TREATMENT OF THE SOFT DRINK COMPANY EFFLUENT OF NORTH-FES (MOROCCO) BY SEQUENCING BATCH REACTOR

FAOUZI M.¹, MERZOUKI M.²*, EL HASSANI F.Z.³, EL KARKOURI A.⁴, EL HASSOUNI M.⁵, BENLEMLIH M.^6
1Unité de Biotechnologie de l’Environnement, Laboratoire de Biotechnologie, Faculté des Sciences Dhar El Mahraz, BP: 1796, Atlas, Fès, Université Sidi Mohamed Ben Abdellah, Fès, Maroc.
²Unité de Biotechnologie de l’Environnement, Laboratoire de Biotechnologie, Faculté des Sciences Dhar El Mahraz, BP: 1796, Atlas, Fès, Université Sidi Mohamed Ben Abdellah, Fès, Maroc.
³Unité de Biotechnologie de l’Environnement, Laboratoire de Biotechnologie, Faculté des Sciences Dhar El Mahraz, BP: 1796, Atlas, Fès, Université Sidi Mohamed Ben Abdellah, Fès, Maroc.
⁴Unité de Génétique Moléculaire des Microorganismes, Laboratoire de Biotechnologie, Faculté des Sciences Dhar El Mahraz, BP: 1796, Atlas, Fès, Université Sidi Mohamed Ben Abdellah, Fès, Maroc.
⁵Unité de Génétique Moléculaire des Microorganismes, Laboratoire de Biotechnologie, Faculté des Sciences Dhar El Mahraz, BP: 1796, Atlas, Fès, Université Sidi Mohamed Ben Abdellah, Fès, Maroc.
⁶Unité de Biotechnologie de l’Environnement, Laboratoire de Biotechnologie, Faculté des Sciences Dhar El Mahraz, BP: 1796, Atlas, Fès, Université Sidi Mohamed Ben Abdellah, Fès, Maroc.
* Corresponding Author : merzoukimo@yahoo.fr

Received : 19-10-2012     Accepted : 22-11-2012     Published : 27-11-2012
Volume : 3     Issue : 1       Pages : 37 - 44
J Biotechnol Lett 3.1 (2012):37-44
DOI : http://dx.doi.org/10.9735/0976-7045.3.1.37-44

Conflict of Interest : None declared

The present work is a study of the microbiological quality of wastewater from the Soft Drink Company of North Fez (SDCN)-Morocco before and after treatment with Sequential Batch Reactor (SBR). The study included monitoring the abundances of faecal indicators (faecal coliforms and faecal streptococci), total coliforms, total germs, staphylococci, yeasts and fungi depending on COD (chemical oxygen demand) and total nitrogen in the effluent. The microbiological analysis showed that the microbiological contamination of raw effluent was highly variable depending on the time and concentration of COD and total nitrogen. After treatment, a removal of fecal coliforms, total coliforms and staphylococci, of respectively 99.65%, 98.6% and 99.33% was obtained. The highest elimination rate was recorded for total bacteria and faecal streptococci, whose turnover rates were respectively 99.4% and 100%. For yeasts and fungi, the elimination rates were of 99.5% and 98%. Thirteen bacterial strains were isolated from activated sludge and effluent before and after treatment with SBR, eight of them were identified by biochemical tests and five by Molecular technics.

Microbiological quality, wastewater, sequencing batch reactor, biochemical identification, molecular identification.

