J . Chern. Tech. Biotechnol. 1993, 56, 155-162

Kinetic Study of an Anaerobic Fluidized Bed Used for the Purification of Fermented Sys tern

Olive Mill Wastewater A. Martin,a R. Borjab & A. Chica" a Department of Chemical Engineering, Faculty of Sciences, Avda San Albert0 Magno s/n, E-14004 Cordoba, Spain bInstitute of Fat and its Derivatives, Avda Padre Garcia Tejero 4, E-41012 Sevilla, Spain

(Received 13 March 1992; revised version received 31 July 1992; accepted 14 September 1992)

Abstract: The anaerobic digestion of olive mill wastewater (OMW) in a fluidized bed, pretreated with Geotrichum candidurn, has been studied. The bioreactor used (volume = 3.5 dm3; biomass concentration = 11.5 g VSS dm-3) maintained satis- factory operation for 4 to 35 days, in terms of hydraulic retention time, and removed 92% of the initial COD. The system was used to develop and test a kinetic model which was subsequently employed to determine growth yield and maintenance coefficient. From the results obtained, the Michaelis-Menten equation accurately described the substrate uptake (i.e. COD removal) in the anaerobic fluidized bed system. Pretreatment of the OMW was found to increase the rate of substrate uptake by a factor of 3.2 when compared to untreated OMW.

Key words : kinetic model, olive mill wastewater. anaerobic digestion, fluidized bed reactor, Michaelis-Menten equation.

AFBR COD E HRT K, K, m MS MSS OLR OMW 4 q C H 4

rCH4

r s

So, S

STP TS

J. Chern.

NOTATION

Anaerobic fluidized bed reactor Chemical oxygen demand Soluble COD removal efficiency (Y COD) Hydraulic retention time Constants of the Michaelis equation Maintenance coefficient (g COD g-l VSS day-') Mineral solids Mineral suspended solids Organic loading rate (g COD dm-3 day-') Olive mill wastewater Volumetric feed flow-rate (dm3 day-l) Flow-rate of produced methane (dm3 CH, day-') Rate of methane production (g COD g-l VSS day-') Rate of substrate uptake per unit mass of microorganism (g COD g-l VSS day-') Substrate concentrations (g COD dm-3) en- tering and leaving the reactor Standard temperature and pressure conditions Total solids

TSS TVFA V VFA vs vss X xe

Y

0 P 0

WCH4

Total suspended solids Total volatile fatty acids Bioreactor volume (dm3) Volatile fatty acids Volatile solids Volatile suspended solids Biomass concentration (g VSS dm-3) Biomass concentration in the effluent (g VSS dm-3) True constant of growth yield per unit COD removed (g VSS g-' COD) HRT (days) Observed specific growth rate (day-l) COD conversion factor of biomass (g COD

COD equivalent of the methane volume STP (g COD dm-3)

g-l VSS)

1 INTRODUCTION

The manufacturing process of olive oil usually yields an oily phase (20 YO), a solid residue (30 YO) and an aqueous

155 Tech. Biotechnol. 0268-2575/93/$06.00 0 1993 SCI. Printed in Great Britain

12.2

156 A . Martin, R. Borja, A. Chica

phase (50 YO), which arises from the water content of the fruit. Such water, combined with that used to wash and process the olives, makes up the so-called 'olive mill wastewater' (OMW) and also contains soft tissues from olive pulp and a very stable oil emulsion.

The annual OMW production of Mediterranean olive growing countries is estimatedl to amount to over 3 x lo7 m". Because of the high organic load of this type of waste, purification by anaerobic digestion is, in principle, very appealing and has been investigated by some authors2-'

In earlier work on the anaerobic digestion of untreated OMW, several authors studied a number of problems arising from the high toxicity and low biodegradability of this effluent, as well as from the acidification of the reactors used.". The process is severely hindered by the combined inhibition caused by high concentrations of aromatic compounds and the build-up of volatile acids.8 These problems were partly solved by diluting the waste, yet the results obtained in this way were not very satisfactory.8

Preliminary experiments demonstrated that treating OMW with Aspergillus niger decreases the toxicity towards methanogenic bacteria and increases methane production and chemical oxygen demand (COD) re- moval in batch anaerobic culture^.^ Similar results were obtained with Geotrichum candidurn."

