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Arab Journal of Nuclear Science and Applications, 46(2),(287-303) 2013

287

Optimizing Degradation of Olive Oil Mill Waste Water Using Paecilomyces

variotii

1O. K. Khatab, 2A. A. Hassan, 3A. B. Abdel El- Aziz, 1A. A. El-Nasr and 3G. H. Zaki

1 Botany and Microbiology Department, Faculty of Science, University of Helwan. 2 Department of clinical laboratory Science- College of Applied Medical- King Saud University.

3 Microbiology Department, National Center for Radiation Research and Technology, Atomic Energy

Authority, Nasr City, Cairo, Egypt.

E-mail: [email protected]

Received: 3/6/2012 Accepted: 6/7/2012

ABSTRACT

Twenty six microbial isolates (ten fungal, nine yeast and seven bacterial

isolates) were isolated from the Olive Oil Mill Waste Water (OOMW) which was

extracted from effluent of olive oil industry factory. All isolates were tested for its

growth on media containing 10% OOMW as sole carbon source. It was found that

(three fungal, two yeast and two bacterial isolates) had the ability to grow on this

concentration. These isolates were identified as Paecilomyces variotii, Ascopus

stercoraris, Aspergillus terrus, Yarowia lipolytica, Candida tropicalis, Lactobacillus

curvatus and Bacillus brevis. The identified isolates were tested for the

biodegradation of phenolic compounds at high concentration of OOMW (25%).

Paecilomyces variotii was the best isolate as it degraded 10.40 % of the phenolic

compounds. The maximum degradation of phenolic compounds and chemical

oxygen demand (COD) decrease percentage was (68.14 and 59.12, respectively)

obtained at 50% dilution of OOMW for 12 days at 37±1˚C, pH 6, supplement the

degradation media with 150 mg/l sucrose, 2.5 g/l yeast extract and 0.070 mmol/l

CuSO4 concentration in aerobic conditions with aeration rate 4:1 (v air: v media),

shaking at 150 rpm and 6 g/l inoculums size. In addition, 0.25 kGy was the best dose

as it led to increase the phenolic compounds biodegradation percent 8.7% than the

optimum conditions previously mentioned. Finally, the biotreated OOMW was

lower toxicity to environment than untreated one.

Keywords: Olive Oil Mill Wastewater, Paecilomyces variotii, Gamma radiation.

INTRODUCTION

Pollution appeared in the last century as a great problem facing the world; most of chemical,

petrochemical and pharmaceutical industries produce harmful pollutants as by- products. Among the

most dangerous and problematic pollutants are phenolic compounds (1). Phenolic compounds produced

from different sources like olive oil industry, polymeric resins industries, oil refining, and coke plants

(2). The olive oil industry is very important in the worldwide due to its high dietetic and nutritional

value (3), so the consumption of olive oil is rapidly increasing. On the other hand, the liquid effluent

from olive presses, left after removal of the olive oil, causes a serious environmental hazard in olive

producing countries, especially around the Mediterranean basin. It has been estimated that 30X106 m3

of olive oil mill wastewater (OOMW) are produced per year. The problems mainly arise from the

effluent’s high level of pollutants such as phenolic compounds leading to a high chemical oxygen

demand (>200 g/ l) (4-5).

The phytotoxic and antibacterial effects of the OOMW have been attributed to the monomeric

phenolic content of OOMW (6). The polyphenolic compounds are the principal cause of the toxicity of

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Arab Journal Of Nuclear Science And Applications, 46(1), 2013

288

the effluent (7). Several chemical and physical methods have been developed for the phenol

degradation such as precipitation, coagulation, ion exchange, ultra filtration but these methods are

expensive and inefficient. The by-products from the chemical and physical methods process possess

toxic compounds (8). Today biodegradation as an emerging tool to remove the environmental

pollutions is used to describe the complete mineralization of the starting compound to simpler ones

like CO2, H2O, NO3 and other inorganic compounds. Parameters such as pollutant concentrations,

temperature, pH and microbial adaptation are the most important parameters that affect phenol

biodegradation rate depends on the period in which the culture was adapted to phenol degrading

microorganism (9).

