الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
Abstract
This work was conducted to study the ability of PGPRs strains to treat the agricultural drainage wastewater from El Mohete drain (Marioteya Canal) for irrigation the Mentha viridis cv. and Gladiolas grandiflorus cv. plants. Studied were focused on:
1-Collectingsamples from different places and different depth levels (summer and winter).
2-Determination of the morphological, chemical and microbiological properties of the water.
3-Counting, isolation and identification of pathogenic bacteria isolates (morphology- physiology- chemically) after each stage of sampling.
4-Determination rates of all the dominant of microbial group.
5-Determination of morphological and chemical properties of contaminated water compared to non-polluting on salinity, acidity, alkalinity, heavy metals, pesticides and nitrate nitrogen component.
6-Studying the ability to remove the pollution using some of (PGPRs) strains e.g. Bacillus megaterium , Bacillus subtillus , Bacillus circulans, Paenibacillus polymyxa , Pseudomonas fluorescens , Serratia sp. and Azotobacter sp. Azotobacter chroococcumAzo.5, Azo.9 and Azo.23 to treat the drainage water for irrigation of the Ment and Gladiolas plant .
a-Studying the optimization of heavy metals removal by (PGPRs).
b-Studying the ability of pesticides degradation by (PGPRs).
c-Studying the optimization of inhibition pathogenic bacteria using (PGPRs).
7-Design a test experimental pot for two crops for two agricultural season and irrigation with treated water and compared with untreated water and determination of the antimicrobial activity types as well as the characteristics morphological, physiological and chemical plants during the growing season.
8-Determination of some special enzymes for microbial activity such as dehydrogenase in soil experimental pots.
Results could be summarized as following:
1-The obtained results revealed that El-Mohate drain EC ranged between 0.993 to 4.29 dsm-1 through winter season meanwhile in summer season the highest value of EC was in location No.3 (6.1 dSm-1), on the other hands locations No.5 (Drain after mixing with drain of Nahia) was the lowest EC (1.3 dSm-1). PH in various sites ranged between7.3 to 7.87 during the summer season. While pH ranged between 7.12 to 7.98 during the winter season.
2-The total solid (TS) and total suspended solid (TSS) during summer season were (in Drain Allpiny) site the highest values where, 2600 and 1620 mg/L in total solids and suspended solids respectively. Mean while, Bridge kerdasa sit recorded the lowest values being 1800 and 370 mg/L total and suspended solid respectively.
3- The total solid (TS) and total suspended solid (TSS) during winter season were in location (No.7) the highest values where, 2130 and 1370 mg/L in total solids and suspended solids were recorded respectively. Meanwhile, location No.2 recorded the lowest values being 1110 and 260 mg/L total and suspended solid respectively.
4- The maximum BOD was 7 mg/L in summer season and the lowest values (1 mg/L) were observed respectively, meanwhile (4-1 mg/L) in winter season.
5-Chemical oxygen demand (COD), increased in site No.4 (Drain Allpiny) giving 50 mg/L while the lowest value of COD was in the first sample location (35 mg/L) during summer season, while in the winter season values are between 21-12 mg/L.
6-The nitrogen sources are different from site to other. Drain Nahia achieved the highest values of nitrite (8.258 ppm) while nitrate showed the highest values with sit No.3 (El-mohate after the Drain of confused Abdel Aal tribal) being 39.82 ppm. Meanwhile Ammonium was low in NO-2it is obliend with Allpiny after the end of the drain branch wind Nazareth sit being 6.113 ppm in winter season. while Nitrogen sources highly varied from one site to another where the values of NO-3, NO-2 and NH4 were lower in summer season than winter season, where NO3-1 recorded 4.29 ppm in Drain Allpiny, NO-2 was highest in Drain Allpiny site (20.18) while NH4 achieved with site No.8 (Allpiny after the end of the drain branch wind Nazareth) being 2.585 ppm.