In this work, the kinetics of anaerobic fermentation of OMW, previously fermented with G. candidum, were studied by using an anaerobic fluidized bed reactor (AFBR). The results were compared with those obtained using unfermented OMW.

2 MATERIALS AND METHODS

2.1 Reactor

2.2 Wastewater

The OMW used was obtained from a continuous process factory. The features of this starting substrate and that fermented aerobically with G. candidum are summarized in Table 1.

The OMW was fermented aerobically with G . candidum at 25°C by using 1 dm3 of air per hour per 1 dm3 of waste. The fungi were isolated from OMW according to Meyers" and deposited in the culture collections of the Instituto Jaime Ferran de Microbiologia (IJFM) of the Centro de Investigaciones Biologicas (CIB) of the Consejo Superior de Investigaciones Cientificas (CSIC) of Spain, as IJFM A 534, and in the University of Alberta Mold Herbarium (UAMH) collection of the University of Alberta (Edmonton, Canada) as UAMH 6257.

The reactor was fed and liquid effluents removed 24 times a day. The released gas was removed continuously, the volume of methane produced being measured after removing CO, by absorption into NaOH.

2.3 Method

Anaerobic digestion was started by using biomass from an OMW storage and evaporation pond as inoculum. The composition of the inoculum is listed in Table 2.

The anaerobic digester was supplemented with 2250 cm3 of distilled water, 1250 cm3 of the inoculum and 30 g dm-s of support. Experiments were preceded by daily feedings of 2&150cm3 of pretreated OMW intended to condition the biomass. The volume of wastewater added was adjusted according to the rate of

TABLE 1 Composition and Features of the Olive Mill Wastewater (OMW) used Before and After Treatment with Geotrichum

candidurn

Fluidized reactors ensure thorough mixing of the substrates. These reactors are, therefore, particularly suitable for kinetic studies. Once a pseudo steady-state (PSS) has been reached, the specific rate of substrate consumption, rs (expressed as g of COD per g of volatile suspended solids (VSS) per day), may be readily calculated" from an expression of the type rs =As>, where S (g CODdm-3) is the concentration of biodegradable substrate in the reactor.

The AFBR used was a methacrylate column (60 cm high, 10 cm i.d.) with a working volume of 3.5 dm3. The system was placed in a temperature-controlled chamber, at a constant 35°C.

Saponite, a fibrous silicate, was added to the biomass (30 g dm ') in the form of 0.5 mm particles to facilitate retention of the bacteria, which have a low specific growth rate, and to increase production.12.'3 The features of this support were described in detail previo~sly. '~

Parameter Untreated O M W Fermented OM W ~- . -

PH 5.0 5.7 COD 50.0 25.0 TS 47.0 22.5 MS 12.0 9.1 vs 35.0 13.4 TSS 11.9 4.1 MSS 2.0 1.9 vss 9.9 2.2 Volatile acidity 0.1 1 0.08

(HAcO) Alkalinity (CaCO,) 0.50 0.45 N (NHJ 0.05 0.03 Polyphenols 0.750 0.045

o-Diphenols 0.085 0.005 (caffeic acid)

(caffeic acid)

All concentrations are expressed in g drn-$.

Anaerobic digestion of olive mill wastewater 157

TABLE 2 Composition and Features of the Biomass (G. candidurn) used

as Inoculum

p H TS VS M S TSS VSS MSS

7.1 40.7 33.5 7.2 33.5 26.8 6.7

All concentrations are expressed in g dm-3.

methane production. The overall duration of this biomass conditioning operation was 85 days.

This preliminary operation was followed by a series of semi-continuous experiments involving fermented OMW as a feed at 100-1 100 cm3 day-' flow-rates, equivalent to 35.00-3.18 days of hydraulic retention time, respectively. The biomass concentration ranged between 11.4 and 11.7 g VSS dm-3 (mean = 11.55 g VSS dm-3) and was virtually identical for all experiments.

Once PSS was approached, the volume of methane produced, pH, volatile acidity and COD of the different effluents were determined at each feed flow-rate. All experiments were performed in duplicate.

