3.1 as agriculture labor.This district is situated

3.1 Study Area:
This Project has “Nalgonda District” as Study area.Nalgonda district lies between in the North Latitudes 16 ° 25′ and 17 ° 50′ and East Longitudes 78° 40′ and 80° 05′. The district forms the southern part of the Telangana Region and is bounded in the north by Medak and Warangal districts,on the south by the Guntur and Mahaboobnagar districts,on the west by Mahaboobnagar and Rangareddy, and on the east by the Khammam and Krishna districts.
Nalgonda has a total of 1,115 villages and 59 mandals.It has an area of 14,240 sq km and a population of 2.8 million.Agriculture is the main occupation with 32 percent of the main workers classified as cultivators; and about 43 percent as agriculture labor.This district is situated in the upper catchment of the watersheds of the tributaries of Krishna river. Only about 40 percent of the district area is under cultivation as against available 70 percent.

Figure 1: Shape File of Study Area( Nalgonda District)
The 4 mandals include such as 1. Peddaa Adiserla Palle
2. Anumula
3. Peddavoora
4. Nidamanur
The Area of 4 mandals occupied: 1497.336 hectors. The Objective of this study is to analyse and to find the flow of water to the some areas of Nalgonda district and to find the percentage of area of cropping per year by collecting the data of consecutive years i.e from 2013-2017.
The following figure shows the drainage map.It indicates the flow dynamics of Nagarjuna Sagar canal system throught out the study area which is to considered.

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3.2Nagarjuna sagar canals:
Bed Width
Depth of
Flow Length of
Branches and

1 Right Canal 203 KM
73.5 M 3.78M 5342 KM 4.75 Lakh Ha.
11.74 Lakh Acres

2 Left Canal 179KM 29 M 6.71M 7722 KM 4.2 Lakh Ha.
10.38 Lakh Acres

Shape file of Study Area:
The folloeing figure shows the shape file of the Nalgonda district with the four mandals with the flow of the Nagarjuna Sagar canals.

Figure: Shape file of Study Area
3.2 Topo sheet data Study Area:
Survey of India is the first scientific department in Government of India, which provide information in terms of Topo-sheets (Maps) as part of mapping for the development of nation from 1767 AD onwards.
Survey of India has two series of Topo sheets
1.Defence series maps (DSM):These are based on WGS-1984 Datum and Lambert conformal conic (LCC) projection.
2.Open series maps (OSM):These are based on WGS-1984 Datum and Universal Transverse Mercator (UTM) Projection.Here datum will be same for b

3.4. Biochemical characterization of free and immobilized enzyme
3.4.1. Effect of temperature and pH on the lipase activity
As shown in Fig. 5A, the maximum activity of free and immobilized lipase was obtained at pH 8.0 and 9.0, respectively. Moreover, relative lipase activity of immobilized lipase was faintly lower than free enzyme in acidic pH, but marginally greater than in basic pH. Therefore, the immobilization process seems to expand the stability of the lipase in strict basic environments. Lipase activity in different temperatures were shown in Fig. 5B. The immobilized lipase showed a broad range of maximum temperature activity about 40-60 °C, compare to free enzyme. These results indicating the development of covalent links between protein and support, which may diminish conformational flexibility and result in preserve lid opening (Perez et al., 2011; Lu et al., 2009).

3.4.2. Thermal stability of free and immobilized lipase
Immobilization method is one of the most promising strategies to improve catalytic activity for the applied application. Consequently, to explore the thermal stability, free and immobilized enzyme were maintained in phosphate buffer (100 mM, pH 7.5) for 3h at 60 °C, and then the remaining activities were measured in the phosphate buffer (100 mM, pH 7.5) with pNPP as substrate. The lipase activity of both free and immobilized lipases was highest up to 45 min of incubation at 60 °C. The remaining activity of the free lipase is 50 % while the immobilized lipase reserved 85 % of its initial activity after 3h of incubation at 60 °C (Fig. 6a). These results evidently designate that the immobilization of lipases into mGO can avoid their conformation transition at high temperature, and improving their thermal tolerance.
3.4.3. Determination of Km and Vmax
Kinetic factors of free and mGO-lipase were investigated by calculating initial reaction speed with different substrate concentrations. As shown in Fig. 6B and Table 1, Vmax values of mGO-CLEA-lipase was slightly upper than free enzyme about 0.1 µmol/min, which directed the rate of pNPP hydrolysis was not significantly changed after mGO-CLEAs-lipase preparation. The same results were also observed for magnetic CLEAs of the other enzyme. In the case of mGO-CLEAs-lipase, the detected lower Km value state a better lipase affinity for the pNPP substrate, about 2.25 folds. It approves that conformational changes by the reason of enzyme immobilization assistance the protein to appropriately turn its active site concerning the substrate (Aytar and Bakir, 2008; Sangeetha and Abraham, 2008; Talekar et al., 2012).

