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Organic matter transformation and detoxification in dry olive mill residueby the saprophytic fungus Paecilomyces farinosus

Inmaculada Sampedro a,b, Tomas Cajthaml c, Sara Marinari b, Maurizio Petruccioli b,Stefano Grego b, Alessandro D’Annibale b,*a Department Microbiologıa, Estacion Experimental del Zaidın, CSIC, Prof. Albareda 1, E-18008 Granada, Spainb Dipartimento di Agrobiologia & Agrochimica, University of Tuscia, Via S. Camillo de Lellis snc, 01100 Viterbo, Italyc Institute of Microbiology of the ASCR, v.v.i., Vıdenska 1083, 14220 Prague 4, Czech Republic

Process Biochemistry xxx (2008) xxx–xxx

A R T I C L E I N F O

Article history:

Received 29 April 2008

Received in revised form 10 September 2008

Accepted 20 October 2008

Keywords:

Dry olive mill residue

Phenols

Paecilomyces farinosus

Humification

Laccase

Phytotoxicity

A B S T R A C T

Dry olive mill residue (DOR), the by-product of the two-phase extraction process, is very rich in organic

matter and nutritionally relevant cations. For this reason, the agronomic use of this waste has been

suggested although DOR exhibits significant phytotoxicity. The objective of this study was to investigate

the impact of Paecilomyces farinosus on both organic matter modification and detoxification of this waste.

Humification ratio in DOR colonized by the fungus for 20 weeks was increased by about 65% with respect

to the abiotic control and humification index reached 0.38, a value that characterizes well-humified

materials. High performance size-exclusion chromatography of humic acids from fungal cultures showed

a marked increase in both weight-averaged and number-averaged molecular weights with respect to

abiotic controls. Water-soluble phenols were reduced by 45% in 20-week-old P. farinosus cultures on DOR

and mass-balance ultra-filtration showed that the relative abundance of the molecular weight fraction of

phenols above 30 kDa increased from 31 to 72% suggesting the occurrence of polymerization.

Experiments performed with alfalfa grown on soils containing 2.5% (w/w) of abiotic controls and fungal-

treated DOR showed that phytotoxicity was totally suppressed in the waste that underwent fungal

treatment.

� 2008 Elsevier Ltd. All rights reserved.

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1. Introduction

The olive oil extraction industry that was formerly confinedwithin the Mediterranean area is gaining an ever increasingeconomic and social relevance at a worldwide level. Largequantities of both solid and liquid wastes are generated by theolive oil manufacturing process [1]. As a consequence, olive oilproducing countries are facing severe environmental contamina-tion problems caused by the accumulation and/or incorrectdisposal of these wastes.

In recent years a new two-phase extraction process wasintroduced in modern mills. In Spain, the most important olive oilproducer in the world, the introduction of this technology wascarried out in 90% of Spanish olive oil factories [1]. This processgenerates two fractions: the olive oil and a solid waste oftenreferred to as ‘‘pomace’’ or ‘‘alperoujo’’ characterized by a high

* Corresponding author. Tel.: +39 0 761 357368; fax: +39 0 761 357242.

E-mail address: dannib@unitus.it (A. D’Annibale).

Please cite this article in press as: Sampedro I, et al. Organic matter tsaprophytic fungus Paecilomyces farinosus. Process Biochem (2008),

1359-5113/$ – see front matter � 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.procbio.2008.10.016

moisture content [2]. This solid waste is generally dried andsubjected to a subsequent extraction with n-hexane to recoverresidual oil thus leading to the formation of a solid waste, termeddry olive mill residue (DOR), the annual production of whichamounts to around 4 million tonnes [3]. This by-product has beenso far mainly employed for energy and co-generation purposes,although some perplexities have been raised due to the presence ofpolyaromatic hydrocarbons in combustion gases [1]. The agro-nomic use of DOR has been also suggested, due to its highconcentration in both organic matter and nutritionally importantcations, such as potassium and calcium. However, the organicfraction includes toxic compounds such as polyphenols, poly-alcohols and volatile fatty acids [1,2] capable of inhibiting bothmicrobial growth [4] and germination and morphogenesis inplants [5]. Consequently, DOR has to be stabilized and detoxifiedprior to its application into soil. In this respect, successful resultshave been obtained by subjecting DOR to composting or co-composting with other agro-residues [5,6]. A possible alternativeto composting might involve the inoculation with saprobe fungiable to both stabilize the waste and to degrade phytotoxic

ransformation and detoxification in dry olive mill residue by thedoi:10.1016/j.procbio.2008.10.016

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compounds such as phenols [3,7]. In a previous study, the ability ofseveral saprobe fungi in degrading both monomeric phenols andphenolic glucosides in DOR was studied; among the fungal speciesinvestigated, Paecilomyces farinosus BAFC F8846 exhibited apeculiar behaviour since it hydrolyzed phenolic glucosides inthe earlier phases of growth and then eliminated phenols, whilethe other fungi simultaneously depleted both monomeric phenolsand phenolic glucosides [7]. Several species belonging to the genusPaecilomyces have economic relevance since some of them cancause food contamination and spoilage while other species,including P. farinosus, are entomopathogenic [8]. In addition, thesame genus is important from an ecophysiological viewpoint dueto its contribution in carbon turnover in both soil and compostenvironments where basidiomycetes are not well established [9].The capability of Paecilomyces species of producing lignin-degrading enzymes and of transforming lignin [10] and chlori-nated lignins released from paper mills [11] have been reported.For this reason, the ligninolytic ability of these saprobe fungi mightbe used to transform the DOR.

Consequently, the present study was aimed at assessing theimpact of P. farinosus BAFC F8846 on both overall chemicalcomposition of DOR and phytotoxicity removal from the waste.Particular focus was devoted to study whether and how organicmatter evolution and stabilization in the waste were affected bythe fungus due to aforementioned ubiquity and ecologicalsignificance of this species in natural ecosystems. To this aim,the possible occurrence of humification upon fungal incubationwas investigated by quantifying the humic substances, namelyfulvic and humic acids, and determining several widely acceptedindices related to the process [12,13]. Unlike the majority ofinvestigations on the fungal conversion of DOR, this studyattempted to evaluate the impact of a fungal species on therelative abundances of both labile and stabilized carbon fractions.In addition, possible structural changes and molecular weightshifts in humic acids from the fungal-treated waste and thecorresponding abiotic controls were comparatively analyzed bypyrolysis with tetramethylammonium hydroxide followed by gas-chromatography mass-spectrometry (TMAH-Py-GC MS) and highperfomance size-exclusion chromatography (HP-SEC), respec-tively. The former technique is widely used to study bothbiopolymers [14,15] and bulk organic matter [16–18]. It involvesthe solvolysis and subsequent methylation of both ether and esterbonds in complex macromolecules thus enhancing the thermalstability of fragments bearing acidic, phenolic and alcoholic groupsthereby allowing their chromatographic detection [14]. To the bestof our knowledge, this is the first study where TMAH-Py-GC MSanalysis was applied to perform a detailed characterization of bothDOR and humic acids purified from the waste.

2. Materials and methods

2.1. Organism and inoculum preparation

P. farinosus strain BAFC F8846 was maintained at 4 8C and periodically sub-

cultured on potato dextrose agar (PDA) added with 2% malt extract (w/v). To

prepare inocula for solid-state conversions of DOR, barley seeds were inoculated

with a 1 cm2 disk of PDA withdrawn from 14-d-old fungal cultures grown at 28 8C.

