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Aspergillus niger P6 and Rhodotorula mucilaginosa CH4 used for olive mill wastewater (OMW) biological treatment in single pure and successive cultures a

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Raja Jarboui , Salwa Magdich , Raja Jarboui Ayadi , Ali Gargouri , Néji Gharsallah & Emna Ammar

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UR Étude et Gestion des Environnements Urbains et Côtiers, École Nationale d'Ingénieurs de Sfax, Université de Sfax, B.P. 1173 - 3038 Sfax, Tunisia b

Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, B.P. 1177 - 3038 Sfax, Tunisia c

Laboratoire de Biotechnologie Microbienne, Faculté des Sciences de Sfax, B.P. 1171 - 3000 Sfax, Tunisia Accepted author version posted online: 11 Jul 2012

To cite this article: Raja Jarboui, Salwa Magdich, Raja Jarboui Ayadi, Ali Gargouri, Néji Gharsallah & Emna Ammar (2012): Aspergillus niger P6 and Rhodotorula mucilaginosa CH4 used for olive mill wastewater (OMW) biological treatment in single pure and successive cultures, Environmental Technology, DOI:10.1080/09593330.2012.710404 To link to this article: http://dx.doi.org/10.1080/09593330.2012.710404

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Aspergillus niger P6 and Rhodotorula mucilaginosa CH4 used for olive mill wastewater (OMW) biological treatment in single pure and successive cultures

Raja Jarbouia, Salwa magdicha, Raja Jarboui Ayadia, Ali Gargourib, Néji Gharsallahc, Emna Ammara*

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UR Étude et Gestion des Environnements Urbains et Côtiers, École Nationale d’Ingénieurs de Sfax, Université de Sfax, B.P. 1173 - 3038 Sfax, Tunisia

b

Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, B.P. 1177 3038 Sfax, Tunisia c

Laboratoire de Biotechnologie Microbienne, Faculté des Sciences de Sfax, B.P. 1171 - 3000 Sfax, Tunisia

* Correspondence to Emna Ammar Tel.: +216 98 41 23 64; Fax: +216 74 27 55 95; E-mail address: [email protected] Raja Jarboui: [email protected] Salwa Magdich: [email protected] Raja Jarboui Ayadi: [email protected] Ali Gargouri: [email protected] Néji Gharsallah: [email protected]

Abbreviations: OMW, olive mill wastewater; EC, electrical conductivity; COD, chemical oxygen demand; BOD, biological oxygen demand; TOC, total organic carbon; TS, total solids; TSS, total suspended solids; VS, volatile solid; Total P, total phosphorus; Total N, total nitrogen, EAE, ethyl acetate extract.

Abstract The aim of this study was to investigate the Rhodotorula mucilaginosa CH4 and Aspergillus niger P6 abilities to purify OMW in single pure and mixed cultures during the treatment. Both fungi were molecularly identified. OMW was used at five dilutions from 5 to 30% with COD ranging from 11600 to 24600 mg l-1. Firstly, each fungus was used separately then, they were successively used to treat the

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OMW. In single pure culture, A. niger showed a better efficiency in OMW purification than R. mucilaginosa. Furthermore, when successively used, the two studied strains exhibited COD, polyphenolic compounds concentration and effluent color decrease improvement. COD removals were of 95.68 to 56.71% by R. mucilaginosa and of 98.02 to 69.51% by A. niger respectively for OMW dilutions varying from 5 to 30%. Both strains showed an important polyphenolic compounds removal: 83 to 45% by R. mucilaginosa and 94 to 58% by A. niger, in accordance with the OMW COD initially used. The COD and phenolic compounds removals fitted simple equation models, with high regression coefficients. The strains growth kinetics decreased according to the OMW concentration, but when successively used, fungal growth was improved allowing efficient effluent treatment.

Keywords: Olive mill wastewater, Aspergillus niger, Rhodotorula mucilaginosa, COD reduction, Decolorization.