The aim of the present study was isolation, identification and selection of the most potent

microbial isolate which can degrade the phenolic compounds, and optimization of factors affecting the

biodegradation of olive oil mill waste water.

MATERIALS AND METHODS

Collection of the OOMW

Samples of the OOMW used in this study were collected from, an olive oil factory, Ismailia,

Egypt-Alexandria Desert Road, Egypt. The OOMW was taken from a modern three-phase (one of the

olive oil extraction methods). OOMW samples were collected in pre-sterilized glasses from a depth of

about 1 meter from the waste water surface.

Isolation, selection and identification of microbial isolates from OOMW

Ten milliliter of OOMW was serial diluted up to 10-7. One ml of each dilution was plated (in

triplicate) on both malt extract agar with 0.05 gm/ml of chloramphenicol at pH 5.5 for 7 days for fungi

and 5 days for yeast at 25˚C and Nutrient agar media with 0.05 gm/ml of amrazole at pH 7.0 for one

day and 37˚C for bacteria. An inoculum from the isolation media was used for inoculation (Malt

Extract Agar medium for fungi and yeast and Nutrient Agar Medium for bacterial isolates),

supplemented with 10% of the OOMW. The selected microorganisms was inoculated in Mineral Salt

Medium for fungi and yeast and Tryptone Glucose Medium for bacterial isolates, supplemented with

25% of the OOMW to know the best isolate for degradation of phenolic compounds and decreasing

COD to complete this study.

Identification of fungi was carried out by the methods of Moubasher (10) and confirmed by the

help of the Micro Analytical Center, Cairo University, Egypt. Identification of yeast isolates was

carried out by the methods of Barnett et al. (11). Identification of bacterial isolates was carried out by

the methods of Holt et al. (12). The electron microscope samples of all microbial isolates (fungi, yeast

and bacteria) were prepared as described by Lorian (13).

Preparation and adaptation of fungal inoculum

Preparation and adaptation of selected fungal isolate inoculums were determined according to

Jacob and Alsohaili (14) and the microbial adaptation of fungus was according to Elsa et al. (15).

Factors affecting the biodegradation of olive oil mill wastewater

Concentrations of OOMW:

Fifty ml of different concentrations of OOMW (25%, 50%, 75% and 100%) v/v with pH 5.5 in

250 ml conical flasks were inoculated with (1.5 g/l) fungal inoculums size at 25˚C for 8 days.


289

Incubation periods:

Fifty ml of optimum concentration OOMW at pH 5.5 in 250 ml conical flasks were inoculated

with (1.5 g/l) fungal inoculums size at 25˚C for different incubation periods (4, 8, 12 and 16 days).

Temperature:

Inoculated flasks contained optimum concentration OOMW were incubated for optimal

incubation period and at different temperature (25±1, 30±1, 37±1, and 45±1˚C).

pH values:

the initial pH of the optimal concentration of OOMW fermentation medium was adjusted with

0.1 N NaOH or 0.1 N HCl to different values ranging from (4.0, 6.0, 8.0 and 10.0), then the inoculated

flasks were incubated optimal incubation period and optimal temperature.

Carbon sources:

The 250 ml conical flasks contained optimal amount of media with optimal initial pH adjusted

and various carbon sources (glucose, fructose, sucrose and starch) with concentration (100 mg/l) were

added to the OOMW fermentation medium. The conical flasks were incubated at optimal temperature

for optimal incubation period. Then, different concentrations (50, 100, 150 and 200 mg/l) of the best

carbon source which recorded from this experiment were investigated.