2.2. Sample preparation and fungal treatment

DOR, withdrawn from an olive oil manufacturer (Sierra Sur S.A., Granada, Spain),

was stored at �20 8C until used. The main physico-chemical characteristics of DOR

were as follows: pH 5.13, total organic carbon 58.5%, total nitrogen 1.87%, total

phosphorus 0.2%, lignin 24.7%, cellulose 18%, hemicellulose 12.8%, total phenols

3.18%, total lipids 0.2%, ashes 9.2%. The most abundant elements were (g kg�1 DOR):

potassium 30.5, calcium 13.6, magnesium 3.8, iron 1.1, sodium 0.17, copper 0.07,

zinc 0.06 and manganese 0.04. The solid residue was added with deionized water to

adjust its moisture content to 25% (w/w) prior to its sterilization (two cycles in

autoclave at 120 8C for 20 min) and subsequent inoculation. One-litre Erlenmeyer

Please cite this article in press as: Sampedro I, et al. Organic matter tsaprophytic fungus Paecilomyces farinosus. Process Biochem (2008),

flasks containing 80 g of steam-sterilized DOR were inoculated with 4 barleys seeds

that had been previously colonized by the fungus for 7 d. Solid-state cultures on

DOR were carried out at 28 8C in the dark under stationary conditions for 2 and 20

weeks. Non-inoculated and sterilized DOR samples were prepared and incubated as

above and will be referred to as abiotic controls from here onwards.

2.3. Analytical assays

Samples were ashed at 600 8C for 12 h and organic matter (OM) obtained by

subtracting ash content from the whole sample weight [19]. OM losses (%) were

calculated from the ash contents of both abiotic control (X1) and corresponding

fungal-treated DOR (X2) according to the following equation [6]:

OM losses ð%Þ ¼ 100� ½X1 � ð100� X2Þ�½X2 � ð100� X1Þ�

� 100 (1)

Total extractable carbon (TEC) was extracted by mechanical shaking for 48 h at

65 8C with 0.1 M NaOH plus 0.1 M Na2P4O7 under a N2 atmosphere and using a 1:50

solid–liquid ratio. The suspension was then centrifuged (8000 � g; 20 min) and the

supernatant filtered through a 0.8 mm membrane (Millipore, USA). This filtered

solution was fractionated into non-humified (CNH) and humified carbon fraction,

the latter one encompassing both humic and fulvic carbon (CHA and CFA,

respectively) [13]. CHA was precipitated from the filtrate by adding H2SO4 up to

pH 2.0, then centrifuging as above and the precipitated fraction was collected and

stored at 4 8C. The supernatant (25 ml) was loaded onto a small column packed with

10 ml of polyvinylpolypyrrolidone previously equilibrated with 0.005 M H2SO4 and

the retained fraction (CFA) was eluted by 0.1 M NaOH and collected into a centrifuge

tube. Total organic carbon (TOC), TEC, CHA and CFA were determined by the method

of Springer and Klee [20]. Non-humic carbon (CNH) was calculated by subtracting

both CFA and CHA from TEC [13]. Degree of humification (DH), humification ratio

(HR) and humification index (HI) were calculated from Eqs. (2) to (4), respectively

[12,13]:

DH% ¼ CHA þ CFA

TEC� 100 (2)

HR% ¼ CHA þ CFA

TOC� 100 (3)

HI ¼ CNH

CHA þ CFA(4)

Klason lignin, cellulose, hemicellulose and lipids contents were determined as

reported elsewhere [3]. Total N, P, K, Fe, Mn, Cu and Zn content of the DOR were

determined as described by Mingorance [21]. Nitrate and ammonium ions were

determined as previously described [22]. Water-soluble phenols (WSP) were

extracted and determined as reported elsewhere [3] using tyrosol as the standard.

Extracts containing WSP from DOR colonized by the fungus or its relative abiotic

controls were subjected to ultra-filtration on a stirred cell (Amicon, Danvers, MA,

USA) equipped with a 30-kDa Diaflo flat membrane (Waters Millipore, Milford, MA,

USA) operated in a dialysis-filtration mode and the concentrations of WSP were

determined both in the permeate and in the retentate fractions. The pH of DOR

samples was measured by using a 1:5 DOR:deionized water (w/v) ratio. The extent

of fungal biomass was indirectly determined by the chemical determination of the

chitin content in the solid substrate [23].

2.4. TMAH-Py-GC MS analyses

Weighed samples (0.5 mg) of either bulk DOR or HAs purified from the waste

were treated by adding an excess of tetramethylammonium hydroxide (25%

aqueous solution), placed on Wolfram wire spirals and then dried in a desiccator

overnight at room temperature. Pyrolysis was performed with a PYR-01 pyrolyzer

(Labio, Prague, Czech Republic) with a quartz sample holder. Each sample on the

Wolfram support was inserted within a coil of platinum filament and this probe was

placed into the injector port of a GC/MS system (Varian 3400/Finnigan ITS 40 ion

trap detector, Palo Alto, CA, USA) where the samples were directly pyrolyzed. The

samples were analyzed in triplicate at 550 8C for 10 s. The GC instrument was

equipped with a split injector (split ratio 1/40), a DB-5MS column (J&W Scientific,

Folsom, CA, USA), was used for separation (30 m, inner diameter 0.25 mm, 0.25 mm

film thickness) and the carrier gas was helium (1 ml min�1). The temperature

program started at 45 8C and the oven was heated to 240 8C at a rate of 5 8C min�1.

The detector delay time was 2 min and it was switched off again after 35 min due to

the consequent elution of only fatty acids. The injector and transfer line

temperature was set to 240 8C. Mass spectra were recorded at 1 scan s�1 under

an electron impact at 70 eV, scanning range 50–450 m/z. The pyrolysis products

were identified by comparing the mass spectra with the data in the NIST 02 library,

and independently by interpreting the fragmentation pattern. The relative

percentages of pyrolysis products were calculated from the relative areas of the

peaks after recalculation according to exact weight of the samples. Reproducibility

of the sample introduction was tested and it was better than 95%. Individual

fragments detected after pyrolysis were divided into the following four main groups

ransformation and detoxification in dry olive mill residue by thedoi:10.1016/j.procbio.2008.10.016

Fig. 1. Glucosamine content (A), ash contents (B), organic matter losses (C) and pH

of aqueous extracts (D) in abiotic controls of dry olive mill residue (DOR) and in the

same waste that had been incubated with Paecilomyces farinosus F8846 for 2 and 20

weeks at 28 8C under stationary conditions. Data are the mean of triplicate

experiments. The significance of differences between distinct treatments of DOR

was tested by the Tukey test. Same letters above bars indicate lack of statistical

significance (P � 0.05). Organic matter losses in fungal-treated DOR were calculated

by Eq. (1) according to Paredes et al. [6]. N.C., Not calculated.

