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Abstract
In this study, the current status of a wastewater treatment plant at Beni-Suef city was investigated to evaluate the efficiency of the treatment process and to study the quantities and diversity of microorganisms in the facility with the objective to make better use of the microbial resources in this interesting environment.
Three sampling runs were carried out to monitor the variation in the physico-chemical parameters in the three main sites of the wastewater plant. Changes in these parameters are presented in Table (2). The pH was around 7 which is normally optimum for activated sludge operations. The influent site recorded high values in all tests; TS, TSS, TDS, COD, BOD5,O&G, NH3 and heavy metals; except in DO, NO3 and NO2. This is due to that the wastewater in this site was not treated yet and the wastewater usually contains complex organic compounds and inorganic substances.
The dissolved oxygen increased from 0 in the influent site to 6 mg/1 in the effluent after chorine treatment by using mechanical surface aerators in the biological treatment tank to ensure the aerobic well-mixed condition. NH3 decreased from 17 mg/1 in the influent to 10 mg/1 in the effluent due to nitrification. This is clearly indicated by the increase in NO3 and NO2 from 0 in the influent site to 4.8 mg/l and 0.452 mg/1, respectively, in the effluent after chorine treatment indicating good biological treatment. The recorded values for NO3 and NO2 are lower than the expected mainly due to anoxic conditions where oxygen competes effectively with nitrate as a final electron acceptor in respiration.
Other physico-chemical characteristics decreased significantly from the influent site to the effluent before and after chorine treatment, as shown in Table (2), where BOD5 decreased from 280 mg/1 to 4.2 mg/1 (98.5% removal), COD decreased from 488 mg/1 to 18.7 mg/l (96.4% removal) and TSS decreased from 248 mg/1 to 16 mg/1 (93.5% removal) indicating good treatment in the plant. It is worthy to mention here that the quantities of the heavy metals entered the plant in the influent site decreased also significantly; even the chromium amount disappeared completely; except in the case of lead.
Numerous colonies of bacteria, actinomycetes and fungi formed on the plates after the incubation period. The greatest total number of microorganisms was 3.85x106 CFU/ml in the influent site on nutrient agar. The smallest total number of microorganisms was 1.34xl03 CFU/ml in the effluent site after chorine on yeast extract-malt extract agar. The average number of microorganisms, in order of 106 CFU/ml, is in agreement with most similar counts in domestic wastewater plants.
Among the total microflora appeared on the plates, 30 different bacterial isolates, 17 different actinomycete isolates and 6 different fungal isolates were isolated from the wastewater samples. Those isolates were then identified by their cultural, morphological and biochemical (in case of bacteria) characteristics and the predominant microorganisms are shown in Table 3 and some representatives are shown also in Fig. 2 . The table lists only the aerobic microorganisms that could be cultured on nutrient media.
The bacterial population was dominated by members of the genera Pseudomonas (20%), Escherichia (16.67%), Bacillus (13.33%), Flavobacterium (6.67%), Staphylococcus (6.67%), Streptococcus (6.67%) and Xanthomonas (6.67%). Our results also showed that members of the nocardioform genera, Nocardia, Gordonia and Rhodococcus are more dominant in addition to Streptomyces and Nocardiodis.
It is clear from the results in Table 3 that the 6 isolated fungal strains belong to only 2 genera, Penicillium and Aspergillus. Some species of protozoa and metazoa, e.g. rotifers, were observed during microscopic examinations of the samples.
The capabilities of the selected actinomycetes for treatment of the wastewater sample collected from Beni-Suef Wastewater Treatment Plant are summarized in Table (4). It is obvious from the results that the starting values of biochemical oxygen demand (BOD5), chemical oxygen demand (COD) and total suspended solids (TSS) in the sample (raw waste before treatment) were 283 mg/1, 498 mg/1 and 416 mg/1, respectively.
The results (Table 4 and Fig. 4) revealed that high BOD5 removal efficiency was achieved by strain C11 followed by the strains M13, C12, B21 and C13. The BOD5 concentration decreased from 283 mg/1 in the raw waste to 13 mg/1 in the effluent after treatment with strain Cll which means a BOD5 removal efficiency of 95.4 %.
Also, the highest efficiency of COD removal was achieved by strain Cll, which decreased the COD concentration from 498 mg/1 in the raw waste to 79 mg/1 in the effluent corresponding to 84.1 % removal efficiency. The remaining COD is the amount of hard and/or non--biodegradable materials.
On the other hand, the highest TSS removal efficiency was achieved by strain B21 followed by strain Cll. The two strains showed TSS removal efficiencies of 98.7 and 96.8 %, respectively.