1. Introduction

In Mediterranean basin, the most serious environmental problem associated with the agroindustrial sector is the olive mill wastewaters (OMW) disposal. In Tunisia, the fourth olive oil producer in the

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world with about 150,000 tons of olive oil extracted, around 7 x 105 tons of OMW are yearly produced [1]. This effluent is characterized by high concentrations of several organic compounds, such as organic acids, sugars, tannins, pectins and polyphenolic substances. The high phenolic compounds concentration seems to be responsible for the OMW phytotoxicity and microbial inhibition [2,3]. Besides, OMW contains inorganic compounds, especially phosphorus and potassium, which might suggest a further OMW use as fertilizer. Because of their instability, OMW polyphenols tend to polymerize into condensed high-molecularweight polymers, difficult to degrade during storage [4,5]. Hence, uncontrolled OMW disposal can generate severe risks to water and soil qualities, and with a less concern, an acute odor problem [6-8]. In the last few decades, several disposal methods (physico-chemical, biological or a combined treatment) have been suggested and developed in order to reduce the OMW ecological impact [9-11]. Nevertheless, with probably the sole exception of land spreading, so far allowed in only a few countries in the world, such as Italy [12], no treatment method seems to be a cost-effective and definitive solution to solve the OMW discharge problem. At present, in the field of OMW valorization, most of the research has focused on the bacterial approach; and in many papers, anaerobic digestion process was used to treat OMW and to produce methane [13-15]. Interestingly, chemical or biological OMW pretreatments were required to reach the process performances. In this respect, fungi have been used to reduce the OMW total polyphenols and their associated toxicity [16,17]. A variety of white rot fungi were used for OMW biodegradation [18-21]. These were efficient in OMW-COD, phenolic compounds and color reductions. In addition, several yeasts were used to treat OMW and showed performances in COD reduction and phenolic compounds removal [22-24].

However, at the author’s knowledge, no previous work considered the application of fungi association to improve the OMW purification while used at high COD. The aim of this study was to investigate the ability of R. mucilaginosa CH4 and A. niger P6 to reduce COD, phenolic compounds concentration and color of OMW used at different dilutions. The performance of the successive treatment by yeast and fungus was studied to improve the OMW purification.

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2. Materials and methods

2.1. Strains isolation and identification

The experimented yeast R. mucilaginosa CH4 and the fungi us A. niger P6 were respectively isolated from the OMW evaporation ponds and the OMW contaminated soil in Sfax (Tunisia). The strains were grown on yeast potato dextrose medium (YPD: 10 g l-1 yeast extract, 20 g l-1 peptone and 20 g l-1 glucose). Both strains were molecularly identified, based on the RNA 18S sequencage. For R. mucilaginosa CH4 identification, two loopfuls of cells grown on the YPD agar were suspended in 30 µl of sterile distilled water and heated at 94 °C for 10 min to release the DNA. Cell debris was eliminated by centrifugation at 12,000 g for 15 min. The supernatant was transferred into a new 1.5 ml tube. The A. niger P6 genomic DNA was isolated from 5-day-old culture mycelia, following the method described by Lahyani and Gargouri [25]. Briefly, about 2 g of frozen mycelia were ground to a fine powder with alumina and mixed in 5 ml of extraction buffer [10 mM Tris–HCl (pH 8.0), 50 mM EDTA, 0.5% sodium dodecyl sulfate (SDS)]. After two extractions with an equal Tris–HCl–phenol volume, followed by overnight dialysis against water, the DNA was precipitated with 2.5 volumes of ethanol and 0.1 volume of 3 M sodium acetate (pH 5.2). The extracted DNA was then visualized by electrophoresis on 1% agarose. The 18S rRNA genes were amplified by using the specific forward primer

NS3

(5’-GCAAGTCTGGTGCCAGCAGCC-3’)

and

NS4

(5’-

CTTCCGGTCAATTCCTTTAAG-3’). The PCR thermal profile was as follows: initial denaturation at 94 °C for 5 min, primer annealing at 50 °C for 30 sec, and extension at 72 °C for 2 min. The final elongation step was extended to 15 min. Under these conditions, a single PCR product of 600 pb was

obtained. The 18S rDNA gene nucleotide sequence has been submitted to the European Nucleotide Archive

which

assigned the accession number FR822395 and FR822396, respectively for R. mucilaginosa CH4 and A. niger P6.

