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Open Access
Synergistic effect of Azotobacter, actinomycetes and phosphate solubilising bacteria on growth and yield of wheat (Triticum aestivum L.)
(Punjab Agricultural University, Ludhiana, 2017) Kiranpreet Kaur; Gangwar, Madhurama
The present investigation was undertaken to evaluate the synergistic effect of four isolates comprising Azotobacter, Sphingobacterium and two actinomycete isolates (Streptomyces badius and W9) for their functionality traits. All the isolates were able to produce Indoleacetic acid (IAA) and gibberellic acid (GA). Actinomycete isolate W9 showed maximum IAA (98.80 µg/ml) and GA (154.6 µg/ml). Similarly all the isolates were tested for siderophore production and phosphate solubilzation where Streptomyces badius exhibited maximum catechol type of siderophore (268 µg/ml) and hydroxamate type of siderophore (252.9 µg/ml). Sphingobacterium sp. isolate exhibited maximum phosphate solubilization activity (37.8±0.9mg/100ml). All the four isolates were screened for their hydrolytic enzymes production. Actinomycete W9 showed maximum amylolytic, chitinolytic, cellulolytic and proteolytic activity with dissolution factor 1.53 cm, 1.58cm, 1.33cm and 1.25 cm respectively. On the basis of the plant growth promoting traits, all the isolates were tested for growth promotion of wheat crop under field conditions. In both the trials, plant growth parameters like shoot and root length, fresh and dry weight of shoot and root, chlorophyll content were found to be significantly enhanced in inoculation with 100% RDF +Azotobacter + W9 over the control. In first trial (2015-16), maximum grain yield was showed by T11 (100% RDF +Azotobacter+ W9) i.e. 51.88 q/ha as compared to control (T2 & T1) was 45.41 & 40.13 (q/ha). Similar trend was observed in second trial (2016-17), where also T5 (100% RDF+ W9+ Azotobacter) showed maximum grain yield (49.5 q/ha) as compared to control (40.3 q/ha). Hence, these actinomycete isolate W9 and Azotobacter are potential candidates for enhancing plant growth that can help in the use of eco-friendly bio-fertilizers.
Open Access
Development of microbial consortia for pretreatment of paddy straw and its utilization for ethanol production
(Punjab Agricultural University, Ludhiana, 2017) Lota, Pardeep; Kocher, Gurvinder Singh
The present study was conducted with the objective of standardization of a fungal consortium from selected ligninolytic cultures for biological pretreatment of paddy straw and its subsequent conversion to bioethnaol. The proximate analysis of paddy straw revealed that 30 mesh straw had highest proportion of cellulose (39.5%) besides, 23.3% hemicellulose and 11.5% lignin. Among different chemical pretreatment methods, acid (1.0% H2SO4) –autoclave (15 psi for 90 min) followed by alkali (2.0% NaOH) pretreatment resulted in 82.61% and 59.23% decrease in lignin and hemicellulose, respectively and 36.60% increase in relative proportion of cellulose. Four fungal strains (viz., LS1, LS2, LCu1 and LCu2) from a total of fifty nine strains isolated from diverse natural habitats, tested positive for the production of laccase (Lacc), lignin peroxidase (LiP) and manganese peroxidase (MnP). Out of three consortia developed on the basis of ligninolytic activities, consortium III comprising Pleurotus ostreatus and Phanerochaete chrysosporium, resulted in Lacc, LiP and MnP activities of 2.40, 37.92 and 62.50 nkat/gds, respectively, significantly higher than the activities of individual fungal strains. Biological pretreatment of paddy straw under Response surface methodology optimized conditions (moisture, 121.0 %; temperature, 31.3°C and log spore count, 8.0 spores/ml) resulted in cellulose, 43.0%; hemicellulose, 12.7%; lignin, 7.0%; total sugars,13.65 mg/gds and reducing sugars, 4.00 mg/gds. The pretreatment of paddy straw with concentrated (10x) ligninolytic enzyme under shake flask conditions using optimized physico-chemical parameters viz. paddy