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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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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.
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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.
A- Degraded phenolic
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.
A- Degraded phenolic
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.
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A- Degraded phenolic
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.
A- Degraded phenolic
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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A- Degraded phenolic
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.
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A- Degraded phenolic compounds
B- Decreased COD
C- Dry weight
Fig. (7) (A-C) Effect of different nitrogen sources on the OOMW phenolic compounds
biodegradation by Paecilomyces variotii
A- Degraded phenolic
compounds
B- Decreased COD
C- Dry weight
Fig. (8) (A-C) Effect of yeast extract concentrations on the OOMW phenolic compounds
biodegradation by Paecilomyces variotii
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.
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A- Degraded phenolic
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.
A- Degraded phenolic
compounds
B- Decreased COD
C- Dry weight
Fig. (10) (A-C) Effect of agitation on the OOMW phenolic compounds biodegradation by
Paecilomyces variotii
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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.
A- Degraded phenolic
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).
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A- Degraded phenolic
compounds
B- Decreased COD
C- Dry weight
Fig. (12) (A-C) Effect of different inoculum size on the OOMW phenolic compounds
biodegradation by Paecilomyces variotii
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.
A- Degraded phenolic
compounds
B- Decreased COD
C- Dry weight
Fig. (13) (A-C) Effect of different doses of gamma rays on the OOMW phenolic compounds
biodegradation by Paecilomyces variotii
Arab Journal Of Nuclear Science And Applications, 46(1), 2013
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.
REFERANCES
(1) Fleeger, J., Carman, K. and Nisbet, M. The Sci. the total environ.; 317 (1-3), 207-233 (2003).
(2) Schleyer, Paul, v. Chem. Rev.; 105, 3433 (2005).
(3) Roig A., Cayuela M. L., Saánchez-Monedero M. A. Waste Management; 26, 960-969 (2006).
(4) Merchichi, T., Sayadi, S. Proc. Biochem; 40, 139–145 (2005).
(5) Della Greca, M., Previtera, L., Temussi, F., Zarrelli, A. Phytochem. Anal.; 15, 184–188 (2004).
(6) Casa, R., D’Annibale, A., Pieruccetti, F., Stazi, S. R., Giovannozzi, S. G., and Lo Cascio, B. Chem.; 50(8), 959–966 (2003).
(7) D’Annibale, A., Quaratino, D., Federici, F. and Fenice, M. Biochem Eng J.; 29, 243- 249 (2006).
parameters Before treatment
After treatment Normal range
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
Chemical oxygen demand (COD) 22800.0 mg/l 6864 mg/l 100.0 mg/l
Oils and grease 2450.0 mg/l 68.0 mg/l 10.0 mg/l
Phenolic compounds 50.0 mg/l 5.80 mg/l 0.05 mg/l
pH 5.5 7.5 6-9
Arab Journal Of Nuclear Science And Applications, 46(1), 2013
302
(8) Rengaraj, S. Moon, S. H. Sivabalan, R. Arabindoo, B. and Murugesan, V. Waste Manag.; 22, 543–548 (2002).
(9) Khazi, M., Aravindan, R., and Viruthagiri, T. Asian J. Exp. Biol. Sci.; 1 (2), 219-234 (2010).
(10) Moubasher, A. Soil fungi in Qatar and other Arab countries. The Center for Scientific and Appl. Research. University of Qatar, Doha, Qatar; (1993).
(11) Barnett, J., Payne, R. and Yarrow, D. Yeasts: Characteristic and Identification 3rd ed. Cambridge university press, New York.; (2000).
(12) Holt, J.G. Krieg, N.R. Sheath, P.H.A. Stanley, J.T and Williams, S.T. Bergy’s Manual of Determinative Bacteriology. 9 th Edn. Williams and wilkins, Baltimore, Maryland, USA. (1994).
(13) Lorian, V. Antibiotics in Laboratory Medicine, 2nd Ed.Williams and Wilkins. USA.; 602 (1986).
(14) Jacob, H. J. and Alsohaili, S.; J. Biol. Sci.; 10 (2), 162-165 (2010).
(15) Elsa, M., Aida, M. and Ana, M.; Electronic J. Biotechnol.; 7 (1), 0717-3458 (2004).
(16) Cary, N. C. One- way analysis of variance (ANOVA) using SAS 6.1., SAS institute (1998).
(17) Ogbo E. M. and Okhuoya J. A.; Afr. J. Biotechnol.; 7(23), 4291-4297 (2008).
(18) Garcia, G. I., Pepa, J., Venceslada, P. R., Martin, M.A., Santos, M.A., and Ramos Gomez, E., Proc. Biochem.; 35,751 (2000).
