Presentation of the study sites
The study was conducted over the five most important wastewater treatment plants in the eastern region of Algeria, which contribute greatly to reducing the impacts of wastewater on the environment [8].
In addition to the purified wastewater produced, more or less important quantities of sewage sludge are produced in parallel accompanying the purification operation. This sludge must find a continuous destination and before valorizing them, it must be ensured that this is possible without any environmental or public health risk.
In this study, we have chosen the five major wastewater treatment plants in the North-East region of Algeria (Fig. 1).
Figure 1: Map of Algeria displaying the Location of the five wastewater treatment plants.
- The wastewater treatment plant of Bourdj Bou Ariridj (BBA) (S1): It is located in South of the city with a surface of 42750 m2 (Fig. 1). Its capacity is 150,000 equivalent inhabitants, with a daily flow rate of 30,000 m3/d.
The BBA WWTP is specialized in the treatment of domestic and storm water discharged by the city using the low-load activated sludge process.
The purified waters of the station are discharged into the OUED K'SOB, it is located about 200 m away from the station which supplies the K'SOB dam in the wilaya of Mila, used for the irrigation of the lands downstream. Moreover, its purified water is recycled in agriculture and the sewage sludge is used as fertilizer. In October 2013, the sludge produced by the WWTP was estimated at 180 m3 [9].
- The station of Sidi Marouane (S2): It is located in the north of the town (12 km northwest of the capital of the wilaya of Milan) (Fig.1). It is an activated sludge treatment plant with a capacity of 137711 equivalent inhabitants. It was constructed to reduce pollution of the Beni Haroun dam, one of the large dams in Algeria. It is called upon to deal with the wastewater of Mila, Grarem Gouga, Sidi-Marouane, Ras- El-Bir, Annouche Ali, Sibari 1, and Sibari 2. Once treated, the water produced by the plant will be discharged into the basin of the dam, which is located right beside it [10]. The amount of sludge produced estimated in November 2013 was24.386 m3 [9].
- The Ibn Ziad wastewater treatment plant (S3) of the wilaya of Constantine: Regionalizes 12 km from the city of Constantine towards the northeast. It is located between 6° 30, 6° 45 north and 36° 15, 36° 30 east. Its surface is estimated by12 hectares [11] (Fig.1). It is an activated sludge treatment plant, designed for 450000 equivalent inhabitants. Today it treats a part of the wastewater discharged by the city of Constantine with a low load.
- The wastewater treatment plant of El Rabta of the wilaya Jijel (S4): It is located west of the city in the region of El Rabta, 2 km from the capital of wilaya, it occupies about 5.9 hectares and was commissioned in June 2008 (Fig.1).The plant is intended to treat domestic wastewater from the city of Jijel and its surroundings. With a capacity of 150000 population equivalent in the first phase, and according to a future extension of the plant as a second phase, with 225000 inhabitants. In October 2013, the amount of sludge produced by the plant was 224 m3 [9].
- The wastewater treatment plant of Guelma (S5): It is located about 1 km north of the city on the right flank of the valley developed by the Oued Seybouse and on the national road No 21, leading to Annaba City, at the exit of the agglomeration.
It is functional since 18 February 2008, and occupies an agricultural land of 8 hectares [9]. The station is characterized by a daily flow of 43 388 m3 /d of wastewater [12], and a capacity of 200000 equivalent inhabitants. It uses the activated sludge process for treatment. The plant produced in October 2013 a quantity of sludge approximately 9288 m3 [9].
SAMPLING
Sampling was conducted from the drying beds of the WWTPs: Bordj Bou Ariridj, Sidi Merouane, Constantine, Jijel, and Guelma.
We carried out an average sampling from three samples from different places of the drying beds and according to the time of stay of the sludge; that is to say, liquid sludge (LS) 00 stay, median sludge (BS1) from 1 to 2 months and dry sludge (BS2) exceeds 6 months. 4 sampling campaigns were carried out for each station, one per season.
The sampling was done while taking our hygiene precautions. It was taken directly from the drying beds. Then, the sample was put in sterile plastic bags, well labeled and deposited in a refrigerated chamber (1 to 4 ° C), and then taken to the laboratory of the CRBt for analysis, in the shortest possible time, to carry out our microbiological analysis with a maximum delay of 8 hours before analysis [13].
The microbiological analyzes of this work were carried out at the level of the microbiology laboratory of the CRBt. While for the parasitology part, the analyzes were carried out at the level of the parasitology laboratory of the “DIDOUCHE Mourad” Hospital in Constantine.
ANALYTICAL METHODS USED
The search for and enumeration of bacteria in a sample to be analyzed or controlled first uses the general dilution technique. It allows the preparation of decimal dilutions of the sample suspension [14].
