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Microbiological Research 161 (2006) 93—101

KEYWORDMicrobialcommunitOlive millwastewatePolyphenoSoil respir

0944-5013/$ - sdoi:10.1016/j.

�CorrespondE-mail addr

www.elsevier.de/micres

Changes in microbial and soil propertiesfollowing amendment with treated anduntreated olive mill wastewater

Ali Mekki, Abdelhafidh Dhouib, Sami Sayadi�

Laboratoire des Bioprocedes,Centre de Biotechnologie de Sfax, BP: ‘‘K’’ 3038 Sfax, Tunisie

Received 8 June 2005; accepted 15 June 2005

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ee front matter & 200micres.2005.06.001

ing author. Tel./fax: +ess: sami.sayadi@cbs.

SummaryWe investigated the effect of untreated and biologically treated olive mill wastewater(OMW) spreading on the soil characteristics and the microbial communities. The waterholding capacity, the salinity and the content of total organic carbon, humus, totalnitrogen, phosphate and potassium increased when the spread amounts of the treatedor untreated OMW increased. The OMW treated soil exhibited significantly higherrespiration compared to the control soil. However, the C-CO2/Ctot ratio decreasedfrom 1.7 in the control soil to 0.5 in the soil amended with 100m3 ha�1 of untreatedOMW. However, it slightly decreased to 1.15 in the soil amended with 400m3 ha�1 oftreated OMW. The treated OMW increased the total mesophylic number while thenumber of fungi and nitrifiers decreased. Actinomycetes and spore-forming bacteriawere neither sensitive to treated nor to untreated OMW. The total coliforms increasedwith higher doses of treated and untreated OMW. A toxic effect of the untreated OMWappeared from 100m3 ha�1. This toxicity was more significant with 200m3 ha�1, wheremicroflora of total mesophilic, yeasts and moulds, actinomycetes, and nitrifiers wereseriously inhibited except for total coliforms and spore-forming bacteria.& 2005 Elsevier GmbH. All rights reserved.

Introduction

The olive mill wastewater (OMW) is a criticalproblem, especially in the Mediterranean area,where the olive cultivation is widespread and hugeamounts of this effluent 30millionsm3 y�1 world-

5 Elsevier GmbH. All rights rese

216 74 440 452.rnrt.tn (S. Sayadi).

wide and 500 000m3 y�1 in Tunisia alone, areannually produced (Sayadi and Ellouz, 1995; Casaet al., 2003). This waste contains an enormoussupply of organic matter, COD between 40 and210 g dm�3 and BOD5 between 10 and 150 g dm�3

(Feria, 2000). Some characteristics of this material

rved.

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Table 1. Chemical and physical properties of untreatedand biological treated OMW

Parameter UntreatedOMW

Treated OMW

pH (25 1C) 5.46 7.6Electric conductivity(25 1C) (dSm�1)

8.7 11.3

Salinity (g l�1) 5.9 9.7Colour (absorbance395 nm)

82 44

UV absorbance 280 nm 368 38BOD5 (g l�1) 34.4 4.5COD (g l�1) 117 21.9Glucose (g l�1) 12 NDResidual solids (g l�1) 26 NDTotal solids (%) 11.4 2.5Total volatiles (%) 9.3 1.42Total suspended solids(g l�1)

8.9 3.5

Volatiles suspendedsolids (g l�1)

6.5 2.7

Nitrogen (g l�1) 1.58 1.72Phosphorous (g l�1) 0.84 1.12Potassium (g l�1) 5.2 4.4o-di-phenols (g l�1) 8.395 1.265Total poly phenols (g l�1) 9.200 1.578Residual oils (g l�1) 9.2 Not detectedToxicity by LUMIStox (%inhibition)

