Meta-analysis of biodynamic (BD) preparations reveal the bacterial population involved in improving soil health, crop yield and quality.

BACKGROUND: Bacterial community found in biodynamic preparations (BD500-BD507) can help improve soil health, plant development, yield, and quality. The current work describes a metagenomic investigation of these preparations to identify the bacterial communities along with the functional diversity present within them. RESULTS: Metagenome sequencing was performed using the Illumina MiSeq platform, which employs next-generation sequencing (NGS) technology, to provide an understanding of the bacterial communities and their functional diversity in BD preparations. NGS data of BD preparations revealed that maximum operational taxonomic units (OTUs) of the phylum Proteobacteria were present in BD506 (23429) followed by BD505 (22712) and BD501 (21591), respectively. Moreover, unclassified phylum (16657) and genus (16657) were also highest in BD506. Maximum alpha diversity was reported in BD501 (1095 OTU) and minimum in BD507 (257 OTU). Further, the OTUs for five major metabolic functional groups viz carbohydrate metabolism, xenobiotic degradation, membrane transport functions, energy metabolism, and enzyme activities were abundant in BD506 and BD501. CONCLUSION: The bacterial communities in BD506 and BD501 are found to be unique and rare; they belong to functional categories that are involved in enzyme activity, membrane transport, xenobiotic degradation, and carbohydrate metabolism. These preparations might therefore be thought to be more effective. The investigation also found a highly varied population of bacteria, which could explain why BD preparations work well in the field. In view of this, the BD preparations may be utilized for unexploited bacterial communities for sustainable agriculture production.

Immobilization of microbial multienzyme preparation on calcium alginate beads as well as lyophilization with mosambi peel matrix improved its shelf-life and stability.

The purpose of the current study was to evaluate the functional activity and storage viability (at 4 °C and 35 °C) of an immobilized as well as lyophilized multienzyme, viz., pectinase, cellulase, and amylase (PCA) that was produced by Bacillus subtilis NG105 under solid state fermentation (SSF) at 35 ℃ for 10 days using mosambi peel as a substrate. After SSF, the culture media was divided into two aliquots. From the first aliquot, the produced ME was extracted, precipitated, and further immobilized on calcium alginate beads (MEICA). In order to immobilize on mosambi peel matrix, the second aliquot was mixed with acetone and subsequently lyophilized (MELMP). Thus, ready MEICA and MELMP extracted 87.5 and 91.5% juice from mango pulp, respectively. In the reusability study, after 5 cycles, MEICA exhibited 23.8%, 24.4%, and 36.5% PCA activity, respectively. The PCA activity of MEICA and MELMP was examined after 60 days of storage at 4 ℃. The result revealed that the PCA for MEICA declined from 100 to 66%, 58.2%, and 64.5%, respectively, while for MELMP, it dropped from 100 to 84.2%, 82.1%, and 69.7%, respectively. Further, after 60 days of storage, the reduction of total protein content (TPC) in free multienzyme (FME), MEICA, and MELMP was 92.2%, 91.5%, and 36.3% observed, respectively. In the localization study, the maximum levels of multienzyme activity were found in cell exudates. This study demonstrated that immobilizing of multienzyme through lyophilization on waste substrates like mosambi peel boosted its stability and shelf-life along with greatly reducing the cost of products.

Mechanism and kinetics of chlorpyrifos co-metabolism by using environment restoring microbes isolated from rhizosphere of horticultural crops under subtropics.