More than two thirds of earth surface is covered by water; yet, fresh potable water free of harmful physical, biological and chemical contamination is not always available at the right time or the right place for human use. Water Pollution comes mainly from human domestic, municipal, agricultural and industrial activities. Worldwide, aquatic ecosystems are the primary means for disposal of wastes, especially effluents, from populations and industries.
Effluents discharged by industries have a great deal of influence on the pollution of the water body and can alter the physical, chemical and biological nature of it [1] . The initial effect of waste discharge is degradation of the physical quality of the water. Later, biological degradation becomes evident in terms of number, variety and dynamic of the living organisms in the water [2] . When the amount of waste released to the ecosystem is low, the water bodies can assimilate waste materials they receive without significant deterioration of some quality criteria due to their assimilative capacity [3,4] . Therefore, the input of waste into water bodies does not always impact negatively the aquatic environment because of the self purification property of lotic systems [5] .
Surface water and ground water degradation are major problems arising from pollution due to discharge of industrial effluents such as those of the industries of the carbonated soft drinks. The Industrial effluent of soft drink companies is a wastewater inappropriately discharged into the environment or receiving streams. Those effluents discharge has led inevitably to alteration in the quality and ecology of receiving water bodies [6,7] . Soft drink effluent consists of wasted soft drinks and syrup, water from the washing of bottles and cans, which contains detergents and caustics, and lubricants used in the machinery. Therefore, the most significant associated pollutants will include total suspended solids (TSS), 5-day biochemical oxygen demand (BOD5), chemical oxygen demand (COD), nitrates, phosphates, sodium and potassium [8] . Biological treatment is the most common method used for treatment of soft drink wastewater because of its organic content. If the wastewater does not have a high organic content, aerobic treatment can especially be used [9] .
This work aims to determine the microbiological quality of wastewater of the Soft Drink Company of North Fez (SDCN) before and after treatment by SBR. Sequencing batch reactors are increasingly used for the removal of organic matter and nitrogen from wastewater [10] . It also allows an assessment of the performance of this method in reducing and eliminating germs, and monitoring the abundance of bacteria, yeasts and fungi in the treated wastewater depending on COD and total nitrogen. This monitoring is accompanied by an identification of some isolates using biochemical and molecular approaches.

In this study, we used activated sludge sampled from the sewage wastewater treatment plant IBIS of Meknes city-Morocco. The samples of wastewater from the SDCN were collected from the point of discharge of the industrial effluent into the municipal sewer system. The industrial effluent is discharged with an average flow of 300 m3 d-1. The samples of the SDCN effluent were taken every day during a week at 8 h, 11 h, 14 h and 17 h then a composite sample was analyzed. The purpose of this analysis is to determine the average concentrations of chemical parameters during the day after samples were taken from the company once a week and kept at 4 °C until use [Table-1] .

The process of wastewater treatment by aeration is sequential in the SBR. It is based on aerobic biological treatment of the effluent due to conduction of an aerobic reactor per cycle, in sequential mode. Every day, a volume of 750 ml of wastewater is introduced into the reactor having a capacity of two liters with a useful volume of 1500 ml of a 1/6 diluted activated sludge, and kept under aeration. After stopping the aeration-agitation, begins a settling phase allowing the separation of sludge from the treated effluent. At the end of the cycle, an amount of excess sludge is removed and a fraction of the treated effluent is withdrawn and it’s volume replaced by a new raw effluent, then a new cycle begins. The SBR reactor used at the laboratory scale consists of a useful volume of two liters. The different phases of feeding, aeration, settling, extraction of treated wastewater and excess sludge removal are performed by programmers [Fig-1] .
The biomass concentration is stabilized between 3 g l-1 and 6 g l-1 of suspended solids by a purging of excess sludge. The SBR operates at low loads with a feed rate of 450 ml d-1 and a COD loading rate of 0.6 g d-1 with a 24 hours cycle.
1, 2, 3- Timers providing the programming aeration (1), input (2) and output (3)
4-Synthetic raw wastewater.
5-Treated wastewater.
6- Stirrer.
7- Valve on the sludge excess.
8- Level of mixed liquor used corresponding to a volume of 1500 ml (the reactor operation).
9- Level of mixed liquor after settling and decanting, which is the volume of settled sludge corresponding to a volume of 750 ml.