A master suspension (MS), solution or emulsion, obtained after a weighed amount (10 g) of sludge to be analyzed (sample) has been mixed for 20 min, if necessary using a homogenizer and observing appropriate precautions, with 100 ml of diluent (sterile distilled water) allowing the large particles to settle, if they exist [15].
After we performed a series of decimal dilutions by transferring 1 ml of the 1/10 dilution into 9 ml of sterile distilled water up to 1/1000 (10-3).
Enumeration of germs
Enumerations were performed using the most probable number (MPN) liquid enumeration method. Enumeration results are determined from the Mac Grady table [13].
We used the liquid media bromocresol purple lactose broth (BCPL), a simple concentration, provided with a Durham bell after seeding we incubated the cultures at 37 °C ± 2°C for 24 h to 48 h.
The E. coli bacterium represents, however, 80 to 90% of the thermo-tolerant coliforms detected [16]. Therefore, this parameter is referred to as E. coli instead of "thermo-tolerant" fecal coliforms.
For each tube of BCPL, showing a positive result, we transfer it to a tube of Schubert medium containing mannitol and incubated it for 24 h to 48 h at 44°C. Mannitol fermented by E. coli (generally in 98% of cases) gives gas under the same conditions as lactose but does not interfere with the production of indole during the degradation of tryptophan. After incubation at 44°C for 24 hours, we added a few drops of Kowacks reagent to the tubes showing cloudiness. A positive reaction corresponds to the formation of a red ring [17].
Fecal streptococci (Enterococcus or D streptococci or intestinal enterococci) are commensals of the intestine. They are considered a good specific indicator of fecal contamination.
Group D Streptococci or Fecal Streptococci are tested in a liquid medium. The technique uses two tests, namely:
- Presumptive test: Reserved for the search for Streptococci on Rothe medium, simple concentration;
- Confirmation test: Tubes found positive on Rothe medium (presence of cloudiness) are transferred to Eva Litsky medium after 24h of incubation at 37°C. The presence of cloudiness in the tubes with the formation of a purple ring at the bottom of the tube, confirms the presence of fecal streptococci [18].
- Salmonella spp.
The enumeration of Salmonella spp. in an SM and often complicated given their low concentration, it is, therefore, necessary to intervene with a method of enrichment. In our case, we used buffered peptone water (BPW), according to Protais et al. [19], we took 25 g of the sample (sludge) and dissolved it in 225 ml of BPW. The incubation was done at 37°C ± 1 for 24 hours, then for enrichment, we took 2 ml of the ETP medium (Pre-enriched solution) and put it in the tube containing the Cysteine Selenite Broth (CSB) medium, and then we incubated it at 37°C for 24 hours. Then, for secondary enrichment and isolation, the previous tubes containing the SFB that was incubated the day before were subjected to secondary enrichment of the new SFBS and isolation on Hektoen and Salmonella Shigella (SS) agar. Incubation was done at 37°C for 24 h [20].
Using the colony counter, the number of colony forming units (CFU) was counted on the plates. The general formula for viable counts on the plates is given in equation (1):
N (CFUs per mL or g) = ∑ C/[V x {n1 + (0.1 x n2)} x d x d] (1)
Where:
N: Number of CFUs per mL or g of the sample, PC: Sum of CFUs counted on all selected plates at two successive dilutions;
V: volume of inoculum added to each plate (mL);
n1: Number of plates selected in the 1st dilution;
n2: Number of plates selected at the 2nd dilution;
d: Dilution factor of the first dilution.
- Spores of anaerobic sulfite-reducing bacteria(Clostridium)
This is an investigation of strictly anaerobic bacteria, especially the genus Clostridium [21]. A temperature of 36°C ± 2 in 24 to 48 hours and on a deep agar, allows the development of this forme by giving white colonies surrounded by a black halo. It is the witness of the reduction of sodium sulfite (Na2SO3) -which is in the medium- in sulfide that in the presence of Fe2+ gives FeS (iron sulfide) and a black color [20].
For the search for parasites in the sludge, we proceeded to three methods of concentration, two techniques of enrichment by flotation [22] with iodomercurate of potassium (d=1.44). We also used the Dada-Lindquist [23] technique with sodium dichromate (d=1.2) for helminth eggs and a concentration technique using the Teleman-Rivas two-phase method modified by Bailenger [24] with ether and acetoacetic buffer for Giardia spp. cysts.
Data analysis
Data comparison was performed using analysis of variance (ANOVA), HDS, and TUKY test using XL stat 2016 and Graph Pad Prism 7 software.