Biomass production and detoxi®cation of wastewaters from the oliveoil industry by strains of Penicillium isolated from wastewater

disposal ponds

Ana Robles, Rosario Lucas, Gerardo Alvarez de Cienfuegos, Antonio G�alvez *

Dpto Microbiology, Fac. Ciencias Experimentales, University of Ja�en, 23071 Jaen, Spain

Received 28 June 1999; received in revised form 12 January 2000; accepted 19 January 2000

Abstract

Olive mill wastewater (OMW) usually has to be diluted before biological treatment. In the present work seven strains of Pen-

icillium isolated from OMW disposal ponds were tested for biomass production and biodegradation of undiluted OMW. Best results

were obtained by using strain P4, which formed 21.50 g (dry weight) of biomass per litre of undiluted wastewater after 20 days of

cultivation. This and other strains also carried out an outstanding reduction of the chemical oxygen demand (COD) and the

phenolic content of OMW, as well as a pH raise. The process could be accelerated by agitation. OMW fermented with Penicillium P4

was devoid of its initial antibacterial activity against Bacillus megaterium ATCC 25848. Ó 2000 Elsevier Science Ltd. All rights

reserved.

Keywords: Olive mill wastewater; Phenols; Penicillium; Biodegradation

1. Introduction

The industry of olive oil extraction is one of the mainsources of coloured phenolic waste olive mill wastewa-ters (OMW) in Mediterranean olive-growing countries.This wastewater has a high pollution potential withchemical oxygen demands (COD) of up to 200 g/l. Theorganic fraction includes sugars, tannins, polyphenols,polyalcohols, pectins and lipids. Some of these sub-stances (mainly sugars and polyalcohols) can serve ascarbon and energy source for growth of suitable mi-croorganisms. The annual OMW production of Medi-terranean regions amounts to over 3� 107 m3 (Borjaet al., 1993).

The phenolic fraction accounts for most of theproblems associated with OMW pollution. First, it ishighly coloured (varying from dark red to black, de-pending on the age and on the type of olives processed).The colour has been attributed to high molecular weightphenolic compounds (Gonzalez et al., 1990), and canstill be detected visually after a 1/1000 dilution in water.Phenolic compounds are also responsible for several

biological e�ects, including antibiosis (Rodriguez et al.,1988; Gonzalez et al., 1990), ovipositional deterrence(Girolami et al., 1981) and phytotoxicity (Capasso et al.,1992).

Because of their antibacterial e�ects, phenolic com-pounds are the main drawback to OMW degradation byeither aerobic or anaerobic processes. To solve thisproblem, several investigations have been carried outusing microorganisms capable of growing aerobically ondiluted OMW with the object of reducing its initial or-ganic load and its phenolic content (Borja et al., 1992,1993, 1995a,b; Borja and Gonzalez, 1994; Vinciguerraet al., 1995; Yesilada et al., 1995; Martirani et al., 1996;Setti et al., 1998). In particular, diluted OMW prefer-mented with Aspergillus terreus or Geotrichum candidumand undiluted OMW prefermented with A. niger havebeen used satisfactorily during a two-stage anaerobicprocess of treatment (Hamdi and Garcia, 1993; Borjaand Gonzalez, 1994; Borja et al., 1995a,b).

Several penicillia, normally soil inhabitants, degradelignin related aromatic compounds as well as otherphenolic compounds similar to those found in OMW(De Jung et al., 1990; Patel et al., 1990; Tillet andWalker, 1990; Hofrichter et al., 1994; Marr et al., 1996;Rodriguez et al., 1996; Wunderwald et al., 1997) and aretherefore good candidates for treatment of phenolic

Bioresource Technology 74 (2000) 217±221

* Corresponding author. Tel.: +34-953-212-160; fax: +34-953-212-

141.

E-mail address: agalvez@ujaen.es (A. GaÂlvez).

0960-8524/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved.

PII: S 0 9 6 0 - 8 5 2 4 ( 0 0 ) 0 0 0 2 2 - 5

e�uents. The purpose of this work was to evaluate sevenstrains of Penicillium spp. isolated from OMW disposalponds for biomass production as well as for their ca-pacity to reduce the organic load and the phenoliccontent of OMW.

2. Methods

2.1. Microorganisms and culture conditions

Seven strains of Penicillium spp. isolated from localOMW disposal ponds were used in this study. Strainswere propagated at 28°C on YM agar (AdsaMicro,Barcelona): 5 g/l peptone, 3 g/l malt extract, 3 g/l yeastextract, 10 g/l glucose, and 17 g/l agar. Strains weremaintained at 4°C by subculturing every three months,or stored at )80°C in 40% glycerol for up to two years.

A liquid growth medium consisting of OMW from acontinuous process ®ltered through Whatman No. 41®lter paper was sterilized by autoclaving and used forbiodegradation studies (OMW broth). The compositionof the medium is shown in Table 1.