straw concentration, 2.5 g; enzyme volume, 8.0 ml; Ca2+ ions (10 mM) and incubation temperature, 45°C in 72 h of enzymatic action resulted in 80.87% and 47.64% decrease in lignin and hemicellulose, respectively and 29.96% increase in relative proportion of cellulose. For saccharification studies, from a total of forty seven fungal strains isolated from diverse natural sources, two strains (Aspergillus sp. CTS1 and Aspergillus sp. CTS2) tested positive for thermophilic and cellulolytic nature. The two strains were used for the development of fungal consortium which revealed filter paper, carboxymethyl cellulase and cellobiase activities of 10.2, 30.0 and 7.9 U/gds, respectively, significantly higher than the activities of individual fungal strains. Under the RSM optimized conditions [temperature, 55°C; pH,4.8 and substrate concentration, 5% (w/v)] and 30FPU/g dry substrate Arrowzyme yielded 476.0 mg/gds reducing sugars from biological pretreated paddy straw which were further increased to 492.0 mg/gds by the addition of Tween 20 at 0.2% (v/v). Enzymatic saccharification of biological pretreated straw by in-house concentrated cellulase (CC1) from thermophilic fungal consortium resulted in 229.0 mg/gds reducing sugars. The saccharification of chemical pretreated straw with Arrowzyme and CC1 resulted in 566.0 mg/gds and 250.0 mg/gds reducing sugar, respectively. Optimization of fermentation under separate hydrolysis and fermentation (SHF) conditions using RSM [S.cerevisiae inoculum size 10.0 % (v/v) and DAHP concentration, 0.2% (w/v)] resulted in 0.172 g/g and 0.193g/g ethanol from biological and chemical pretreated hydrolysate, respectively. Similarly, the CC1 saccharified hydrolysate resulted in an ethanol content of 0.076 g/g (biological pretreated) and 0.085 g/g (chemical pretreated). Fermentation of non-enzymatically saccharified biological and chemical hydrolysate by S.cerevisiae produced 0.013 g/g and 0.041 g/g ethanol, respectively, whereas sequential fermentation of chemical hydrolysate with S.cerevisiae and Pachysolen tannophilus produced 0.047g/g ethanol. The combined ethanol production from enzymatically and non-enzymatically saccharified biological and chemical pretreated paddy straw hydrolyaste was observed to be 0.185 g/g and 0.240 g/g, respectively.
Open Access
Production of extracellular proteases from Pleurotus florida (Mont.) Sing. and Volvariella volvacea (Bul. Fr.) Sing.
(Punjab Agricultural University, Ludhiana, 2017) Prabhjot Kaur; Sodhi, H.S.
Pleurotus florida and Volvariella volvacea are grown widely in subtropical and tropical areas. Proteases are a group of fundamentally hydrolytic enzymes that acylate and deacylate to peptide bonds. P.florida showed higher mycelial dry weight (13.4g/L) and wheat straw gave maximum yield for P.florida (72.5kg/q). In qualitative estimation fruit body of V.volvacea showed maximum halo diameter (1.17 cm). In quantitative analysis, for serine proteases in crude and partially purified extract, mycelium of P. florida (28.1 nmol/min/mg) and fruit body of P.florida (148.3 nmol/min/mg), showed maximum specific activity. The maximum specific activity of cysteine proteases in crude and partially purified extract was observed in fruit body of V.volvacea (28.1 nmol/min/mg) and fruit body of P.florida (145.2 nmol/min/mg). The maximum specific activity of aspartic proteases in crude and partially purified extract was observed in mycelium of P.florida (42.2 nmol/min/mg) and fruit body of P.florida (318.5 nmol/min/mg). Maximum molecular weight of protein was 50.2 kDa recorded in all the samples. The FTIR (Fourier transform infrared spectroscopy) assay indicated that the spent substrate of V.volvacea was devoid of Amide A bond, aromatic amino acids and β-sheet conformation. Arginine, lysine and histidine were present in all the samples, whereas sulfur containing amino acids were present in some of the samples. The proteases production potential is more in P.florida than V.volvacea.