2.4 Analyses

Analyses were carried out according to the guidelines of the Standards Methods for the Examination of Water and Wastewater."

Volatile fatty acids (VFA) were identified and quantified by gas chromatography using a 0.8 m glass column of 0.6mm i.d. packed with 0 3 % (w/w) Carbowax and 0 1 % (w/w) H,PO, on Carbopack support of 60/80 mesh. The chromatographic conditions used were as follows:

- Oven temperature: 5 min at 70°C; 10°C min-' ramp up to 110°C; 2 min at 110OC; 5°C min-l ramp up to 180°C.

- Injector and detector temperature: 300°C (a nitrogen stream was passed at a rate of 40 cm3 min-').

The total phenol content was determined by the Folin-Ciocalteau method, while o-diphenols were quantified with sodium molybdate and sodium nitrite."

3 RESULTS AND DISCUSSION

3.1 Operational parameters

The results obtained from the reactor at the various flow- rates tested are shown in Table 3 . The pH remained

within the optimal working range for anaerobic digesters (6.6-7.8) throughout the studies. However, a volumetric flow-rate above 1100 day-l, the maximum rate examined, might result in acidification of the reactor and hence an increased COD content in the product, together with decreased methane production. This is demonstrated in the data presented in Fig. 1, which shows the variation of the total volatile fatty acid concentration (TVFA) as a function of the hydraulic retention time (HRT). Both the pH and the TVFA concentration remained constant for HRT values be- tween 16 and 35 days. At lower HRT values, the TVFA

TABLE 3 Experimental Conditions used for the Study of Purification of Fermented Olive Mill Wastewaters in an Anaerobic Fluidized

Bed Reactor

Flow-rate HRT p H G I 4 [CODI,,,, (cm3 day-') (days) (cm3 day-') ( g d w 3 )

100 200 300 400 500 600 700 800 900

1000 1100

35.00 7.3 17.50 7.2 1166 7.3 8.75 7.2 7.00 7.2 5.83 7.1 5.00 7.0 4.37 7.1 3.88 7.0 3.50 6.8 3.18 6.6

895 1785 2675 3560 4440 5305 6190 7040 7900 8700 9500

1.10 1.15 1.20 1.30 1.35 1.40 1.45 1.50 1.60 1.75 1.95

3401

3201 4 300 I \

5 10 15 20 25 30 35 * 2000

HRT (days)

SO

7.5

7.0 I,

6.5

6.0 1

Fig. 1. Variation of the total volatile fatty acid (TVFA) concentration (a) and pH values (0) with the hydraulic retention time (HRT) of an anaerobic fluidized bed system employed for the purification of olive mill wastewater (OMW).

A . Martin, R . Borja, A . Chica 158

loor--

0

20: .L ~

I I a gy 7 E

1

0 5 10 15 20 25 30 3 5 40

a gy 7 E 1

0 5 10 15 20 25 30 3 5 40

HRT (days)

Fig. 2. Variation of the volatile fatty acid (VFA) concentration with the hydraulic retention time (HRT). 0, C,; 0, C,; V, C,;

V, C,; 0, iC,.

,->

90' 0 5 10

1

15 20 25 30 35

HRT (days)

Fig. 3. Effect of HRT on the efficiency of COD removal.

concentration started to increase gradually and rose sharply below H R T = 4, concomitant with a decrease in the pH.

The variation of the VFA concentration with HRT is shown in Fig. 2. The proportion of these acids remained virtually constant over the HRT range 6-35 days, below

which the content of propionic and butyric acids increased markedly at the expense of acetic acid, which is clearly indicative of unbalanced populations of acidogenic and methanogenic bacteria.

According to Hill and Bolte,'' an acetic acid con- centration above 800 mg dm-3 points to the impending failure of swine manure digestion. Hill also believes that long-chain VFA, such as butyric and valerianic acids and particularly in their isomeric forms, are accurate indicators for stress conditions preceding complete fa i1~re . l~ The maximum levels for impending failure and complete failure are 5 and 15 mg dm-3 for isomers iC, and iC,, respectively; complete failure is defined as methane production below 0.25 dm3 CH, per gram of volatile solids (VS) added. In the present work, iC, was 5 % (w/w) at the maximum flow-rate assayed; since the TVFA concentration at that flow-rate was 320 mg dm-3, the iC, concentration emerging from the digester was 16 mg dm-3, i.e. slightly higher than the suggested limit. However, as shown below, no significant decrease in methane production was observed. Other authors2"," have proposed a ratio of 1.4 between the propionic and acetic acid concentrations as the failure criterion, which. judging from Fig. 2 , was never reached under the present experimental conditions.