Nitrogen sources:

1 g/l of different nitrogen sources (sodium nitrate, urea, peptone and yeast extract) were

investigated. The best nitrogen source was determined from different concentrations of (0.50, 1.0, 1.5,

2.0, 2.5 and 3 g/l) supplemented in250 ml conical flasks and incubated in optimal incubation period,

optimal temperature, pH and carbon source concentration determined from the above experiments.

Aeration rate:

To stimulate different aeration conditions, three levels of medium volume were used (1:1, 4:1

and 9:1) volume air: volume media in 250 ml conical flasks were incubated in optimal incubation

period, optimal temperature, pH, carbon source concentration, nitrogen source concentration and

optimal nitrogen source determined from the above experiments.

Agitation and static condition:

Different agitation speeds (100, 150, and 200 rpm) used in 250 ml conical flasks incubated in

optimal incubation period, optimal temperature, pH, carbon source concentration, nitrogen source

concentration, optimal nitrogen source and optimal aeration determined from the above experiments.

Mineral salts:

Various mineral salts (copper sulphate, zinc sulphate and ferric chloride) (0.070 mmol/l) were

added to the biodegradation medium used in 250 ml conical flasks incubated in optimal incubation

period, optimal temperature, pH, carbon source concentration, nitrogen source concentration, optimal

nitrogen source and optimal aeration and optimal agitation determined from the above experiments.

Inoculum size:

Different inoculums (0.75, 1.5, 3, 6 and 12 g/l) of selected fungus, was added to 250 ml conical

flasks incubated in optimal incubation period, optimal temperature, pH, carbon source concentration,

nitrogen source concentration, optimal nitrogen source and optimal aeration, optimal agitation and

optimal mineral salts determined from the above experiments.


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Effect of Gamma irradiation on the biodegradation of phenolic compounds

Three replicated tubes containing 10 ml optimum inoculums size were exposed to various low

doses of gamma radiation (0.0, 0.25, 0.50, 0.75 and 1.00 KGy). 3 ml was transferred in 250 ml conical

flasks at previous optimum conditions.

Analytical methods:

Dry weight: the dry weight was determined by dry cell weight (DCW, g/l). 10 ml culture broth was

dried at 60˚C to constant weight.

Determination of pH: the pH of the broth media was measured at 25˚C with glass electrode pH

meter (type pioneer 10 portable pH meter with a fisher pencil probe).

Growth: growth was measured by optical density of microbial growth using spectrophotometer at 600

nm.

Scanning electron microscopy: the samples of all microbial isolates (fungi, yeast and bacteria)

were prepared as described by Lorian(13).

Radiation process: Irradiation process was carried out at National Center for Radiation Research

and Technology, Nasr City, Cairo, Egypt. The irradiation facilities used were an experimental Co60

Russian gamma chamber. INDIA. The average dose rate of this gamma radiation source was 2.5

kGy/1hr at the time of the experiments.

Determination of phenolic compounds concentration: phenolic compounds concentrations were

determined by kits of (Nanocolor® phenol 12.05), Cat. No.91875 and Test 1-75.

Determination of Chemical oxygen demand (COD) concentration: COD concentration was

determined by kits of (Nanocolor® COD 300), Cat. No. 985 033 and Test 0-33.

Statistical analysis: the data are the means of 3 independent experiments. Experimental data were

subjected to analysis of variance (ANOVA) using SAS program (16).

RESULTS AND DISCUSSION

Biodegradation is an important mean to eliminate toxic wastes from the environment (17). Fungi

(Phanerochaete chrysosporium, Aspergillus niger and Aspergillus terreus) are found to be good

candidates, (18). Our study is based on biodegradation of the phenolic compounds in OOMW by

Paecilomyces variotii (Penicillium divericatum) at optimum conditions.

Physico-chemical characterizations of the freshly untreated OOMW

Table (1) showed a major complexity of OOMW especially phenolic compounds and chemical

oxygen demand (COD) reached 50.0 and 22800.0 mg/l which were higher levels than normal range

(0.05 and 100 mg/l) , respectively. Paredes et al. (19) reported that the total phenolic compounds in

OOMW ranged from 10.32-30.99 mg /l. Dincer et al. (20) reported that OOMW has high values of

COD equaled to 21000 mg/l.