2.2. Culture conditions

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The OMW sample was collected in February 2008, from the last pond of the evaporation-ponds system, located at Agareb, a village in the South-West of Sfax (Tunisia) and was stored at 4°C until use (Jarboui et al., 2010). The OMW sample was physico-chemically characterized (Table 1). The OMW-based media had the following composition (w/v): sucrose, 0.5%; (NH4)2SO4, 0.1%; CaCl2, 0.1% and diluted OMW, 1l. The OMW was used at different dilutions: 5, 10, 15, 20 and 30%, corresponding to initial soluble COD of 11600, 14200, 16800, 19400 and 24600 mg l-1. The OMWbased media was sterilized at 121 °C during 20 min. For the inoculum preparation, fungi-spores were sampled from 7 day-old culture by flooding with 10 ml sterile distilled water and scraping off the agar plates. The OMW-based media inoculation was made by 2 ml of A. niger P6 spore solution, having 5 g l-1 dry matter. For R. mucilaginosa CH4, 2 ml pre-culture of 5 day-aged colonies with OD at 600 nm of 14.56 were used for the yeast single pure culture, corresponding to 6 g l-1 of dry matter. For the successive yeast-fungus treatment, the inoculum OD at 600 nm was 12.50 (4.60 g l-1 dry matter), 2 ml were used to inoculate the same OMW-based media. Cultures were grown in 500 ml flasks containing 200 ml of OMW-media and stirred at 200 rpm; the temperature was 30 °C. The incubation was held 6 days and samples were collected daily for analysis. The strains growth was assessed by the dry matter determination after the final medium culture centrifugation at 10,000 rpm for 15 min. In the successive treatment, the OMW-base medium previously treated by the first fungus strain during 6 days was sterilized at 121 °C during 20 min. To this solution, 2.5 g l-1 sucrose sterilize solution was added and finally used as a culture medium for the second experimented strain.

2.3. Physico-chemical analyses

Electrical conductivity (EC) and pH were measured directly in the sample. Chemical oxygen demand (COD) was measured by the Knechtel method [26]. Soluble COD was determined after OMW centrifugation at 4000 rpm for 15 min. BOD5 was determined using respirometric method. The total suspended solids (TSS) were gravimetrically assessed after crude OMW centrifugation at 4000 rpm for 15 min. The total solids (TS) were gravimetrically determined by drying the OMW sample overnight at 105 °C [27]. The volatile solids (VS) content was deduced after weighting the incinerated dry OMW at 550 °C for 6 h [27]. The ash content was determined as the difference between TS and VS contents.

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The ethyl acetate extracts (EAE) were determined after extraction with three aliquots of ethyl acetate. Polyphenolic compounds contents were measured with the Folin-Ciocalteu reagent (Merck), using catechol as the standard [23]. The OMW decolorization was assessed by the absorbance measurement at 395 nm. Total phosphorus (Total P) was determined colorimetrically as a molybdovanadate phosphoric acid complex [27]. The oil content was gravimetrically determined after soxhlet solid/liquid extraction using n-hexane as solvent. Total nitrogen (Nt) and NH4+ concentrations were determined using the Kjeldhal method. All the analyses were performed in triplicate.

2.4. Statistical analysis

The analysis/determination mean was reported using ANOVA software. Statistical significance was defined for p < 0.05.

3. Results and discussion

3.1. OMW physico-chemical characterization

During this study, the OMW -collected from the reception evaporation pond of the serial natural evaporation ponds [5]- was characterized by acidic pH, reflecting the OMW richness in polyphenolic and fatty acids [5,11] (Table 1). The OMW showed a high organic and mineral contents (COD, TSS, TS, fats, BOD, VS, Nt, ash and EC) and high polyphenols concentration, responsible for antimicrobial and phytotoxic activities [3,23,28]. Furthermore, the OMW had phosphorus and nitrogen high contents,

which may enhance further microbial growth. Moreover, its CODs/BOD5 rate was lower than 2, reflecting its easy biodegradability [29]. Several authors found the OMW pH varied between 4.0 and 6 [3, 17, 23, 28, 30, 31]. The same authors mentioned very high COD (40-200 g l-1), BOD (12-60 g l−1), total solids content (40-150 g l−1) and high polyphenols content, up to 30 g l−1.