I. Sampedro et al. / Process Biochemistry xxx (2008) xxx–xxx 3

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PRBI-8531; No of Pages 10

according to their origin: lignin-related compounds (L), carbohydrates (C), fatty

acids (F) and nitrogen-containing compounds (N). When possible, fragments

belonging to the first group were assigned to lignin basic structures: H, p-

hydroxyphenyl; G, guaiacyl (3-methoxy-4-hydroxyphenyl); and S, syringyl (3,5-

dimethoxy-4-hydroxyphenyl). The syringyl/guaiacyl ratio (S/G) was calculated by

dividing the sum of peak areas from syringyl derivatives by the sum of peak areas

from guaiacyl counterparts as described by Vane et al. [24]. The relative percent

abundances of H, G and S subunits were calculated by dividing the areas of peaks

pertaining to each subunit by the total area of pyrograms. Guaiacyl and syringyl

acid/aldehyde ratios, namely (Ad/Al)G (Ad/Al)S, respectively, were calculated from

the 3,4-dimethoxybenzoic ME/3,4-dimethoxybenzaldehyde and 3,4,5-trimethox-

ybenzoic acid ME/3,4,5-trimethoxybenzaldehyde area ratios as described by other

investigators [18].

2.5. High performance size-exclusion chromatography

The molecular weight (Mw) distribution of HA samples was determined by high

performance size-exclusion chromatography. To this aim, 5 mg HA were dissolved

in 50 mM sodium nitrate at pH 7.0 (eluent A) and 10 ml sample were injected in a

PWXL TSK G-3000 (Supelco, Bellefont, USA) column (300 mm � 7.8 mm i.d.)

equilibrated with eluent A at a flow rate 0.6 ml min�1. The elution profile was

monitored by a 9065 diode array detector (Varian) set at 278 nm. The

chromatographic behaviour of both standards and HA fractions was defined in

terms of the distribution coefficient Kav which was calculated using the following

expression: Kav = (Ve � Vo)/(Vt � Vo), where Vo is the void volume, Ve the elution

volume of the peak and Vt is the total column volume [25]. Vo and Vt were

determined by the elution volumes of blue dextran (Mw > 250 kDa, Kav = 0) and

acetone (Mw = 0.068 kDa, Kav = 1), respectively. The column was calibrated with

catalase (Mw = 200 kDa, Kav = 0.05), lactate dehydrogenase (Mw = 145.8 kDa,

Kav = 0.09), bovine serum albumin (Mw = 66 kDa, Kav = 0.14) ovalbumin

(Mw = 43 kDa, Kav = 0.18), carbonic anhydrase (Mw = 29 kDa, Kav = 0.26), ribonu-

clease (Mw = 13 kDa, Kav = 0.59), aprotinin (Mw = 6 kDa, Kav = 0.69). The Mw of HA

peaks were calculated at their highest intensity from the calibration curve obtained

by plotting log Mw of the aforementioned standards vs. their related Kav values. On

the other hand, weight-averaged (Mwav) and number-averaged (Mn) molecular

weights of the whole HA profiles were calculated by Eqs. (5) and (6), respectively.

Mwav ¼PN

i¼1 hi�MiPN

i¼1 hi(5)

Mn ¼PN

i¼1 hiPN

i¼1 hi=Mi(6)

where hi and Mi are the intensity and estimated Mw of each fraction eluted at

volume i, respectively. The polydispersity was calculated from the Mwav/Mn ratio.

2.6. Biochemical determinations

Extra-cellular enzymes were extracted from DOR, dialysis-filtrated and 20-fold

concentrated as previously reported [3]. Endo-b-1,4-xylanase, b-xylosidase, endo-

b-1,4-glucanase and cellobiohydrolase activities were determined as described

elsewhere [26]. The activities of b-glucosidase and a-arabinofuranosidase were

determined according to the methods of Hayano and Tubaki [27] and MacKenzie

et al. [28], respectively. Esterase and lipase activities were determined spectro-

photometrically at 35 8C using either b-naphtylacetate or b-naphtylmyristate,

respectively, as substrates as described elsewhere [3]. Tannin-acyl hydrolase was

assayed as described elsewhere [29]. Mn-peroxidase, Mn-independent peroxidase,

laccase and aryl alcohol oxidase activities were assayed according to the method of

Saparrat et al. [30]. The mono-phenolase activity of tyrosinase, i.e. the hydroxyla-

tion of monophenols to ortho-diphenols, was assayed according to the method of

Espın et al. [31]. Appropriate controls were performed with heat-denatured

extracts. Enzyme activities were expressed as IU (g DOR)�1. One IU was defined as

the amount of enzyme producing 1 mmol product min�1 under the assay

conditions.

2.7. Phytotoxicity experiments

The characteristics of the soil employed in phytotoxicity experiments were

reported elsewhere [3]. Both fungal-treated and abiotic controls of DOR were

sterilized and added to soil pots at concentrations of 25 g kg�1 soil. The same soil in

the absence of DOR was used as the control. Alfalfa (Medicago sativa L.) was used as

the test plant due to its importance as a perennial crop. The soil, that had been

previously steam-sterilized, was mixed with sterilized quartz sand in a 1:1 ratio (v/

v) and added to 0.3 L pots. Plant seeds were pre-germinated and selected for

uniformity prior to planting. Plants were grown in a greenhouse with natural light

supplemented by Sylvania incandescent and cool-white lamps giving

400 nmol m�2 s�1 in the wavelength range of 400–700 nm; there was a 16–8 h

light–dark cycle at 25–19 8C and 50% relative humidity. Plants were watered from

below, and fed with a nutrient solution as previously reported [3]. Plants were

Please cite this article in press as: Sampedro I, et al. Organic matter tsaprophytic fungus Paecilomyces farinosus. Process Biochem (2008),

harvested after 4 weeks and the dry weights of both shoots and roots were

measured.

3. Results

3.1. Mycelial growth on DOR

P. farinosus initially grew slowly on DOR and colonization wasnot complete within the second week of cultivation, while it wasextensive after 20 weeks incubation as inferred by visualinspection. To obtain a quantitative, albeit indirect estimation offungal biomass, the chitin content of the solid substrate wasdetermined. Fig. 1A shows that after 2 weeks incubation, the chitincontent amounted to 1.6 mg glucosamine mg�1 DOR and markedly

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I. Sampedro et al. / Process Biochemistry xxx (2008) xxx–xxx4

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increased in 20-week-old cultures (5.3 mg glucosamine mg�1

DOR). The significant increase in ash concentration that wasobserved as the incubation time was prolonged from 2 to 20 weeksclearly showed that effective organic matter degradation occurredthroughout fungal growth (Fig. 1B). Accordingly, OM losses werethree-fold higher in 20-week-old cultures than those grown for 2weeks (38% vs. 12%, respectively) (Fig. 1C). The pH values inaqueous extracts of DOR which had been colonized by the fungusfor 2 weeks was lowered with respect to the related abiotic control(4.8 vs. 5.3, respectively) while a marked alkalinization of thesubstrate was observed as incubation was extended to 20 weeks(Fig. 1D).