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Table (1). Physico-chemical characters of untreated olive oil mill wastewater.

Isolation, selection and identification of microorganisms

Ten fungi, nine yeast and seven bacterial isolates were isolated and grew on 10% OOMW.

Similar results have been found by Gupta and Mukerji (21) reported that three fungi, two yeast and two

bacterial isolates have been grown on 10% OOMW.

Isolated fungi were identified according to Moubasher (10) and confirmed by the help of the

Micro Analytical Center, Cairo University, Egypt. They were Paecilomyces variotii, Ascobolus

stercorarius, and Aspergillus terreus. Our results were in agreement with that recorded by Aissam et

al. (22) who found that Penicillium sp. and Aspergillus niger from OOMW cause a decrease in phenolic

compounds and COD.

Isolated yeasts were identified according to Barnett et al.(11). They were Yarrowia lipolytica and

Candida tropicalis. Similar results were obtained by Martinez et al. (23) who isolated and identified

Candida tropicalis from OOMW.

Isolated bacteria were identified according to Holt et al. (12). They were Lactobacillus curvatus

and Bacillus brevis. This is in agreement with Eusebio et al.(24) who isolated many bacterial species

from OOMW. They isolated and identified isolates belonged to the Bacillus genus (with predominance

of Bacillus megaterium, Bacillus sphaericus and Brevibacillus brevis).

Selection of the most potent phenolic compounds degrading microorganisms

As shown in Table (2), the results indicated that all microbial isolates were degraded phenolic

compounds in OOMW. The maximum phenolic compounds reduction was 10.40% by Paecilomyces

variotii, while the minimum phenolic compounds (6.00%) were obtained by Lactobacillus curvatus.

Paecilomyces variotii was selected to complete the study. Fadil et al. (25) found that Paecilomyces

variotii was a biodeterioration causal agent of palm oil, leather, cosmetics, jute and paper, and it was

isolated from different substrates like compost, cocoa beans, rice flour, synthetic rubber, jute fiber and

also from kerosene storage tank.

Parameters Measured

(mg/l) Normal range

(mg/l)

Total suspended solids (TSS) 94000.0 2000.0

Total dissolved solids (TDS) 22000.0 60.0

Chemical oxygen demand (COD) 22800.0 100.0

Oils and grease 2450.0 10.0

Phenolic compounds 50.0 0.05

pH 5.5 6.0-9.0


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Table (2): Comparison between the potentialities of phenolic compounds degrading isolates

* MSM supplemented with 25% OOMW ** TSA supplemented with 25% OOMW.

***%=100- [(Remained *100)/Control]

Remained= Control- degraded phenolic compounds, Control =12.5 mg/l

Factors affecting the biodegradation of phenolic compounds by Paecilomyces variotii

Concentration of OOMW

The results presented in Fig. (1) illustrated that the maximum phenolic compounds reduction

was 4.46 mg/l (17.84 %), and the maximum decreased COD 123.9 g/l (10.87%) were obtained at 50%

olive oil wastewater. The dry weight of Paecilomyces variotii increased by decreasing the OOMW

concentration from 100% to 50%, reached 3.30 g/l and 4.78 g/l, respectively, so 50% olive oil

wastewater concentration was the optimum in the biodegradation of OOMW by Paecilomyces varioti.

Yousuf et al. (26) showed that Lipomyces starkey was able to survive and proliferate in the presence of

OOMW, they found that the preliminary dilution of OOMW enhanced the reduction of polluting

components of OOMW, leading to lower levels of residual phenolic compounds. Peixotoa et al. (27)

reported that Candida oleophila grew in a diluted and sterilized OOMW and caused a significant

decrease in the total tannins content but no significant alteration was observed in phenolic acid and

fatty acid content.