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3.2. COD removal

To treat OMW, the isolated R. mucilaginosa CH4 and A. niger P6 were used in single pure and successive cultures revealing a COD decrease during both treatment types (Fig. 1). In single pure culture, R. mucilaginosa CH4 COD removals were 81.7; 64.7; 41.3; 36.6 and 31.2%, while A. niger P6 showed COD removals of 89.6; 71.8; 62.1; 53.0 and 45.2%, respectively for OMW dilutions 5, 10, 15, 20 and 30%. While comparing the COD removals by both fungi in single pure culture, no significant difference was noted (p > 0.05). For COD-effluent exceeding 5000 mg l-1 (dilution > 10%), the treatment revealed that A. niger P6 was 1.5 folds more perferment in OMW pollution reduction than R. mucilaginosa CH4. For the treatment based on successive fungi growths, the preliminary tests made showed that both studied strains had weak growth. To improve the growth rate, 2.5 g l-1 of sucrose were added in the OMW. As a consequence, the initial COD values increased (Fig. 1), and after six days, the COD removal improved reaching 95.68; 90.18; 75.09; 61.75 and 56.71% for R. mucilaginosa CH4 and 98.02; 93.97; 84.82; 76.53 and 69.51% for A. niger P6 used in a second growth step, in accordance with OMW dilutions. These results confirmed the successive treatment efficiency in decreasing the OMW organic load. It seems that R. mucilaginosa CH4 was able to use organic compounds, enhancing A. niger P6 accessibility to available organic matter and improving organic compounds reduction, while using first the yeast then the fungus. In the two treatment processes, COD variation models fitted a simple order equation for the two studied strains (Table 2). The regression coefficients showed high values, ranging from 0.850 to 0.980 in single pure treatment and from 0.905 to 0.987 in the successive treatment, which confirmed the models validities. However, there was no significant difference while comparing COD removal in the

two treatment processes (single pure and successive treatments) and also both experimented strains activities (p > 0.05). Referring to Table 2, the time required to eliminate all the initial COD was presented in Figure 2. The modelization of this progress fitted first order equations with high regression coefficients varying from 0.957 to 0.997. These equations were: t = 0.692 x CODi - 0.445 (R² = 0.957), for R. mucilaginosa CH4; t = 0.631 x CODi - 1.065 (R² = 0.997) for A. niger P6;

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t = 0.534 x CODi - 0.195 (R² = 0.979) when yeast was used after fungus treatment; t = 0.495 x CODi - 0.073 (R² = 0.980) when fungus was used after yeast treatment. These models showed A. niger P6 performance while used in the second step-treatment unlike R. mucilaginosa CH4. Indeed, in the successive treatment, 4 days may be saved if the yeast was used first for initial COD exceeding 14 g l-1. Previous studies reported R. mucilaginosa and A. niger efficiency and specific abilities to degrade several phenolic compounds found in OMW. These two isolated strains confirmed their performance in the effluent treatment considering their COD removal after single pure or successive treatments. Indeed, A. niger performances in OMW treatment was studied by several authors. Cereti et al. [32] evidenced the ability of immobilized A. niger cells to reduce 35 to 64% of initial COD of OMW supplemented with rock phosphate, with a limited phenolic compounds reduction. Moreover, more recently, Alaoui et al. [20] in a five-day experiment found that A. niger reduced only 26.7% of COD and 6.1% of phenolic compounds concentration when the fungus was used on OMW based media, with initial COD and phenolic compounds concentrations of 92 and 6.1 g l-1 respectively. The OMW successive treatment was studied by De Felice et al. [33] who used firstly Yarrowia lipolytica ATCC 20255 to reduce OMW COD and to produce a lipase, and secondly Pseudomonas putida to reduce phenolic compounds concentration. In the present study, A. niger P6 showed an important COD and polyphenolic compounds reductions comparing to the previously published works, and the reduction was well improved by the successive OMW treatment using first R. mucilaginosa CH4 and then A. niger CH4.