7-The heavy metals observation was found among Zn, Co, Ni and Cu which recorded higher concentrations at locations 1, 8 and 10, to be 225.4, 177.5 and 107.7 µg Zn/L, 50, 48 and 46 µg for Co/L, 50, 47 and 48 µg for Ni/L, 50, 48 and 49 µg for Cu/L, respectively, in spite that they didn’t exceed upper limit concentration recommended by European Union for irrigation water (EU limit) previously noted in materials and methods. Besides, Cd concentration was undetectable in most locations but was found to be exceeding double maximum limit recommended by EU (3 µg/L) at location 10, recording 6.2 µg/L. On the other hand, Hg was absent in most locations reaching maximum at locations 2 and 8 recording 0.50 and 0.57 µg/L, respectively.
8- The residues of pesticides were ortho-phenyl phenol (OPP), Malathion (Mal), Chloropyrifos (ChPy) and Piperonyl butoxide (PiBu). In all samples the amount of OPP ranged from 0.03 to 0.14µg/L and wasn’t found in location 5. Mal wasn’t detected in samples of locations 1, 2, 4, 6, 7 and 9, whereas in samples from locations 3, 5, 8 and 10 Mal pesticide was detected in variable values of 0.1, 0.13, 0.09 and 0.07µg/L, respectively. On the other hand, ChPy was only detected in sample of location 5 recording 0.9µg/L. Concerning samples from locations 6, 7, 8 and 9, the PiBu was found at levels of 0.1, 0.09, 0.16 and 0.05µg/L, respectively.
9-Most of pathogenic counts increased in summer than in winter, Location No.4 (Drain Allpiny) recorded the highest counts for both total and fecal coliform being 28x102 and 20.8x102cfu/ml respectively. Samonella and Shigella were obtained 10x101cfu in location No.8 (Allpiny after the end of the drain branch wind Nazareth). On the other hand, total counts of bacteria (not pathogen) location No.2 (Drain of the El-mohate from the top of the bridge traffic Ncla) recorded the highest number during the winter season. The lowest numbers of total and fecal coliform were with location number 7 where, it recorded 2.3 and 0.8 (1x102cfu/ml), Salmonella and Shigella achieved the lowest numbers in both location No.1 and 2 (1x101cfu/ml). While total count of bacteria were the smallest value with location No.7 (1x103cfu/ml). Total coliform and fecal coliform during the summer season recorded the highest counts with location No.4 (Drain Allpiny), There are 33 and 27x102cfu/ml respectively. Meanwhile the lowest numbers of pathogen bacteria were 3 and 2x102cfu in location No.7 (Drain of the El-mohate from the top of the bridge traffic Ncla). Pathogen bacteria (Salmonella and Shigella) were founding in location No.2 5x101cfu/ml but location No.7 achived the lowest numbers 1x102cfu/ml. Location No.4 (Drain Allpiny) was the highest number of total count of bacteria being 31x103cfu/ml, while location No.3 was the lowest (9x103cfu/ml).
Application of some processes to removing of pollutants in agricultural waste water in vitro:
1- Different concentration of thiourea solvent in agricultural wastewater led to decrease NO2 and NO3, while it increased NH4 and the best result was being 0.5g/100ml drainage.
2- The concentration of heavy metals as in control treatment (untreated) exceeded the critical limit with Copper 301.7 mg/L according to Urbian Standers whereas the concentrations of zinc, nickal and cobalt were the same or below the critical range (50 mg/L). There is no detection for both mercury and cadmium. The PGPRs could reduce the concentration of copper compared to control where the average Cu reduction was 19.7 % by (Azo.3) and 20.6 % by Serratia and 53.6 % by (Azo.5) and ranged between 85.42 to 86.74 with other PGPRs. For Mercury and Cadmium there is No detection in control and other treatments. The reduction of zinc, nickal and cobalt ranged between 2-20 % compared to control by all other Treatments. The mixture of PGPRs treatment is considered the best one as it resemble agricultural waste water sample.