In general, high quality effluent was delivered by treatment with the strains Cll, C12, C13, B21 and M13 with respect to the measured three parameters (BOD, COD and TSS). The ultimate function of the biological treatment is to remove the organic matter to meet the corresponding discharge standards set by the local government. According to the local standards applied for Beni-Suef Wastewater Treatment Plant, the final effluent should have less than 60 mg/1 of biochemical oxygen demand (BOD5), 80 mg/l of chemical oxygen demand (COD) and 50 mg/1 of total suspended solids (TSS) (Law 48/82 Discharge into non potable surface water). Therefore, the isolated strain C11 is found to be the most efficient organism in the biological treatment and the only organism met the standards as it produced a final effluent with 13 mg/1 of biochemical oxygen demand (BOD5), 79 mg/1 of chemical oxygen demand (COD) and 10 mg/1 of total suspended solids (TSS). This means that the organism Cll is highly efficient in removal of BOD5 and TSS, but less efficient in removal of COD.
Capabilities of the selected isolated strains for removal of heavy metals:
The results in Table (5) and Fig. (5) indicated that a significant decrease in the concentrations of the five tested heavy metals (Cu, Fe, Mn, Pb and Zn) is achieved by the actinomycete strains under study. The percentages of removal are 3.3% - 77.5%, 51.3 - 95.5%, 65.5% - 90.9%, 2.3% - 56.5% and 46.1 - 80.7% for Cu, Fe, Mn, Pb and Zn, respectively.
The highest Cu, Mn and Zn removal efficiencies (76 %, 84.8 % and 80.7%) were achieved by strain Cll (Table 5 and Fig. 5). On the other hand, the highest Fe removal efficiency (95.5%) was achieved by strain B21, while the highest Pb removal efficiency (53.3%) was achieved by strain M13.
In conclusion, strain C11 is the most efficient organism in respect to biological treatment of the wastewater sample and decrease of heavy metals’ concentrations in the sample.
One of the main objectives of the present study was to screen the actinomycete strains from the wastewater samples for productions of industrially important enzymes, and then purification and characterization of the most interesting produced enzymes were carried out. In this regard, the isolated ten actinomycete strains were screened for their amylolytic, cellulytic, lipolytic and proteolytic activities.
The results showed that the wastewater actinomycetes have good capabilities for the production of the tested enzymes. It was found that all the ten actinomycete isolates gave moderate amylolytic and high proteolytic activities; nine isolates gave weak cellulytic activities, whereas only seven isolates recorded very weak lipolytic activities. The results of the enzymatic activities are shown in table (6) and the standard curves used in determination of amylolytic, cellulytic and proteolytic activities are given in fig. 6 and 7, respectively.
Data recorded in table (6) revealed that the protease production was very high comparing to the other tested enzymes and that the actinomycete strains Cll was the most potent organism in protease production. Therefore, Cll was selected for further studies including the production of protease, purification and characterization of the enzyme and characterization of the strain.
The best incubation period for protease production by C11 was investigated and 4 days was found to be the best incubation period for maximum enzyme production.
The results of the effect of temperature on protease production by Cll showed that the protease under study is a mesophilic enzyme which has a good enzymatic activity in the temperature range of 25 - 45°C. The optimum temperature for enzyme production is 30°C, while the enzymatic activity is very low in the range of 10- 20°C and decreased sharply after 30°C till 60°C; the maximum temperature
The results revealed that the protease enzyme under investigation has a wide pH range for activity. The maximum enzyme activity was obtained at pH 8 and the activity declined dramatically with the increase or decrease below 7 in the pH value. Also, the optimum pH value for maximum protease production by C11 was attained at pH 8.
It is obvious from the recorded results in Fig. (11) that the optimum casein concentration (substrate concentration) for protease production by Cll was 0.6 g/1. The highest enzyme productivity was in the range of 0.3 to 0.9 g/1 of casein which followed by a decline at higher concentrations.
The results shown in Fig. (12) clearly indicated that starch was the best carbon source for protease activity followed by glucose and the other tested carbon sources. Then, the effect of starch concentration on enzyme production was evaluated. The results (Fig. 13) showed that maximum enzyme production occurred in the presence of 1% starch concentration. The enzyme activity declined with further increase or less starch concentrations. Sodium chloride usually affects the growth and enzyme activity. The results (Fig. 14) revealed that the maximum protease activity was recorded at 0.4% NaCl, while less activity was recorded at lower or higher percentages of NaCl.
Production of the protease enzyme to be used for further studies, including purification and characterization of the enzyme, were performed by culturing Cll on starch casein broth medium prepared based on the optimal concentrations reported above.
The purification process includes essential steps as preparation of the cell free filtrate; precipitation of protein using ammonium sulphate or other precipitants as low molecular weight alcohols; dialysis; and then passing the enzyme preparation through a gel using different column chromatography like sephadex G200 column.