3.2. Effect of fungal growth on chemical composition and organic

matter evolution in DOR

Total carbon losses in solid substrate cultures are usuallyassociated with a decrease in OM due to mineralization (which isdirectly related to respiration). Table 1 shows that the highestcarbon losses were observed in 20-week-old P. farinosus cultureson DOR where it amounted to 13.3% with respect to thecorresponding abiotic control. Table 1 also shows that the largemajority of total nitrogen content in DOR, accounting for18.7 g kg�1, was organic nitrogen. In fact, low levels of ammoniumand nitrate were detected in the waste in agreement with otherstudies [32]. After 2 weeks colonization, total nitrogen (TN)content in fungal-treated DOR did not significantly differ from thatof the abiotic control. After 20 weeks incubation, TN was increasedby around 16%. C/N ratios after 2 and 20 weeks incubation werereduced by 8 and 25.8% in P. farinosus cultures on DOR with respectto the related abiotic controls (28.5 vs. 31 and 23 vs. 31,respectively). Total lipids, the concentration of which wassignificantly reduced in 2-week-old P. farinosus cultures on DOR,were almost depleted after 20 weeks fungal incubation. The mostabundant biopolymer in DOR at start was lignin, the content ofwhich was around 24.7 � 0.6%, while hemicellulose and cellulosecontents were 12.8 � 1.5% and 18 � 1%, respectively. Table 1 showsthat the contents of these biopolymers in DOR that had beencolonized for 2 weeks were not substantially affected. In 20-week-oldcultures, P. farinosus reduced the hemicellulose and cellulose contentsby 14.7 and 15%, respectively, with respect to the correspondingabiotic control. By contrast, the lignin content in fungal-colonizedDOR was not affected, regardless of the incubation time. Thermo-chemolysis of DOR abiotic controls released more than 45 fragments,most of which were identified as methyl ethers and esters of naturalcompounds. The majority of these compounds, the identification and

Table 1Main properties of dry olive mill residue (DOR) colonized by the fungus Paecilomyces fa

Parametera DOR abiotic control

2 weeks 2

Total organic carbon (g kg�1) 585 � 10.6b

Total nitrogen (g kg�1) 18.7 � 0.3a

NH4+-N (mg kg�1) 11 � 0.4a

NO3�-N (mg kg�1) 351 � 30b

C/N ratio 31 � 0.3c

Lignin (%) 24.3 � 0.8a

Cellulose (%) 18.3 � 0.7b

Hemicellulose (%) 12.4 � 0.3b

Total lipids (g kg�1) 21.9 � 0.5c

TEC (g kg�1) 281.4 � 26.9b 2

CNH (g kg�1) 133.8 � 20.0c

CHA (g kg�1) 112.5 � 17a 1

CFA (g kg�1) 35.1 � 6.1c

a Data are the mean � standard deviation of three replicates and row means followed b

(P � 0.05); TEC, total extractable carbon; CNH, non-humic carbon; CHA, humic acid carbon;

Please cite this article in press as: Sampedro I, et al. Organic matter tsaprophytic fungus Paecilomyces farinosus. Process Biochem (2008),

relative abundances of which are reported in Table 2, were lignin-related compounds. Contrary to indications arising from chemicalanalyses low amounts of carbohydrates were found among pyrolysisproducts in agreement with other studies [17,18]. Among fragmentsreconducible to polysaccharides, the most abundant were 1,2,4-trimethoxybenzene and methoxyacetic acid methyl ester. The majoraromatic fragments in 2-week-old abiotic control were 3,4-dimethoxyphenyl acetone (11.3%), 1,4-dimethoxybenzene (7.7%)and 3,4-dimethoxybenzoic methyl ester (7.1%) and these relativeabundances were not significantly modified as incubation time wasextended to 20 weeks (Table 2). In fungal cultures, the relativeabundance of 3,4-dimethoxybenzoic acid methyl ester tended todecrease with time while syringyl-related fragments showed anopposite trend (Table 2).

Regardless of the incubation time, abiotic controls of DORexhibited similar percent abundances of syringyl, guaiacyl and p-hydroxyphenyl subunits, the related values being in the range 7.5–8.0, 35.0–37.4 and 16.4–18.0, respectively (Table 3). In the samecontrols both acid/aldehyde ratios did not significantly differ eachone another and very low S/G ratios were evident. In DOR that hadbeen incubated for 20 weeks with P. farinous, the relativeabundances of lignin subunits were extensively modified. Mostrelevant changes were observed for S units the abundance of whichwas nearly doubled with respect to the relative abiotic control witha consequent increase in the S/G ratio (0.48 vs. 0.23, respectively)(Table 3).

Table 4 shows that the water-soluble phenols (WSP) content in2-week-old DOR abiotic control was 31.8 � 0.6 mg g�1. In addition,a mass-balance performed by ultra-filtration showed that the percentabundance of WSP contents in Mw fractions higher than 30 kDa (F1)and lower than 30 kDa (F2) were about 33 and 67%, respectively. Theextension of incubation time, i.e. 20 weeks, in the abiotic control led toa 16% reduction in WSP content while the relative amounts of theseMw fractions did not significantly differ from those of the 2-week-oldabiotic control. The WSP content in DOR that had been colonized for 2weeks by P. farinosus was slightly reduced while the relativeabundances of Mw fractions were not affected with respect to thecorresponding abiotic control (Table 4). By contrast, after 20 weeksincubation the fungus led to a 45% reduction in WSP and a markedshift in the relative abundance of F1 and F2 fraction. In particular, thepercent abundance of the F1 fraction increased from 31 (abioticcontrol) to 72%. By contrast, the F2 fraction decreased from 69 (abioticcontrols) to about 28% (Table 4).

In order to assess a possible impact of the fungal treatment onthe stabilisation of the carbon pools of DOR, some parametersrelated to the humification process were determined. Table 1

rinosus BAFC F8846 for 2 and 20 weeks at 28 8C and in its related abiotic controls.

DOR colonized by P. farinosus for

0 weeks 2 weeks 20 weeks

577 � 13.4b 526 � 0a 500 � 18.9a

18.7 � 0.3a 18.4 � 0.2a 21.7 � 0.8b

16 � 1.8b 9.1 � 0.2a 15.6 � 2.0b

374 � 11b 328 � 48.6b 220 � 13.1a

31 � 0.6c 28.5 � 0.1b 23 � 0.9a

25.2 � 0.6a 24.1 � 0.9a 26.4 � 1.3a

17.9 � 0.6b 18.1 � 1.2b 15.2 � 0.1a

12.9 � 0.1c 13.1 � 0.4c 11.0 � 0.1a

21.7 � 1.4c 3.2 � 0.2b 0.8 � 0.1a

26.6 � 11.1a 231.5 � 6.4a 233.6 � 6.9a

88.4 � 7.4b 96.4 � 14.1b 64.7 � 4.4a

16.9 � 14.5a 118.6 � 14.7a 146.8 � 8.4b

21.3 � 1.9b 16.4 � 3.7a 22.5 � 3.0b

y the same superscript letter did not significantly differ as assessed by the Tukey test

CFA, fulvic acid carbon

ransformation and detoxification in dry olive mill residue by thedoi:10.1016/j.procbio.2008.10.016

Table 2Identification of pyrolysis products, grouped according to their origin, and changes in their relative percentages based on peak area in DOR colonized by P. farinosus (PF) for 2

and 20 weeks and in relative abiotic controls (AC).