Open Access
Enzymatic debittering and aroma enhancement of kinnow juice
(Punjab Agricultural University, Ludhiana, 2017) Manmeet Kaur; Sahota, Param Pal
The processing of acidic fruits to beverages has encountered commercial restrictions due to development of delayed and inherent bitterness by limonoids and flavonoids. The present study has been undertaken with purified debittering enzymes; limonin dehydrogenase, naringinase and β-glucosidase to meet the consumer palatability of citrus juices. The limonin dehydrogenase enzyme has been purified to 1.14- fold with 33.98% recovery, 29.25 IU activity and 0.525 IU/mg specific activity resulted in 86.43% degradation of limonin in juice by catalyzing oxidation of limonin –A-ring lactone to the non-bitter 17dehydroxylimonoate. The debittering naringinase enzyme 24.8IU activity,0.442 IU/mg specific activity, purified to1.68 fold could reduce 57.93% naringin which is hydrolysed by the enzyme into non-bitter bioactive compound naringenin.. The flavour enhancing βglucosidase with activity 14.68 IU, specific activity 0.344 IU/mg purified to 1.18 fold resulted in the increase in glucose content upto 4.25µg/ml in kinnow juice. The debittered fermented low alcoholic naturally carbonated (LANC) beverage has been developed by optimizing the bioprocess conditions as TSS 13ºB, yeast inoculum @ 0.75%(v/v), incubation temperature 28±2ºC and time 36hrs.The physicochemical changes were recorded as TSS 13ºB, acidity 0.313%, naringin 181.13 ppm, limonin 4.73 ppm, glucose 1.3µg/ml, total sugars 42.97 µg/ml, ascorbic acid 34.22 mg/ml in fermented kinnow beverage after 25 days of storage at 4ºC. In kinnow juice, maximum reduction in bitter component limonin 87.34%, naringin 58.41% whereas increase in glucose upto 4.38 µg/ml, acidity 30.13% and total sugar content 42.97 µg/ml were observed. The nutraceuticals recorded in fermented kinnow beverage were flavonoids (mg/L)- gallic acid 0.075, caffeic acid 0.002, rutin 0.001, ferulic acid 0.001; Organic acids (mg/L) – oxalic acid 0.243,tartaric acid 3.698, mallic acid 0.018, citric acid 0.432; Fat soluble vitamins (mg/L)-Vitamin A (Retinol) 0.060, cholecalciferol (D3) 0.038, α-tocopherol (Vitamin E) 0.001, Vitamin K 0.018 ; water soluble vitamins (mg/L)thiamine 0.782, pyridoxine hydrochloride 0.040, pantothenic acid 0.002, riboflavin 0.001, biotin 0.011; Protein 6.42g/100g,Carbohydrate 40.95g/100g.
Open Access
Bioprospects of microalgal isolates from water logged area of Punjab for biogas production
(Punjab Agricultural University, Ludhiana, 2017) Dar, Rouf Ahmad; Phutela, Urmila Gupta
The present study was aimed at isolation, identification, screening and characterization of microalgae from the waterlogged area of south-west Punjab, India. Optimization of cultural conditions of potential microalgal strains, their biogas production potential and anaerobic co-digestion studies were also conducted. Nineteen microalgal cultures (BGLR1-BGLR19) were isolated and were screened using different culture media for their growth kinetics. Isolate BGLR6 followed by BGLR5 showed the highest growth and biomass production in Algae culture medium and BG-11 medium respectively. These isolates upon molecular identification were found to be Asterarcys quadricellulare BGLR5 (MF661929) and Spirulina subsalsa BGLR6 (MF191711). The cultivation conditions for the enhanced production of biomass and other functional components (chlorophyll, carbohydrate, lipid and protein) of BGLR5 and BGLR6 were first screened by Plackett–Burman design and then the significant factors were optimized by Central Composite design (CCD). The optimal cultural conditions for BGLR5 and BGLR6 as per the model were pH of 9.92 & 11.5; temperature of 21.84 & 20°C; light intensity of 80.99 & 81 μmol m-2 s-1; growth period of 25 days (both); 15.00 mM NH4Cl/ CaCl2; 12.28 & 5.00 mM NaNO3 and 7.09 & 2.00 mM K2HPO4 respectively. Under these conditions, the response variables generated a desirability index of 84.10 and 94.91% for BGLR5 and BGLR6 respectively. A 0.42 and 1.60-fold increase in dry cell biomass yield was achieved in BGLR5 and BGLR6, compared to the basal condition (0.8858 and 1.0890 g biomass L-1) respectively. The biogas potential of the microalgal biomass under controlled temperature (35 ± 2°C) conditions revealed that in case of BGLR5, the highest biogas yield (51.712 Lkg-1algal biomass) was obtained in the hydrothermally (100°C 30min) pretreated biomass whilst in BGLR6, the highest biogas yield (42.73 Lkg-1algal biomass) was obtained in enzymatically (10% 24hr) pretreated biomass. Further, co-digestion of 50% paddy straw and 50% BGLR5 biomass (replacement of paddy straw by equal amount of microalgal biomass) produced 168.32 L biogas kg-1 feedstock with the ultimate biogas yield potential of 361.81 mLg-1 VS, reduced lag phase (λ:2.81 d) and increased rate of biogas production (Rm: 8.19 mLg-1d -1) which was higher not only in comparison to the anaerobic digestion of paddy straw and algal biomass individually but also to that of co-digestion of BGLR6 biomass with paddy straw. Likewise, 50 and 100% supplementation of BGLR5 biomass led to enhanced biogas production. Utilization of algal biomass cultivated in the open pond for co-digestion study with paddy straw in field scale digesters resulted in an increase of 17.27% biogas compared to control. The enhancement of methane content from 46.4% (control) to 66.5% (digester containing algal biomass) was also achieved. Hence, the present study signifies that the Asterarcys quadricellulare BGLR5 and Spirulina subsalsa BGLR6 biomass could be utilized as a cofeedstock with paddy straw for biogas production.
Open Access
Effect of biofertilizers and inorganic fertilizers on soil health and growth of Poplar under nursery conditions
(Punjab Agricultural University, Ludhiana, 2017) Khipla, Neha; Gosal, S.K.
A pot experiment was conducted to study the effect of biofertilizers and inorganic fertilizers on soil microbial dynamics, nutrient content of soil and plant and plant growth parameters. Significantly higher bacterial (195 x 108 CFU/g of soil), fungal (29 x 104 CFU/g of soil) and PGPR (186 x 105 CFU/g of soil) population was recorded in treatment having consortium biofertilizer + N100P100 whereas actinomycetes population (51 x 105 CFU/g of soil) in treatment having inorganic fertilizers only. Highest diazotroph (77 x 105 CFU/g of soil) and PSB (42 x 104 CFU/g of soil) population was recorded in treatment having Azotobacter and PSB inoculation respectively, with inorganic fertilizers. Maximum soil dehydrogenase (14.35 μg TPF formed/min/g of soil), alkaline phosphatase (2.95 μg PNP formed/ h /g of soil) and urease (394.3 μg urea hydrolysed/ h/ g of soil) activity was observed where consortium biofertilizer + N100P100 was applied. Soil organic carbon (0.33%), available N (171.5 Kg/ha), P (21.3 Kg/ha) and K (142.0Kg/ha) were found to be significantly higher in treatment with consortium biofertilizer + inorganic fertilizers. Plant N (0.74 g/plant) and K (0.68 g/plant) uptake was recorded highest under Azotobacter inoculation whereas highest P (0.16 g/plant) uptake was recorded under consortium inoculation at N100P100 respectively. Plant growth parameters viz shoot fresh and dry weight, shoot length, leaf number per plant were significantly higher in treatment having Azotobacter + N100P100 while stem collar diameter and root length were recorded highest under consortium + N100P100. Treatment having Azotobacter or consortium biofertilizer with N100P75 varied non significantly for highest root fresh and dry weight among the treatments. The effect of treatments having AM cultures and inorganic fertilizers resulted in significantly higher bacterial (132 x 108 CFU/g of soil), diazotroph (49 x 105 CFU/g of soil) population in treatment having AM1 + N100P75 while PSB (32 x 104 CFU/g of soil) and PGPR (142 x 105 CFU/g of soil) in treatment having AM1 + N75P75. Inoculated treatment having AM1 + N75P100 was recorded with maximum fungal population (24 x 104 CFU/g of soil) whereas actinomycetes population (51 x 105 CFU/g of soil) was found highest in control. Highest soil phosphatase (1.92 μg PNP formed/ h /g of soil) and urease (399.5 μg urea hydrolysed/h/g of soil) activity was observed under AM1 + N100P75 whereas dehydrogenase activity (14.78 μg TPF formed/min/g of soil) under AM1 + N75P75. Maximum soil organic carbon (0.34%), available N (157.1 Kg/ha) and P (17.0 Kg/ha) content was recorded in treatment having AM1 + N100P75 while available K content (135.5 Kg/ha) in treatment having AM1 + N75P75. Significantly higher plant N (0.70 g/plant), P (0.15 g/plant) and K (0.54 g/plant) uptake was recorded in treatments with AM1 inoculation and different doses of inorganic fertilizers. Plant growth parameters viz. root- shoot biomass and length, leaf number per plant, collar diameter were recorded highest in treatment having AM1 inoculation + recommended dose of N and P fertilizer. Hence the combined effect of biofertilizers and inorganic fertilizer boosts soil health and growth parameters of Poplar.
