الفهرس 
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Abstract
Extracellular polysaccharides (EPSs) are naturally produced by microorganisms into the environment facilitating their recovery. In nature, these gums are produced by microbes as exudates that provide them with defense against environmental influences. Environmental and soil textural conditions play a key role in plant establishment. One of the highly effective methods to enhance and improve soil structure is addition of conditioners such as synthetic and natural polymers that improve soil properties and plant growth. The present study was undertaken to produce different microbial extracellular polysaccharides varying in their structures under optimal nutritional and environmental conditions and evaluate their efficiency as soil conditioners on microbiological and physical properties of soil and plant growth .
Results could be summarized as follows:-
1- Isolation and selection of extracellular polysaccharide producing cultures:
Isolation of microbes capable of producing diversified types of extracellular polysaccharides was investigated; Xanthan, was produced from the Xanthomonas spp; dextran from the Leuconostoc spp; alginate from Azotobacter spp.; pullulan from Aurebasidium Pullulans, curdlan from Rhizobium. Sixteen bacterial isolates belonging to genus Xanthomonas were isolated on yeast malt agar and YGC medium from rotten vegetable samples. Twenty five bacterial isolates belonging to genus Leuconostoc sp.were isolated on McCleskays agar medium from vegetable and fruit samples. Twenty isolates of genus Azotobacter were isolated on Ashby’s agar medium from rhizosphere samples. Three fungal strains of Aurebasidium pullulans have the ability for pullulan production, and twelve bacterial isolates belonging to genus Rhizobium, were obtained from the Unit of Biofertilizers, Fac. Agric, Ain Shams Univ.
2. Screening of the potent cultures :
Screening was carried out based on their efficiency of extracellular polysaccharide production as a culture viscosity of liquid media. The most efficient cultures of Xanthomonas (Xc6, Xc11, Xc9, Xc13, Xc15), leuconostoc isolates (Lm11, Lm21, Lm24), Azotobacter isolates (Az5, Az10, Az15, Az16) . Rhizobium sp.isolates (R3, R7, R10) and three strains of Aurebasidium pullulans were selected for further studies.
3. selection of suitable media for extracellular polysaccharides production
The most efficient cultures for each one of studied exopolysaccharides were examined on different production media to select the suitable ones for polymer production. For xanthan production; med. A5 gave the highest production of xanthan and cell dry weight by Xanthomonas isolates (xc6, xc9, xc11, xc13, xc15). The highest values of dextran production and cell dry weights were obtained by Leuconostoc isolates (Lm11, Lm21, Lm24) on med B4. The maximum cell dry weights and alginate production were observed on med. C2 by Azotobacter isolates (Az5, Az10, Az15, Az16).The highest production of pullulan was achieved on D3 medium by tested strains .Med E2 gave the highest values of curdlan production and cell dry weights by Rhizobium isolates (R3, R7, R10).
4. Nutritional factors for producing extracellular polysaccharides:
The highest values of cell dry weights and xanthan production were obtained by Xanthomonas isolates (Xc6, Xc11, Xc13) on productive med. A5 containing sucrose (45gl-1) ammonium nitrate at C/N ratio of 27. The corresponding values for Xanthomonas xc6, Xanthomonas Xc11 and Xanthomonas Xc13 were 2975, 4540 and 3500 cP for culture viscosities respectively. The isolates Luecononstoc (Lm11, Lm21 and Lm24) achieved the highest values of dextran and cell dry weights on productive med. B4 containing sucrose (150 gl-1) and yeast extract + peptone as nitrogen source at C/N ratio of 31.5. The corresponding values of culture viscosity for Leuconostoc isolates Lm11, Lm21 and Lm 24 were 2650, 3220 and 4250 P respectively. The highest alginate production and cell dry weight were obtained by Azotobacter isolates (Az5, Az10 and Az15) grown in productive med. C2 supplemented with sodium citrate (60 gl-1) and yeast extract + ammonium sulfate as nitrogen source at C/N ratio of 9.68. The corresponding values of culture viscosities were 720, 1459 and 940 cp by Azotobacter Az5, Az10 and Az15 respectively. The highest culture viscosities and cell dry weight of Aurebasidium pullulans strains were achieved in productive med. D3 containing sucrose ( 50 gl-1 ) and glutamic acid + yeast extract + ammonium sulfate as nitrogen source with C/N ratio of 63.79. The corresponding values of culture viscosities by Aurebasidium pullulans 1, Aurebasidium pullulans 2, Aurebasidium pullulans 3 were 2800, 4680, 3180 cP, respectively. The efficient isolates of Rhizobim (R10, R7, R3) achieved the maximum curdlan production in productive med. E2 with glucose at (25 gl-1) and yeast extract at C/N ratio of 31.25 .The values of culture viscosities for Rhizobim R10, R7, R3 were 5060, 7298 and 9855 cP respectively.