3- The organophosphours pesticides like, Atrazine, Propamocarb, Piperonyl butoxide, Chloropyrifors are found in untreated waste water sample in concentrations, 0.1, 0.14 and 0.05 mg/kg for Atrazine, Propamocarb and Chlorpyrifos respectively. All PGPRs could induce biodegration of pesticides and B.megaterium, B.subtilius, Serratia, Azotobacter 3 and Azotobacter 5 could completely degrade and remove the residues of Atrazine whereas Bacillus polymyxa, B.circulans and Azotobacter 23 partially degraded Atrazine comparing to control in different ration. Regarding to propanocarb, all PGPRs could totally degrade and remove this pesticide and changing it to save products, whereas Pseudomonas flourscens reduced the concentration to 0.13 mg/kg compared to control. The same trend was observed with chlorpyrifs as B.polymxa, B.subtilis and B.circulans reduced the concentration to 0.05, 0.02 and 0.02 mg/kg respectively. Other PGPRs could completely reduce and remove the residues effect of this pesticide.
4- Antagonistic action between single strain of PGPRs and pathogenic bacteria (E.coli and Salmonella sp), the PGPRs exhibited antibacterial potential by inhibiting the growth of both pathogens. The highest inhibition action was Serratia marcescens, Bacillus sabtillas, Bacillus megateriuom and Pseudomonas fluorescens being 77.7, 77.7, 74.4 and 72.2 % with E.coli and 77.7, 72.2, 75.5 respectively, while the highest action being 83.3 % with Pseudomonas fluorescens against Salmonella sp.
5- Antagonistic action between dual strain of PGPRs and pathogenic bacteria (E.coli and Salmonella), The highest inhibition action are (Pseudomonas fluorescens + Serratia marcescens), (Pseudomonas flouracens + Bacillus subtilis), (Serratia marcescens + Bacillus megateriuom), (Serratia marcescens + Paenibacillus polymyxa), (Serratia marcescens + Bacillus circulans), ( Azotobacter chroococcum 9+ Bacillus sabtillas), (Azotobacter chroococcum 3+ Bacillus sabtillas), (Bacillus subtilis + Paenibacillus polymyxa) are found to be 77.7, 77.7, 75.5, 75.5, 75.5, 77.7, 77,7 and 77.7 % respectively with E.coil, and (Pseudomonas fluorescens + Serratia marcescens), (Serratia marcescens + Azotobacter chroococcum 5), (Serratia marcescens + Bacillus megateriuom),(Azotobacter chroococcum 9+ Azotobacter chroococcum 3),( Azotobacter chroococcum 9+ Paenibacillus polymyxa), (Azotobacter chroococcum 3+ Paenibacillus polymyxa) are detected 75.5, 75.5, 75.5, 74.4, 74.4 and 74.4 % receptively against Salmonella sp. respectively.
6- The antagonistic action between five mixtures of PGPRs being the best result it is exhibited antibacterial potential by inhibiting the growth of both pathogens. The highest inhibition action of (Serratia sp. + Azotobacter chroococcum 9 + B.megaterium + P.fluorescens + B.subtilis) was detected high against E.coli (88.8%) and biggest 96.6 % against Salmonella spp.
Bioremediation of homogenized drainage water using physical, chemical and biological treatments before application in pot experiments:
1- That water pH in various treatments ranged between 7-7.7. The salinity (Electric conductivity) EC (dSm-1) in different treatments was high in T1 5.4 dSm-1, followed by T4 and T5, whereas T2 and T3 gave 4 dSm-1. The total solids (TS) and total suspended solids (TSS) were the highest in T3 and T7 being 2975 – 2956 and 1842 -1845 mg/L respectively. The lowest value was 933mg/l TS and 320 TSS in treatment T10. On the other hand the biochemical oxygen demand (BOD) gave the highest value in case of T3, T7 and T8 treatments being 14 mg/L. whereas the lowest value T10 was detected in (2 mg/L). The chemical oxygen demand (COD) exhibited the highest values in T2, T3, T6, T7, T7 treatments and gave approximetly the same.
2- The drainage water treated with chemical (thiourea) gave the best reduction of NH4 being 0.63and 0.88 ppm while the treatment content PGPR and chemical (thiourea) achieved the lowest values of NO-2 being 0.95 and 0.97 ppm and 0.76 and 0.78 ppm NO-3. This may be due to thiourea has a role as nitrification inhibitors. Also, PGPRs have a role in utilization of NO-3 as source of nitrogen for growth.