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3.4.4. Reusability assay
Reusability of immobilized lipase preparation is a dominant factor for its commercial use in biotransformation reaction. The reusability of mGO-CLEAs lipase was measured up to 8 cycles. Enzyme activity of mGO-CLEAs lipase was the highest up to 5 cycles, but it continuously decreased over 5 cycles (Fig. 7a). Protein leaking was also investigated throughout reusability tests of mGO-CLEAs lipase. Results exhibited no lipase activity was detected in reaction mixture up to 4 cycles of lipase reusability test. These results recommend that suitable cross-linking of enzyme and mGO nanomaterials produced stable MGO-CLEAs lipase (Talekar et al., 2012).
Storage tolerant of both free and mGO-CLEAs lipase were also examined by storing them at 4 °C and checking the lipase activity. Results displayed mGO-CLEAs-lipase reserved about 75 % of its original activity after 30 days of incubation, wherein free enzyme missed its preliminary activity at the similar time (Fig. 7b). These results verified that mGO-CLEAs lipase had chief protection on the storage stability of lipase. These results designated that an active mGO-CLEAs lipase prevents protein leaking from mGO-CLEAs nanomaterials (Yong et al., 2008).

3.5. Biodiesel production from non-edible
Nowadays, non-edible oil resources as a favorable source for biodiesel synthesis has been admired for researchers. Ricinus communis is a small and fast-growing tree which is a highly productive and precocious maker of toxic seeds. In addition, it is very adjustable to diverse situations and has been broadly dispersed. The highest biodiesel synthesis (26 %) from R. communis oil was gained at room temperature after 24 h of incubation by Entrobacter Lipase MG10 (10 mg) (Fig. 8). Mehrasbi and co-workers described using of free C. antarctica lipase B (100 mg) constructing 34% of biodiesel from waste cooking oil at 50 °C after 72 h of incubation (Mehrasbi et al., 2017). Some excellent properties of MG10 lipase such as methanol-tolerant, and short time reaction make it capable as a latent enzyme for biodiesel creation from non-edible oils.
Remarkably, mGO-CLEAs lipase formed the highest biodiesel construction (78 %) from R. communis oil after 24 h (Fig. 5). Besides, the immobilized MG10 lipase enriched biodiesel construction from R. communis oil about 3.1 folds at diverse time of incubation, compare to free lipase (Fig. 5). De los Ríos reported 42% of biodiesel fabrication by consuming immobilized lipase of C. antarctica (De los Ríos et al., 2011).
As mentioned formerly, construction of several links between lipase and support, could reserve protein in open conformation and improved the enzyme rigidity with affiliate making of a protected micro-environment. Furthermore, it made a further active lipase cross-linking in mCLEAs lipase which evades enzyme leaking from composite and shield it against methanol solvent and the other by products (Talekar et al., 2012; Aytar and Bakir, 2008; Sangeetha and Abraham, 2008).

4. Conclusion
Lipase MG10 is a high potent lipase (thermostable, inducible, high methanol-tolerant, and short time reaction rate) which was isolated from Gehver hot spring. The CLEA of lipase MG10 was immobilized on the mGO. The lipase immobilization considerably developed the thermal tolerant, storage stability and the lipase reusability. In addition, the obtained nanocomposite displayed a shift to acidic pH, which is outstanding possessions for biodiesel construction. Biodiesel fabrication was also attained by 75% recovery from R. communis oil as non-edible oil feedstock which would have prospective in green and clean construction methods.

The authors express their gratitude to the Research Council of the Shahid Bahonar University of Kerman, Kerman (Iran) for financial support during the course of this project.