100 38

A. Mekki et al.94

are favourable for agriculture since this effluent isrich in organic matter, nitrogen (N), phosphorous(P), potassium (K) and magnesium (Mg). Theorganic fraction of this wastewater includes sugars,tannins, poly phenols, poly alcohols, pectins, lipids,and proteins (Mulinacci et al., 2001; Lesage-Meessen et al., 2001). For these reasons, increasingattention has been given to find the best me-thods to spread OMW on agricultural lands andto recycle both the organic matter and thenutritive elements in the soil crop system. More-over, agricultural irrigation with wastewater efflu-ents became a common practice in arid andsemiarid regions, where it was used as a readilyavailable and inexpensive option to fresh water(Angelakis et al., 1999; Oved et al., 2001). FreshOMW was used as a fertilizer in the horticultureand in the olive cultivation (Cox et al., 1997;Ben Rouina et al., 1999; Ammar and Ben Rouina,1999; Cereti et al., 2004). However, biodegra-dation of this waste in the nature is difficultbecause it contains a strong antibacterial effectexerted, by various phenolic compounds (Yesiladaet al., 1999; Sayadi et al., 2000; Rinaldi et al.,2003). Before its utilization in the irrigation, OMWwas treated by several processes such as aerobictreatment, anaerobic digestion and compostingprocess (Sayadi and Ellouz, 1992, 1995; Ehaliotiset al., 1999; Paredes et al., 2000; Kissi et al.,2001; Marques, 2001; Casa et al., 2003; D’Annibaleet al., 2004).

Some Mediterranean countries established lawsabout soil capability to endure the OMW applica-tion, particularly in Italy (Law N1 574, 1996). Themaximum amount of OMW tolerated in the fields is80 and 50m3 ha�1 for OMW obtained by centrifugeand pressure extraction techniques, respectively(Law N1 574, 1996).

The addition of such compounds may causesignificant shifts in the structure and the functionof the microbial community, which in turn mayinfluence the viability of the soil for agriculture.The effect of the OMW on the physical and chemicalcharacteristics of the soil are well documented(Cabrera et al., 1996; Cox et al., 1997; Sierra et al.,2001; Zenjari and Nejmeddine, 2001; Rinaldi et al.,2003). However, these studies did not deal with theeffect of this waste on the microbial community ofthe soil (Moreno et al., 1987; Paredes et al., 1987;Kotsou et al., 2004).

An integrated approach using a pre-treatment ofthe OMW with the white-rot fungus Phanerochaetechrysosporium followed by an anaerobic digestionwas developed in our laboratory in order to reusethe effluent in agriculture (Sayadi and Ellouz,1995). Thus, the aim of our work was to investigate

the effect of untreated and biologically treatedOMW on the soil characteristics and on themicrobial communities.

Materials and methods

OMW origin

The fresh OMW was taken from a three-phasediscontinuous extraction factory located in Sfax,Tunisia.

Biological treatment of OMW

The treated OMW was obtained with an inte-grated process based on aerobic fungal pre-treat-ment using P. chrysosporium DSMZ 6909 followed bya decantation step then anaerobic digestion (Sayadiand Ellouz, 1995).

The characteristics of the treated and untreatedOMW are given in Table 1.

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Changes in microbial and soil properties following amendment 95

Study sites and sampling

The study area consisted in a field of olive treeslocated in Chaal at 60 Km to the South-West ofSfax, Tunisia, North latitude 341 30, East longitude101 200. The mean annual rainfall is 200mm (BenRouina et al., 2001). The field was divided in fiveplots. Three experimental plots P1, P2, and P3 wereannually amended in February with 50, 100, and200m3 ha�1 of untreated OMW respectively (BenRouina, 1994). The plot P4, was annually amendedwith 400m3 ha�1 of the treated OMW. The fifthplot, plot C, was not amended and served ascontrol.

Soil samples were collected from different partsof each plot from 0 to 10 cm deep, using a soilauger. All soil samples, taken from each plot werethen mixed, air-dried, sieved with a mesh size of450 mm and stored at 4 1C prior to use. Watercontent was immediately determined before air-drying the sample.

Physicochemical analyses

Analysis of ortho-diphenols: The ortho-diphenolsconcentration in the OMW was quantified by meansof Folin-Ciocalteau colorimetric method (Box,1983) using caffeic acid as standard. The absor-bance was determined at l ¼ 765 nm.