3- Total, fecal coliform, Salmonella and Sigella In homogenized wastewater were shown the T1 derange water contemned 56x102 cfu/ml. total coliforme, 47x102 cfu/ml fecal coliform, 11x101 cfu/ml SS, and 11.9x104 cfu/ml total bacterial count. The treatment contents chemical addition (thiourea) were still highly counts in total, fecal coliforme, SS and total count bacteria either diluted water or undiluted water, where total and fecal coliform recoded 47, 56 and 48, 57 cfu/ml without dilution in T4, T5 respectively. Meanwhile, SS was 12 and 10 cfu/ml respectively. T.C showed 21.5, 21.2 x104 cfu/ml. respectively. Also the same treatments after dilution (T10 and T11) recorded high bacterial counts (all counts) in that diluted treatment or tap water. It was 22, 27, 6 and 61 cfu/ml of T10 while 24, 20, 6,70 cfu/ml T11 treatment for total, fecal coliform, SS and T.C respectively. And the growth of total count of PGPRs during 48 h for T2, T3, T6 and T7 where the counts reached 8.592, 8.611, 8.594 and 8.6 OD at 48 h respectively.
Two pot experiments were used:
1-The first Experiment was Gladiolus grandiflorus cv.
a- Marked influences on plant height were observed due to supplying plants with the different treatments in one season, where T9 exhibited better plant height (104 cm) and length of cut spike (94 cm) than all other treatments. Control treatment recorded the biggest diameter (1.92 cm) of the first floret and the highest floret number (14.33) and the highest leaves number (10.33). Number of days from planting to flowering was 132 days for T4, Corm circumference 16 cm for T9 and number of cormlets/corm was 19.66. Spike length in all treatments gave the lowest value for T1 flowed by T5. Whereas T8 and T 13 recorded the highest spike length value (89.66 and 85.66 cm) respectively.
b- Chlorophyll a, b and carotenoids during first month from experiments where, wastewater diluted by tap water (50:50) with inoculants and chemical treatment plus 50% NPK (T12) gave the highest values of chlorophyll a and b (9.24 and 9.95 mg/g) in first month from experiment. Meanwhile treatment number 13 was high load from carotenoids pigment. In the flowering stage of experiment treatment (which contents wastewater diluted and incubated with PGPR and NPK 100%) was superior treatment in content of chlorophyll a and b (9.94 and 10.82 mg/g) respectively, but wastewater untreated (T2), wastewater undiluted with PGPR and 100 NPK( T3) gave the best content in Gladiolas carotenoids being 5.0 and 5.08 mg/g respectively.
c- Contents of NPK in spike stem T8 and T9 treatments were high significant of nitrogen content, being 2.53 and 2.50% respectively and not significant between them. On the other hand, first treatment was the lowest of nitrogen percentage. Therefor phosphorus content in Gladiolus plant achieved highly significant percentage of phosphorus either with treatment wastewater plus microbial and chemical with NPK 100% or wastewater diluted with PGPR and 50% NPK treatment being 0.89 and 0.86 % respectively. The some previous treatments were the prefect in potassium content in plants (2.13 and 2.12 %). and carbohydrates content in plant (44.79 and 43.65 %) respectively.
d- soluble nitrogen in soil and in the drainage irrigation filtrates varied from one treatment to another where in general a remarkable increase of NH+4 in the drainage irrigation filtrate than in soil and the highest ammonium values were obtained with the treatments 3A 1.75, 3B 1.81, 6A 1.22, 6B 1.4 in soil and 1.871 in irrigation filtrate and 8B 1.110 in soil and 0.691 ppm in irrigation filtrate. Treatment 6B and 5B exhibited the optimum NO-2 where they recorded 1.1 in soil and 1.23 ppm in irrigation filtrate respectively. In concern, Total nitrogen in soil 8B obtained higher valve 0.073 ppm than all other treatments whereas in the drainage irrigation filtrate Total nitrate for 4B treatment recorded 1.91 ppm more than all other treatments. The increase of NH4-N in the drainage filtrate more than in soil.
e- The pathogenic bacterial counts are reducing with PGPRs treatments 8B (T8= Wastewater dilution with water 50:50 microbial treatment with PGPR and using fertilizer (NPK) 50%) in irrigation water filterate and in soil respectively.
f- The dehydrogenase values increased to maximum values specially with treatment T3 (Wastewater microbial treatments using fertilizer (NPK) 50%) which recorded 507.2 mg cfu/g dry soil/day.