Fragment RTa (min) Origin Percent relative area

AC PF

2 weeks 20 weeks 2 weeks 20 weeks

Benzaldehyde 7.477 L1 1.4 1.1 1.0 1.5

2-Methoxy-phenol 11.286 G1 4.8 4.1 3.8 4.2

Benzoic acid ME 11.450 H1 2.1 2.1 2.1 2.3

1,2-Dimethoxy-benzene 12.960 G2 1.5 1.6 1.0 1.5

1-Ethenyl-4-methoxy-benzene 13.161 H2 4.4 4.6 2.9 3.0

1,4-Dimethoxy-benzene 13.490 H3 7.7 9.2 9.9 2.3

?-Methoxy-?-methylphenolb 14.387 L2 0.5 0.5 0.6 0.8

3,4-Dimethoxytoluene 15.714 G3 1.7 1.7 1.3 1.4

1,4-Dimethoxy-2-methyl-benzene 16.916 H4 0.7 0.8 0.7 1.3

1,2,3-Trimethoxybenzene 17.861 S1 1.2 1.5 1.0 1.3

2-Methoxy-4-vinylphenol 17.929 G4 1.7 2.0 1.8 2.5

3-Methoxy benzoic acid ME 18.683 L3 0.8 0.7 1.1 1.6

2,6-Dimethoxy-phenol 18.960 S2 1.8 1.9 2.0 3.6

4-Ethenyl-1,2-dimethoxy-benzene 19.374 G5 3.5 3.0 3.7 4.3

1,2,4-Trimethoxybenzene 19.454 C1 6.7 6.3 8.2 5.0

4-Methoxy-benzoic acid ME 19.750 H5 0 0 0.3 0.4

2-Methoxy-4-(1-propenyl)-phenol 21.602 G6 0 0.6 1.2 0.9

3,4-Dimethoxy-benzaldehyde 22.413 G7 1.7 1.5 1.3 2.4

1,2-Dimethoxy-4-(1-propenyl)-benzene 22.791 G8 1.2 1.0 1.1 1.2

1-(4-Hydroxy-3-methoxyphenyl)-2-propanone 23.774 G9 0.8 1.3 1.1 1.4

1-(3,4-Dimethoxyphenyl)-ethanone 24.589 G10 1.6 1.9 1.8 3.1

3,4-Dimethoxyphenylacetone 24.839 G11 11.3 9.4 6.8 4.3

3,4-Dimethoxy-benzoic acid ME 25.226 G12 7.1 6.9 5.1 4.2

3,4,5-Trimethoxybenzaldehyde 25.512 S3 0.9 1.0 1.1 2.1

3,4-Dimethoxy-benzeneacetic acid ME 26.077 G13 0.7 0.7 0.7 1.3

1,2,3-Trimethoxy-5-(1-propenyl)-benzene 26.699 S4 1.5 1.5 2.6 3.9

3-(4-Methoxyphenyl)-2-propenoic acid ME 27.198 H6 2.1 2.2 2.5 3.2

1-(3,4,5-Trimethoxyphenyl)-ethanone 27.369 S5 0.7 0.7 0.9 1.7

3,4,5-Trimethoxy-benzoic acid ME 28.293 S6 1.0 1.3 1.1 2.8

1-(4-Hydroxy-3,5-dimethoxyphenyl)-ethanone 28.604 S7 0.2 0 0 0.4

3-(3,4-Dimethoxyphenyl)-2-propenoic acid ME 31.838 G14 1.4 1.1 1.7 1.3

Methoxy-acetic acid ME 3.202 C2 2.5 3.3 1.5 2.8

Unidentified aliphatic 4.911 C3 0.2 0.2 0.2 0

1-(2-Furanyl)-ethanone 6.199 C4 1.0 0.7 0.7 0.9

1,?-Cyclopentadioneb 6.560 C5 0.5 0.5 0.6 0.4

Unidentified aliphatic 7.910 C6 0.7 1.0 0.8 0.9

1-Methoxy-4-methyl-benzene 9.254 H7 0.9 0.7 0.9 1.0

3-Methyl-1,2-cyclopentadione 9.445 C7 1.8 1.6 1.5 1.5

butanedioic acid DME 9.568 FA1 1.5 1.7 1.8 0.8

3-Methyl-2-hexenoic acid ME 10.573 FA2 0.9 0.8 1.2 0.9

3-Ethyl-2-hydroxy-2-cyclopenten-1-one 10.878 C7 2.4 2.3 1.7 1.8

2,3-Dihydro-benzofuran 15.337 C8 0 0 3.1 0.9

Unidentified aliphatic 15.905 C9 1.5 1.6 1.3 0.9

4-Methyl 4-cyclohexene-1,2-dicarboxylic acid DME 20.244 C10 1.6 1.8 2.9 6.2

1,4-Dimethyl pyrazole 4.420 N1 2.9 2.4 2.7 2.4

1H-Imidazole-4-carboxylic acid ME 7.627 N2 0.5 0.5 0.4 0.7

N,N,2-Trimethyl-benzenamine 13.959 N3 0.6 0.6 0.6 0.8

1,3-Dimethyl-2,4(1H,3H)-pyrimidine dione 14.939 N4 2.5 2.3 1.9 0.7

Unidentified N-containing compound 15.203 N5 5.3 5.6 5.4 5.3

Data are the mean of three determinations. Standard deviation of data was �9%. L, Lignin-related; G, guaiacyl lignin; H, p-hydroxyphenyl lignin; S, syringyl lignin; C,

carbohydrate; FA, fatty acids; N, nitrogen-containing compounds; ME, methyl ester; DME, dimethyl ester.a RT, Retention time.b Mass spectra did not allow the determination of the exact position of substituents.

I. Sampedro et al. / Process Biochemistry xxx (2008) xxx–xxx 5

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shows that CHA content in DOR which had been incubated for 2weeks with P. farinosus did not differ from that of the relatedabiotic control. By contrast, in 20-week-old DOR incubated withthe fungus, CHA content was significantly higher than that of therelative abiotic control (146.8 g kg�1 vs. 116.9 g kg�1, respectively)and CNH content was decreased by about 27% (64.7 g kg�1 vs.88.4 g kg�1, respectively). The tendency of CHA to accumulate alongincubation time in P. farinosus cultures on DOR was also confirmedby the increases in HR value that was significantly higher thanthose of the related abiotic control (34% vs. 20%, respectively)(Fig. 2A). HI, expressing the ratio between non-humic and humiccarbon, reached a value of 0.38 in DOR that had been colonized byP. farinosus while in the related abiotic control a value of 0.67 was

Please cite this article in press as: Sampedro I, et al. Organic matter tsaprophytic fungus Paecilomyces farinosus. Process Biochem (2008),

observed (Fig. 2B). By contrast, DH values of fungal-treated DORdid not significantly differ from those of the corresponding controls(Fig. 2C).

TMAH-Py-GC MS analyses of HA resulted in the identification ofthe same pyrolysis products observed in bulk DOR albeit withdifferent relative amounts. The most abundant products in abioticcontrols were 3-(4-methoxyphenyl)-2-propenoic acid, 3,4-dimethoxybenzoic acid methyl ester and 1-ethenyl-4-methox-ybenzene, the relative abundances of which were in the range12.6–13.2, 5.7–5.8 and 6.7–7.0%, respectively (data not shown).Most relevant differences between HA from fungal cultures on DORand corresponding abiotic control were detected after 20 weeksincubation where the relative abundances of the majority of

ransformation and detoxification in dry olive mill residue by thedoi:10.1016/j.procbio.2008.10.016

Table 3Relative percent abundances of syringyl (S), guaiacyl (G) and p-hydrophenyl (H)

units, syringyl/guaiacyl (S/G) and acid/aldehyde ratios [(Ad/Al)G and (Ad/Al)S] in

bulk DOR colonized by the fungus Paecilomyces farinosus BAFC F8846 for 2 and 20

weeks at 28 8C and in its related abiotic controls.