The indiscriminate use of organophosphate insecticide chlorpyrifos in agricultural crops causes significant soil and water pollution and poses a serious threat to the global community. In this study, a microbial consortium ERM C-1 containing bacterial strains Pseudomonas putida T7, Pseudomonas aeruginosa M2, Klebsiella pneumoniae M6, and a fungal strain Aspergillus terreus TF1 was developed for the effective degradation of chlorpyrifos. Results revealed that microbial strains were not only utilizing chlorpyrifos (500 mg L-1) but also coupled with plant growth-promoting characteristics and laccase production. PGP traits, that is, IAA (35.53, 45.53, 25.19, and 25.53 µg mL-1), HCN (19.85, 17.85, 12.18, and 9.85 µg mL-1), and ammonium (14.73, 16.73, 8.05, and 10.87 µg mL-1) production, and potassium (49.53, 66.72, 46.14, and 52.72 µg mL-1), phosphate (52.37, 63.89, 33.33, and 71.89 µg mL-1), and zinc (29.75, 49.75, 49.12, and 57.75 µg mL-1) solubilization tests were positive for microbial strains T7, M2, M6, and TF1, respectively. The laccase activity by ERM C-1 was estimated as 37.53, 57.16, and 87.57 enzyme U mL-1 after 5, 10, and 15 days of incubation, respectively. Chlorpyrifos degradation was associated with ERM C-1 and laccase activity, and the degree of enzyme activity was higher in the consortium than in individual strains. The biodegradation study with developed consortium ERM C-1 showed a decreased chlorpyrifos concentration from the 7th day of incubation (65.77% degradation) followed by complete disappearance (100% degradation) after the 30th day of incubation in the MS medium. First-order degradation kinetics with a linear model revealed a high k -day value and low t 1/2 value in ERM C-1. The results of HPLC and GC-MS analysis proved that consortium ERM C-1 was capable of completely removing chlorpyrifos by co-metabolism mechanism.

Microbial production of multienzyme preparation from mosambi peel using Trichoderma asperellum.

Fruit and vegetable wastes create unhygienic conditions and pose a environmental pollution. The utilization of such wastes as carbon sources for production of enzyme with microbial intervention could be an ecofriendly and profitable approach, apart from diminishing the waste load. The present investigation focused on the feasibility of using mosambi (Citrus limetta) peel as substrate for multienzyme production (pectinase, cellulase and amylase) through microbial intervention. Fifteen fungi were isolated from organic waste and screened in vitro their potential of biodegradation of mosambi peel through enzymes production. The best performing isolate was selected and identified as Trichoderma asperellum NG-125 (accession number-MW287256). Conditions viz. temperature, pH, incubation time and nutrient addition were optimized for efficient enzymes production. The maximum enzyme activity (U ml-1 min-1) of pectinase (595.7 ± 2.47), cellulase (497.3 ± 2.06) and amylase (440.9 ± 1.44) were observed at pH 5.5, incubation temperature of 30 °C after 10 days of fermentation. Moreover, macro-nutrients such as ammonium sulfate (0.1%) and potassium-di-hydrogen-ortho-phosphate (0.01%) further also enhanced the production of enzymes. The SDS-PAGE analysis of purified pectinase, cellulase and amylase using showed molecular mass of 43, 66 and 33 kDa, respectively. The enzyme retention activity (ERA) of aforesaid enzymes was also tested with four different natural fiber matrices viz., bagasse, rice husk, paddy straw and wheat straw. Among these, the maximum ERA was observed on bagasse matrix (pectinase-56.35%, cellulose-77.68% and amylase 59.54%). Enzymatic juice clarification yield obtained with test enzyme was 75.8%, as compared to 80.5% of commercial enzyme. The result indicates that T. asperellum may be exploited as multifaceted biocatalysis.

Indian goose berry fortified, anti-oxidant rich bael (Aegle marmelos) fermented beverage.

