Resources, Conservation and Recycling, 9 ( 1993 ) 201-211 201 Elsevier Science Publ ishers B.V.

Anaerobic digestion of olive mill wastewater pretreated with Azotobacter chroococcum

R. Borja a, A. Martin b, L.F. G6mez c and A. R a m o s - C o r m e n z a n a c

aInstituto de la Grasa y sus Derivados, Seville, Spain bDepartamento de lngenieria Quimica, Facultad de Ciencias, Universidad de Crrdoba, Spain

CDepartamento de Microbiologia, Facultad de Farmacia, Universidad de Granada, Spain

(Accepted 18 January 1993 )

ABSTRACT

We carried out a kinetic study of the anaerobic digestion of olive mill wastewater that was previ- ously fermented with Azotobacter chroococcum. The process was carried out batchwise in a bioreactor containing sepiolite as support for the mediating bacteria. The anaerobic digestion process was found to follow first-order kinetics and the experimental methane volume ( G ) - t i m e ( t) data to conform to an equation of the form G=Gm[ 1 - e x p ( - K o t ) ], from which the apparent rate constant, Ko, was calculated. This kinetic constant (average value 1.21 day -~ ) was found to be scarcely influenced by the substrate concentration over the studied COD range (viz. 4.2 - 8.5 g/l ). The specific rate of meth- ane production was substantially higher than that obtained in the anaerobic digestion of untreated olive mill wastewater, and the process seemingly involved no inhibition phenomena as the biotoxicity of the waste was reduced by 30% as a result of the pretreatment. Finally, the average COD fraction removal was 73% and the yield coefficient was 313 ml CH4 STP/g COD.

I N T R O D U C T I O N

Among other organic compounds, the composition of olive mill wastewater (OMW) includes some aromatic substances that are difficult to degrade (e.g. lignins) as they consist chiefly of simple or polymerized phenolic com- pounds. These compounds are biologically active and have antimicrobial and phytotoxic properties [ 1 ], which poses serious problems to the purification of OMW by anaerobic digestion [ 2 - 6 ].

Azotobacter chroococcum can grow in the presence of various OMW con- centrations [ 7 ], but optimally in 15% OMW, while preserving its nitrogenase activity. The Azotobacter genus can also grow on substrates containing aro- matic compounds [ 8,9 ], which it can readily metabolize [ l0 ].

Notwithstanding the potential interest of the above-mentioned treatment,

Correspondence to: R. Borja, Ins t i tu to de la Grasa y sus Derivados, Avda Padre Garcia Tejero 4, E-41012 Sevilla, Spain.

0921-3449/93/$06.00 © 1993 Elsevier Science Publishers B.V. All rights reserved.

2 0 2 R. BORJA ET AL.

it fails to completely remove the COD; on account of its high organic load, lower toxicity and lower content of phenolic compounds, the remaining resi- due might be suitable for anaerobic treatment.

The aim of this work was to investigate the feasibility of purifying anaerob- ically olive mill wastewater previously digested aerobically with Azotobacter chroococcum.

MATERIAL A N D M E T H O D S

Aerobic fermentation Experiments were carried out in a Braun Biostat-M fermenter of 1.5 l, of

which 1.2 1 was used as working volume. The fermenter was thermostated at 30°C and the pH automatically adjusted to 6.8 [ 11 ] by means o f a pH-meter and delivery pumps loaded with 0.5 N HC1 and 0.5 N NaOH. Aeration was adjusted to 10% dissolved oxygen by using an oxygraph [ 12 ] that was cali- brated under the above working conditions at a constant stirring speed of 500 rpm, with N2 saturation at 0% and 100% obtained by means of synthetic air (22% O2, 78% N2).

The culture med ium used was olive mill wastewater from the 1991 season that contained an initial load of 212.2 g COD/1 and was stored at - 2 0 ° C . Prior to the experiments, it was diluted to 15% with Burk medium.

The pre-inoculum used was Azotobacter chroococcum H23 from previously isolated strain cultures in a liquid Burk medium containing 0.5% glucose. The suspension was stirred at 100 rpm and 30°C for 48 h, after which cells were washed by centrifugation and re-suspended in saline up to an optical density A55o=0.22. A volume of l0 ml of the resulting cell suspension (i.e. roughly 10% of the working volume) was used to inoculate the fermenter. Under these conditions, bacterial growth was moni tored for 10 days.