The variation of removed COD as a function of the HRT was quite similar (Fig. 3). The percentage COD removed for HRT values between 10 and 35 days remained virtually constant at 95%. For an HRT shorter than 10 days, the amount removed was somewhat smaller. Finally, for an HRT of 5 days, COD removal was much less efficient, which indicates the increasing difficulty of methanogenic bacteria to degrade VFA.

3.2 Kinetic model

Because of the occurrence of thorough mixing in the AFBR, a substrate (COD) balance provides the fol- lowing equation :

d( VS)/dt = qS, - q S - Y, V X

I , = (S" - S ) / X B

(1)

In the steady state, d( VS)/dt = 0, so rs will be given by

(2)

where 8, the hydraulic retention time (in days), is defined by the ratio V/q.

While the biomass is retained in the system, HRT are relatively short compared to biomass retention times. The system is unable to reach a steady state as regards the solid phase, but steady-state conditions are applicable to both the soluble phase and the substrate balances. Variations in the biomass concentration during the time the experiments were performed were negligible in relation to the biomass content of the bioreactor, so they do not contribute to the system dynamics. In such a PSS, soluble and solid material balances are solved with dS/dt = 0 and dX/dt + 0 by assuming the volumetric

Anaerobic digestion of olive mill wastewater

3.0

159

-

-

TABLE 4 True Growth Yield ( Y ) and Maintenance Coefficient (m) for Anaerobic Bacteria in

Treatment of Wastewater

Y m Substrate Reference (g YSS g-' COD) (g COD g-' VSS day-')

~~

0.074 0.080 0.127 0.139

0.033 0'08-023

0.0033 Fermented OMW This work 0.0375" Glucose 24 0.089 Sucrose 23 0.076 Sucrose 23

Carbohydrates 25 0.06" Glucose 26 -

a Calculated from data supplied by the authors.

rate of biomass build-up to be constant over the PSS interval considered. Under these conditions, a biomass balance yields

d( VX)/dt = fiXV-qX, (3) The COD balance defined by eqn (1) may be rewritten

for PSS conditions as follows:

q s O qs+ WCHO qCH4 + OqXe + d( VX)/dt (4) Combination of eqns (2), (3) and (4) yields

rs = T C H 4 + wfi

where rCH4( = wCH4qCH4/XV) is the rate of methane production (g COD g-' VSS day-'). According to Pirt,"

( 5 )

r, = (p/ Y ) + m (6) where Y is a true constant of growth yield per unit COD removed (g VSS g-' COD) and m is the so-called 'main- tenance coefficient' (g COD g-l VSS day-').

Combination of eqns (5) and (6) gives,

rCH4 = (1 -wY)v,+wmY (7) When w , Y and m are constant for a given system, there is a linear relationship between rCH4 and rs. Although, strictly speaking, the experimental arrangement used here was not continuous and the working conditions were not strictly steady-state continuous, the slow evolution of the system allowed the assumption that these two conditions were approximately met. In fact, fitting the rcH4 vs rs data pairs by least-squares linear regression yielded a correlation coefficient of 0.999, a slope of 0.8952 and an intercept of 0.00033. These data allowed m and Y to be calculated once w was known (w = 1.41 g COD g-' VSS). Table 4 lists these parameter values and others compiled from the l i t e~a tu re .~~- '~ As shown, while the Y value lay within the range of typical literature values, the m value found was somewhat smaller than reported elsewhere.

3.3 Kinetics of substrate uptake

In the above equations, S denotes the concentration of biodegradable substrate. However, the experimental

V n 0 V In v

, 0 0.1 0.2 0.3

HRT" (day-?