Analysis of total polyphenols: OMW was centri-fuged at 7000 rpm for 20min. The supernatant wasextracted three times with ethyl acetate. Thecollected organic fraction was dried and evapo-rated under vacuum. The residue was extractedtwo times with dichloromethane in order to removethe non-phenolic fraction (lipids, aliphatic, sugars).The liquid phase was discarded while the washedresidue was weighed and analysed by gas chroma-tography coupled with the mass spectroscopytechnique to confirm the phenolic structure of theextracted compounds.

COD was determined according to Knechtel(1978) standard method.BOD5 was determined by the manometric meth-od with a respirometer (BSB-Controller Model620 T (WTW)).Dry weight and moisture content were deter-mined by weighing samples before and afterdrying overnight at 105 1C.Organic matter was determined after furnacingsamples at 550 1C for 4 h.Total carbon and nitrogen were determined bydry combustion (TOC Analyser multi-N/C 1000).Ca, K, Na, and Mg analyses, the air-dried soil was

extracted with 1M solution of ammonium acet-ate (pH 7) using a soil/solution ratio of 1/10 (w/v). The suspension was analysed with an induc-tively coupled plasma atomic emission spectro-meter (ICP-AES, ARL 3580).

Microtoxicity determination

The microtoxicity test consisted in the inhibitionof the bioluminescence of Vibrio fischeri LCK480using the LUMIStox system (Dr. Lange GmbH,Duesseldorf, Germany) and according to ISO11348-2 (1998). Percentage inhibition of the biolu-minescence was achieved by mixing 0.5ml of OMWand 0.5ml luminescent bacterial suspension. After15min exposure at 15 1C, the decrease in lightemission was measured. The toxicity of the OMWwas expressed as the percent of the inhibition ofbioluminescence (%IB) relative to a non-contami-nated reference. A positive control (7.5% NaCl) wasincluded for each test.

Respirometric test

Biological activity in the soil was achieved bymeasuring CO2 evolution in the aerobic condition(Ohlinger, 1995). The soil sample was humidified to50% of its water holding capacity and incubated at30 1C in the dark. The CO2 evolved was trapped inan NaOH solution and titrated with HCl.

Microbial estimation

Ten grams of the soil sample was suspended in anerlenmeyer flask containing 90ml of a sterilesolution (0.2% of sodium polyphosphate (NaPO3)nin distilled water, pH 7.0) and 10 g of sterile glassbeads (1.5mm diameter). The flask was shaken at200 rpm for 2 h. Serial 10-fold dilutions of thesamples in a 0.85% NaCl solution were plated intriplicate on PCA at 30 1C for total bacterial counts,on Sabouraud containing chloramphenicol at 25 1Cfor yeasts and moulds, on DCL at 37 1C for totalcoliforms, and on soil extract agar at 30 1C foractinomycetes.

Soil extract agar was prepared as follows: 1 kg ofsoil was added to 1 l of distilled water and agitatedenergetically. Supernatant was filtered. Its pH wasadjusted to 7 and sterilized at 121 1C for 20mintwice. A 200ml of this extract and 20 g of Agar-agarwere added to 800ml of distilled water andsterilized at 121 1C for 20min. Penicillin G, Cyclo-heximide, Ampicillin and Nistatin were dissolved inwater and sterilized by filtration (0.22 mm) and

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A. Mekki et al.96

were added at final concentration of 1, 50, 10 and50mg l�1, respectively.

For spore-forming bacteria counts, aliquots wereheated for 10min at 80 1C before spreading on PCAand incubation at 37 1C.

Ammonia and nitrite-oxidizing bacteria wereenumerated by the most probable number (MPN)procedure (Trolldenier, 1995). Culture tubes sup-plemented either with ammonium or nitrite wereinoculated with serially diluted soil suspension.After an extended incubation of 4 weeks at 28 1C,acidification of the medium was recorded by takingcolour change as an indication for growth ofammonium oxidizers and the absence of nitrite asan indication for growth of nitrite oxidizers.Subsequently, the MPN was calculated in accor-dance with the table of MPN values. The totalnitrifiers count was the sum of the oxidizers ofammonium and of nitrite.