Chemical Oxygen Demand (COD): The determination of COD was performed by the method of potassium dichromate. The method is based on oxidation to a boil (150°C for two hours) of oxidizable matter with an excess of potassium dichromate in acid medium and in presence of silver sulphate (catalyst) and sulphate of mercury (complexing). COD values were determined by spectrophotometer type UV / visible brand JENWAY6105 and a wavelength of 585 nm [11] .
Total Nitrogen: Nitrogen compounds present in the effluent are oxidized to nitrate by alkaline persulphate solution in an autoclave (120°C/15 min). Then, the nitrates are determined by the method of sodium salicylate. The positive reaction produces a yellow colored product named paranitro sodium salicylate which is detectable at a wavelength of 415 nm [11] .
Microbiological Analysis: The wastewater samples are collected monthly in the input and output of the reactor. Microbiological analysis focused on the enumeration of total germs, total coliforms, fecal coliforms, fecal streptococci, staphylococci, yeasts and fungi. These germs are detected by the averaging method on agar plates. The culture media used and incubation conditions of the various organisms sought are listed in [Table-2] .

The identification of 13 bacterial isolates was performed by biochemical and molecular approaches. Eight isolates were identified by biochemical tests using the API 20 E, API 20 NE, API STAPH [12] and BBL Crystal Gallery [13] .

Five isolates were identified by amplification and sequencing of 16S DNAr. After DNA extraction and amplification by PCR (polymerase chain reaction), sequencing was conducted at the Regional University Center of Interface Fez (RUCI)-Morocco. PCR of 16S DNAr was performed using universal primers fd1 and rp2 which amplify a 1.5 Kb fragment of 16S DNAr of eubacteria [14] .
fD1: 5’-AGA GTT TGA TCC TGG CTC AG-3’
rp2: 5’-TAC GGC TAC CTT GTT ACG ACT T-3’

To perform PCR, an aliquot of 1 ml of fresh overnight culture is centrifuged for 5 min at 8000 x g at 4°C. The pellet was washed and suspended 3 times in 1 ml of distilled water. After centrifugation at 8000 x g at 4°C, the pellet is then resuspended in 100 µl of distilled water. The suspension is subjected to thermal shock: 3 minutes at -20°C followed by 3 minutes at 95°C. This sudden change in temperature allows a breakdown of cells and a release of components including DNA. After centrifugation at 12000 x g at 4°C for 5 min, 2 µl of supernatant containing the DNA was used for PCR reaction.

PCR was performed in a volume of reactional mixture of 20 µl containing 1.5 mM MgCl2, 0.2 mM of each dNTP, 1 µM of each primer, 0.1 units of Taq polymerase (Promega) and approximately 20 ng of DNA extract. The reaction starts with a step of DNA denaturation at 95°C for 2 minutes followed by 35 cycles each comprising a denaturation phase at 94°C for 30 seconds, a phase of hybridization at 55°C for 30 seconds and an elongation phase at 72°C for 1min, 30 seconds. The PCR reaction ended with a final extension step of 2 minutes at 72°C. The PCR tubes are carried in the thermal cycler (Techne Cambridge Ltd, 1996). The control PCR tube contained water instead of DNA.

The agarose gel was prepared by dissolving 1g of agarose in 100 ml of buffer solution TAE 1X. Ethidium bromide was added to a final concentration of 0.5 mg ml-1. The buffer was added to 1/5th of the sample. The size of the used marker was 1kb DNA Ladder.

The results show that the effluent pH is basic, requiring neutralization by adding acetic acid before biological treatment. The characteristics of the raw effluent of SDCN show that the organic pollution is essentially in a soluble form and that the CODS is representing 78.5% of CODT. The average ratio of COD/BOD5 which is of 2.5 suggests that the biological treatment of this effluent is possible. The report BOD5/N/P being of 100/2.3/2.3 shows that the aerobic treatment could be done without nutritional supplementation [Table-1] .

Temperature is a critical factor as it has a mean control of biological processes. The temperature in the SBR ranged between 23°C at the start of the cycle and 25°C at the end of it. During the treatment, we have noticed an increase in pH with time while the pH at the entrance of SBR is equal to 7 and at the end of treatment equal to 8.7. A likely source of increase in pH is ammonification of organic nitrogen [15] .