Erlenmeyer ¯asks (250 ml) containing 50 ml of OMWbroth were inoculated with 1 ml of a spore suspension insterile saline solution (to give a ®nal concentration of ca1:0� 106 spores/ml). Spore suspensions were preparedfrom lawns of sporulated mycelia previously grown onYMA-OMW agar plates consisting of YMA plus 10%OMW (by volume). Incubation was carried out at 28°Cwithout agitation, or under agitation in an orbitalshaker (Rotaterm P; Selecta, Barcelona) at 120 rpm.

2.2. Analytical procedures

The dry weight of fungal mass was obtained by ®l-tering the contents of each ¯ask through preweighedWhatman No. 41 ®lter paper and drying the solids toconstant weight at 70°C. The mycelia-free ®ltrates werefurther centrifuged at 10,000 ´ g for 20 min before beingused for analytical determinations. Results are themeans of three replicates.

The pH of the medium was measured using a pHmeter. OMW decolourization was determined accordingto the method of Sayadi and Ellouz (1993), with slightmodi®cations. Tenfold diluted supernatants were ad-justed to pH 7.0 and their absorbance was measured at465 nm against distilled water, using a Beckman DU 640spectrophotometer. The total phenol content was de-termined according to a modi®cation of the Folin±Ci-ocalteau method (Swain and Hillis, 1959). The CODwas determined according to the recomendations of theAmerican Public Health Association (APHA, 1985).Reducing sugars were determined by the Nelson method(Nelson, 1944). Soluble protein was determined by themethod of Bradford (Bradford, 1976).

2.3. Assay of antibacterial activity

The antibacterial activity of OMW was determinedby spot test, using B. megaterium ATCC 25848 as in-dicator strain. The samples to be tested were previously®ltered through 0.45 mm ®lters under sterile conditionsand spotted (10 ll each) on the surface of tryptic soyaagar (TSA) plates seeded with ca 1:0� 106 colony-forming units (CFU)/ml. After 24 h of incubation at37°C the plates were examined for halos of inhibitedgrowth around the spots.

3. Results

3.1. Biomass production and OMW biodegradation

The growth capacity of Penicillium spp. strains fromOMW disposal ponds on OMW and the changes in thephysico-chemical properties of the medium after culti-vation were studied. All seven strains grew e�ciently inOMW broth, forming a pellicle that covered the liquidsurface. After 20 days of cultivation, most of the strainsproduced a considerable amount of biomass (Fig. 1).The highest and lowest values corresponded to strainsP4 and P1, respectively.

At the end of the incubation period, the pH of cul-tured broths was always much higher than the initialpH, ranging from 6.22 to 7.62 (Table 2). Modi®cation ofother parameters depended on the strain used for cul-tivation and its capacity to grow on OMW (Table 2).The best results were always obtained with strain P4.The COD of OMW fermented with this strain was re-duced to 60.75% of its initial value, while the totalphenol content was reduced to 54.50%. Cultivation ofother strains also caused a substantial reduction of theCOD and phenolic content. In all cases, the amount ofreducing sugars present in OMW decreased markedlyafter cultivation.

Table 1

Physical and chemical characteristics of OMW broth used in this

studya

Colour (OD at 465 nm) 14:7� 0:89

pH 4:8� 0:1COD 114:0� 10:47

Total protein 0:7� 0:11

Total sugars 20:4� 1:42

Reducing sugars 4:3� 0:77

Total phenolic compounds 1:6� 0:18

a The data represent the average of three determinations. Concentra-

tions are given as grams per litre.

218 A. Robles et al. / Bioresource Technology 74 (2000) 217±221

3.2. Time-course of OMW degradation by Penicillium P4

The strain Penicillium P4 was cultivated on OMWwithout or with agitation in order to follow the evolu-tion of di�erent parameters along the incubation period.Under resting conditions, maximum biomass concen-tration was achieved at day 18 (Fig. 2(A)). Consumptionof reducing sugars and reduction of the COD followed asimilar pattern as biomass production and, in bothcases, highest levels of reduction were achieved at days16±18.

Reduction of the phenolic content and the pigmen-tation of the broth was not observed until the late phaseof growth (days 16±20), once reducing sugars had beenconsumed and a signi®cant amount of biomass formed.

Growth of Penicillium P4 was favored markedly byincubation under agitation (Fig. 2(B)). The highestvalues of biomass production, substrate utilization andcolor reduction were obtained much earlier (at days 12±14 of incubation). As in the previous case, reduction ofthe phenolic content and the pigmentation of the me-dium took place mainly at the end of the exponential

growth phase. The ®nal values of the di�erent parame-ters did not di�er much from those obtained underresting conditions, though they were slightly higher.