Each soil sample was analysed in duplicate andthe dilution series were plated in triplicate for eachmedium. All these counts were expressed as colonyforming units (CFU) per gram of dried soil (24 h at105 1C). The total nitrifiers count was expressed asMPN per gram of dried soil.

Results

Characterisation of the effluents

Untreated OMW totally inhibited V. fischeri(Table 1). This toxicity was essentially due to its

Table 2. Results of the air-dried soils characterization

Characteristics C P1

Particle size

distribution

in control soil

9>=>;ð%Þ

Sand

clay

Silt

8><>:

89.82 ND7.44 ND2.74 ND

Moisture content (%) 1.14 1.1pH (KCl) 7.9 7.9Salinity (mg kg�1) 69 240P (mg g�1) 0.02 0.0P (water soluble) (mg g�1) 0.001 0.0K (mg g�1) 0.14 1.0Mg (mg g�1) 0.25 0.3Na (mg g�1) 0.02 0.1Ca (mg g�1) 14.70 19.8Ntot (mg g�1) 0.23 0.5N-NH4 (mg g�1) 0.0312 0.0Ctot (mg g�1) 2.001 8.0Humus (mg g�1) 4 16C/N 8.7 14.2

P1, P2, and P3: Soils amended with 50, 100, and 200m3 ha�1 of untreatreated OMW. The plot C was not amended and served as control. N

high content of phenolics (9.2 g l�1). This toxicitywas reduced to 38%IB in treated OMW whichcontained only 1.58 g l�1 of phenolics. The COD(21.9 g l�1) of treated OMW remained high and farexceeded the standard for direct discharge to anatural water body. Several costly steps arenecessary if we want to reach the Tunisian standard(0.09 g l�1). The treated OMW contained appreci-able concentrations of N, P, and K. This effluent wasfree of pathogens, relatively not toxic and con-tained low concentrations of heavy metals. Apartfrom COD, BOD5 and black colour, the quality oftreated OMW was high and could be used forirrigation after field tests.

Analytical results of soils profiles

A darker soil colour was observed in the plotsamended with OMW. After drying, the amendedsoils showed higher compactness and hardness.Soils were sampled and analysed in a particularlydry year in Tunisia. Only weak precipitations wererecorded in February, May and November. The soilwater content was very weak and it varied between0.8% and 1.15% in the samples collected inSeptember (Table 2). The pH increased to 9.2 whenP4 soil was amended with treated OMW and slightlydecreased to 7.4 when P3 soil was amended withraw OMW. Table 2 shows also that salinity of theamended soil increased proportionally with quan-tity of treated or untreated OMW. The content ofthe nutrients as total carbon (Ctot), total nitrogen(Ntot), P, K, Mg and humus increased after spreading

P2 P3 P4

ND ND NDND ND NDND ND ND

3 1.15 1.07 0.827.6 7.4 9.2

336.5 447.5 4733 0.08 0.08 0.0503 0.016 0.12 0.0275 1.60 1.80 2.425 0.40 0.37 0.337 0.03 0.04 0.310 16.20 15.80 14.706 0.95 0.91 0.4544 0.055 0.089 0.08802 15.504 16.999 4.001

31 34 89 16.32 18.68 8.89

ted OMW respectively; P4: Soil amended with 400m3 ha�1 of theD: not done.

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18

C-CO2/CtotCtot

1.8

1.6

1.4

1.2

1

0.8

0.6

0.4

0.2

0C P1 P2

Soil

P3 P4

16

14

12

10

8

6

4

2

0

C-C

O2

and

Cto

t (m

g g-

1 )

C-C

O2/

Cto

t

C-CO2

Figure 2. Specific respiration C-CO2/Ctot, cumulative C-CO2, and total carbon Ctot of the soil samples studied.