Dissolved oxygen is an essential element used by nitrifying bacteria to carry out the oxidation reactions. The ventilation system must be designed to maintain periods of ventilation at a minimum concentration of 3 mg O2 l-1. The concentration of dissolved oxygen in the SBR ranged from 4.2 mg O2 l-1 at the start of the cycle to 7.8 mg O2 l-1 at the end of it and 4.6 mg O2 l-1 at the end of sedimentation. These results are similar to those of other authors [16,17] who used a dissolved oxygen concentration of 0.7 mg O2 l-1 at the start of aeration. After 30 minutes of aeration, the dissolved oxygen concentration increased to 5 mg O2 l-1, then it increases gradually up to 6 mg O2 l-1 after 23 hours of aeration and 2.2 mg O2 l-1 at the end of the settling. This increase is explained by [17] by an endogenous respiration and biodegradation of organic matter.

We conducted a physical-chemical analysis of sludge before and after inoculation of SBR for an adjustment period of two months. The results of these tests show a remarkable difference in the concentration of chemical parameters. This means that during the biological treatment, there is an assimilation and transformation of organic matter in the effluent by aerobic microorganisms in mineral matter and new microbial biomass reflecting:
• Reduction of the concentration of chemical parameters,
• Growth of biomass sludge,
• Treatment of wastewater to be treated.
The amount of suspended mater oscillates between a minimum value of 3.12 g l-1 and a maximum value of 4.41 g l-1. Values obtained are within the standards because the design process requires SBR between the amounts of 3 and 6 g l-1 TSS.

The average concentrations of total bacteria, total coliforms, bacteria of fecal contamination, staphylococci, yeasts and fungi in sewage of SDCN before and after treatment are presented in [Table-3] .
In raw SDCN sewage, the number of fecal coliforms is 100 cfu 100 ml-1 and 40 cfu 100 ml-1 for fecal streptococci. This load is lower than the abundances generally found in urban sewage which varies between 4.9 106 and 4.1 107 cfu 100 ml-1 [18-22] . The enumeration of FC and FS provides guidance on fecal microbial pollution origin [23] . Their number is correlated with the presence of pathogens that are harmful to public health, thus, those Indicators are widely used to determine the microbiological quality of waters especially treated wastewater destinated to irrigation.
In Morocco, as in other countries, the concentration of FC has been introduced in quality standards [24] . In the case of surface water for irrigation of crops eaten raw, the number of FC should not exceed 103 per 100 ml (with absence of salmonella in 5 l and vibrio cholera in 450 ml). Treated wastewater by the SBR carries a relatively lower bacterial load than raw sewage. Fecal indicators and fungi are less abundant in the treated wastewater, their abundances are of about 2 cfu 100 ml-1 for fecal coliforms, 3 cfu 100 ml-1 for fecal streptococci and 1 cfu 100 ml-1 for fungi. The SBR therefore contributes to an important reduction of germs, mainly FC with a percentage reduction of 98%. Hence, the treated effluent meets discharge standards for liquids to irrigate vegetable crops eaten raw. The elimination of micro-organisms takes place by three mechanisms: i) the settling of these germs which adsorb large quantities of suspended solids in the sludge and settle during the settling phase, ii) the biotic phenomenons of predation, antagonism and competition between different bacteria existing in the mixed liquor of SBR and iii) abiotic factors such as the decrease in concentration of total nitrogen and COD.