3.3. OMW decolorization and phenolic compounds removal

During the two treatments, the highest color rate removals were observed with the highest OMW dilutions used (5 and 10%) (Table 3). In single pure culture, A. niger P6 showed the best OMW decolorization. The successive treatment allowed an increase in the color reduction and consequently, ethyl acetate extraction (EAE) concentrations decreased (Table 4). There was no significant difference (p > 0.05) while comparing color rate removals in single pure and successive treatments by both experimented strains. The OMW toxicity and biodegradability studies showed that polyphenolic compounds are responsible for the wastewater black color and light toxicity. Moreover, these aromatics

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are not easily biodegradable. The two strains performances in OMW toxicity elimination were daily studied by determining polyphenolic compounds concentrations reduction (Fig. 3) and final ethyl acetate extract (EAE) concentration (Table 4). The results showed polyphenolic compounds elimination by both tested strains during the two experimented treatments. Indeed, from one dilution to another, the percentage of polyphenolic compounds elimination decreased, this would be due to the OMW phenolic inhibitor effect on both strains. On the other hand, for all the OMW dilutions studied an increase of the polyphenolic compounds elimination was observed while considering the progress in the same dilution, this could be due to the studied strains capacities to reduce the OMW toxicity. Although A. niger P6 showed the most important polyphenolic removal rate, the yeast R. mucilaginosa CH4 showed an important percentage of polyphenolic compounds elimination 67, 55, 52, 40 and 27% during the single pure treatment and 83, 73, 64, 57 and 45% during the successive treatment, respectively with different OMW dilutions and the initial polyphenolic compounds concentrations (200, 400, 600, 800 and 1200 mg l-1). In the successive treatments, the initial polyphenolic concentrations were 58.49; 150.91; 260.05; 402.03 and 784.47 mg l-1 for R. mucilaginosa and 68.90; 176.50; 290.48; 480.00 and 873.60 mg l-1 for A. niger respectively, in accordance with the OMW dilutions. The polyphenolic compounds removal variation was not significantly different (p > 0.05) during the two treatments process and by the two experimented strains. During the single pure and the successive treatment, a decrease of the final ethyl acetate extracts (EAE) concentrations was noted (Table 4). Indeed, EAE removals of 57, 49, 43, 37 and 31% were obtained by R. mucilaginosa CH4 and 61, 54, 49, 40 and 35% by A. niger P6 during the single pure treatment; while these removals were 86, 78, 73, 64, 57% and 88, 80, 74, 66, 58% during the successive treatment respectively for the two strains. The EAE removals were no significantly different for all OMW

dilutions and by the two experimented strains (p > 0.05). The performance of A. niger P6 is due to the fact that moulds have a capacity to produce a wide variety of extracellular proteins, organic acids and other metabolites, with a specific adaptation capacity to extreme environments more perferment than yeasts and bacteria [34,35]. The A. niger P6 growth led to a decrease of the OMW color intensity, possibly due to the degradation of some phenolic compounds and to the adsorption of the polyphenols and tannins on the fungal mycelium. This adsorption may be due to the hydrogen bound between phenolic compounds and proteins or to mycelial wall chitin which has a strong coagulant effect [36].

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Howerver, previous work showed A. niger ablity to catabolise catechol which is an important intermediary of aromatic compounds aerobic catabolism, thank to a catechol 1,2-dioxygenase [37]. Furthermore, A. niger was used to metabolize many phenolic compounds identified in OMW, such as protocatechuic acid, p-hydroxyphenylacetate, quercitin, oleuropein [37], benzoic, coumaric and cinnamic acids [38]. Until now, no previous work reported Rhodotorula sp. use for OMW treatment. Nevertheless, this species expressed a significant capacity to degrade phenol, catechol, cresol, resorcinol, 3-methoxybenzoic acid and hydroquinones [39-41].

3.4. Strains growth

Both studied fungi strains growth determined by final dry weight after single pure and successive treatments are presented in Table 5, the results showed a progressive growth in accordance with the OMW dilutions. This could be due to the OMW toxic effect exerted on the two studied strains. However, an improved growth of both strains was noted during the successive treatments. In the OMW, it is well known that the biomass formation depends on the COD and TSS concentrations and on the inoculum size [42]. The biomass growth resulted from the metabolism of the OMW organic content (sugars, lipids, pectins, tannins, anthocyanins and aromatic compounds). The strains growths were associated with the COD and color decreases. During the two treatments, R. mucilaginosa CH4 biomass was higher than that of A. niger P6. Furthermore, the Student-test showed that unlike A. niger P6, R. mucilaginosa CH4 growth differed significantly (p < 0.05) during both treatment types. This can be due to the medium accessibility by the two studied strains. During the single pure treatment, both pure strains growth were not significantly different (p > 0.05), while, when the strains were used in successive cultures, the strain exhibited a noticeable difference.