3.3. Depletion of antibacterial activity

The e�ect of Penicillium P4 on the antibacterial ac-tivity of OMW was followed during cultivation at restand under agitation, using B. megaterium ATCC 25848as sentive strain (Table 3). The levels of antibacterial

Fig. 2. Biomass production and detoxi®cation during the course of

growth of Penicillium P4 in OMW broth at rest (A) or under agitation

(B). The biomass dry weight (d) and the percentage reduction of the

COD (�), reducing sugars (n), total phenols (m) and colour (optical

density at 465 nm; s) of the cultured broth are shown.

Table 2

Changes in the physico-chemical characteristics of OMW after incubation with di�erent strains of Penicillium sppa

Strain pH Pigmentation (%) COD (%) Total phenols (%) Reducing sugars (%)

P1 6:22� 0:25 92:20� 4:20 74:75� 5:25 67:82� 4:85 17:50� 3:28

P2 6:83� 0:25 90:70� 3:25 69:90� 3:82 65:20� 5:42 6:75� 3:75

P3 7:11� 0:32 87:40� 3:67 65:50� 4:80 59:50� 6:48 14:20� 2:10

P4 7:62� 0:24 80:20� 2:30 60:75� 5:90 54:50� 4:30 9:89� 2:55

P5 7:28� 0:25 87:78� 4:40 61:82� 4:42 53:90� 4:85 10:22� 1:50

P6 7:48� 0:61 86:87� 2:75 63:50� 2:20 52:60� 5:82 11:43� 0:85

P7 7:53� 0:41 87:34� 5:20 61:82� 5:45 55:72� 3:32 10:35� 1:70

a Incubation was carried out at 28°C for 20 days. Percentages were calculated taking the values of untreated OMW as 100%.

Fig. 1. Biomass production by di�erent strains of Penicillium P4 on

OMW broth. The dry weight was estimated after incubation at 28°C

for 20 days without agitation. The data represent the average of three

experiments.

A. Robles et al. / Bioresource Technology 74 (2000) 217±221 219

activity naturally present in untreated OMW decreasedgradually as growth of Penicillium P4 proceeded. Max-imum depletion of antibacterial activity occurred duringthe late phase of growth, concomitantly with removal ofphenolic compounds. At late stages of cultivation (day12 in cultures under agitation and day 16 in cultures atrest), the cultured broths were completely devoid ofantibacterial activity against the test strain.

4. Discussion

The olive oil extraction industry generates toxicwastewaters that are often eliminated by evaporation inopen ponds. These provide a good substrate for isola-tion of microbial strains with selected biodegradativepotentials, like the Penicillium strains used in this study.

The data presented here suggest that strains of Pen-icillium (and specially strain P4) can use OMW e�-ciently for biomass production. This seems to occurmainly at the expenses of reducing sugars present inOMW. Nevertheless, the pH increase observed suggeststhat organic acids are also being used for growth.

The processes of biomass production and OMW de-gradation by Penicillium P4 can be shortened by the useof agitated broths, but the results as far as total amountof biomass and modi®cation of physico-chemical pa-rameters of the e�uent could not be improved sub-stantially. This was probably due to the fact thatsu�cient aeration was already being provided by thesurface pellicle formed by the Penicillium.

Although Penicillium P4 carries out a relatively smallreduction (ca 45%) of the total phenolic content, theantibacterial activity of OMW is depleted, suggestingthat toxic phenolic compounds are e�ciently degradedor adsorbed to the fungal biomass. Nevertheless, weshould also consider that the use of organic acids in

OMW (other than the phenolic acids) as carbon andenergy source by this mould should result equally in adecrease of antimicrobial activity (exerted both by theorganic acid molecules and by the low pH). The increasein the pH of the medium after cultivation of Penicilliumspp. supports this possibility.

Strains of Penicillium have been reported to carry outdegradation of phenolic compounds like ferulic acid(Tillet et al., 1990), phloroglucinol (Patel et al., 1990),veratryl alcohol (De Jung et al., 1990), di¯uorinatedphenols (Wunderwald et al., 1997), halogenated phenols(Hofrichter et al., 1994; Marr et al., 1996) as well asnatural lignins and lignocellulosic substrates (Rodriguezet al., 1996). Therefore, there is great interest in use ofPenicillium strains for treatment of phenolic-rich e�u-ents.

The development of integrated aerobic/anaerobicprocesses for OMW biodegradation is an attractive ap-proach. The main purpose of aerobic treatments is(besides lowering the COD) detoxi®cation, becausephenolic compounds associated with complex lipids inOMW are very stable and highly inhibitory to metha-nogenic consortia. The results obtained with PenicilliumP4 are comparatively similar to results reported forother molds on undiluted (Hamdi and Garcia, 1993) ordiluted OMW (Borja et al., 1993; Vinciguerra et al.,1995; Yesilada et al., 1995; Martirani et al., 1996; Settiet al., 1998). Recently, a strain of P. cyclopium isolatedfrom OMW-irrigated soils was also able to grow onundiluted OMW (Tardioli et al., 1997). The capacity ofPenicillium P4 for growth and detoxi®cation of undi-luted OMW should be of advantage in integrated bio-degradation processes.

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