/H,O along perpen- dicular directions. The plates were finally sprayed with diazotized p-nitroaniline or Arnow’s reagent (o-diphenols).”

The total phenol content was determined by the Folin-Ciocalteau method, while o-diphenols were assayed with sodium molybdate and sodium ni- trite.l’

RESULTS

Tables 4 and 5 indicate the variation of the volume of methane accumulated as a function of time in the anaerobic digestion of the OMWs listed in Table 2.

Tables 6 and 7 show results analogous to those in Tables 4 and 5; except that the process water in the former was previously subjected to aerobic fer- mentation and then to anaerobic digestion. The initial and final COD values obtained in these experiments are listed in Tables 8 and 9.

Table 10 lists the phenols found in the OMW and

Table 4. Volume of Methane Accumulated (ml) as a Function

of Time (days) by Using Different Feed Volumes of OMW (ml) in the Digestor Containing Pansil as Support

Time (days)

10

1 2 3 4 5 6 7 8 9

10

11 12

13 14 15 16

131

191

216 226

228 236 243

247 250

241

36Y 429 464

482 518 520

273

488 718 803

898 928 945

957 967 910

120

243 256 438 442

650 672 895 952

1123 1145 1249 1399 13x4 1659 1414 1883 1690 2028 1744 2160

1759 2328 1783 2508 1 x02 2554

1823 2590 1831 2608 1839 2618

Table 5. Volume of Methane Accumulated (ml) as a Fun&on

of Time (days) by Using Different Feed Volumes of OMW (ml) in the Rcfcrcncc Digcstar

2 3 4

5 6 7

8 9

10 I1 12

13 14 15

16

Feed volume (mf)

10 20 40 80 120

190 163 183 213 302 265 293 333 418 494 305 408 503 658 714 330 478 693 963 1064 347 528 818 1248 1494 369 570 838 1575 I779 392 577 856 1610 lY11 407 870 1640 208 1 422 870 1660 2187

884 1682 2397 1713 2537 174-Z 2640 1766 2696 1770 2751 1774 2771 1774 2772

234 R. Borja, A. Martin, R. Maestro, J. Alba, J.A. Fiestas

Table 6. Volume: of Methane Accumulated (ml) as a Function of Time (days) by Using Diffcrcnt Feed Volumes of Aerobically Pretreated OMW (ml) in the Digestor Containing a Pansil Support

I

60 160 80 260

100 365 120 452 140 555 160 605 180 638 200 614 220 578 240 571 260 6X0 280 790 300 803 320 II51

2

280 465 595 742 895 970 998

1079 1048 1101 1198 1205 1245 1543

3 4

349 354 358 359 470 58-I 602 603 683 727 750 752 802 850 865 871

1001 1044 1048 1049 1136 1189 1217 1237 1248 1342 1400 1414 1344 1509 1540 1576 1323 1573 1627 I636 1351 1576 1656 1684 1462 16SO 1771 1 x39 1498 1695 1830 1932 1510 1720 1873 1995 1801 1979 2120 2218

5 6 Y 10

603 752 871

1049 1242 1415 1614 1641 1691 lXR2 1999 2090 2292

752

1242

1629 1658 1751 1912 2060 2171 2351

1698 1701 1755 - 1933 2098 2230 2408 2483

Table 7. Volume of Methane Accumulated (ml) as a Function of Time (days) by Using Different Feed Volumes of Aerobically Pretreated OMW (ml) in the Reference Digestor

I 2 3 4 7 8 9 IO

100 120 140 160 180 200 220 240 260 280 300 320

138 236 320 365 369 369 225 397 487 522 535 542 29s 515 606 689 714 717 355 595 695 795 822 837 412 697 841 976 1018 1031 443 768 910 1030 1100 1116 441 776 946 1078 1220 1295 475 870 1115 1290 1375 1395 436 X46 1126 12x1 1429 1465 442 892 1192 1392 1497 1532 550 976 1210 I390 1507 1608 551 979 1245 1430 15x1 1693 720 1172 1401 1580 1712 1820

1011 1325 1540 1705 1840 1920

548 729 837

1031 1123 1 Z4O 1400 1406 1410 1533 I543 1553 1540 1604 1641 I682 1736 1760 1769 1787 1 X42 1888 1907 1903 1959 2008 2041 2002 2072 2131 2190

731)

1123

reflects the changes undergone on being subjcctcd

to aerobic fermentation followed by anaerobic 1. _.