Open Access
Effect of different biofertilizers and inorganic fertilizers on soil health and growth of Eucalyptus clone 413
(Punjab Agricultural University, Ludhiana, 2017) Dhanjal, Sukhmani; Gosal, S.K.
A pot experiment was carried out to study the effect of biofertilizers and inorganic fertilizers on soil health and growth of Eucalyptus clone 413. Significantly higher bacterial population (190 × 108 CFU/g of soil), PGPR population (86 ×105 CFU/g of soil) and soil dehydrogenase activity (8.83μg TPF formed/hr/g of soil) was observed in treatment having consortium + N100P100 while the maximum fungal population (22 ×104 CFU/g of soil) and diazotrophic population (89 ×105 CFU/g of soil) was found in treatments having Azotobacter inoculation. PSB population (48 ×104 CFU/g of soil) and soil alkaline phosphatase activity (3.01 μg pNP formed/ h /g of soil) was observed to be maxmum in treatment having PSB inoculation. Actinomycetes population and urease activity showed varying trend as actinomycetes population (48 ×105 CFU/g of soil) was observed to be significantly higher in control and urease activity (394.6 μg urea hydrolysed/ hr/ g of soil) in treatment having consortium + N100P100. Soil organic carbon (0.33%) and available N (171.34 Kg/ha), P (21.0 Kg/ha), K (152.4 Kg/ha) were found to be significantly higher in treatment having consortium + N100P100. Significantly higher plant N( 1.20 g/plant) and K (0.92 g/plant) uptake was found in treatment having Azotobacter + N100P100 while, plant P uptake(0.22 g/plant) was highest in treatment having consortium + N100P100. Plant growth parameters viz root shoot biomass, leaves, branches and root shoot length were found to be significantly higher in treatment having Azotobacter + N100P100while, collar diameter was found to be highest in consortium + N100P100. The present study was also done by using two cultures of Glomus fasiculatum as AM1 and AM2. Significantly higher bacterial population (138 × 108 CFU/g of soil), PSB population (86 ×105 CFU/g of soil), diazotrophic population(58 ×105 CFU/g of soil), soil dehydrogenase activity (7.67 μg TPF formed/hr/g of soil) and alkaline phosphatase activity (3.01 μg pNP formed/ h /g of soil) were observed in treatment having AM1 and inorganic fertilizer combinations while, the maximum fungal population (24 ×104 CFU/g of soil) and PGPR population (64 ×105 CFU/g of soil) was found in treatments having AM2 and inorganic fertilizer combinations. Actinomycetes population and urease activity showed varying trend and urease activity (367.7 μg urea hydrolysed/ hr/ g of soil) in treatment having AM2 + N-100% P-100%. Soil organic carbon (0.33%), available K (144.7 Kg/ha) and plant K uptake (0.83 g/plant) were found to be significantly higher in treatment having AM2 and inorganic fertilizers combinations while significantly higher available N (162.9 Kg/ha), available P (18.6 Kg/ha), plant N (0.92 g/plant) and plant P (0.14 g/plant) uptake were found in treatment having AM1 and inorganic fertilizer combinations.. Plant growth parameters viz root shoot biomass, leaves, branches and collar diameter were found to be significantly higher in treatment having AM2 and inorganic fertilizer combinations while, root and shoot length was found to be highest in AM1 and inorganic fertilizer combinations. Hence the combined effect of biofertilizers and inorganic fertilizers boost the soil health and growth parameters of Eucalyptus.