Evolution of the rate of removal of bacteria, yeasts and fungi depending on the rates of COD removal are shown in [Fig-2] , [Fig-3] and [Fig-4] .
The results show that the performance of SBR in terms of bacteria removal is important because the average turnover rate of microorganisms during the ten months of study exceeds 90.5%. The highest elimination rate was recorded for total bacteria and fecal streptococci, which abatement rates are respectively 99.4% and 100% during the month of April [Fig-2] .
During this month, we obtained the highest removal rate with a concentration of COD in the SBR input of 1030 mg l^-1 and an output of 38 mg l^-1, which corresponds to a reduction rate of 96.31%. Thus, we noted that the highest rates of removal of fecal coliforms, total coliforms and staphylococci were of 99.65%, 98.6% and 99.33% respectively [Fig-2] and [Fig-3] .
They are recorded at average input concentrations of COD of 1100 mg l^-1 and output of 70.4 mg l^-1 of COD, with a removal rate of 93.6%. This rate varies progressively according to the variation of COD removal.
The removal rate of yeasts and fungi with one cycle per day, which corresponds to an aeration time of 23 hours showed that the highest treatment efficiency of these organisms is recorded at concentrations of COD in the inlet and the reactor outlet of 1030 mg l^-1 and 57 mg l^-1 respectively, corresponding to a COD removal rate of 94.46%, reaching an average turnover rate of 99.5% for yeasts and 98 % for fungi [Fig-4] .
This difference in performance is explained by the rate of COD removal. By increasing this rate, the concentrations of bacteria, yeasts and fungi at the exit of the reactor decrease. It’s due to degradation of organic matter that increases the pH of the reactor, what acts negatively on the survival of enteric microorganisms. These results of abatement rates are consistent with the values found in literature and showing that biological processes can reduce the abundance of coliforms according to the type of process [25] . Removal rates are about 90% for activated sludge [26,27] and 99% for extended aeration activated sludge reactors and fixed culture [25] . These rates are related to the concentration of nutrients and organic matter in the reactor, the settling time, the hydraulic residence time and the quality of the raw sewage [25] .

The effectiveness of the reduction of microorganisms, abundances in biological treatment can undergo changes in the concentration of total nitrogen. It depends on the treatment system and the concentration of microorganisms. In general, the rate of elimination of different microorganisms sought is influenced by the increase of the removal rate of total nitrogen [Fig-5] , [Fig-6] and [Fig-7] .
According to the results recorded, increasing the removal rate of total nitrogen to 81.5% leads to an increased turnover rate of total germs, total coliforms and yeasts of 99.4%, 98. 6% and 99.5% respectively with mean concentrations of total nitrogen input of 4.2 mg l-1 and an output of 0.77 mg l-1. In the case of fecal coliforms, staphylococci and fungi, the highest removal rates are 99.65%, 99.33% and 96.66% respectively and are obtained with an average concentration of total nitrogen of the effluent at the inlet of 4.7 mg l-1 and at the output of 0.84 mg l-1 in the reactor, with a total nitrogen removal rate of 82%. The highest elimination rate was recorded for fecal streptococci during the month of April and reached 100%. During this month, we recorded the highest removal rate for total nitrogen (83%), its average concentration was about 4.3 mg l-1 in the input and 0.731 mg l-1 in the output. This result of reduction could be explained by the fact that the extension of the aeration phase promotes the oxidation of total nitrogen from the effluent by microorganisms in the mixed liquor suspended solids. The total nitrogen found in the effluent as ammonium, which is oxidized to nitrites in a reaction of nitration carried out notably by the bacteria of the genus Nitrosomonas. The nitrites ions are then oxidized to nitrates ions by a reaction of nitration carried out notably by members of the genus Nitrobacter during the process of nitrification [28] . The aeration phase is followed by a settling phase, which lasts 60 minutes and during which the nitrates formed during nitrification are denitrified to molecular dinitrogen by a facultative anaerobic flora (Pseudomonas sp.). Therefore, the decrease in turnover rate of the microorganisms studied is due to the phenomenon of competition between nitrifying bacteria (Nitrosomonas and Nitrobacter) and the pathogens studied. The nitrifying bacteria consume the total nitrogen which is a food for microorganisms. On the other hand, during the nitrification process, there is release of OH- ions that increase pH [15] . OH-ions are released into the outside environment, which promotes the increase of pH to 9.5 and acts negatively on the concentration of microorganisms. These results are consistent with the values found earlier by [29] . According to these authors, a biological treatment of wastewater by activated sludge eliminates 60-90% of bacteria, but on the other side, the sludge had little effect on elimination of protozoan cysts and helminthes eggs. According to [30] , an activated sludge process removes 90% of enteric bacteria. The elimination of germs takes place mainly through sedimentation and competition between micro-organisms in activated sludge.
The treatment efficiency obtained in our study may be explained by richness of the activated sludge of microorganisms endowed with great purification performance. This prompted us to try to identify these germs using the biochemical pathway and the molecular identification.