Table 9. Initial and Final COD Values (g OJlitre) Obtained in the Experiments Carried out with OMWs Previously Subjected to Aerobic Fermentation with Geotrichum cundidum

aigesuon.

The influence of the support on the total phenol

and o-diphenol contents measured after the aerobic

fermentation and anaerobic digestion processes is - indicated in Table 11.

Feed volume Initial COD Final COD (m0

Reference Pansil Reference Pansii

60 7.1 5.7 5.8 4-4 80 7.6 6.0 5.8 4.3

100 7.9 6.7 5.7 4.5 120 8.4 7.0 5.8 4.4 140 8.9 7-6 5.8 4.5 160 9.0 8-O 5.5 4.5 180 9.3 8-6 5.4 4.6 200 9.4 8.9 5.0 4.5 220 I@1 9.2 5.3 4.4 240 10.6 9.6 5.3 4-4 260 1 o-9 10-l 5.2 44 280 11-4 10-5 5-2 4-3 300 11.9 11 .o 5.2 4.4 320 12-Z 11.5 5-2 4-4

Table 8. Initial and Final COD Values (g O,/litre) Obtained in the Experiments Carried out with the OMW whose Features are Summarized in Table 2

Feed volume Initial COD Final COD (ml)

Ryfermcr Punsil R~fermce Punsil

IO 6-4 4.9 5.8 4.3 20 7-o 5.4 5.8 4.2 40 8-2 6.7 5.8 4.3 80 10-6 9.0 5.8 4.2

Enhuncement qf‘ the anaerobic digestion of olive mill wasrewater 235

Table 10. Phenolic Compounds Identified in the OMW and Changes Undergone Throughout the Aerobic Fermentation- Anaerobic Digestion Processes

I AE CAN), (AN),

Apigenin R Luteolin R R R Quercetin R R R rruns-Calkc acid p-Coumaric acid ci.s-Caffcic acid Protocatcchuic acid R Vanillic acid R Hydroxythyrosnl R Thyrosol Oleuropein 1 -CafTeylglucose R R Hydroxythyrosol R R R

monoglucoside

1, initially present in the OMW; AE, after aerobic fermentation ; R, remains ; (AN),. (AN),, after aerobic fermentation and anaerobic digestion in the Pansil and reference digestor. respectively.

Table 11. Influence of the Microorganism Support on the Final Phenol and o-diphenol Contents of OMW Successively Subjected to Aerobic Fermentation and Anaerobic Digestion

Reference Pansil

Total phenol content (ppm caffeic acid) o-Diphenol content (ppm caffeic acid)

6.6 w4 1.1 0.3

DISCUSSION

The accumulated volume of methane at the end of the anaerobic digestion process (Tables 4-7) and the COD consumed (Tables 8 and 9) were used to determine a yield coefficient (Y,) for each ex- periment. This was corrected for the effect of temperature (25 “C) and the presence of steam accompanying the gas. Table 12 lists the average y, values obtained for each digestor and OMW used. Such values are of the same order of magnitude as those reported in the literature.r3 It is significant that the coefficient varies with the features of the treated water, probably as a result of the change in the composition of the metabolized substrate, which

Table 12. Yield Coefficient Y, (ml CH,/g COD) of the two Types of OMW Studied

Untreated OMW Treated OMW

R~fCV-LW CC Pansil

363 366 280 303

increases the average oxidation state of carbon and hence results in a decreased CH,/CO, ratio in the biogas produced.”

In order to characterize each experiment kinetic- ally and thus facilitate comparisons, the model described below was developed. The anaerobic digestor used can be considered to be a bioreactor where the nutrients, expressed as COD, react with microbial sludge of concentration X.r5 The rate of nutrient removal will be given by:

-dS/dt = K-S.X (1)

Because of the small value of the cellular yield coefficient (Y,) in the anaerobic digestion (0.022 0.06 g cells/g COD),“‘. ” and taking into account that the COD varied very little throughout the experiment, X can be assumed to remain roughly constant. In fact, as noted earlier, this hypothesis was fulfilled by all the digestors and in all experiments. Integration of eqn (1) on this as- sumption yield :

S = S;exp(-K.X.t) (2)

If one defines a yield coefficient, Y,, for the product formed (methane) such that

Y, = -dG/dS (3)

then one obtains:

G = G,,;[l -exp(-KK,-t)] (4)

where G, = S; Y, and K, = K- X. The fact that the biomass concentration in the

digestors remains virtually constant helps interpret- ation of the results; in fact, on comparing the aforementioned apparent kinetic constants, K,, it is seen that all of them are multiplied by the same factor (X).