5. Polymer production by the most efficient cultures under optimal nutritional conditions.
Xanthomonas Xc11 recorded 12.69 gl-1 of xanthan dry weight, 28.2% of biopolymer yield, sugar utilization efficiency of 95.17% and conversion coefficient 29.76% and 0.18gl-1h-1 of productivity after 6 days incubation period. The biopolymer dry weight , yield, sugar utilization efficiency and productivity of Leuconostoc Lm 24 were 16 gl-1 , 11.77%, 95.33% and 0.33 gl-1h-1 respectively. Azotobacter Az10 grown in modified medium with optimal nutritional conditions for 6 days record 10.7 gl-1 biopolymer dry weight, 17.83% yield, 85.37% of sugar utilization efficiency, 20.91% of conversion coefficient and 0.07 gl-1h-1 productivity.
Aurebasidium pullulans 2 gave 15 gl-1 of biopolymer dry weight, biopolymer yield 30%, sugar utilization efficiency 86.48% and 0.10gl-1h-1 of productivity after 6 days . Rhizobim R3 was grown in modified medium to recorded 9.75gl-1 of biopolymer dry weight, 39% biopolymer yield, 90.04% of sugar utilization efficiency and 0.17 gl-1h-1 of productivity after 6 days of incubation period .
6. Evaluation of extracellular polysaccharides as soil conditioners:-
In this experiment, two types of soils were treated by studied biopolymers ( xanthan, daxtran, alginate, pullulan and curdlan ) at different ratios; 0.0, 0.4, 0.6, 0.8, 1, 1.5, 0.2 ,0.3 and 0.5% for improving the soil properties under un-leaching and leaching conditions
a- Cumulative water added:
Data show significant differences between all bio-treatments and control (without polymer) in sand clay loam (A) and sandy loam (B)soils without and with leaching process. Under un-leaching conditions: the soil without any polymer required 750.2 cm3 and 811.3 cm3 of adding water for soil (A) and soil (B) respectively during the experimental period using 50% water depletion of field capacity whereas adding water for biopolymer treated pots ranged from 745 to 530 cm3 / pot for soil (A) and from 800 to 678 cm3 for soil (B) during 60 days. Dextran and alginate recorded the lowest amounts of added water at ratios of 2 and 3% with two types of soil. Under leaching conditions the quantities of water were 865.7 and 913.8 cm3 for soil (A) and (B) respectively during the experimental period. Dextran and alginate improved the water holding capacity indicated by the values of adding water which were reduced by them at ratios of 2 and 3% by 14 , 19.1% and 9.83 ,17.00% decrease in respective order for soil (A) but in soil (B), the reduction percentage were 16.32 and 16.32% for dextran treatment at the same ratios respectively compared to control treatment.
b- Soil porosity :
Under un-leaching conditions, there were significant differences between all bio-treatments and control for tested soils. In soils (A) and (B), the bacterial polymers; dextran and alginate amendment showed comparatively the highest increases in porosity especially at ratios 3 and 5%.The highest values of porosity (%f) were recorded by dextran and alginate with the two ratios(3 and 5%) being 40.7 and 37.0% in soil (A) 34.9 & 31.8% in soil (B) respectively. Under leaching conditions dextran showed the highest efficiency in the porosity whereas pullulan and curdlan gave the lowest values of porosity. The highest values were recorded by dextran at 2, 3 and 5% being 36.3, 36.9 and 36.9% in soil (A) but 31.2% was recorded with these ratios in soil (B).