Anaerobic digestion

Apparatus. The experimental set-up used for the anaerobic treatment of OMW which had been pretreated with Azotobacter consisted of an anaerobic diges- t ion unit (ADU) composed of a 1-1 Pyrex flask with two inlets at the top that were used to load it and carry the biogas, respectively. The flask was modified by including two further inlets in the neck, one for the incoming inert gas required for unloading and the other for the outgoing effluent. The ADU was provided with magnetic stirring and was placed in a thermostated chamber at 37°C.

The methane volume produced in the process was measured by using a 1-1 Boyle-Mariotte reservoir that was fitted to the digester. A tightly closed bub- bler holding an NaOH solution intended to collect the CO2 produced in the process was intercalated between the two elements. The methane produced

ANAEROBIC DIGESTION OF OLIVE MILL'WASTEWATER PRETREATED WITH A. CHROOCOCCUM 203

displaced a given volume of water from the reservoir which allowed the for- mer to be readily determined.

The microorganisms effecting the process were supported on sepiolite, a micronized fibrous silicate that retains methanogenic bacteria preferentially [ 4,15 ]. On account of their slowest growth, these bacteria are to be retained in the bulk reactor so as to avoid any reduction in the overall rate of digestion. The most salient features and chemical composition of the support were de- scribed elsewhere [ 3 ].

Inoculum. The starting inoculum used was slurry from an OMW pond that was appropriately diluted and neutralized. The solid contents of the biomass were as follows: total suspended solids, 13.0 g/l; volatile suspended solids, 10.4 g/l; and mineral suspended solids, 2.6 g/1.

Olive mill wastewater. The composition and features of both untreated OMW (diluted in Burk medium ) and OMW pre-fermented with Azotobacter chroo- coccum that was used in the anaerobic experiments are gathered in Table 1. We should emphasize that the pretreatment resulted in a reduction of the ini- tial COD by 52%.

Experimental procedure The A D U was initially loaded with 750 ml of distilled water, 250 ml of the

above-described inoculum and 10 g of sepiolite. While larger amounts of sup-

TABLE 1

Features and composition of the diluted OMW and Azotobacter chroococcum-fermented OMW used in the anaerobic digestion experiments

Diluted OMW Fermented OMW

pH 5.3 6.2 COD (g /L) 32.00 15.30 TS (g /L) 19.20 15.95 TMS (g /L) 5.70 5.35 TVS (g /L) 13.50 10.60 TSS (g /L) 14.85 7.05 MSS (g /L) 4.50 2.45 VSS (g /L) 10.35 4.60 VA (g HAcO/L) 0.62 0.37 Alkalinity (g CaCO3/L) 0.40 0.59 N-NH + (g /L) 0.45 0.40 TP (ppm) 1800 175 OD (ppm) 160 15

COD=chemical oxygen demand; TS=total solids; TMS=total mineral solids; TVS=total volatile solids; TSS=total suspended solids; MSS=mineral suspended solids; VSS=volatile suspended sol- ids; VA-- volatile acidity; TP = total phenols; OD-- o-diphenols.

204 R. BORJA ET AL.

port increased the biomass concentration, they also increased the apparent viscosity of the medium, thereby hindering mass transfer and decelerating the process. Before the experiments were started, the ADU was loaded with in- creasing volumes of the OMW between 10 and 50 ml for 2 months in order to condition the biomass. The loaded volume was changed successively after methane production was found to have finished.

After this preliminary step, the experiments proper were performed by suc- cessively loading the ADU with 20, 40, 80, 120, 160, 200, 240, 280, 320, 360 and 400 ml ofAzotobacter-pretreated OMW. Each experiment was allowed to develop until the load used was fully biomethanized and involved daily de- terminations of the methane volume produced and measuring the COD, pH and volatile acidity at the end. Before the above volumes were loaded, iden- tical volumes were withdrawn from the digester after 2 h in order to avoid biomass losses. The amount of biomass present remained virtually constant at 5.2 g volatile suspended solids per litre throughout. All experiments were carried out in duplicate.

Analyses The characteristic parameters of the OMW (COD, pH, volatile acidity, al-

kalinity, ammonia nitrogen, and total and mineral and volatile suspended solids) were determined according to the recommendations of the American Public Health Association (APHA, 1985 ) [ 16 ].

The total phenol and o-diphenol contents were determined by the Folin- Ciocalteau method and the molybdate-sodium nitrite test, respectively [ 17 ].