According to eqn (4), the methane production conforms to a first-order kinetic mode1.l’

By plotting the experimental data listed in Tables 4-7 as G versus t, we could obtain curves coinciding with the predictions of eqn (4). The parameters G, and K_., were calculated by using standard softwarer8 using the (G. t) value pairs for each experiment and the equation to which they must be fitted.4 The programme calculated G, and K,, as well as various statistical parameters required to evaluate the fit. Once the statistical significance of the fittings was checked, KA was plotted as a function of the average COD obtained in each experiment, I?&. the arith- metic mean of the initial and final COD (Fig. 1). This graph allows us to draw two salient conclu- sions : (a) At a given COD, the apparent kinetic constant

236 R. Borja, A. Martin, R. Maestro, J. Alba, J.A. Fiestus

of the aerobically treated OMW was larger than that of the unpretreated OMW.

(b) Whether or not the OMW had been pretreated aerobically, the apparent kinetic constant de- creased markedly with increasing COD, which indicates that, though efficient, the pretreatment applied fails to remove all inhibitors present in the OMW.

The increased anaerobic digestion rate of the OMW that was pretreated aerobically can be ascribed to its lower phenolic content (Tables 2 and IO). In order lo conlirm this hypothesis, the OMW was subjected to aerobic fermentation under the established working conditions and the resulting product was supplemented with caffeic acid-one major phenol in OMW-up to a composition identical with the original OMW. A 1X0 ml feed volume of this OMW was then added to a digester with Pansil support. The results obtained (Table 13) yielded an apparent kinetic constant of O-22/day (Fig. l), which is of the same order of magnitude as

Table 13. Volume of Methane Accumulated (ml) as a Function of Time (days) by Using a 180 ml Feed Volume of Aerobically Pretreated OMW in the Pansil Digestor

1 2 3 4 5 6 7 8 9 10 6 191 322 511 714 838 910 924 942 962 965

Total phencl content (added) = 300 ppm can‘ac acid. Initial COD = 8-6 g O,/litre. Final COD = 4-6 g OJlitre.

that obtained by direct anaerobic treatment of the OMW. The removal of phenolic compounds there- fore increases the kinetic constant of the process.

The hehaviour of the reference digestor and that containing the Pan.41 support was not significantly different (Fig. 1); (K,, COD) pairs are grouped around two curves where the only significant variables appear to be the COD of the OMW and whether or not the wastewater was pretreated.

As far as the behaviour of the support is concerned, the only salient fact apparent from the results listed in Tables 9, 11 and 12 is that wastewater effluent features a lower COD and phenolic concentration, and is degraded with a high yield coefficient.

The increased degradation rate of the pretreated OMW can also be demonstrated via the following route. The mean rate of methane production can be calculated from the overall accumulated volume and the corresponding time; in as much as the amount of biomass present in each reactor is known, the methanogenic activity (MA) may be calculated from the following equation :

MA = G/(t .xX’. V. Y,) (5)

Figure 2 shows the plot of MA for the reference digestor against the average COD. The methan- ogenic activity increased linearly with COD, both in the untreated and in the pretreated OMWs through- out the substrate concentration range studied. The slope of the two lines is rather different however and

!

2 4 s 8 10 12

COD (g/lit&

Fig. 2. Variation of the methanogenic activity with the substrate concentration in the reference reactor.

Enhancement of the anurrobic digestion qf olive mill wastewater 237

,’ /m /’ /

0 2 4 6 8 10 12

COD (g/litre)

Fig. 3. Variation of the methanogenic activity with the substrate concentration in the Pansil reactor.

at a given identical COD, the MA of the pretreated OMW is significantly greater, the difference in MA increased with increase in the COD. As can be seen from Fig. 3, the Pansil reactor bchavcd identically in this respect.

CONCLUSIONS

Olive mill wastewater contains various phenolic compounds which decreased methanogenic activity and hence resulted in a diminished apparent kinetic constant of the anaerobic digestion process. Re- moval of such compounds by aerobic pretreatment with Geotrirhum candidurn substantially increased the rate of the anaerobic process. The apparent rate constant was inversely proportional to the COD of the OMW with or without pretreatment.

The support used (Pansil) yields wastewaters with lower COD values and phenolic contents.

ACKNOWLEDGEMENT

The authors wish to express their gratitude to the Consejeria de Educacidn y Ciencia dc In Junta de AndaZuct’a (Spain) for financial support granted for the realization of this work.

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