Parameter DOR abiotic control P. farinosus on DOR

2 weeks 20 weeks 2 weeks 20 weeks

S (%) 7.5 � 0.3a 8.0 � 0.7a 8.7 � 1.5a 15.8 � 0.6b

G (%) 37.4 � 0.2a 35.0 � 1.2a 31.1 � 1.5b 32.7 � 1.0b

H (%) 16.4 � 0.8a 18.0 � 2.1a 17.9 � 1.9a 12.0 � 0.6b

S/G 0.20 � 0.01ac 0.23 � 0.02a 0.28 � 0.03ad 0.48 � 0.01e

(Ad/Al)G 4.2 � 0.3a 4.6 � 0.2a 3.9 � 0.1b 1.8 � 0.2c

(Ad/Al)S 1.2 � 0.08a 1.3 � 0.13a 1.1 � 0.03a 1.3 � 0a

Data are the mean � standard deviation of three replicates and row means followed by

the same superscript letter did not significantly differ, as assessed by the Tukey test

(P � 0.05).

Fig. 2. Humification ratio (A), index of humification (B) and humification degree (C)

in abiotic controls of dry olive mill residue (DOR) and in the same waste that had

been incubated with Paecilomyces farinosus F8846 for 2 and 20 weeks at 28 8C under

stationary conditions. Data are the mean of triplicate experiments. Same letters

above bars indicate lack of statistical significance (P � 0.05) as assessed by the

Tukey test.

I. Sampedro et al. / Process Biochemistry xxx (2008) xxx–xxx6

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syringyl-related pyrolysis products increased while the one of 3-(4-methoxyphenyl)-2-propenoic acid methyl ester was halved.

Table 5 shows that the relative composition in G, S and H-related compounds of HA differed from those observed in the bulkwaste. In particular, HA was characterized by a higher amount in Sunits and a lower amount in G units. The most relevant changesdetected in HAs purified from DOR that had been incubated withthe fungi were observed after 20 weeks incubation and mainlyregarded a significant increase in the S/G ratio (0.53 vs. 0.40) aspreviously observed with bulk DOR.

Fig. 3 shows the HP-SEC elution profiles at 278 nm of HAisolated and purified from both abiotic controls and related fungalcultures on DOR. HAs from 2- and 20-weeks abiotic controlsexhibited the presence of two distinct peaks with either similar oridentical Mw, the former one being eluted at Kav values of 0.10–0.11 (Mws equal to 128.1 and 126.1 kDa, respectively) and thelatter at Kav of 0.28 (about 36 kDa). Comparison of the HP-SECprofile of HA from 2-week-old P. farinosus cultures with that fromthe related abiotic control showed that fungal incubation led to aslight polymerization associated with a higher intensity of thosepeaks (Fig. 3C vs. Fig. 3A, respectively). However, the most relevantchanges were evident in HA samples from 20-week-old fungalcultures where the former peak was eluted at a Kav of 0.055(Mw = 185.5 kDa) and exhibited a higher intensity than that of thecorresponding abiotic control (Fig. 3D vs. Fig. 3B, respectively).Similarly, the Mw of the second peak was markedly higher thanthat of the related control (67.8 kDa vs. 36 kDa, respectively). Suchdifferences are also confirmed in Table 6 showing that fungalcolonization led to significant changes in the molecular sizedistribution (expressed by Mwav, Mn and polydispersity) withrespect to the related abiotic controls.

Table 4Water-soluble phenols (WSP) contents and percent relative abundances of WSP in

F1 (Mw > 30 kDa) and F2 (Mw < 30 kDa) aqueous fractions in DOR colonized by

Paecilomyces farinosus BAFC F8846 for 2 and 20 weeks and in relative abiotic

controls.

Sample Water-soluble

phenolsa (mg g�1 DOR)

Percentage of relative

abundance of WSP in

F1 fraction F2 fraction

Abiotic control (2 weeks) 31.8 � 0.6c 33a 67b

Abiotic control (20 weeks) 28.6 � 1.9b 31a 69b

P. farinosus (2 weeks) 27.8 � 0.2b 30a 70b

P. farinosus (20 weeks) 15.3 � 0.6a 72b 28a

a Data are the mean � standard deviation of three replicates and column means

followed by the same superscript letter did not significantly differ, as assessed by the

Tukey test (P � 0.05).

Please cite this article in press as: Sampedro I, et al. Organic matter tsaprophytic fungus Paecilomyces farinosus. Process Biochem (2008),

3.3. Extra-cellular enzyme production on DOR by P. farinosus

In order to assess the possible involvement of lignin-modifyingenzymes in the previously mentioned partial dephenolization, P.

farinosus solid-state cultures on DOR were assayed for the presenceof laccase, mono-phenolase, aryl alcohol oxidase and Mn-dependent peroxidase. Both laccase and mono-phenolase activ-ities were detected in DOR that had been colonized by the fungusand were found to increase with incubation time (Table 7). Tannin-acyl hydrolase activity was also detected in P. farinosus cultures onDOR and maintained a value of 7.0 IU kg�1 in both incubationtimes. Several hydrolytic activities involved in the depolymeriza-tion of plant cell wall polysaccharides were also detected andincreased as the incubation time was extended from 2 to 20 weeks.With regard to these glycosyl hydrolases, the highest activity levelswere observed in 20-week-old solid-state P. farinosus cultures forendo-b-1,4-glucanase (6224 IU kg�1 DOR). High levels of lipaseand esterase activities were detected and were found to increase

ransformation and detoxification in dry olive mill residue by thedoi:10.1016/j.procbio.2008.10.016

Table 5Relative percent abundances of syringyl (S), guaiacyl (G) and p-hydrophenyl (H)

units, syringyl/guaiacyl (S/G) and acid/aldehyde [(Ad/Al)G and (Ad/Al)S] ratios in

humic acids (HA) purified from DOR colonized by the fungus Paecilomyces farinosus

BAFC F8846 for 2 and 20 weeks at 28 8C and from its related abiotic controls.

Parameter HA from DOR abiotic control HA from P. farinosus cultures

on DOR

2 weeks 20 weeks 2 weeks 20 weeks

S (%) 11.8 � 0.1a 11.5 � 0.7a 12.1 � 0.0a 18.5 � 1.6b

G (%) 28.3 � 0.3a 28.7 � 0.4a 28.4 � 0.5a 34.3 � 2.1b

H (%) 26.0 � 0.1b 26.8 � 0.6b 27.1 � 0.3b 20.6 � 0.8a

S/G 0.42 � 0.0a 0.40 � 0.02a 0.43 � 0.01a 0.53 � 0.02b

(Ad/Al)G 2.2 � 0.4a 2.0 � 0.4a 1.6 � 0.2a 1.9 � 0.1a

(Ad/Al)S 2.6 � 0.2a 2.4 � 0.2a 2.7 � 0.3a 2.4 � 0.3a

Data are the mean � standard deviation of three replicates and row means followed by

the same superscript letter did not significantly differ, as assessed by the Tukey test

(P � 0.05).

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with the incubation time reaching 21,210 and 24,800 IU kg�1 DOR,respectively, in 20-week-old cultures.