Isolates

Phenolic compounds

Degraded phenolic

compounds (mg/l)

Degraded phenolic

compounds (%) ***

*Ascopus stercoraris 1.15 9.20

*Paecilomyces variotii 1.30 10.40

*Aspergillus terrus 0.95 7.60

*Yarowia lipolytica 1.08 8.60

*Candida tropicalis 1.05 8.40

**Lactobacillus curvatus 0.75 6.00

**Bacillus brevis 0.80 6.40


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A- Degraded phenolic

compounds

B- Decreased COD

C- Dry weight

Fig. (1) (A-C) Effect of olive oil wastewater at different concentrations on its biodegradation by

Paecilomyces variotii.

Incubation periods

The results presented in Fig. (2) showed that the degraded phenolic compounds concentration

increased gradually untill 12th day and decreased thereafter. It was evident from the results that the

optimum fermentation time was 12 days, when degraded phenolic compounds concentration reached

(4.93 mg/l), decreased COD was 1303.6 mg/l and dry weight was 5.7 g/l. Tsioulpas et al. (28) observed

that various strains of Pleurotus had the ability on removal of 69–76% phenolic compounds after 12–

15 days in shake flasks. Afify et al. (29) found that Aspergillus wentii, P. ostreatus and Aspergillus

niger obtained the maximum COD 74.5%, 43% and 13.8% after two weeks, three weeks and first

week, respectively. Also, they observed that Aspergillus wentii, P. ostreatus and Aspergillus niger

obtained the maximum phenolic compounds reduction 81.3%, 88% and 64.1% after two weeks, two

weeks and first week, respectively.


compounds

B- Decreased COD

C- Dry weight

Fig. (2) (A-C) Effect of incubation periods on the OOMW phenolic compounds biodegradation

by Paecilomyces variotii.

Incubation temperature

From Fig. (3), it was noticed that the optimum incubation temperature for Paecilomyces variotii

was 37±1˚C with degraded phenolic compounds (5.82 mg/l), decreased COD (1769.33 mg/l),

respectively and maximum growth (6.82 g/l), Dry weight of microorganism increased with increasing


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in temperature, accordingly Paecilomyces variotii could be specified as a thermo tolerant. Pettersson

and Baath (30) reported that the temperature could affect the biodegradation of organic compounds

through direct effects on enzyme activity. They found that the most effective temperature for

biodegradation by Pseudomonas sp. strain ST-4 was 30˚C. Agarry et al. (31) observed that the phenol

degradation rate increased with the increase in temperature up to 37±1˚C, but above 37±1˚C it

decreased. It was observed that the growth of Pseudomonas aeruginosa increased up till 37±1˚C and it

decreased thereafter.


compounds

B- Decreased COD

C- Dry weight

Fig. (3) (A-C) Effect of different temperature on the OOMW phenolic compounds

biodegradation by Paecilomyces variotii.

pH values:

The results represented in Fig. (4) showed that initial pH values appeared to have an obvious

effect on the dry weight of selected fungi (Paecilomyces variotii), degraded phenolic compounds, and

COD. Degraded phenolic compounds concentration showed noticeable increase with rising pH till

reached the maximal value at pH 6 which reflected the great ability of Paecilomyces variotii to grow

and efficiently degrade phenolic compounds over range of pH values. pH 6 was optimum as it gave

the highest degraded phenolic compounds (8.55 mg/l), also maximum decreased COD (2180 mg/l)

and dry weight (8.89 g/l ). This is in agreement with the results of Kai and Jing (32) who reported that

the extra cellular pH plays an important role in biodegradation processes and metabolic process. Reda

et al. (33) found that the optimum pH for the microbial degradation in the range 6 to 8 and the solubility

of phosphorous, an important nutrient in biological systems, was maximized at pH 6.5. They found

that the optimum pH value for both Bacillus subtilis- EPRIS12 and Bacillus laterosporus –EPRIS41

was 7 at which these bacterial strains gave high growth rate on toluene biodegradation.