Changes in microbial and soil properties following amendment 97

the treated or untreated OMW. The C/N ratioremained constant in the soil amended withtreated OMW while it increased proportionally inthe soils amended with untreated OMW.

Phenolic compounds migrated in soil according totheir molecular mass. Polyphenols were adsorbed inthe soil upper layers while monomers migrated indepth. Indeed phenolic monomers were detectedat 1.2m depth 1 year after irrigation withuntreated OMW (data not shown).

Soil respiration

A respirometric test was achieved on soilssampled in September. CO2 production increasedwith OMW amendment (Fig. 1). For the treatedOMW, a more pronounced CO2 production rate wasshown since the first week of incubation. However,for the untreated OMW, the start-up of the CO2

production was delayed to the 3rd week ofrespiration.

The specific respiration rate expressed as theratio of C-CO2/Ctot for the different soil samples isshown in Fig. 2. The amendment of the soil with200m3 ha�1 increased the carbon content to17mg g�1 while the specific respiration remainedvery low. However, the amendment with400m3 ha�1 of treated OMW did not much affectthe specific respiration of the soil.

Effect on soil microbiology

Viable mesophilic microfloraGenerally, the total microflora increased with

the soil humidity. OMW enhanced the water holdingcapacity of the soil. The soil water contentincreased when the OMW dose increased (data not

0

2

4

6

8

10

12

0 5 10 15 20 25 30

C P1 P2 P3 P4

mg

C-C

O2 g

(d

ry s

oil)

-1

Time (d)

Figure 1. Cumulative respiratory activity as mg CO2 g�1

dry soil of different samples incubated over 28 days at28 1C in the dark.

shown). An increase in the total microflora countwas observed in P1, P2 and P4 in all dates ofsampling (Table 3). However, at 200m3 ha�1 ofuntreated OMW, the total bacterial counts re-mained much higher compared to the control soil,but lower compared to the other doses of OMW.

Viable yeasts and moulds microfloraIn comparison with the control soil, an overall

high CFU of fungi in the soil amended withuntreated OMW was found (Table 4). In all datesof sampling, the fungal CFU number decreasedwhen OMW increased but remained much higherthan the control soil except for biologically treatedOMW which had a lower CFU than the control. Wenoted that the pH of OMW leaving the anaerobicreactor ranged between 7.6 and 8. This pHincreased to 8.7 during its storage at ambienttemperature. After amendment with treated OMW,the pH of the soil increased to 9.2. Such pH value isconsidered as detrimental for the fungal growth.

Viable total nitrifiersSoils C, P1 and P2 showed broadly comparable

nitrifier MPN numbers. However, a decrease inviable nitrifiers count was observed in P3. Thisdecrease was more significant in P4 (Table 5).

Viable actinomycetesThe actinomycetes CFU number increased when

OMW doses increased up to the dose of 100m3 ha�1.At 200m3 ha�1 of untreated OMW, the CFU numberremained higher than C and P1 (50m3 ha�1).However, it was lower than P2 (100m3 ha�1)(Table 6).

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Table 3. Aerobic heterotrophic bacteria counts CFU (� 104) g�1 in the different plots

Feb May Jun Sep Nov

C 3472 6974.05 24.571.44 1570.88 21.571.26P1 8574.95 7774.52 45.572.67 48.572.85 8374.88P2 12177.11 8975.32 57.573.38 65.573.85 16179.46P3 6673.9 7274.23 5773.35 43.572.55 9275.4P4 9075.3 10175.93 7974.64 6073.52 15879.29

Data expressed as mean value (three replicates) and standard deviation for colony forming units per gram of dried soil.