3.1.1 Cultures used in the study
The Neurospora crassa cultures (wild-type and mutant cultures having genetic markers) used during the study were obtained from Fungal Genetic Stock Center, Kansas City, USA. The following cultures were obtained during the study:
S. No. FGSC# Genotype Mating Type
1 FGSC #2489 74-OR23-IVA A
2 FGSC #4200 ORS6a a
3 FGSC #3661 alcoy csp-2 A
4 FGSC #3434 alcoy csp-2 a
5 FGSC #1206 al-1;arg-5 A
6 FGSC #4347 fl a
7 FGSC #4317 fl A
8 FGSC #2997 pyr-4 arg-12 A
9 FGSC #2998 pyr-4 arg-12 a
10 FGSC #7192 thr-2 arg-5 A
11 FGSC #7193 thr-2 arg-5 a
12 FGSC #8596 eas trp-3 A
13 FGSC #1290 cys-3 arg-5 A
3.1.2 Growth and maintenance of cultures
Vogel’s minimal medium having 1.5% dextrose and 1.5% agar was used for growth and maintenance of Neurospora cultures (Vogel 1956, 1964). The Vogel’s medium was prepared as follows:
Vogel’s minimal medium
Dextrose1.5 g
Agar1.5 g
Vogel’s stock solution (50X)2 ml
Distilled water100 ml
The method of preparation of stock solution is described below. While preparing stock solutions, salts were added in serial order and each salt was dissolved by continuous stirring before adding the next salt. CaCl2 was predissolved in 2 ml distilled water and added drop wise.

Composition of (50 X) Vogel’s stock solution
dH2O 75 ml
Na3citrate.2H2O12.672 g
KH2PO4 (anhydrous)25.00 g
CaCl2 (fused)0.378g
Biotin stock (5 mg biotin in 0.5 ml
100 ml of 50% ethanol)
Trace element stock0.5 ml
The final volume of the stock solution was made 100 ml using dH2O and 0.5 ml chloroform was added for preservative. The stock solution was kept in brown bottle at room temperature. The composition of Trace element stock was as follows
Composition of Trace element Stock Solution
dH2O47.5 ml
Citric acid. 1H2O2.50g
ZnSO4. 6H2O2.34g
Fe(NH4)2(SO4)2. 6H2O 0.662 g
CuSO4. 5H2O 0.125g
MnSO4. 1H2O 0.025 g
H3BO3 0.025 g
Na2MoO4. 2H2O0.025 g
Chloroform (0.5 ml) was added for preservation and final volume was made 50 ml by adding distilled water. The solution was stored in at 4-8oC in refrigerator. The cultures were grown at 34±2oC unless otherwise mentioned and stored at 4 oC for short term preservation and -20 o C for long term.

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3.1.3 Mutagenesis
The macroconidia of seven day old wild-type Neurospora culture (FGSC #2489 ‘A’) were mutagenised using Ethyl Methane Sulphonate (0.1 M). The mutagenesis was done using the modified method described by Mukati et al., 2015. The suspension was prepared in 0.05 M phosphate buffer and contained 42.5 x 106 conidia/ ml. Conidial count was done in Neubauer counting chamber. Conidia were treated with EMS for different time intervals (0 h, 0.30 h, 1 h, 1.30 h, 2 h and 2.30 h) after which centrifugation was done. Conidial suspension was washed twice with phosphate buffer and stored in refrigerator at 4±2°C. Mutagenised conidia were enriched by cold treating at 7-8°C for two days. The filtrate containing enriched mutagenised conidia was plated on 1% sorbose containing Vogel’s agar media and incubated at 34±2°C for 48 h. Ninety well isolated colonies were picked and transferred to Vogel’s minimal agar medium slants after 48 hours at 34±2oC. The slants were kept 34±2oC for 4-5 days and well growing Neurospora cultures were obtained. The subculturing was done in same medium for purifying the cultures.
3.1.4 Determination of mating type
Mating type of mutant cultures were determined in Synthetic crossing medium by Spot inoculation method. The fluffy culture (FGSC #4347 ‘a’) of known mating type was allowed to grow on Synthetic Crossing Medium for 5 days at 25oC. After 5 days, mutant cultures whose mating was to be determined was rubbed in circular manner on the grown fluffy plate. The plate was observed after two days of incubation at 25oC. The opposite mating type cultures (‘A’) had reacted with the fluffy culture (mating type ‘a’) and developed black colored ascospores while the same mating type did not show any change. Hence, the mating type of unknown mutant cultures were determined. The mating type reaction is shown in Figure . The composition of Synthetic Crossing medium is described below.