295


compounds

B- Decreased COD

C- Dry weight

Fig. (4) (A-C) Effect of different pH values on the OOMW phenolic compounds biodegradation

by Paecilomyces variotii

Carbon sources

The results in Fig. (5) showed that all added carbon sources (100 mg/l) had enhancement effect

on phenolic compounds degradation. Sucrose was the superior carbon source since it gave the lowest

residual phenolic compounds concentration and the highest degraded phenolic compounds (12.22

mg/l), the highest decreased COD (2801 mg/l) and the highest fungal growth (12.95g/l). Sucrose was

added at different concentrations (50, 100, 150 and 200 mg/l). The results represented in Fig. (6)

showed that the significant degraded phenolic compounds concentration was linearly increased with

increasing sucrose concentration till reached its maximum value at 150 mg/l. Conversely, decrease in

the degraded phenolic compounds with increasing the concentration of sucrose than the optimum

value was observed. Zissi and Lyberators (34) observed that the presence of glucose as carbon and

energy source for the microbes, enhancing their activity to utilize the resistant aromatic amines. In

addition, Benito et al. (35) reported that adding sucrose (0.3%) enhanced in removing COD (77%) of

wastewater by Trametes versicolor.


compounds

B- Decreased COD

C- Dry weight

Fig. (5) (A-C) Effect of different carbon sources on the OOMW phenolic compounds

biodegradation by Paecilomyces variotii


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compounds

B- Decreased COD

C- Dry weight

Fig. (6) (A-C) Effect of different sucrose concentrations on the OOMW phenolic compounds

biodegradation by Paecilomyces variotii.

Nitrogen sources

The results in Fig. (7) showed that yeast extract (1 g/l) was the superior nitrogen source and the

most suitable organic nitrogen source for the biodegradation (degraded phenolic compounds was

14.55 mg/l, decreased COD reached 4844 mg/l and the dry weight reached 16.18 g/l. Yeast extract

was added to MSM media in concentration equivalent to 0.5, 1, 1.5, 2, 2.5 and 3 g/l. The results in

Fig. (8) showed that after twelve day of fermentation, the degraded phenolic compounds concentration

decreased with increasing the yeast extract concentration and reached its maximum value at yeast

extract concentration equivalent to 2.5 g /l. It was also clear from the data that the highest degraded

phenolic compounds (15.45 mg/l), decreased COD (5644 mg/l) and dry weight (17.59 mg/l) were

obtained in concentration 2.5 g/l. Then the degraded phenolic compounds and COD concentration

decreased with concentration of yeast extract 3 g/l. Hamdi et al. (36) reported that nitrogen was crucial

for the growth of Aspergillus niger and if ammonium and sulphate were not added, the spores of A.

niger did not germinate.

Krishnaswamy and Namasivayam (37) found that the nutrients, particularly nitrogen are required

to improve the aromatic compounds biodegradation. They found that the presence of 0.01% yeast

extract at pH 7 in medium contained 6 bacterial strains of Bacillus cereus, Arthrobacter sp., Bacillus

licheniformis, Halomonas salina, Bacillus subtilis and Pseudomonas aeruginosa showed the highest

phenolic compounds removal efficiency (99 %) but when urea was substituted for yeast extract the

efficiency reduced to 75 %. They showed that replacement of yeast extract with tryptone and urea

affected the degradation efficiency of phenolic compounds. This might be due to the structure of yeast

extract as it is readily available as amino acids in the mineral salts medium. They concluded that using

yeast extract as nitrogen source was more efficient in degrading phenolic compounds.