Bael (Aegle marmelos Correa), an important fruit of Indian subtropics, traditionally utilized in the preparation of preserve, candy, squash, RTS, etc. has immense therapeutic potential. An attempt was made to develop a bael based low alcoholic sweet fermented beverage ( may be called as bael cider), anti-oxidant fortified with 0.25 per cent bael leaf or 10 per cent Indian goose berry (Emblica officinalis Gaertn.) juice in separate batches. Unfortified bael pulp based drink was kept as control. The ameliorated pulp was fermented at 30 ± 2ºC using Saccharomyces cerevisiae. The gooseberry blended bael fermented beverage had higher anti-oxidant content in the form of phenolics (323 mg/100 ml) than leaf extract added fermented beverage (265 mg/100 ml) and control (266 mg/100 ml). Sensory evaluation of product revealed that gooseberry blended fermented beverage scored higher (8.2/10) than bael leaf blended fermented beverage (7.9/10) and control (7.0/10). Twelve months maturation study of beverage revealed increase in reducing sugars and decrease in phenolic content in all the treatments. Bael fermented beverage with gooseberry blend retained highest phenolic content (257 mg/100 ml) and sensory score 7.8/10. The study inferred that an acceptable quality fermented drink could be prepared from bael-gooseberry blend, which could be stored for one year with higher antioxidant value and minimum deterioration in the quality.

Influence of imidacloprid on bacterial community diversity of mango orchard soil assessed through 16S rRNA sequencing-based metagenomic analysis.

Imidacloprid, used against mango hopper, is a persistent insecticide in soil. Microbes have the ability to remove toxic pesticides from soil surface. Metagenomic is an approach for understanding the diversity and related metabolic activities in any environmental sample without culturing the microbes. Metagenomic analysis of mango orchard soil was carried out using 16S rRNA gene sequencing to understand the impact of imidacloprid on soil microbial population. In control and imidacloprid applied soil samples, representative sequences clustered were 0.142930 and 0.082320 million, respectively. At the kingdom level, 85 and 88 percent represented to bacteria, 2 and 1 percent to archaea, and 13 and 11 percent to unassigned for control and treated metagenomes, respectively. At phylum level, 16 and 17 percent of OTUs (operational taxonomic units) were assigned with Proteobacteria, while 13 and 11 percent of OTUs were unassigned in control and imidacloprid-treated samples, respectively. The other abundant phyla in both the samples were Planctomycetes, Bacteroidetes, and Actinobacteria. At class level, 9 and 11 percent of OTUs were assigned with Planctomycetia in control as well as imidacloprid-treated samples, respectively. A number of OTUs present in control and imidacloprid applied samples are 31,173 and 21,909, respectively, with 18,018 number of OTUs shared between the two samples. The genus Gemmata totally disappeared in imidacloprid applied soil, while those belonging to class Phycisphaerae, genus Prevotella and species copri were identified in imidacloprid treatment. Bacterial community transformation was evident from this study indicating possible microbial bioremediation of imidacloprid in mango orchard soil.

Maternal and severe anaemia in delivering women is associated with risk of preterm and low birth weight: A cross sectional study from Jharkhand, India.

BACKGROUND AND OBJECTIVES: Haemoglobin content is the well accepted indicator for anaemia assessment. The high prevalence of anaemia, maternal health care issues and adverse delivery outcome in Jharkhand, we investigated whether delivering women with anaemia would present a modifiable risk of preterm (PTB) and low birth weight (LBW). METHODS: A facility-based cross-sectional study involving pregnant women, with screening for pregnancy endpoints and haemoglobin assay, were conducted. Anaemia was classified according to World Health Organization's definition of anaemia in pregnancy. Confounding variables were adjusted in a logistic model. The adjusted odds ratios (AORs) with 95% confidence intervals (CIs) were used for analyzing the association among maternal anaemia, PTB and LBW. RESULTS: We observed a high prevalence of anaemia (78.45%) in delivering women, whereas high prevalence of preterm birth (34.75%) and LBW (32.81%) in delivering women overall. In the adjusted analysis, overall anaemia in pregnancy was strongly associated with preterm birth (OR, 3.42; 95% CI, 1.98-5.88; P ≤ .0001) as compared to LBW (OR, 1.12; 95% CI, 0.65-1.61; P = .0003). The risk of PTB and LBW were dependent on the stratification of the anaemia group, as the strongest association was observed in severe (OR, 4.86) followed by mild (OR, 3.66) and moderate (OR, 3.18) anaemia in PTB; whereas risk of LBW was found in severe (OR, 2.5) followed by moderate (OR, 1.11) and mild (OR, 0.57) anaemia. The risk of PTB and LBW across six pregnancy haemoglobin groups were compared, haemoglobin of 10-10.9 g/dl (OR, 1.25) and ≤ 8 g/dl (OR, 1.03) have shown association with PTB and LBW, respectively. However, high haemoglobin concentration was not associated with either PTB or LBW. CONCLUSIONS: Anaemia in delivering women was associated with an elevated risk of PTB and LBW and the risk increased with the severity of anaemia in pregnant women.