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Decolorization of synthetic dyes using Pleurotus spp.spent mushroom substrate
(Punjab Agricultural University, Ludhiana, 2017) Pawandeep Kaur; Kapoor, Shammi
Five Pleurotus spp. (P. florida, P. sajor-caju, P. ostreatus, P. sapidus and P. eryngii) were evaluated for dye decolorization potential using the lignolytic enzymes from spent mushroom substrate (SMS). All the five species showed potential to decolorize three synthetic dyes viz. Amido Black, Congo Red and Remazol Brilliant Blue R (RBBR) when supplemented in solid and liquid medium. The mean decolorization of all the three dyes (@25-200 ppm) in agar medium was maximal for P.sajor-cajuand least for P. sapidusafter 10th day. In liquid culture, dye decolorization potential of Pleurotusspecies ranged from 67-78% for Amido Black, 8395% for RBBR and 67-77% for Congo Red by 8th day.Cultivation of all the Pleurotus species was carried out on wheat straw substratein polybags for the generation of spent mushroom substrate (SMS) and lignin modifying enzyme production potential in SMS were tested at different growth stages i.e. 10 days interval from the day of opening of bags.Among the lignin modifying enzymes, maximum activity was obtained for laccase enzyme followed by manganese peroxidase and Lignin peroxidase.The crude enzyme extracts from SMS of three Pleurotus species (P.eryngii, P.ostreatus and P.sajor-caju) were tested for their dye decolorization potential at five different growth stages of the crop. For Amido black, maximum decolorization(94%) was observed with crude enzyme extracts of P.ostreatus (94%), and for RBBR (82%)and Congo red (88%) with P.sajor-caju. Optimization studies showed that LME medium supplemented with sucrose as C-source, ammonium oxalate as Nsource with a pH of 6.5 and incubated at 25°C resulted in maximum decolorization of 50 ppm of Amido Black dye using P. sajor-caju SMS.
Open Access
Production and partial purification of cellulases from Penicillium roqueforti NCIM 712 using poultry dropping based biodigested slurry
(Punjab Agricultural University, Ludhiana, 2017) Versha Suman; Phutela, Urmila Gupta
The present study was aimed at production and partial purification of cellulases from Penicillium roqueforti NCIM 712 using poultry dropping based biodigested slurry. The proximate and chemical analysis of biodigested slurry indicated the presence of sufficient amount of cellulose for using it as a substrate for cellulase production. Cellulolytic enzyme production was studied under autoclaved and unautoclaved conditions. Unautoclaved poultry droppings based biodigested slurry showed higher activities of endoglucanase, exoglucanase and β glucosidase. Optimized cultural conditions for cellulase production were inoculum size of 107 spores/ml, 30°C of incubation temperature, 7 days of incubation period, pH of 5.0, and slurry concentration of 100%, where 145.76 U/ml of endoglucanase, 46.46 U/ml of exoglucanase and 11.61 U/ml of β glucosidase were obtained under unautoclaved conditions and 62.91 U/ml of endoglucanase, 41.31 U/ml of exoglucanase and 9.98 U/ml of β glucosidase were obtained under autoclaved conditions. The cellulases produced under optimum conditions were partially purified by ion exchange chromatography. Two isoforms were observed in endoglucanase i.e. Pool I and Pool II which showed 8.54 and 9.96 fold purification respectively. The exoglucanase was purified by 11.45 folds and β glucosidase by 10.8 folds. The exoglucanase was further studied for its temperature and pH stability. Exoglucanase exhibited maximum stability at 45°C and pH 5.0. Production of biogas was studied from paddy straw supplemented with animal wastes including poultry droppings and biodigested slurry. Highest biogas production (65.19 litres/ kg paddy straw) was observed in soaked paddy straw supplemented with cellulolytic enzymes from P. roqueforti NCIM 712 produced in poultry droppings based biodigested slurry. There was 22% enhancement in biogas produced from paddy straw mixed with enzymatically activated poultry droppings biodigested slurry than paddy straw mixed with poultry droppings based biodigested slurry. Hence poultry droppings based biodigested slurry was found to be suitable substrate for cost effective cellulase enzyme production which can be further used for increasing biogas production from paddy straw.

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