Determination of biotoxicity The toxicity of the biomass was assessed by means of the luminescent ma-

rine bacteria (Photobacterium phosphoreum) test using a Microtox R system and the recommended reagents and procedure [ 18 ]. The samples were pre- viously diluted to 1.5 -0 .75% in 0.9% saline and the effective relative concen- tration (%) that decreased the bacterial luminescence by 50% (ECso) was determined after 5 minute's contact, as were the corresponding toxic units (TU). The results obtained were as follows: • Prior to inoculation with Azotobacter: EC5o=0.64 ( T U = 156.9 ). • Upon fermentation: ECso = 0.92 (TU = 108.2 ).

R ESULTS A N D D I S C U S S I O N

Figure 1 shows the variation of the methane volume produced in the differ- ent experiments as a function of time. As expected, the amount of gas pro- duced increased with increasing volume of OMW used. On the other hand, unlike in the anaerobic digestion of untreated OMW with similar COD values [ 19 ], the process never reached a stand-still. The decrease in the slope with

ANAEROBIC DIGESTION OF OLIVE MILL WASTEWATER PRETREATED W I T H A. CHROOCOCCUM 205

A m E

0

1600 ~ 4o0 mJ

1400 ~ A p ~ j ~- : 360ml

1200 / / ~ ~ 320ml

looo ~ / - ~ ~ ,~2,om,

6°° 1 / / , -

0 ~ ' I I I I I 0 1 2 3 4 5 6

Time (days) Fig. 1. Variation of the methane volume produced as a function of time.

time can be ascribed to the gradual decrease in the concentration of biode- gradable substrate.

We characterized the anaerobic digestion of OMW fermented with Azoto- bacter chroococcum on the basis of a kinetic model based on the Contois equa- tion, namely

It= itm[ S / (#X-I- S) ] ( 1 )

where It (day - 1 ) is the specific growth rate of the microorganisms, Itm the maximum # value, S (g COD/ l ) the substrate concentration, p a constant and X (g VSS/1) the microorganism concentration.

For dilute enough solutions, S<< pX, so

It= ItmS/flX (2)

The microorganism yield coefficient is defined as

Yx= - d X / d S (3)

Hence:

- Yx(dS/dt) = dX/dt

Taking into account that

dX/ dt = # X

a combination of Eqs (2), (4) and ( 5 ) yields

(4 )

(5)

2 0 6 R. BORJA ET AL.

- d S / S = K o d t (6)

where

Ko = ltml flYx (7)

Integration of Eq. (6) on the limiting condition t = 0 at S = So yields

S=Soexp( - Kot ) (8)

where So is the initial substrate concentration in g COD/1. The yield coefficient of the product formed (methane) is defined as

Yp = - d G / dS (9)

which, on integration on the limiting condition G = 0 at S=So yields

G= Y p ( S o - S ) ( lO)

By solving Eq. (10) for S and substituting it into (8) one has

G=So Yp[l - e x p ( - g o t ) ] ( 11 )

Hence:

G = G m [ 1 - e x p ( - K o t ) ] (12)

where Gm = So Yp, G (ml) being the accumulated methane volume as a func- tion of time, Gm (ml) the maximum methane volume obtained at an infinite digestion time, Ko (day- i ) the apparent rate constant and t (days) the diges- tion time.

Equation (8) is coincides with that derived by McCarty and Mosey [ 20 ], while Eq. ( 12 ) is analogous to that developed by Roediger [ 21 ] in order to relate the volume of gas produced in a batchwise anaerobic digestion process to time.

Taking natural logarithms in Eq. (12) and rearranging yields

In [Gm/ ( G m - G ) ]=Kot (13)

Accordingly, by plotting In [Gin~ (Gin-G)] against t one should obtain a straight line of slope Ko and zero intercept. Figure 2 shows such a plot for a Gm value equal to the methane volume obtained at the end of each experi- ment. In accordance with Eq. ( 13 ), the plot is a straight line of virtually zero (0.005) intercept, which allows the experimental data to be fitted to the pro- posed model.

Once the accuracy of the model used was checked, we used the methane volume-time data from Fig. 1 to calculate the specific rate constant, Ko, ana- lytically by non-linear regression [ 22 ]. The mean value of this kinetic con- stant was 1.21 +_0.08 day -~ (P<0.05) . Such a value was virtually the same for all the experiments, which reflects the occurrence of no substrate inhibi-

A N A E R O B I C D I G E S T I O N O F O L | V E M I L L W A S T E W A T E R P R E T R E A T E D W I T H A. CHROOCOCCUM

LnIGmI(Gm-G)] 8 f 1

207

I I I I I

0 1 2 3 4 6

Time (days)

Fig. 2. Plot of In [G,~/(Gm-G) ] as a function of time.

tion. Previous work on untreated OMW revealed the occurrence of a marked inhibitory effect: Ko decreased from 0.76 to 0.06 day-1 on increasing the digester COD from 0.6 to 9.6 g COD/1 [3]. This enhancing effect on the kinetics of anaerobic digestion of OMW was-to be expected from the results obtained in the biotoxicity tests. According to previous results [23 ], it can be ascribed to the decreased phenolic content resulting from the pretreatment; thus, the initial caffeic acid concentration at the same COD was diminished from ca. 1800 ppm to 175 ppm upon treatment.

