الفهرس 
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Abstract
Irresponsible disposal of industrial wastewater effluents which contain phenols into the environment has serious adverse effects on the ecosystem and public health. Phenolic compounds can persist in the environment for a long time, due to their stability and capability for bioaccumulation. Phenols are listed as priority pollutants by the US Environmental Protection Agency (EPA) because of their toxicity and possible carcinogenic and mutagenic effects on humans. The different phenolic compounds have different ecological effects, behave differently, and have different toxicities. Therefore, it is important goal to study each compound of phenols individually. Also, it is mandatory to reduce the level of these phenols in industrial wastewater to environmentally acceptable limits by an effective and environmentally benign process before discharging into the environment. Biological treatment is the most cost effective and environmentally friendly method as well as it is more efficient in the removal of phenolic compounds. So, isolating bacterial strains able to degrade phenols efficiently is a main goal. Hence, in this study:
35 bacterial isolates capable of growth on phenolic compounds as the sole source of carbon and energy were
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isolated from activated sludge. These bacterial isolates were 15 isolate capable of growth on phenol, 11 isolate capable of growth on 2, 4- dichlorophenol and 9 isolate capable of growth on 4- methoxyphenol.
All these isolates were submitted to screening for utilization of different concentrations of its phenolic substrate (phenol, 2, 4- dichlorophenol and 4- methoxyphenol). Two isolates were selected for each compound as the most efficient degraders. PL4 & PL5 were selected for phenol; DL2 & DL3 were selected for 2, 4- dichlorophenol and MA3 & ML1 were selected for 4- methoxyphenol.
These bacterial isolates were identified by biolog system and 16s rDNA sequence. The isolates PL4, DL2 and MA3 were identified as Providencia stuartii, Lysinibacillus macroides and Staphylococcus arlettae with accession No. KY848366, KY848367 and MF445101, respectively. The three isolates PL5, DL3 and ML1 have the same morphological characters and were identified as Pseudomonas aeruginosa with probability nearly about 100 % by Biolog system. So one of them was submitted for sequencing with code PDM and also was identified as Pseudomonas aeruginosa (accession No. MF445102).
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A mixture of PL4 Providencia stuartii KY848366 with PDM Pseudomonas aeruginosa MF445102 at the ratio of 1:1 was used to complete the remaining part of this study on phenol biodegradation. A mixture of DL2 Lysinibacillus macroides KY848367 with PDM Pseudomonas aeruginosa MF445102 at the ratio of 1:1 was used to complete the remaining part of this study on 2, 4- dichlorophenol biodegradation. Also, a mixture of MA3 Staphylococcus arlettae MF445101 with PDM Pseudomonas aeruginosa MF445102 at the ratio of 1:1 was used to complete the remaining part of this study on 4- methoxyphenol biodegradation.
Each bacterial consortium was able to degrade its phenolic substrate over a wide range of pH with different efficiencies of degradation. The maximum degradation and the highest dry cell weight (DCW) for the three studied phenolic compounds occurred at pH 7 whereas the lowest removals were at pH 5 and 9.
The increase in volumes of the bacterial suspensions led to increase the degradation of the three studied phenolic compounds and the maximum degradation occurred in the shortest duration was at 6 % (v/v) of the bacterial biomass for the three studied phenolic compounds. Whereas, 5 % (v/v) of
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the bacterial consortium has degradation efficiency nearly equivalent to the efficiency of 6 % concentration.
Different nitrogen sources were tested to optimize the used medium. All supplied sources led to the enhancement of the biodegradation of the three studied phenolic compounds. However, it was observed that DCW increased with using the organic nitrogen sources (peptone and yeast extract) but the highest and fastest degradation (complete removal) occurred with using ammonium chloride as a nitrogen source for the three studied phenolic compounds.
The increase in the concentrations of ammonium chloride from 500 to 2000 mg L-1 led to a marked increase in DCW and degradation of the three studied phenolic compounds. The complete removal was achevied at concentrations 1500 and 2000 mg L-1 of ammonium chloride but the fastest removal was at 1500 mg L-1 for the three phenolic compounds. A marked decrease in DCW and the biodegradation of the three phenolic compounds have been occurred at concentration 2500 mg L-1 of ammonium chloride.
The effect of different vitamins on the biodegradation of phenolic compounds was studied. The results indicated that
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the tested vitamins have no effective induction (nearly close to the removal efficiency of control without any vitamins) for the biodegradation process of phenol except biotin (which increased the degradation of phenol by 5.6 % within 6 h of incubation compared to control) and riboflavin (which increased the degradation by 3.8 % compared to control). Also, riboflavin increased the degradation of 2, 4- dichlorophenol and 4- methoxyphenol by 3.7 % and 3.2 %, respectively within 72 h of incubation (compared to control without any additions of vitamins). Also biotin increased the degradation for the two compounds by 3 % and 1.2 %, respectively.
The effect of different concentrations of a mixture of riboflavin and biotin on the biodegradation of phenolic compounds was studied. The results indicated that the fastest removal compared to control (without any additions of vitamins) was with using a mixture of 25 mg L-1 of riboflavin and 25 mg L-1 of biotin for the three studied phenolic compounds.