2- The Second Experiment was Mentha viridis cv.
a- There were significant differences among treatments of wastewater using in irrigation plants. Inoculation with plant growth promoting rhizobacteria significantly increased the morphological parameters of mint plants. T9 possessed the highest leaves number (203.33), plant dry weight (12.9 g) and Root length (43 cm). plant height the results being T5, T8 and T11 statistically increased plant height of Mentha viridis plants and the superiority was for T5 which recorded optimum mint height 37.66 cm.
b- Chlorophyll A was superior with treatment T5, it was 9.16 mg/g while the lowest treatment (T4) it obtained 8.31and 6.52 mg/g for Chlorophyll A and B respectively. The highest value of Chlorophyll B with T9 in content of chlorophyll B where it was 8.06 mg/g, on the other hands treatment (T8) was the best in carotenoids (3.36 mg/g) during the first stage after 10 days. After 20 days from sowing treatment which content mix of microbes and chemical treatment with NPK 100% as recommended dose for Mint achieved the best content in chlorophyll A 9.66 mg/g compared with control (Tap water) it obtained 8.01 mg/g from chlorophyll A. The same treatment (T9) was highest value of chlorophyll B (10.82 mg/g) but treatment (T4) which content chemical treated with 50% NPK achieved 4.7 mg/g from carotenoids. After 40 days where, treatment was water diluted with tap water (50:50) with inoculants of PGPR with NPK 100% (T10) was better than all treatments in content of chlorophyll A and B being 9.11 and 8.06 mg/g respectively. Meanwhile treated with microbes and chemical plus NPK 100% was the best treatment in carotenoids pigment in Mint plant (2.67 mg/g).
c- Content of NPK in spearmint plants in both treatments number T8 and T9 were obtained the best significant value of spearmint nitrogen being 4.05 and 4.15 % respectively. Also, the same treatments (T8 and T9) recorded the highest significant values of phosphorus percentage (0.59 and 0.55 %) but treatment which content wastewater with PGPR and NPK 50% waste was the best. Potassium in spearmint was more significant with both treatments T8 and T9. It was 3.06 and 3.12 % respectively. On the other hand, treatment contents wastewater only was the lowest value of potassium percentage (1.69%), carbohydrates (8.05 %) respectively.
d- soluble nitrogen in soil and in the drainage irrigation filtrates varied from one treatment to another where in general a remarkable increase of NH+4 in the drainage irrigation filtrate than in soil and the highest ammonium values were obtained with the treatments 3A (1.75), 3B (1.81), 6A (1.22), 6B (1.4) in soil and 1.871 in irrigation filtrate and 8B (1.110) in soil and (0.691) ppm in irrigation filtrate. Treatment 6B and 5B exhibited the optimum NO-2 where they recorded 1.1 in soil and 1.23 ppm in irrigation filtrate respectively. In concern, total nitrogen in soil (8B) obtained higher value (0.073 ppm) than all other treatments, whereas in the drainage irrigation filtrate, total nitrate of 4B treatment recorded (1.91) ppm more than all other treatments.
e- The dehydrogenase activity is increased to maximum values especially with treatment T3 (Wastewater microbial treatment using fertilizer (NPK) 50%) that recorded 507.2 mg Tpf/g dry soil/day.
f- The percentage of oil content /100 g in mint plant was marked higher in both T8 and T9 where they both obtained the highest percent 1.6 %. All other treatments exhibited less percentage.
g- Regarding to the effect of irrigation with treated derange water on the chemical composition of Mentha viridis volatile oil, it could be concluded that, the maximum linalool and limonene content (64.22 and15.01) recorded with T9.
The PCR sequence was determined to identification of two pathogenic isolates they were detected to Salmonella enteric and Escherichia coli.