c- Pore size distribution:-
Under un-leaching conditions, dextran improved the soil properties; in soil (A), water holding pores (WHP %) were increased by 45.8% whereas reduced the values of total drainable pores (TDP%) by 52.7% by using ratio of 2% compared to control treatment. Dextran amended soil(B) gave WHP% higher than the control by 34.02% whereas TDP% recorded values less than control by 47.22%. Under leaching conditions dextran amended soil (A) had the maximum WHP% and lowest TDP% at ratio of 2%. In soil (B), dextran achieved the highest efficient for Fcp%, WHP% and TDP% being 15.28, 34.3 and 50.4% respectively.
d- Available water:
Under un-leaching conditions after 60 days, available water of soil treated by biopolymers was significantly greater than that available water of control treatment (without polymer). Dextran treatment generally showed the highest records value of available water at all applied ratios comparing to other biopolymers. The highest values of available water were 22.4 and 24.6 cm3 for dextran at ratios of 2 and 5%. The same trend was observed on soil (B). Under leaching condition in soil A available water of soil treated by biopolymers increased for all bio-treatments with increasing the applied ratios. These parameters were greatest for dextran treatments followed by alginate and xanthan treatments which increased the available water by 39.67 and 66.94% when using ratios at 2 and 3% respectively. In Soil B; the same observation was recorded to insure that by increasing in the used of biopolymers ratios available water was increased. Generally, the values of available water of soil (A) were greater than those of soil B probably due to the differences in soil structures between them.
e- Stability and distribution of soil aggregates
Under un-leaching conditions in sandy clay loam soil (A) : biopolymer treatments improved the percentage of soil stable aggregates which increasing the aggregates with diameter above 1 mm to 10 mm and decreased the granules with diameters below 1 mm. Dextran amendment at ratio 2% gave increased the percentage of soil stable aggregates to record 23.6, 9.0 and 6.2% for aggregate with diameter from 1 - 2, 2 - 5 and 5 to 10 mm, respectively. In soil (B) dextran amendment at ratio 2% recorded high values of this parameter being 16.16, 4.69 and 4.0% for aggregates with diameters of 1-2, 2-5 and 5-10 mm respectively. The MWDwet showed significant differences between all bio-treatments and control. The beneficial order of enhancement of aggregate stability (MWDwet) was dextran> alginate>xanthan> pullulan > curdlan respectively in soil (A) and (B).Under leaching condition in soil(A): Dextran amendment at 2% gave increased the percentage of soil stable aggregates to record 20.66, 3.67 and 2.4% for aggregate with diameters from 1 to 2, 2 to 5 and 5 to 10 mm, respectively. In soil (B) dextran amended soil at 2% concentration gave increased the percentages of soil stable aggregates to be 12.21, 1.91 & 2.1 at 2.1% for aggregate with diameter ranged from 1 to 2, 2 to 5 and 5 to 10 mm, respectively. In soil (B), dextran (2%), alginate (3%) and xanthan (3%) enhanced the MWDwet of soil by 60.78%, 23.53% and 17.64% increases compared with control treatment.
7. Effect of environmental factors on extracellular polysaccharides production.
From, the previous experiment, the resultants indicated that dextran, xanthan and alginate showed very significant positive effect on soil structure. Therefore, in these experiments, some environmental factors were tested to study their effect on these on these parameters.
a- Incubation temperature
- For xanthan production, results indicated that biopolymer production increased by increasing temperature till 30°C which was more efficient on xanthan production and bacterial growth by Xanthomonas Xc11 after 6 days. The corresponding figures for culture viscosity and cell dry weight were 4200 cP and 4.78 gl-1, respectively at 30°C.
- For dextran production, the highest values by Leuconostoc Lm24were recorded at 25°C being 4560 cP and 4.25 gl-1 for culture viscosity and bacterial growth (cell dry weight ) respectively.