3.4. Phytotoxicity removal

In the present study, toxicity was inferred by the amount ofplant biomass reduction in M. sativa plants grown in soil in thepresence of 2.5% of either the fungal-treated DOR or the relatedabiotic control with respect to plants grown in the absence of thewaste (Table 8). In this respect, the waste proved to be highlyphytotoxic and this effect did not decrease significantly in abioticcontrols as the time was extended from 2 to 20 weeks. Inparticular, the percent growth inhibition of shoots and roots inalfalfa plants grown in the presence of 20-weeks DOR abioticcontrols were about 68 and 54%, respectively. Table 8 shows that in2-week-old DOR samples where fungal incubation did not lead to asubstantial reduction in WSP content, phytotoxicity was notsignificantly removed. By contrast, biomass production in plantsgrown in the presence of the waste that had been incubated withthe fungus for 20 weeks was significantly increased compared withthat obtained in the absence of the waste.

4. Discussion

Dry olive mill residue has been suggested to have a potentialfertilizer value in virtue of its chemical composition. However, itsapplication onto soil might lead to a variety of negative effects,including phytotoxicity [3], bacteriostatic activity [33] andstimulation of widespread plant pathogens [34]. The biologicalconversion of DOR prior to its application into soil has beenproposed as a valuable approach that might open the door to a safeagronomic use of this waste [1].

In the present study, the capability of the saprobe fungus P.

farinosus BAFC F8846 to both detoxify and upgrade DOR wasstudied. The selection criteria were based on both the peculiardegradation behaviour of this strain towards phenolics [7] as wellas on the reported capability of Paecilomyces species to degradelignin [10]. To gain evidence on the feasibility of this approach,DOR did not undergo either nutrient supplementation or additionof bulking agents and gaseous exchanges were allowed only bypassive aeration. Under these conditions, the strain under studyshowed the ability to grow on DOR albeit at slow rates as indicatedby low glucosamine contents and limited organic matter losses inDOR within the first two weeks incubation. Lignin breakdown didnot appear to take place in P. farinosus cultures on DOR, regardlessof the incubation time. By contrast, both cellulose and hemi-cellulose contents were partially reduced in DOR that had beencolonized by the mycelium for 20 weeks. The compact structure

Please cite this article in press as: Sampedro I, et al. Organic matter tsaprophytic fungus Paecilomyces farinosus. Process Biochem (2008),

and the subsequent scarce porosity of DOR [33] might account forthe failure of the fungus to degrade lignin, the breakdown of whichis predominantly an oxygen-dependent process [19,35]. Toinvestigate whether the failure of the fungus in degrading ligninmight be due to structural reasons, TMAH-Py-GC MS analyses ofthe bulk waste were performed. Although lignin from dicotyle-donous angiosperms are mainly composed of guaiacyl and syringylmonolignol moieties [36], a significant presence of p-hydroxy-phenyl subunits was observed in bulk DOR. In addition, a very lowS/G ratio was observed in this waste. This ratio has been suggestedto be an important determinant for the susceptibility of a givenlignocellulosic material to be delignified ether by means ofchemical or biological agents [18,24]. In fact, S units in ligninare typically linked via relatively labile ether bonds and arecharacterized by a lower redox potential than G units [24]. On theother hand, G monolignols form more highly condensed ligninwith a preponderance of more stable biphenyl and other carbon–carbon linkages that are less prone to biodegradation [36].Therefore, the failure in waste’s delignification observed with thisstrain as well as with other fungal strains [3,22,37] and microbialconsortia [2] might be explained by the presence of a highlycondensed lignin in olive drupes. The most relevant changes inDOR that had been incubated with P. farinosus BAFC F8846involved a preferential decay of p-hydroxyphenyl subunits and anunexpected increase in the S/G ratio. With this regard, P. farinosus

showed a distinct behaviour with respect to ligninolytic basidio-mycetes, the wood-degrading activity of which generally involvesa decrease in the S/G ratio [16,24]. However, it has to be taken intoaccount that the mechanism of thermochemolysis by TMAH isselective for cleavage of b-O-4 bonds when there is an a-hydroxylgroup on the propenyl side chain [38]. Although the b-O-4 bond isthe most abundant inter-unit linkage in lignin, regardless of theplant source, an aliquot of monomers of lignin in olive’s drupesmight be connected via different linkages, such as b-5, b-b and b1bonds. Consequently, and as suggested by other studies conductedwith other lignocellulosic materials [24,38], thermochemolysisproducts from DOR might be only partially representative of thewhole lignin. It has been suggested that large increases in theguaiacyl and syringyl acid/aldehyde ratios in lignin degraded byboth fungi [24,38] and microbial consortia [18] might indicate theoccurrence of the oxidative cleavage of the Ca–Cb bonds located onthe propenyl side chain, one the main mechanisms involved inlignin depolymerisation. Therefore, the slight decrease or theunsuccessful modification of these ratios appeared to confirm thefailure of P. farinosus in affecting the content of lignin in DOR(Table 1).

The above mentioned low macroporosity of DOR has beenidentified as one of the main technical constraints encountered inDOR bioconversions thus both hindering colonization of micro-organisms and negatively affecting ligninolysis [5,33]. This has led,for instance, to the development of co-composting processes ofDOR employing lignocellulosic wastes as bulking agents [2,5].Moreover, although a lignin-like fraction has been recently isolatedfrom olive pulp [39], the large majority of lignin is located withinthe endocarp of the drupe, that is often referred to as olive stone[39]. In this respect, the limited lignin biodegradation in olive millsolid wastes was suggested to be due to the low surface area ofchopped stones exposed to microbial attack [2,33]. In addition, it isalso worth mentioning that the genus Paecilomyces belongs tothose anamorphic genera of Ascomycetes that are characterized bypolysaccharide-degrading activity rather than by a significantligninolytic behaviour [9]. For instance, Paecilomyces inflatus

strains, isolated from distinct ecological niches, generally exhib-ited a cellulolytic behaviour when they were allowed to colonizeeither annual or woody plant residues [9].

ransformation and detoxification in dry olive mill residue by thedoi:10.1016/j.procbio.2008.10.016

Fig. 3. Size-exclusion chromatograms at 278 nm of humic acids from 2- and 20-weeks abiotic controls (A and B, respectively) of DOR and from Paecilomyces farinosus F8846

cultures conducted on the waste for the same incubation times (C and D, respectively). Elution times of molecular weight standards are indicated by the arrows above plots.

Table 6Weight-averaged (Mwav) and number-averaged (Mn) molecular weights and

polydispersity (Mwav/Mn) in humic acids purified from 2 and 20 weeks abiotic

controls of DOR and in the related Paecilomyces farinosus cultures conducted on the

same waste.

Sample MWava (kDa) Mn

a (kDa) Polydispersitya

Abiotic control (2 weeks) 52.1 � 3.1a 37.8 � 2.1a 1.38 � 0.005a

Abiotic control (20 weeks) 70.7 � 5.7b 48.6 � 3.8b 1.46 � 0.003b

P. farinosus on DOR (2 weeks) 82.1 � 6.0b 56.0 � 3.8b 1.47 � 0.008b

P. farinosus on DOR (20 weeks) 112.9 � 7.7c 82.7 � 5.5c 1.37 � 0.002a

a Data are the mean � standard deviation of three replicates and column means

followed by the same superscript letter did not significantly differ, as assessed by the

Tukey test (P � 0.05).