Fig. 4. Estimation of the amount of non-biodegradable matter contained in the processed waste. Results obtained: S,,, biod =

1.03: corr. coeff. = 0.99.

method used to determine the substrate concentration (i.e. the COD determination) does not distinguish between biodegradable and non-biodegradable sub- strate. The experimental values in Table 3 should therefore be corrected by subtracting the fraction of non- biodegradable substrate. A graphical estimation of such a fraction, on the basis of the linear relationship between In (S)exp and l/9, is presented in Fig. 4.

The corresponding r,-Sbiod pairs are shown in Fig. 5. The shape of the curve indicates that the process may conform to a Michaelis-type equation of the form

rs = K S / ( K , + S ) (8) which is confirmed by the straight line obtained by plotting 1 / r , against 1 /S,,,,, (Fig. 6). The K and K, values

160 A. Martin, R. Borja, A. Chica

0.5 1 ,'

10 P

Fig. 5. Variation of thc rate of substrate uptake with the biodegradable substrate concentration in the steady-state

effluent.

TABLE 5 Parameters of Eqn (8) and Confidence Limits

Parameter Estimate Lower limit Upper limit

K 1.54 0.97 2.10 K. 1.22 0.56 1.87

Fig. 6. Linewcaver-Burk plot of ratc of substrate uptake per unit mass microorganisms against biodegradable substrate

concentration, expressed as reciprocal values.

in Table 5, which also lists the confidence limits obtained for a likelihood of 95 %, were estimated by a weighted least-squares procedure applied as a multivariate non- linear modeL2'

Even with the relatively wide confidence interval of both K and K,, substitution of the central value (first column in Table 6) into eqn (8) provides an estimated rate of substrate uptake withinf 10 */a of the actual value (Fig. 7). A similar equation was used by Shieh et a1.21 to

TABLE 6 Comparison of the Behaviour of Various Anaerobic Digesters used to Process Olive Mill

Wastewater (OMW)

Process Volume Load Eficiency References (dm") (g COD dm-3 day l ) (YO COD)

______ ~- - ~

Contact 2 600 1.55 70 2 70 000 2.55 80 3

Fixed bed 21" 2.80 83 4 300" 8.00 87 4

I Ob 2.50 60 5 lo' 2.50 55 5 1 I d 3.00 65 5 1 I ' 3.00 60 5 21 2.70 65 28 29 440 75 28

Fluidized bed 3.5 7.80 92 This work

Packings used : a polyurethane ; prisms; 'cubes ; T30 cylindrical plugs ; TR30 cylindrical plugs; fplastic; gclay.

Anaerobic digestion of olive mill wastewater 161

0.8

0.6

U - 0.4

L

0.2

, I I

/ /

V , I

0 0.2 0.4 0.6 rs exp

Fig. 7, Comparison between the experimental rates of substrate uptake and those predicted by eqn (8).

describe the anaerobic digestion in a fluidized bed of synthetic wastewater, containing glucose as the sole carbon source, by using activated carbon particles (4 = 0 6 mm) as microorganism support.

A comparison of the kinetic behaviour of OMW treated with G . candidum and untreated substrate may now provide useful data. The process undergone by treated OMW at biodegradable substrate concentrations below 0.6 g COD dm-3 may be described by the following first-order kinetic equation

(r,) = 0.94s (9)

By using a similar experimental arrangement, untreated OMW and the same range of substrate concentrations in previous the degradation process was found to conform to a first-order kinetics. Hence,

which points to the advisability of pretreating the OMW from a kinetic point of view.

Analogous conclusions (Table 6) may be derived on comparing the organic loading rates (OLR) and effici- encies (E) obtained in this work and those reported in the literature."6, 28 The purification efficiency obtained here (92%) exceeded all the reported values, whilst the OLR was similar to the maximum reported value and at least twofold the others. This may be attributed to the fact that treatment with G . candidum removes 94% of the

phenols present in the OMW (Table I) , which are known inhibitors of the anaerobic pro~ess . '~ -~ l

ACKNOWLEDGEMENTS

The authors wish to thank Dr Aldo Gonzalez, of the CIB (CSIC), Madrid (Spain), for kindly supplying the G. candidum inoculum used in this work. Financial support from the CSIC and the Consejeria de Educacion y Ciencia de la Junta de Andalucia is also gratefully acknowledged.

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