4. Conclusion

The single pure and successive OMW treatment by R. mucilaginosa CH4 and A. niger P6 were studied. The two strains were efficient in reducing the OMW toxicity and its inherent pollution. Consequently, they showed interesting COD, color and polyphenolic compounds reduction rates. A high treatment performance was obtained with A. niger P6 and it seems that R. mucilaginosa CH4 facilated the OMW

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assimilation for A. niger P6 during the successive treatment. The strains prospective application would be the exploration of the studied microorganisms’ performances in OMW pre-treatment and reuse, considering their high performance evidenced.

Acknowledgements The authors are indebted to Mr. Ahmed Ayadi the General Manager of the Tunisian Service Society, for providing the material and allowing exploration of the site. They are also grateful to Mrs Hela Chaabouni Fourati from Sfax, for proofreading.

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Figures Caption

Fig. 1 OMW COD progress during Rhodotorula mucilaginosa CH4 and Aspergillus niger P6 single pure culture (a, b) and successive treatment (c): OMW base-media firstly treated by R. mucilaginosa CH4 and secondly treated by A. niger P6; (d): OMW base-media firstly treated by A. niger P6 and secondly treated by R. mucilaginosa CH4 according to the OMW initial dilutions; ( * ) 5%; ( (

) 15%; (

) 10%;

) 20%; ( ) 30%.

Fig. 2 Time when the COD annulled by Rhodotorula mucilaginosa CH4 and Aspergillus niger P6 during single pure and successive cultures. Fig. 3 OMW polyphenolic compounds removal by Rhodotorula mucilaginosa CH4 and Aspergillus niger P6 during single pure culture (a, b) and successive treatment (c): OMW base-media firstly treated by R. mucilaginosa CH4 and secondly treated by A. niger P6; (d): OMW base-media firstly treated by A. niger P6 and secondly treated by R. mucilaginosa CH4 according to the OMW initial dilutions; ( * ) 5%; (

) 10%; (

) 15%; (

) 20%; ( ) 30%.

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Fig. 1 (a)

(c) - First treatment by R. mucilaginosa CH4 - Second treatment by A. niger P6

(b)

(d) - First treatment by A. niger P6 - Second treatment by R. mucilaginosa CH4

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Fig. 2

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Fig. 3 (a)

(c) - First treatment by R. mucilaginosa CH4 - Second treatment by A. niger P6

(b)

(d) - First treatment by A. niger P6 - Second treatment by R. mucilaginosa CH4

Table 1 OMW physico-chemical characterization (February, 2008). Parameters

Value

Basic parameters pH

4.87 ± 0.10 -1

EC (mS cm )

14.15 ± 0.20

-1

TSS (g l )

37.55 ± 0.50

-1

TS (g l )

72.93 ± 1.26 -1

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COD (g l )

125.00 ± 0.80

-1

51.80 ± 1.42

-1

31.80 ± 0.66

CODs (g l ) BOD5 (g l ) CODs/BOD5

1.63 -1

Organic content (g l ) Fats

6.78 ± 0.54

Nt

1.40 ± 0.08

VS

58.03 ± 1.08

EAE

28.26 ± 0.74

Polyphenolic compounds

4.00 ± 0.09

Inorganic content (g l-1) Ash NH4

14.90 ± 1.00 +

Total phosphorus

0.56 ± 0.02 1.02 ± 0.02

EAE: Ethyl acetate extracts; CODs: Soluble chemical oxygen demand; Nt: Total nitrogen.