The different behaviour of the pretreated waste towards anaerobic diges- tion can also be accounted for in an alternative way. Thus, the specific rate of methane production, Rp, can be determined from

Rp = G / t X V ( 14 )

provided the accumulated methane volume (G) at each t ime t, the biomass concentration (X) and the reactor volume (V) are known.

Figure 3 shows the variation of R e (ml C H 4 / d a y g VSS) after 6 days of digestion as a function of the initial substrate concentration (So, g COD/ l ) for both the untreated and the pretreated OMW. As can be seen, the specific rate of methane product ion increases linearly with the substrate concentra- t ion in both cases. On the other hand, the slopes of the two lines are rather

208 R. BORJA ET AL.

Rp (mL methane/day/g V88) 60

50

40

3O

2O

10

0 i 0 2 4 8 8 10 12 14 le

COD (g/L)

* FermenWd OMW - '~ OMW ~

Fig. 3. Variation o f R v after 6 days of digestion as a function of the initial substrate concentration for the untreated and pretreated OMW.

different; thus, at a given initial COD, the specific rate is significantly higher for the pretreated OMW than it is for the untreated waste. The difference between the two rates increases with increase in the substrate concentration.

Biodegradability Table 2 gives the COD values measured at the beginning of each experi-

ment. Taking into account that the COD level in the effluents remained vir- tually constant at 4.0 g/l, the average fraction of biodegraded substrate was 73.5%, which testifies to the efficiency of anaerobic digestion as a purification procedure for this type of waste.

On the other hand, the pH of the effluents remained virtually unchanged in every instance and averaged at 7.1. The volatile acidity (average 265 ppm caffeic acid) scarcely varied with the volume of pretreated OMW used. Its constancy and small value show the suitability of the biomethanation process for this type of waste.

Yield coefficient Inasmuch as the methane volume produced (G) and initial and final COD

in each experiment were perfectly known, the methane yield coefficient, Yp,

ANAEROBIC DIGESTION OF OLIVE MILL WASTEWATER PRETREATED WITH A. CHROOCOCCUM

TABLE 2

Initial COD value for each experiment

209

Volume (mL) CODinit (g /L)

20 4.20 40 4.45 80 4.90

120 5.35 160 5.80 200 6.25 240 6.70 280 7.15 320 7.60 360 8.05 400 8.50

G (rnL) 1 8 0 0

1600

1400

1 2 0 0

1000

8OO

600

40O

2 0 0

C-" i i i i 0 1 2 3 4 6

COD (g/L)

~( FermenWd OMW o OMW /

Fig. 4. Var ia t ion of the me thane vo lume produced with the chemical oxygen demand .

could be readily calculated. By linear least-squares fitting of G - COD uptake pairs we determined this coefficient from the slope obtained, which provided Yp=313 ml CH4 STP/g COD (Fig. 4). This value is 20% greater than that obtained for untreated OMW (Yp=260 ml CH4 STP/g COD) [6], which

210 R. BORJA ET AL.

again shows the positive effect of the prior fermentat ion with Azotobacter on the anaerobic purification process.

CONCLUSIONS

Olive mill wastewater which has been previously fermented with Azotobac- ter chroococcum can be readily degraded anaerobically with COD fraction removal higher than 73%.

The degradation process conforms to a first-order kinetics and the apparent rate constant, Ko (average 1.21 d a y - 1 ) , is scarcely influenced by the substrate concentrat ion over the COD range studied ( 4 . 2 - 8.5 g / l ) .

The specific rate of methane production is substantially higher than ob- tained for untreated OMW. In addition, no inhibition phenomena are seem- ingly involved since the biotoxicity of the waste is reduced by 30% upon treat- ment. Finally, the yield coefficient obtained was 313 ml CH4 STP/g COD, viz. 20% higher than that provided by untreated OMW.

ACKNOWLEDGEMENTS

The authors wish to express their gratitude to the CSIC (Spanish Council for Scientific Research) and the Consejeria de Educaci6n y Ciencia de la Junta de Andalucia for financial support granted for the realization of this work. Valuable experimental help from Ms Carmen S~lnchez is also gratefully acknowledged.

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