In this study, fractional precipitation of the produced protease enzyme was carried out firstly by ammonium sulphate, since it is highly soluble in water, cheap and has no deleterious effect on protein structure. After precipitation and during purification, the protein content was determined with the reference standard curve given in Fig. 15.
The results revealed that increasing the concentration of ammonium sulphate resulted in an increase in the specific activity of protease up to 80% saturation and then the specific activity decreased at higher concentrations (Table 7).The protein preparation, after ammonium sulphate precipitation, was dissolved in the least amount of tris-buffer (pH 9). Then, it was dialyzed against distilled water to get rid of the sulphate ions followed by concentration by dialysis against sucrose crystals to reach a minimum volume resulting in raising the purification fold for the precipitated enzyme many times.
The dialyzed crude enzyme preparation was applied onto column packed with sephadex G200. Data recorded in table 8 and represented graphically in figure 16 showed that there were one active peak in fractions 4-10, and the maximum specific activity, 7361.4 U/mg protein, was reached at fraction number 7.
The produced and purified protease enzyme was checked for purity and homogeneity by running on SDS-PAGE. The purity and homogeneity of the enzyme was confirmed as it gave only a single band on the gel with a molecular weight of about 50 kDa (Fig. 17).
It is important to study enzymes in a simple system (only with small ions, buffer molecules, cofactors, etc.) for understanding its structure, Kinetics, mechanisms, regulations and role in a complex system. Beside the numerable properties of protease, it was considered essential to investigate further on the potential of the protease, particularly the stability towards temperature, surfactants, and requirements of divalent cation (Ca2+) for its activity. Thus, it was important to study the stability of the purified enzyme under different conditions to understand its properties and expect its future applications.
The results showed that the purified enzyme is highly stable at 50°C and can resist and work well up to 65°C which means that this enzyme is moderately thermostable (Fig. 18). It was found also that the enzyme is highly stable at the slightly alkaline pH 7- 8 and moderately stable at pH 6 and 9(Fig. 19).
The effects of various metal ions on the stability of the protease enzyme under study were tested. The results showed that the enzyme activity increased gradually with the increase of NaCl concentration up to 4% and then decreased sharply (Fig. 20), which means that it is stable at NaCl concentration up to 4%.
The data showed also that cobalt chloride (Fig. 21) and ferric chloride (Fig. 23) greatly affected the enzyme stability negatively as the enzyme activity decreased with the increase in the salt concentration. On the other hand, increase in the concentration of cupper sulphate (Fig. 22) and lead acetate (Fig. 24) to certain value affected the enzyme activity positively and then the activity decreased with further increase in the concentration. The enzyme activity and stability was not much affected in the case of manganese sulphate (Fig. 25). from the recorded results, the protease enzyme produced by the wastewater actinomycete strain Cll under investigation with the reported characteristics can be used in many biotechnological applications which need further experiments to be confirmed.
The ten selected wastewater actinomycete isolates were characterized for better understanding of the diversity of actinomycetes in the wastewater habitats. The cultural characteristics of the actinomycete isolates All, A13, B21, B22, B31, Cll, C12, C13, Ml and M13 are summarized in Table (10).
The selected actinomycete strains were characterized by studying their cultural, morphological and biochemical characteristics which revealed that strain All is similar to members of genus Nocardia, strain A13 is similar to members of genus Rhodococcus, strain B21 has similarity with members of genus Nocardia, strain B22 is similar to members of genus Rhodococcus, strain B31 showed the characteristics which are typical for members of Nocardia, strain Cll has similar characteristics of
Streptomyces, strain C12 is typical for members of genus Streptomyces, strain C13 showed characteristics are also typical for members of genus Streptomyces, strain Ml has characteristics are typical for genus Gordonia, strain M13 showed characteristics are similar to those of members of genus Nocardiopsis.
The actinomycete strain C11 was selected for its interesting activities in treatment and enzymes productivities to be identified to species level, the most potent actinomycete strain C1l contained LL-diaminopimelic acid as the characteristic diamino acid of the peptidoglycan in the whole-cell hydrolysates, contained galactose as the only sugar. The polar lipid pattern revealed the presence of phosphatidyl ethanolamine, phosphatidyl inositol mannosides, diphosphatidyl glycerol. It is clear now that strain Cll has the same morphological and chemotaxonomical features of genus Streptomyces.
The membership of this strain to genus Streptomyces was also confirmed by the phylogenetic analysis of the 16S rDNA gene sequence as obvious from the phylogenetic tree (Fig. 28). It is evident from the phylogenetic data that strain C11 formed a distinct phyletic line of the Streptomyces 16S rRNA gene tree and it could be formally recognized as a novel species of the genus Streptomyces. This conclusion needs to be confirmed by further comparative studies with the closest phylogenetic lieighbours.