- For alginate production, the highest values of production and bacterial growth were obtained at 30°C. The corresponding data of culture viscosity and cell dry weight at 30°C were 1570 cP and 4.12 gl-1.
b- Inoculum age
For xanthan production, at 24 hrs. inoculum age, the highest values of culture viscosity and cell dry weight were recorded to be 4350 cP and 4.39 gl-1 by Xanthomonas Xc11 respectively. For dextran production, maximum culture viscosity (4460 cP) and biomass (4.3 gl-1) of Leuconostoc Lm 24 were found at 20 hrs of inoculum age; beyond that inoculum age, these parameters decreased to be 4250 cP and 4.15 g/L, respectively. For alginate production, the maximum culture viscosity of Azotobacter Az10 was achieved when inoculated with inoculum age of 24 hrs .
c.Inoculum size
For xanthan production, with increasing of inoculum size, culture viscosity and cell dry weight of Xanthomonas Xc11 increased to record maximum values at 10% inoculum size (5460 cP and 5.02 gl-1) whereas 1% inoculum size recorded the lowest values of xanthan production .
For dextran production, the inoculation with 10% standard inoculum gave the highest amount of dextran of 4370 cP for culture viscosity and 4.21 gl-1 for cell dry weight. For alginate production, inoculated medium by 10% active inoculum gave maximum culture viscosity and cell dry weight being 1467 cP and 4.01 gl-1.
8. Using industrial byproducts for extracellular polymers production:
All tested industrial by-products were used as sole carbon source for producing dextran, xanthan and alginate, in modified medium under optimal environmental conditions compared with the production on synthetic medium.
For xanthan production, Xanthomonas Xc11 exhibited the highest efficiency of xanthan production on glucose syrup as carbon source in growth medium but it was less than that produced on modified medium (synthetic medium) by 56%. For dextran production, glucose syrup gave the highest culture viscosity and cell dry weight (2950 cP, 3.55 gl-1) followed by sugarcane juice compared with other tested by-product.
For alginate production, molasses and glucose syrup were the most suitable as carbon source for production and biomass by Azotobacter Az10 ; they supported the maximum production of alginate and biomass after 6 days being 700 cP, 2.70 gl-1 and 750 cP, 2.8 gl-1 in respective order. Thevalues of culture viscosities were 44% and 40% less than those found on synthetic medium, respectively.
9. Molecular analysis of extracellular polysaccharides producing bacteria
The most efficient isolates for xanthan, dextran and alginate production, which attained the highest effects as soil conditioners, were identified. The selected bacterial isolates were identified by their cultural and morphological properties as a preliminary classification. Then the identity was confirmed by molecular identification using the universal 16 S primers.
Amplified sequences showed 97% similarity between Xanthomonas xc11 and Xanthomonas campestris strain ATCC 33913.The similarity level was 88% between leuconostoc Lm24 and leuconostoc mesenteroides strain ATCC 8293. The similarity level was 83% between Azotobacter Az10 and Azotobacter vinelandii strain NBRC 1026/2.
10. charcterization of extracelleular polysacchairide:
a- 13C NMR analysis , For xanthan production, xanthan showed six prominent 13CNMR resonances at 100 MHZ being; 92.12, 82.5, 79.18, 78.98, 78.65 and 73.0 ppm which are characteristic of linear xanthan that revealed to C-1, C-4, C-3, C-2, C-5 and C-6 respectively. For dextran production, the signal at 65.5 (64.55ppm) and the peak at 97.86ppm (98.3ppm) region indicated that the glucose residents in dextran are linked by α (1-6) glucosidic bond The assignments for different resonances of 13CNMR namely; 98.3, 71.59, 75.47, 69.59 ، 70.25 and 64.5 revealed to C-1, C-2, C-3, C-4, C-5 and C-6 respectively. For alginate production, the 13CNMR spectrum showed at 100 MHZ: 97.32 (C-1), 73.64 (C-3), 71.99 (C-2), 71.02 (C-5), 70.39 (C-4) and 61.60 (C-6) ppm, which are characteristics of linear alginate. The peaks which were observed in the region from 75 to 85 ppm indicated for alginate branched at C-2, C-3 or C-4.
b- FTIR analysis: the type of linkages and the functional groups of the most efficient biopolymers (xanthan, dextran and alginate) were characterized by FTIR spectroscopic analysis.