Given the importance of bacteria in the process of sewage treatment and in order to determine the kind of bacterial germs contained in the mixed liquor (LM1, LM2, LM3), the raw sewage (BE1, BE2, BE3) and the treated effluent (BS1, BS2), we conducted a characterization of the form, Gram and some biochemical characteristics of germs [Table-4] .

BE1 and BE2 isolates were isolated on the Chapman medium which is a selective medium for staphylococci. About morphology, we found that these germs are in the form of cocci grouped in clusters. These bacteria are Gram-positive, catalase positive, immobile, hence they likely belong to the family of Micrococcaceae. The profile of identification obtained by the API STAPH corresponds to the species Staphylococcus lentus (isolate BE1) and Staphylococcus sp. (isolate BE2). We conducted a characterization of the form, Gram and some biochemical characteristics of BE3 isolate [Table-5] .

The isolate BS1 is a Gram-negative, stationary, strictly aerobic, strict respiratory metabolism, catalase positive, oxidase negative. The isolate did not reduce nitrates to nitrites in complex mediums. It is characterized by a negative response to tests LDC, ODC, ADH, producing hydrogen sulfide, indole, beta-galactosidase and DNase. The isolate has a fermentative and oxidative metabolism. It ferments D-glucose, L-arabinose, D-cellobiose, lactose, alpha-D-melibiose, D-mannose, D-xylose, L-rhamnose. He likens adipate, L-arginine, 4-aminobutyrate, azelate, citrate, L-glutamate and Glutarate. Also, it is characterized by the assimilation of malonate, Oxoisocaprate, phenylacetate, pimelate and suberate. According to the identification key, isolate BS1 is the specie Acinetobacter lwoffi.
The BS2 isolate has the general character of the Enterobacteriaceae family. It is a Gram-negative bacillus, motionless, encapsulated, aero-anaerobic and oxidase negative. About enzyme tests, we note positive reactions with NO2, ONPG, LDC and VP. This means that the isolate reduces nitrate, metabolizes lactose (ONPG +), degrades amino acids by the enzyme lysine decarboxylase (LDC +) and produces acetoin (VP +). The negative reaction indicates that the isolate does not metabolize citrate (CIT) and amino acids requiring the enzymes ornithine decarboxylate and arginine hydrolase Di-(ODC-andADH) neither tryptophan, nor sulfur amino acids (H2S-). The isolate ferments glucose with gas production, D-mannitol, innositol, L-rhamnose, D-sucrose, D-melibiose, amygdalin and L-arabinose but doesn’t oxide, D-sorbitol. The key mentioned that this strain corresponds to Klebsiella terrigena.

The isolate BLM1 consists of a cocco-bacillus, Gram negative, stationary, non capsulated, catalase and oxidase positive, nitrate reductase generally positive, nitrite reductase positive, aero-anaerobic acidifies glucose and ribose without gas production, doesn’t produce urease, lysine decarboxylase, ornithine decarboxylase, phenylalanine deaminase and galactosidase. It does not produce indole or acetoin or DNase and does not liquefy gelatin. The production of hydrogen sulfide is negative on TSI medium. The isolate correspond to Neisseria saprophyte bacteria. BLM2 is a Gram-positive, catalase positive, strictly aerobic and immobile. Its biochemical characteristics are as follows: Enzymatic hydrolysis of the amide bond in particular, 4 MU-phosphate, enzymatic hydrolysis of Proline and Leucine-p-nitroanilide with the release of p-nitroaniline by the positive result of PLN (1H +), hydrolysis p-nitrophenyl-phosphate, PHO (4I +) and can metabolise arginine, ARG (1J +). A negative character is observed for other enzyme tests as Urea (4J-), Esculin (2J-) and L-tryptophan-AMC (4C-). The obtained ID profile corresponds to Corynebacterium jeikeium.
The isolate BLM3 is a Gram-negative, catalase positive and Gram-negative. His respiratory pattern is a strictly aerobic bacterium. The oxidase test was positive for the mobility of this organism (presence of the enzyme phenylenediamine oxidase), what shows that this bacterium belongs to the family of Enterobacteriaceae. The use of mid-Hajna Kligler showed that the isolate did not ferment glucose, while reaction with citrate simons was positive, that is to say, the isolate used citrate as carbon source. The bacterium isolated on ordinary agar is then subjected to identification by API 20 NE which has identified it as Pseudomonas putida.