Application of response surface methodology for optimization of polygalacturonase production by Aspergillus niger.

Polygalacturonase (PG) degrades pectin into D-galacturonic acid monomers and is used widely in food industry especially for juice clarification. In the present study,. fermentation conditions for polygalacturonase production by Asgergillus niger NAIMCCF-02958, using mango peel as substrate, were optimized using the 2(3) factorial design with central composite rotatable experimental design (CCRD) of response surface methodology (RSM). The maximum PG activity 723.66 U g(-1) was achieved under pH 4.0, temperature 30 degrees C and 2% inoculum by response surface curve. The experimental value of PG activity wkas higher 607.65 U g(-1) than the predicted value 511.75 U g(-1). Under the proposed optimized conditions, the determination coefficient (R2) was equal to 0.66 indicating that the model could explain 66% of the total variation as well as establish the relationship between the variables and the responses. ANOVA analysis and the three dimensional plots also confirmed interactions among the parameters.

Production and characterization of alpha-amylase from mango kernel by Fusarium solani NAIMCC-F-02956 using submerged fermentation.

Microbial production of enzymes using low valued agro industrial wastes is gaining importance globally. Mango is one of the major fruit processed into a variety of products. During processing 40-50% of solid waste is generated in form of peel and stones. After decortications of mango stone, kernel is obtained which is a rich source of starch (upto 60%). It was utilized as a substrate for alpha-amylase production using Fusarium soloni. Maximum alpha-amylase production (0.889 U g(-1)) was recorded using a substrate concentration of 5% (w/v), pH-4 and temperature 30 degrees C on 9th day of incubation. Supplementation of production medium with micronutrients viz., Ca2+, Fe2+ or Mg2+ improved the enzyme production while, Zn2+, B3+ or Mn2+ ions exhibited inhibitory effect. The extracellular protein was precipitated by ammonium sulphate up to 70% saturation, dialyzed and purified (27.84 fold) by gel-exclusion (Sephadex G-75) chromatography. Protein profiling on 12% SDS-PAGE revealed three bands corresponding to 26, 27 and 30 kDa molecular sizes. The optimum amylase activity was achieved at pH 5.0 at 40 degrees C. The Michaelis constant (KM), Vmax and activation energy (-Ea) were found to be 3.7 mg ml(-1), 0.24 U mg(-1) and 42.39 kJ mole(-1), respectively.

Production and characterization of carboxymethyl cellulase from Paenibacillus polymyxa using mango peel as substrate.

Mango peel, a solid mango processing waste, comprises 15-20% of total fruit weight. This, being a rich source of lignocelluloses, was used as substrate for carboxymethyl cellulase (CMCase) production using Paenibacillus polymyxa. Maximum CMCase production (7.814 U mg(-1)) was observed in a medium containing 7% mango peel (w/v) with 1.5% ammonium sulphate (w/v) at 37 degrees C and pH 5.5. Purification to an extent of 28.24 fold was achieved by affinity column chromatography. Bands corresponding to 26.5 and 34.0 kDa molecular sizes were observed on 12% denaturing Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) while of 72 kDa on 10% non-denaturing Native-PAGE, proving its heteromeric multienzyme nature. The enzyme was stable over a range of 20-60 degrees C and pH of 4.0-7.5. Michaelis-Menten equation constant (Km and Vmax) values of purified CMCase were 8.73 mg ml(-1) and 17.805 mM ml(-1) min(-1), respectively.