I. Sampedro et al. / Process Biochemistry xxx (2008) xxx–xxx8

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PRBI-8531; No of Pages 10

Please cite this article in press as: Sampedro I, et al. Organic matter tsaprophytic fungus Paecilomyces farinosus. Process Biochem (2008),

The microbial conversion of lignocellulosic residues might leadto the formation of several OM fractions, some of which may beassigned to humic substances, having lignin as the main core[19,40]. In P. farinosus cultures on DOR, the humic carbon contentincreased over incubation time although no apparent lignindegradation had taken place. With this regard, it has beensuggested that both amounts of CHA and humification efficiencyare not necessarily highest in those fungal cultures where ligninhad been significantly degraded. In fact, the relative amounts ofCHA and CFA in solid-state cultures of fungi have been shown to bevariably affected by the balance between lignin depolymerisation/polymerisation reactions [41]. In addition, humic acids have beenshown to be efficiently depolymerised by fungi with subsequenteffects on CHA/CFA ratios [42] and the taxonomically related species

ransformation and detoxification in dry olive mill residue by thedoi:10.1016/j.procbio.2008.10.016

Table 7Extracellular enzyme activities produced by Paecilomyces farinosus BAFC F8846

after 2 and 20 weeks incubation at 28 8C on dry olive mill residue.

Enzyme Enzyme activitya IU (kg DOR)�1

2 weeks 20 weeks

Laccase 34.3 � 2.1 216.5 � 12.4

Mono-phenolase 14.8 � 0.8 128.3 � 7.8

Aryl-alcohol oxidase n.d.b n.d.b

Mn-independent peroxidase n.d.b n.d.b

Mn-dependent peroxidase n.d.b n.d.b

Endo-b-1,4-glucanase 1280.5 � 75.7 6224.3 � 345.6

Cellobiohydrolase 423.1 � 19.5 1340.9 � 92.3

b-Glucosidase 487.4 � 23.4 813.5 � 33.7

Endo-b-1,4-xylanase 451.0 � 22.8 920 � 11.8

b-Xylosidase 305.6 � 15.3 602.4 � 28.6

a-Arabinofuranosidase 23.1 � 0.8 84.2 � 1.7

Tannin acyl-hydrolase 7.0 � 0.4 7.0 � 0.2

Lipase 2408.2 � 134.4 21210.0 � 520.6

Esterase 3412.0 � 112.9 24800.0 � 384.3

a Data are the mean � standard deviation of three determinations.b n.d.: Not detected.

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P. inflatus was found to degrade and mineralise natural humic acidsin a compost environment [10]. In the present study, HR was foundto be increased by about 65% with respect to the correspondingabiotic control. In addition, HI, the value of which has been shownto be lower than 0.5 in well-humified materials [12], was 0.38 in20-week-old P. farinosus cultures on DOR. HP-SEC analyses showedthat MW distribution was markedly affected in HA purified fromthe waste that had been colonized with the fungus where bothMwav and Mn were significantly increased with respect to thosedetected in abiotic controls.

The unsuccessful dephenolization observed in DOR colonizedfor 2 weeks by P. farinosus (present study) confirmed previousresults where salidroside depletion was counterbalanced by anincrease in both tyrosol and hydroxytyrosol due to the hydrolysisof their related glucosides, namely hydroxytyrosol-4-b-D-gluco-side and oleuropein [7]. By contrast, a 45% removal in WSP wasevident in 20-week-old P. farinosus BAFC F8846 cultures on DOR.Although the dephenolization extents were largely lower thanthose achieved on DOR by other fungi [22,37] or by microbialcommunities in compost [33], the fungus under study significantlyaffected the relative abundances of WSP fractions with molecularweight higher and lower than 30 kDa by largely increasing theformer one. These results suggest the occurrence of polymerisationphenomena possibly promoted by phenol-oxidizing enzymes [42].Among these oxidases, laccase activity was detected in P. farinosus

cultures on DOR (present study). This was not unexpected since insolid-state cultures of other Paecilomyces species the presence of

Table 8Production of shoots and roots biomass by alfalfa (Medicago sativa L.) plants grown

in soil for 4 weeks in the absence of DOR or added with 2.5% (w/w) of either the

waste that had been colonized by P. farinosus BAFC F8846 for 2 and 20 weeks at

28 8C or its abiotic controls.

Plant grown in Biomass productiona (mg)

Shoots Roots

Soil alone 156.3 � 3.1b 37 � 2.3b

Soil + 2.5% 2-week-old DOR abiotic control 54.0 � 3.5a 19.6 � 0.8a

Soil + 2.5% 20-week-old DOR abiotic control 49.0 � 6.2a 21 � 1.3a

Soil + 2.5% 2-week-old P. farinosus-treated DOR 62.0 � 7.4a 18.6 � 1.5a

Soil + 2.5% 20-week-old P. farinosus-treated DOR 222.6 � 5.2c 78.3 � 1.8c

a Data are the mean � standard deviation of triplicate experiments. Column means

related to each plant part and sharing the same superscript letter were not significantly

different as assessed by the Tukey test (P � 0.05).

Please cite this article in press as: Sampedro I, et al. Organic matter tsaprophytic fungus Paecilomyces farinosus. Process Biochem (2008),

laccase activity was reported [10]. In addition, activity of mono-phenolase, the enzyme responsible for the ortho-hydroxylation ofmono-phenols, was also detected in P. farinosus cultures on DOR(present study).

The high phytoxicity of DOR was even evident when alfalfaplants were grown in soil added with 2.5% (w/w) and this effect didnot decrease with time in DOR abiotic controls. By contrast, DORthat had been colonized by P. farinosus for 20 weeks did not displayany toxicity and led to a significant and unexpected increase inplant biomass. These findings are in agreement with a previousstudy where phytotoxicity reduction from DOR by fungaltreatments required long colonization times, regardless of boththe species employed and their related growth abilities on othersolid substrates [43]. Soil saprobic fungi are widespread compo-nents of the soil rhizosphere and P. farinosus is reported to be anubiquitous species. Although there are no experimental evidencesthat might account for the reported biomass increase in alfalfa, itshould be borne in mind that several fungal species significantlyaffect plant growth via their participation in nutrients mobilizationand production of bioactive substances. In this respect, P. farinosus

is an active producer of an array of secondary metabolites, thebiological activities of which have to be fully elucidated [44,45].

The long colonization time required by P. farinosus in theremoval of phytotoxicity from DOR is undoubtedly a remarkableconstraint for a possible transferability of the process andintermediate incubation times (i.e. 10 weeks) were not sufficientto detoxify the waste (data not shown). However, this studysuggests both a possible explanation for the widely reported lowsusceptibility to biodegradation of DOR’s lignin [2,3,22,33] andprovides evidence for the capability of the strain under study topromote the humification of this waste.

Acknowledgements

One of the authors (I.S.) wish to acknowledge the Ministerio deEducacion, Ciencia y Tecnologıa for funding a research grant. Thiswork was also supported by the Ministry of Education, Youth andSports of the Czech Republic (LC06066 and grant 2B06156) and bythe Institutional Research Concept AV0Z50200510 of the Instituteof Microbiology, ASCR.

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