To ascertain the taxonomic position of the bacteria isolated from activated sludge, we amplified and sequenced the 16S rDNA. These sequences were compared with sequences in the database. The program used is BLASTN 2.2.14 through the National Center for Biotechnology Information (NCBI). The results are given as a percentage of identity [Table-6] .
The molecular definition of gender states requires that the homology of 16S rDNA should be greater than or equal to 97%. Homology greater than or equal to 99% allows identification of species, while a score below 97% of homology does not allow identification [31] . [Table-6] shows the plausible species or genus for the different isolates studied and their percentage of homology with sequences of the 16S RNA. The five isolates corresponded to genera Pseudomonas sp., Streptomyces, Flavobacterium sp., Alcaligenes sp. and Neisseria sp. Among the bacteria isolated from activated sludge, the Pseudomonas isolate is a nitrifying bacteria.

The performance of the SBR increased significantly with increasing rates of reduction of COD. The highest elimination rate was recorded for total bacteria and faecal streptococci, with reduction rates of 99.4% and 100% respectively with highest rate of COD removal of 96.31%. Thus, we noted that the highest rates of removal of fecal coliforms, total coliforms and staphylococci were 99.65%, 98.6% and 99.33% respectively and were recorded at a rate of COD removal of 93.6%. The treatment of yeasts and fungi by a cycle per day which corresponds to an aeration time of 23 hours showed that increasing the treatment efficiency of these organisms is recorded when the rate of COD removal is 94.46%, reaching an average turnover rate of 99.5% for yeasts and 98% for fungi. According to these results, increasing the removal rate of total nitrogen to 81.5% leads to an increase in removal of total germs, total coliforms and yeasts to 99.4%, 98.6 % and 99.5% respectively. For fecal coliforms, staphylococci and fungi, removal rates are the highest; 99.65%, 99.33% and 96.66% respectively, recorded at a rate of total nitrogen removal of 82%. The higher reduction of microorganisms abundances of 100% was recorded for fecal streptococci, with an elimination rate of total nitrogen of 83%.
The identification of eight isolates from the bioreactor revealed species such as Staphylococcus lentus, Staphylococcus sp, Micrococcus sp, Acinetobacter lwoffi, Klebsiella terrigena, saprophytic Neisseria, Corynebacterium jeikeium and Pseudomonas putida. The identification by molecular pathway of the five bacterial isolates were deduced from the taxonomic position of each isolate belonging to the genera Pseudomonas and Streptomyces with 99% of homology, genus Flavobacterium with homology of 98% and genera Alcaligenes and Neisseria with homology of 97%.

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	Fig. 1- Schematic of the SBR process at laboratory scale
	Fig. 2- Evolution of the removal rate of total germs, staphylococci and fecal streptococci according to the rate of COD removal
	Fig. 3- Evolution of the removal rate of total coliforms and fecal coliforms according to the rate of COD removal
	Fig. 4- Evolution of the removal rate of yeasts and fungi according to the rate of COD removal
	Fig. 5- Evolution of the removal rate of total germs, staphylococci and fecal streptococci based on the removal rate of total nitrogen
	Fig. 6- Evolution of the removal rate of total coliforms and fecal coliforms based on the removal rate of total nitrogen
	Fig. 7- Evolution of the removal rate of yeasts and fungi based on the removal rate of total nitrogen
	Table 1- Physico-chemical characterization of the raw effluent rejected by the Company of Soft Drinks of North of Fez
	Table 2- Culture media and incubation conditions
	Table 3- Mean abundances of microbial groups before and after effluent treatment by SBR
	Table 4- Biochemical characteristics of bacteria in the mixed liquor before and after treatment
	Table 5- Biochemical identification of BE3 isolated from the effluent at the entrance of SBR
	Table 6- Identification of species and percentage of homology