(19) Paredes, C., Cegana, J. Roig, A., Sanchez-Monedero, M. A. and Bemal, M. P. Bioresource Technology; 67, 111-115 (1999).
(20) Dincer, A. R., Karakaya, N. and Gunes, E. Global NEST J.; 10(1), 31-38. (2008).
(21) Gupta, R., Mukerji, K. G. Bioremediation: Past, Present and Future. In: Role of microbes in the management of environmental pollution. Tewari R, Mukerji KG, Gupta JK, Gupta LK. (eds). A.P.H. Publishing Corp, New Delhi.; 73 – 81 (2001).
(22) Aissam, H., Penninckx, M. J., Benlemlih, M.; World J. Microb. and Biotechnol.; 23(9), 1203-1208 (2007).
(23) Martinez G., Johnson, A., Bachmann, R. , Williams, C., Burgoyne, A. and Edyvean, R. J Hazard Mater.; 30, 164,(2-3) 1398-405 (2009).
(24) Eusebio, A.; Mateus, M.; Baeta-Hall, L., Saagua, M. C.; Tenreiro, R.; Almeida-Vara, E.; Duarte, J. C. Inter. Biodet. and Biodeg.; 59(3), 226-233 (2007).
(25) Fadil, K., Chahlaoui, A., Ouahbi, A., Zaid, A., Borja, R. Inter. Biodet. and Biodeg.; 51(1), 37-41 (2003).
(26) Yousuf, A.; Sannino, F.; Addorisio, V.; Pirozzi, D. J. Agricultural and Food Chem.; 58 (15), 8630-8635 (2010).
(27) Peixotoa, F., Martinsa, F., Amaralb, C., Gomes-Laranjob, J., Almeidac, J., . Palmeira , C. M. Ecotoxicology and Environmental Safety.; 70, 266–275 (2008).
(28) Tsioulpas, A., Dimou, D., Iconomou, D., Aggelis, G. Bioresour Technol.; 84, 251– 257 (2002).
(29) Afify, A., Mahmoud, M., Emara, H. and Abdelkreem, K. Australian J. Basic and Appl. Sci.; 3 (2), 1087-1095 (2009).
(30) Pettersson, M., Baath, E.; FEMS Microbiol. Ecol.; 45, 13-21 (2003).
(31) Agarry, S. E., Solomon, B. O. and Layokun, S. K. Afr.; J. Biotechnol.; 7 (14), 2409-2416 (2008).
(32) Kai, C.L. and Jing, S. W.; Biodeg.; 8, 329-338 (1998).
(33) Reda, A. Bayoumi and Ashraf, T.Abul-Hamd.; J. Appl. Sciences Research; 6(8), 1086-1095 (2010).
(34) Zissi, U. S., Lyberators, G. C. Water Environ. Res.; 71, 43-49 (1999).
(35) Benito, G., Pena, M., Rodriguez, D. Biore- source Technol.; 61, 33 – 37 (1997).
(36) Hamdi, M., Bouhamed, H. and Ellouz, R. Appl. Microbiol. and Biotechnol.; 36(2), 285-288 (1991).
(37) Krishnaswamy, V. and Namasivayam, V. Inter. J. Biotechnol. Biochem.; 6(5), 783-791 (2010).
(38) Oboirien, B. O., Amigun, B., Ojumu, T. V., Ogunkunle, O. A., Adetunji, O. A., Betiku, E., Solomon, B. O. Biotechnol.; 4(1), 56-61 (2005).
(39) Fulya, E., Sayit, S., Gaye, O., Fazilet, V. Inter. Biodet. and Biodeg.; 63, 1–6 (2009).
(40) Khleifat, Khaled M. Biorem. J.; 11, 103-112 (2007).
(41) Yesilada, O., Sik, S., Sam, M. World J. Microbiol. Biotechnol.; 14, 37–42 (1998).
(42) Galhaup, C., Wagner, H., Hinterstoisser, B., Haltrich, D. Enzyme Microbiol. Technol.; 30, 529–536 (2002).
Arab Journal Of Nuclear Science And Applications, 46(1), 2013
303
(43) Nazareth, S., Marbaniang, T. J. Basic Microb.; 48, 363-369 (2008).
(44) Abo-State, M. A. M., Hammad, M. A. M., Swelim, M. and Gannam, R. B. American- Eurasian J. Agric. Environ. Sci.; 8(4), 402-410 (2010).
(45) El-Batal, A. I. and Abo-State, M. A. Egypt. Rad. Sci. applic.; 19, 139-156 (2006).
(46) Meleigy, S.A. N. Egypt. J. Microbiol.; 9, 59-76 (2004). (47) Kapellakis, K., Iosif, E., Tsagarakis, P., John, C. and Crowther. Rev. Environ. Sci. Biotechnol.; 7,
1–26 (2008).