Table 2

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COD variation model fitting Rhodotorula mucilaginosa CH4 and Aspergillus niger P6 OMW treatment (Single pure and successive). Rhodotorula mucilaginosa CH4

Aspergillus niger P6

Initial CODs (g l-1)

Single pure culture (6 days)

11.60 14.20 16.80 19.40 24.60

Successive treatment1

Single pure culture

Successive treatment2

(6 days)

-1458t + 10113

-605t + 3925

-1667t + 10153

-780t + 4902

(R² = 0.925)

(R² = 0.978)

(R² = 0.912)

(R² = 0.957)

-1405t + 12963

-1065t + 7388

-1552t + 12536

-1115t + 7757

(R² = 0.947)

(R² = 0.926)

(R² = 0.930)

(R² = 0.960)

-1341t + 16744

-1115t + 8787

-1590t + 15124

1172t + 10066

(R² = 0.980)

(R² = 0.925)

(R² = 0.927)

(R² = 0,905)

-1317t + 18003

-1197t + 11926

-1542t + 17474

-1273t + 13598

(R² = 0.926)

(R² = 0.987)

(R² = 0.899)

R² = 0.984

-1492t + 23662

-1366t + 16526

-1770t + 22941

-1413t + 18124

(R² = 0.935)

(R² = 0.955)

(R² = 0.934)

(R² = 0.976)

1: Yeast (6 days) then fungus (6 days); 2: Fungus (6 days) then yeast (6 days); CODs: Soluble chemical oxygen demand.

1

Table 3

2

OMW coloration removal percentage by A. niger and R. mucilaginosa during single pure culture (6 days)

3

and successive treatments (6 + 6 days). OMW dilutions (%)

OMW treatment 5

10

15

20

30

R. mucilaginosa

74.0 ± 1.0

65.0 ± 0.7

54.0 ± 0.6

42.7 ± 0.5

32.5 ± 0.5

A. niger

80.3 ± 1.2

68.8 ± 0.8

58.0 ± 0.7

50.3 ± 0.5

44.0 ± 1.0

89.0 ± 1.4

81.0 ± 1.0

66.3 ± 0.7

59.3 ± 0.4

51.9 ± 0.6

81.9 ± 1.1

77.1 ± 0.9

61.9 ± 1.1

53.0 ± 0.7

Single pure culture

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Successive cultures 1- R. mucilaginosa* 2- A. niger 1- A. niger* 2- R. mucilaginosa

4

46.4 ± 0.6

*: Sterile supernatant from the first single pure culture inoculated with the second fungus.

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

21 22

Table 4

23

OMW ethyl acetate extracts (EAE) concentration (g l-1) after the two studied strains purification during 6

24

days (single pure culture) and 12 days (successive treatment). Strains

OMW dilutions % (initial EAE concentration (g l-1)) 5 (1.40)

10 (2.80)

15 (4.20)

20 (5.60)

30 (8.40)

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Single pure culture R. mucilaginosa

0.60 ± 0.05

1.44 ± 0.04

2.40 ± 0.08

3.50 ± 0.04

5.82 ± 0.01

A. niger

0.55 ± 0.06

1.30 ± 0.06

2.13 ± 0.08

3.34 ± 0.07

5.47 ± 0.01

0.17 ± 0.09

0.55 ± 0.05

1.09 ± 0.07

1.90 ± 0.06

3.50 ± 0.03

0.20 ± 0.04

0.61 ± 0.01

1.14 ± 0.05

2.00 ± 0.06

3.54 ± 0.02

Successive culture 1- R. mucilaginosa* 2- A. niger 1- A. niger * 2- R. mucilaginosa

25

*: Sterile supernatant from the first single pure culture inoculated with the second fungus.

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 22

42

Table 5

43

Final dry weight of the two studied strains during pure and successive culture.

44

Final dry weight (g l-1) OMW

Single pure culture

dilutions (%)

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R. mucilaginosa

51

A. niger

Successive culture

45

1- R. mucilaginosa*

1- A. niger*

2- A. niger

2- R. mucilaginosa

5

9.61 ± 0.49

8.40 ± 0.42

12.67 ± 0.12

9.95 ± 0.66

10

8.98 ± 0.65

7.44 ± 0.15

11.01 ± 0.54

8.35 ± 0.45

15

6.02 ± 0.44

5.38 ± 0.12

10.41 ± 0.25

8.61 ± 0.44

20

5.86 ± 0.34

4.70 ± 0.45

9.87 ± 0.87

6.49 ± 0.34

30

4.72 ± 0.38

2.10 ± 0.65

6.78 ± 0.55

5.21 ± 0.16

46 47 48 49 50

*: Sterile supernatant from the first single pure culture inoculated with the second fungus.

23