The effect of additional carbon sources on biodegradation of the three phenolic compounds was studied. The results indicated that the highest degradation of phenol (complete removal) in the shortest incubation period (6 h) was achieved
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by 400 mg L-1 of lactose. The highest degradation of 2, 4-dichlorophenol (complete removal) in the shortest incubation period (48 h) was achieved by 600 mg L-1 of sucrose. Also, the highest degradation of 4- methoxyphenol (complete removal) in the shortest incubation period (48 h) was achieved by 600 mg L-1 of lactose.
Phenol was degraded completely up to 1500 mg L-1 within 58 h by its bacterial consortium with lag phase for 10 h. While, 2, 4- dichlorophenol was degraded completely up to 100 mg L-1 within 120 h by its bacterial consortium and this consortium was able to grow in the presence of 150 mg L-1 of 2, 4- dichlorophenol with lag phase for 72 h. Also, 4- methoxyphenol was degraded completely up to 150 mg L-1 within 120 h and its bacterial consortium was able to grow in the presence of 200 mg L-1 of 4- methoxyphenol with lag phase for 48 h.
The optimization process proved that, it is an effective way to improve the ability of the isolated bacterial consortiums to overcome the inhibition effect of the three phenolic compounds. At the same time to reduce the overall cost of using vitamins and additional carbon source in biodegradation process, another acclimation period was done. At the end of this acclimation period each bacterial
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consortium was able to degrade high concentration of its substrate completely (1500 mg L-1 of phenol, 150 mg L-1 of 2, 4- dichlorophenol, and 200 mg L-1 of 4- methoxyphenol) without any vitamin or additional carbon source.
Immobilization of microbial cells on various inert support materials for the biodegradation of high concentrations of phenols has been proven efficiently viable option. Support materials are very critical to any successful immobilization process. The results of this study proved that, attachment and entrapment of bacterial cells in PUF seemed to be the best immobilization used technique compared to immobilization by adhesion and biofilm formation on Nylon sheet and by encapsulation using alginate beads. The results also indicated that the immobilization by encapsulation using PVA- alginate beads is somewhat considered a good method of immobilization. Hence, the both of the two carriers PVA- alginate and PUF in immobilization process are applicable to phenolic compounds biodegradation process.
Phenol solutions at concentrations 1500, 2000 and 2500 mg L−1 were completely degraded by the immobilized PVA- alginate after 58, 96, and 120 h of incubation, respectively. Also, the immobilized PUF degraded these concentrations (1500, 2000 and 2500 mg L−1) completely after 48, 72, and 120 h of
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incubation, respectively. On the other hand, the freely suspended bacterial consortium degraded phenol completely only at the lower initial loading (1500 mg L−1) and the efficiency of degradation decreased to only 76 % at 2000 mg L−1 of the initial loading of phenol.
The immobilized PUF degraded concentrations 75, 150 and 200 mg L−1 of 2, 4- dichlorophenol completely after 48, 96, and 192 h of incubation, respectively. Also, the immobilized PVA- alginate degraded 75 and 150 mg L−1 of 2, 4- dichlorophenol completely after 72 and 120 h of incubation, respectively and degraded 93 % of 200 mg L−1 2, 4- dichlorophenol within 192 h. On the other hand, the freely suspended bacterial consortium degraded 2, 4- dichlorophenol completely at the lower initial loading (75 mg L−1 and 150 mg L−1) and the efficiency of degradation decreased to only about 82.5 % of 200 mg L−1 of 2, 4- dichlorophenol after 192 h of incubation.
The solutions of 4- methoxyphenol at concentrations 200 and 300 mg L−1 were completely degraded by the immobilized PVA- alginate after 96 and 144 h of incubation, respectively. Also, the immobilized PUF degraded these concentrations (200 and 300 mg L−1) completely nearly within 72 and 120 h of incubation, respectively. On the other hand, the freely
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suspended bacterial consortium degraded 4- methoxyphenol completely only at the lower initial loading (200 mg L−1) and the efficiency of degradation did not exceed 85 % at the higher initial loading (300 mg L−1).
The degradation of these phenolic compounds by the immobilized PVA- alginate beads and by the immobilized PUF was much higher than that of free cells, even at higher loadings. However, the immobilized PUF had higher degradation efficiency than the immobilized PVA- alginate.
A combination between suspended bacterial biomass and PUF immobilized biomass (for all consortiums) was used in two unit hybrid sequencing batch reactor (HSBR) for the treatment of synthetic and industrial effluent. The results indicated that the total removal efficiency in the first unit of the bioreactor was about 62 % for amixture of the three phenolic compounds (7.5 mg L-1 of 2, 4-dichlorophenol; 75 mg L-1 of 4- methoxyphenol and 375 mg L-1 of phenol) and was about 99.8 % in the second unit. Also, the results of treatment of real pharmaceutical industrial wastewater indicated that phenol was successfully eliminated by HSBR