Fig. 1 : Mating type determination by Spot inoculation
Synthetic Crossing Medium stock solution composition (Westergaard and Mitchell, 1947)
dH2O950 ml
KNO31.0 g
K2HPO40.7 g
MgSO4. 7H2O0.5g
CaCl2 (Predissolved in 20 ml dH2O)0.1 g
Trace element stock (same as used in 0.1 ml
Vogel’s minimal medium)
Biotin stock (5 mg biotin in 100 ml0.1 ml
of 50% ethanol)
The final volume was made 1000 ml with distilled water.

3.1.5 Characterization of mutants
The morphological studies of mutant cultures were done by:
1. Observing colony characteristics- The mutant cultures were grown on Vogel’s minimal agar medium and incubated at 25oC and 34oC. After 24 h of incubation the colony morphology was observed and noted.

2. Microscopic observation- The mutant cultures were grown on Vogel’s minimal agar medium, incubated at 25oC and 34oC at different magnification (10X, 40X and 100 X). The defects in tip growth, hyphal morphology, branch initiation and branch formation were observed and recorded.

3. Growth rate- The growth rates of wild-type and mutant cultures were determined in race tubes (Ryan et al., 1943).

3.1.6 Inheritance of defects
The mutants were reciprocally crossed with the wild-type (FGSC #4200) culture. A month after crossing, random ascospores were individually picked, heat shocked at 60oC for 30 minutes and incubated at 34oC for 24 h so that a single ascospore grows into a mature culture. Twenty viable progeny were studied under the microscope to see the inheritance of defects in mutant cultures. The ratio of parental and mutant progeny was calculated. The mutant progeny was again reciprocally crossed with wild-type (FGSC #4200) to confirm the results. Twenty viable progeny were again studied to see the inheritance of defects in mutant progeny. The cross tube is shown in Figure 1.

Figure 1: Wild-type (FGSC #4200), cross tube (centre) and mutant (VU13-16)
3.1.7 Linkage group analysis
The tester strain, alcoy was used for determining the position of an unmapped gene on chromosome by a single cross. This strain contains four phenotypic markers which can be clearly. For assisgning the gene on the two chromosomes, it has three translocations, each translocation tagged with a phenotypic marker. The right arm of chromosome I and II was tagged by the marker albino-1 (al-1), right arm of chromosome IV and V was tagged by the marker colonial temperature sensitive-1 (cot-1), right arm of chromosome III and VI was tagged by the marker yellow-1 (ylo-1) and the linkage group VII was tagged by the marker conidial separation-2 (csp-2).

The cross PS #57 was set up in which mutant, PS-4-11 (progeny of VU13-16) was used as a male parent and the alcoy tester strain (FGSC #3434) was used as a female parent. After a month of crossing, 132 random ascospores were isolated and studied to determine the linkage group on which the mutant gene was present.

3.1.8 Follow up cross
After determining one of the four linkage group, next step was to determine the chromosome number on linkage group on which the mutant gene was present. For this, the cross #PS-98 was set up in which the mutant strain (PS-4-11) was used as a female parent and the tester strain (FGSC #1206) was used as a male parent. After a month of crossing, random ascospore analysis was done.
3.1.9 Fine mapping
3.1.10 Amylase, Protease and Lipase assay
The wild-type and mutant cultures were grown in starch agar, milk agar and Tween 80 agar plates for 24 hours to determine the protease, amylase and lipase activity in mutant cultures. Compositions of starch agar, milk agar and tween 80 agar medium is described below:
Composition of Starch agar medium (100 ml)
dH2O100 ml
Starch0.5 g
Agar1.5 g
Vogel’s salt2 ml
Composition of Milk agar medium (100 ml)
dH2O100 ml
Sterilized milk5 ml
Agar1.5 g
Vogel’s salt2 ml
Composition Tween 80 agar plates
dH2O100 ml
Tween 801 ml
Agar1.5 g
Vogel’s salt2 ml
3.1.11 HPLC analysis
Well grown wild-type (FGSC #2489 and FGSC #4200) and mutant cultures (VU13-16, PS-4-9, PS-4-11, VU13-37, PS-142-1, VU13-12, AM-232-10 and VU-82) were inoculated and allowed to grow in Vogel’s liquid medium (50 ml) in conical flasks for 5 days in static condition in sets of duplicates. After 5 days of incubation at 25oC the mycelium and culture filtrate were harvested and sent for HPLC analysis in sterilized screw capped tubes to PG Tech Laboratory, Indore.


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