11. Effect of biopolymers as conditioners on growth of Lactica sativa plant and soil structure under greenhouse conditions:
A pot experiment was conducted at the Unit of Bio-fertilizers, Ain Shams Univ, using iceberg lettuce (Lactuca Sativa ) seedlings as test plant grown in sandy clay loam soil amended by biopolymers. The measured parameters refer to:
a. Growth performance
Fresh and dry weightS of Lactuca sativa plant were increased significantly from 4.70 and 1.75 g/plant for fresh and dry weight respectively in un-amended soil (control) to 6.55 and 2.85 g/plant for dextran and to 5.60 and 2.11 g/plant for alginate treatments respectively. Dextran at ratio of 2% exhibited the highest effect on fresh and dry weights of plants followed by alginate (3%) treatments after 55 days of transplanting.
b. Plant nutrient content
The most pronounced effects of this application were manifested in plant nutrient uptake contents grown with dextran treatment giving 0.03 gN /plant ، 0.02gP /plant and 0.05 gK /plant for plant uptake. Synthetic polymers at different ratios recorded significantly the lowest values of nitrogen, phosphorus and potassium uptake which ranged from 0.003 to 0.005 , 0.004 to 0.007 and 0.01 to 0.007 g/plant respectively.
c.Soil microbial activity:
CO2 evaluation increased significantly in all biopolymer amendments especially with dextran (2%) treatment. Synthetic polymers gave low respiration rate compared to biopolymer and control treatments. Dextran recorded the maximum respiration rate of 286 mg CO2/100g which increased by 85.71% over control treatment, and by 271.43% over polyacrylamide (2%) treatment.
The highest dehydrogenase activity was recorded with dextran treatment at 2% followed by alginate and xanthan treatments. Dextran treatment gave the superiority of dehydrogenase activity in the rhizosphere soil after 55days of transplant being 43.89 µg TPF/100g soil. It improved the microbial population (dehydrogenace activity) by 4.31 fold comparied with control treatment.
d.Soil structure as affected by the polymers :
1. Cumulative of water added
Data show that the lowest value of added water was observed by dextran. Generally, the order of reducing cumulative water added was dextran> alginate>xanthan> synthetic polymers 3% daiper polymer> 2% daiper polymer> 3% polyacrylamide>2% polyacrylamide in decreasing order.
2. Soil porosity.
The highest values of porosity were recorded by dextran being 39.13%, increasing by 11.1% than control. On the other hand, the lowest values were recorded by daiper polymer.
3.Pore size distribution
The lowest values of total drainable pores (TDP %) were recorded by dextran being 28.7%, against 58.29% for control treatment. On the other hand, the highest values of TDP% were recorded by synthetic polymers ranging from 50.65% with 3% to 52.15 with 2% of diaper polymer (DIP) .
4.Available water content (AW)
The highest values of AW % were recorded by dextran (24.71%) increasing by 90.08% than control; on the other hand, the lowest values were recorded by synthetic polymers ranged from 2.4, with 3% DIP to 3.13 with 2%DIP.
5. Stability and distribution of soil aggregates
Biopolymers treatments increased the percentage of soil stable aggregates especially the aggregates with diameters between 1 mm to 10 mm and decreased the aggregates with diameters below 1 mm. Dextran amendment increased the percentage of soil stable aggregates. Their corresponding figures were 31.24, 32.6 and 5.66% for aggregates with diameters in diameter 1- 2, 2 -5 and 5 to 10 mm, respectively. The highest values of MWDwet were recorded by dextran increasing by 128.4% than control followed by alginate and xanthan. On the other hand, the lowest values of MWD were recorded by synthetic polymers in ranged from0.66 to 0.81 mm.