297

A- Degraded phenolic compounds

B- Decreased COD

C- Dry weight

Fig. (7) (A-C) Effect of different nitrogen sources on the OOMW phenolic compounds



compounds

B- Decreased COD

C- Dry weight

Fig. (8) (A-C) Effect of yeast extract concentrations on the OOMW phenolic compounds


Aeration rate:

From the results presented in Fig. (9), it was clear that the lowest biodegradation rate was in

anaerobic conditions but there was marked increase in the degraded phenolic compounds, COD and

dry weight in aerobic conditions from (13.18 mg/l, 4104.00 mg/l and 15.26 g/l, respectively) to (15.45

mg/l, 5644 mg/l and 17.67 g/l, respectively) with increasing the air to medium ratio in the

fermentation medium from 1:1 to 4:1, but in the fermentation medium ratio 9:1, there were decrease in

degraded phenolic compounds, COD and dry weight of fungus as they reached 15.09 mg/l and 5387

mg/l and 17.59 g/l respectively.

Oboirien et al.(38) reported that the biodegradation rate of phenol by using freely suspended P.

aeruginosa NCIB 950 increased with increase in aeration rate from 1.5 to 2.5 v/v/m Fulya et al. (39)

found that all simple phenolic compounds disappeared from the medium after the 8th day of cultivation

at 0.25 v/v/m aeration rate by using the white-rot fungi Trametes versicolor.


298


compounds

B- Decreased COD

C- Dry weight

Fig. (9) (A-C) Effect of aeration rate on the OOMW phenolic compounds biodegradation by

Paecilomyces variotii

Agitation and static conditions

It is evident from Fig. (10) that the maximum degraded phenolic compounds (16.49 mg/l) and

decreased COD (6011 mg/l) with dry weight was (18.22 g/l) at 150 rpm. The results in Fig. (10)

indicated that the shaking condition accelerated the biodegradation process of OOMW in comparison

with the static condition. In the static condition, degraded phenolic compounds, decreased COD and

dry weight (15.45 mg/l, 5644.33 mg/l and 17.67 g/l), respectively. Khleifat and Khaled(40) found that

the phenol were degraded by Actinobacillus species at pH 7, the incubation temperature ranged from

35 to 37˚C, and the agitation rate of 150 rpm were the optimal conditions for achieving the higher

degradation of phenolic compounds. Yesilada et al. (41) found that the optimal agitation values for

biodegradation of OOMW by Coriolus versicolor and Funalia trogii were around 150 and 200

rpm/min. They observed that the favored COD removal and decolonization could be attributed to

increased amounts of dissolved O2 as a result of agitation.


compounds

B- Decreased COD

C- Dry weight

Fig. (10) (A-C) Effect of agitation on the OOMW phenolic compounds biodegradation by

Paecilomyces variotii


299

Mineral salts

The results represented in Fig. (11) showed that CuSO4 could enhance phenolic compounds

degradation (degraded phenolic compounds was 16.88 mg/l), decreased COD was 6643.67 mg/l and

dry weight was 20.47 g/l . Furthermore, ZnCl2 showed a noticeable delaying in degradation process

(low degraded phenolic compounds concentration reached 13.39 mg/l) comparable to that of control

(16.46 mg/l). The results in Fig. (11) showed that the addition of Cu2+ caused activation in phenolic

compounds degradation. Galhaup et al. (42) reported that the enzymes which were responsible for

phenolic compounds degradation activated by cobber ions.

Zissi and Lyberators (34) selected four halotolerant Penicillium that were isolated from

mangroves and salterns, on the basis of the variations in morphology of the penicillial heads and by

their resistance to lead, copper and cadmium salts. They reported that all four isolates could resist Pb

(II) at a concentration of 7.5 mM, with no decrease in growth up to 5 mM and with minimal changes

in growth pattern and morphology compared to that of Cu (II) and Cd (II). Three of the strains were

resistant at least to 2 mM of Cu (II). The cultures grown in presence of metals showed striking

variations in colony appearance, morphology, sporulation and in pigment production as compared to

the controls; these changes were more pronounced with increasing metal concentrations.


compounds

B- Decreased COD

D- Dry weight

Mineral salts was added at concentration 0.070 mmol/l

Fig. (11) (A-C) Effect of different mineral salts on the OOMW phenolic compounds biodegradation by Paecilomyces variotii