Table 4. Fungi counts in the different plots CFU (� 104) g�1

Feb May Jun Sep Nov

C 3.571.11 370.95 370.95 1.370.41 2.770.85P1 1073.17 11.573.64 1775.38 15.574.9 14.774.65P2 5.571.74 571.58 1574.75 1073.17 13.574.27P3 7.572.37 4.671.45 1173.48 4.171.29 11.273.55P4 1.8270.57 1.8570.58 2.8570.9 1.7470.55 1.3570.42

Table 5. Nitrifiers counts MPN (x104) g�1 in the different plots

Feb May Jun Sep Nov

C 3.670.47 4.770.62 3.270.42 2.870.37 3.870.5P1 2.670.34 4.570.59 3.470.45 2.470.31 3.170.41P2 270.26 4.270.55 2.670.34 2.470.31 2.870.37P3 1.970.25 2.870.37 1.770.22 1.170.14 1.170.14P4 0.4670.06 0.4870.063 0.5170.07 0.0770.009 0.970.11

Table 6. Actinomycetes counts CFU (� 104) g�1 in the different plots

Feb May Jun Sep Nov

C 0.670.08 2.170.28 270.26 270.26 3.570.46P1 770.93 14.571.93 871.06 1071.33 12.871.7P2 1171.46 18.572.46 15.572.06 10.571.39 17.272.29P3 570.66 1872.39 1271.59 5.570.73 14.771.95P4 13.2971.77 17.572.33 15.672.07 12.2971.63 15.772.09

A. Mekki et al.98

Viable spore-forming bacteria and total coliformsThe spore-forming bacteria increased with

the increase of OMW doses (data not shown). ForP4 soil, it shifted from the CFU g�1 number rang-ing from 0.28 to 1.12� 104 in the control soilto CFU g�1 number ranging from 1.1 to 2.12� 104

in P4 amended with 400m3 ha�1 of treated OMW.Total coliforms are well known as contaminantindicator bacteria in wastewater and soil. Thenumber of the total coliforms was very low in thecontrol soil. It increased when the treated oruntreated OMW quantity increased (data notshown).

Discussion

This study attempted to demonstrate that soilamended with different concentrations of OMWshowed modification of its structure and its texture.The acidity of the untreated OMW was compensatedby the soil carbonate alkalinity. The carbonates at thesame time became bicarbonates, moved and accu-mulated in deeper horizons as was shown by Sierra etal. (2001). The increase of the salinity in the soilcould result from the main ionic species, sodiumchloride and sulphate, coming from the treated oruntreated OMW. This is in line with previous finding

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Changes in microbial and soil properties following amendment 99

(Paredes et al., 1987; Sierra et al., 2001). Hence, inlong-term applications, replacement of the soilcalcium by the cations of Na, K and Mg could leadto the degradation of the soil structure and theformation of saline soils as was suggested earlier byZenjari and Nejmeddine (2001).

Biologically treated OMW had a pH48, and thealkalinity of this waste was not regulated (buf-fered) by the soil components. Soil porosity wasreduced by the combined effect of the suspendedsolids and the COD formed by highly polymerisedpolyphenolic compounds such as humic acid-likesubstances (Cox et al., 1997). Consequently, soilplugged and became impermeable which led to areduction of the soil aerobic community such asfungi and actinomycetes. This finding confirms thereported correlation between the soil pH and thechange in community composition (Frosteg(ardet al., 1993; Perkiomaki and Fritze, 2002).

The increase of nutrient contents, Ctot, Ntot, P, Mgand K at all OMW treated plots, may have a beneficialeffect on the soil fertility. The OMW treated soilexhibited a higher respiration rate compared to thecontrol soil. Nevertheless, when taking into accountthe added organic carbon, this activity was not inproportional ratio. Specific respiration expressed asC-CO2/Ctot decreased from 1.7 in the control soil to0.5 in the soil amended with 100m3ha�1 of untreatedOMW. Yet, it slightly decreased to 1.15 in the soilamended with 400m3ha�1 of treated OMW. This canbe explained by the fact that the phenolic com-pounds may inhibit the soil respiration, especially inthe high OMW doses, and thus neutralize thefavourable influence of its higher nutrient contentsas was demonstrated by Sierra et al. (2001), Cox etal. (1997), Cabrera et al. (1996), and Paredes et al.(1987). In simple terms, the inhibition of soilrespiration could be caused by the fact that the bigamount of carbon added to the soil was unavailableto the microflora under the effect of its strongadsorption or its reaction with the components of thesoil. This disproportion could not be due to the addedsalt because despite the high content of salt in P4(473mgkg�1) compared to that in P3 (447.5mgkg�1),the former had a nearer specific respiration rate tothe control plot C which contained only (69mgkg�1).