Inoculum size

The results presented in Fig. (12) showed that degraded phenolic compounds (17.04 mg/l) and decreased COD (6740.33 mg/l ) with a maximum dry weight (21.05g/l) were reached by using 6.0 g/l inoculums size. On the other hand, the use of high inoculum size (12.0 g/l) resulted marked decrease in dry weight, degraded phenolic compounds and COD. Yesilada et al. (41) investigated the relationship between inoculum size and biodegradation. They found that the optimal inoculum size of white rot fungi Coriolus versicolor and Funalia trogii were 5 ml for COD removal. They found that higher inoculum size affected phenol removal by these fungi. Fulya et al. (39) found that all simple phenolic compounds disappeared from the medium after the 8th day of cultivation at 0.25 v/v/m aeration rate by using the white-rot fungi Trametes versicolor. Temperature was maintained at 26˚C and inoculum size used was 10% (v/v).


300


compounds

B- Decreased COD

C- Dry weight

Fig. (12) (A-C) Effect of different inoculum size on the OOMW phenolic compounds


Gamma irradiation

The data recorded in Fig. (13) showed that under optimizing culture condition maximum

degraded phenolic compounds, decreased COD and dry weight were 19.21 mg/l and 6864 mg/l and

21.72 g/l, respectively at radiation dose of 0.25 kGy. The degraded phenolic compounds, COD

concentration and dry weight decreased with increasing the irradiation dose above 0.25 kGy. Meleigy (46) exposured cells of Auerobasiduim pullulans to 0.25 KGy and found that this dose induced the

formation of non-pigment mutants with increasing the productivity of pullulan production.

Abo-State et al. (44) found that the viable count of Aspergillus terreus MAM-F23 and MAM-F35

gradually decreased as the gamma radiation dose increased. Doses 5.0 and 4.0 kGy reduced the viable

count of Aspergillus terreus MAM-F23 and MAM-F35 completely.

El-Batal et al. (45) found enhanced productivity in CMCase, EPase, Avicelase, xylanase, and

pectinase by gamma irradiation at dose 1.0 kGy with increased percent 8%, 20%, 10%, 31% and 34%,

respectively as compared with unirradiated microorganism control.


compounds

B- Decreased COD

C- Dry weight

Fig. (13) (A-C) Effect of different doses of gamma rays on the OOMW phenolic compounds



301

The physico-chemical characteristics of OOMW before and after treatments

It is evident from the results in Table (3) that the total content of phenolic compounds is reduced from 50.0 mg/l to 5.8 mg/l by Paecilomyces variotii. Similar results were previously reported by many investigators. Kapellakis et al. (47) found that fungal species as Geotrichum candidum, Phanerochaete chrysisporium and Panus tigrinus are used to reduce the phenolic content of OOMW. Fadil et al. (25) have found that using Geotrichum sp., Aspergillus sp. and Candida tropicalis enables the removal of polyphenolic compounds in percentages of 46.6%, 44.3%, and 51.7%, respectively. The results in Table (3) indicated that COD was high but after the experiments became lower (from 22800.0 mg/l to 6864mg/l) by Paecilomyces variotii.

Table (3): Physico-chemical composition of untreated and treated OOMW

CONCLUSION

This study describes the biotreatment olive oil mill wastewater factory by Paecilomyces

variotii. The biodegradation treatment is less expensive than other technologies that are used for

cleanup of hazardous waste and safe to environment comparing with chemical and physical methods.

The current study suggested that 88.4% of phenolic compounds and 70% COD were removed in olive

oil mill wastewater samples containing phenolic compounds to get the opportunity of using the treated

effluent in different purposes such as irrigation.

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parameters Before treatment

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Total suspended solids (TSS) 94000.0 mg/l 860.0 mg/l 60.0 mg/l

Total dissolved solids (TDS) 22000.0 mg/l 4000.0 mg/l 2000.0 mg/l

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