Addition of the untreated or the biologicallytreated OMW to the soil created some modificationsin the average values for total number of micro-organisms and their repartition. Results showed aninitial increase in the numbers of CFU in mostmicroflora groups after the OMW amendment,excepted for nitrifiers which decreased. In linewith this finding, Paredes et al. (1987) reportedalso an increase in the total viable counts in the soilpolluted with OMW. The overall low CFU number

observed in the P3 soil could be explained by theOMW dose becoming high and toxic (Capasso et al.,1995). The chemolithotrophic ammonia-oxidizingbacteria (AOB) are responsible for the first rate-limiting step in nitrification in which ammonia(NH3) is transformed to nitrate (NO3

�) via nitrite(NO2

�). The AOB play a critical role in the naturalnitrogen cycle (Oved et al., 2001; Mendum andHirsch, 2002). This microflora could be affected bya variety of chemical conditions including aromaticcompounds and salts. Indeed, the number ofnitrifiers shifted from the CFU g�1 number rangingfrom 2.8 to 4.7� 104 in the control soil to CFU g�1

number ranging from 0.46 to 0.9� 104 in P4amended with 400m3 ha�1 of treated OMW. Someauthors reported that higher pH is not favourablefor some phylogenetic groups of nitrifying bacteria(Kowalchuk et al., 2000). Moreover, some residualpolyphenolic compounds present in treated OMWmay be toxic for this sensitive category of micro-organisms (Peredes et al., 1987).

Actinomycetes and spore-forming bacteria play asignificant role in the organic matter cycle innature, by virtue of their considerable powers andability to break down complex organic molecules.Actinomycetes counts were strongly enhanced bytreated and untreated OMW amendment. Theintroduction of organic pollutants, which canpotentially act as toxic substances and nutrientsources, was shown to preferentially stimulatespecific populations (Atlas et al., 1991). Theincrease of the CFU count of spore-forming bacteriawere in accordance with the earlier investigationsof Paredes et al. (1987) who reported an increase inspore-forming bacteria counts but a decrease in theproportion of this population in the communityfrom 10% to 12% in the control soil to 0.02% in thepolluted soil with OMW.

Fungi populations are known by their considerabledepolymerising enzymes and their resistance torecalcitrant substances. The OMW enhanced fungi,the most important organisms decomposing lignin andpolyphenols (Scheu and Parkinson, 1994; Borken etal., 2002). Consequently, this population was fa-voured in plots P1, P2 and P3 where pH and C/N ratiowere also more favourable compared to the control.This observation confirms previous findings by Perkio-maki and Fritze (2002) and Joergensen et al. (1995).

Conclusion

Based on previous studies and our results, wesuggest that the effect of the long-term use of OMWin the ferti-irrigation on the soil microbial commu-

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A. Mekki et al.100

nity, the soil fertility and the soil physico-chemicalproperties remain unclear. Yet, specific attentionmust be devoted to the irrigation potential oftreated OMW with explicit reference to the majorcrops of agricultural interest.

The following guidelines should be adhered tothe OMW spreading on soil

�

do not exceed 50m3 ha�1 y�1 of untreated OMWand to decrease the dose of treated OMW up to100m3 ha�1 y�1 to avoid the increase of the soilsalinity. � integrate a polishing tertiary treatment of OMW

for reducing the residual coloration and toxicityof the effluent.

� frequently till and avoid dry soil conditions to

maintain a maximal activity of the soil micro-flora.

Acknowledgments

This work was supported by Inco-med project‘‘Mediterranean usage of biotechnological treatedeffluent water’’ ICA3-CT-1999-00010.

The authors would like to thank: ‘‘Institut del’Olivier de Sfax’’ and Dr Bechir Ben Rouina for theirpermission to use the experimental plant of OMWamendment at Chaal farm.

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