Kinetics of fungal extracellular alpha-amylase from Fusarium solani immobilized in calcium alginate beads.

Extracellular alpha-amylase mass produced by Fusarium solani using mango kernel as substrate was immobilized in calcium alginate beads through entrapment technique. Maximum enzyme immobilization efficiency was achieved in 2 mm size beads formed by 6.5% (w/v) of sodium alginate in 2% (w/v) calcium chloride. The catalytic properties of the immobilized alpha-amylase were compared with that of free enzyme (soluble). The activity yield of the immobilized enzyme was 81% of the free enzyme. The immobilized enzyme showed optimum activityat pH 4.5-6.0 and temperature 40 degrees C, in contrast to the free enzyme at 5.5 and 30 degrees C, respectively. Thermal stability of the immobilized enzyme was found to be more than the free enzyme over a longer time interval. The immobilized enzyme retained activity upto 20% of optimum even after 180 min. While the free enzyme lost its 80% activity after 60 min and lost total activity down to zero by 120 min. The kinetic constants, viz., K(M) (Michaelis constant), V(max) and activation energy were affected by immobilization. However, the immobilized alpha-amylase in calcium alginate beads supports its long-term storage which has immense industrial applications.

Isolation and purification of fungal pathogen (Macrophomina phaseolina) induced chitinase from moth beans (Phaseolus aconitifolius).

OBJECTIVE: Chitinase (EC 3.2.1.14) is one of the major pathogenesis-related proteins, which is a polypeptide that accumulates extracellularly in infected plant tissue. An attempt was made to isolate and purify the chitanase enzyme using moth beans as an enzyme source. MATERIALS AND METHOD: The enzyme was isolated and purified from moth beans against the fungal pathogen Macrophomina phaseolina strain 2165. The isolation and purification was done in both in vitro and in vivo conditions. Purification of chitinase was carried out to obtain three fractions, viz. 50°C heated, ammonium sulfate precipitated and sephadex G-25 column-eluted fractions. The molecular mass of Chitinase was directly estimated by sodium dodecyl sulfate-polyacryamide gel electroresis (SDS-PAGE). RESULT: The yield is sufficient for initial characterization studies of the enzyme. The molecular study of the enzyme shows the possibility of generating the defense mechanism in plants in which it cannot occur. Chitinase was purified by gel filtration chromatography with 20.75-fold and 32.78-fold purification in the in vitro and in vivo conditions, respectively. The enzyme shows a maximum activity after 90 min with 0.1 ml of colloidal chitin as a substrate and 0.4 ml of crude chitinase extract. The optimum pH of 5.0 and an optimum temperature of 40°C was found for maximal activity. The molecular weight of purified chitinase was estimated to be 30 kDa by SDS-PAGE. CONCLUSION: The chitinase isolated in both in vitro and in vivo conditions is stable andactive.

First Report of Black Soft Rot of Indian Gooseberry Caused by Syncephalastrum racemosum.

Indian gooseberry (Emblica officinalis Gaertn.) is a medicinal plant with high nutraceutical value. During November and December 2003, soft rot was noticed on harvested and stored (20 ± 5°C and 65 ± 5% relative humidity) fruits at the experimental farm in Rehmanhera, Lucknow, India (26°50'N, 80°54'E). These fruits had numerous, minute brown necrotic lesions showing white mycelial growth. A pronounced halo of water-soaked, faded tissue surrounded the lesion between the fringe of mycelium and healthy tissues. The rotted surface was covered with a black, powdery layer of spores. On Czapek yeast extract agar, fungal colonies were blackish grey, moderately dense, and covered the entire petri dish. The fungus produced aseptate mycelium. The sporangial heads were 30 to 50 µm in diameter with sporangiospores found linearly within cylindrical sacs (merosporangia) borne on spicules around the columella. Sporangiospores, spherical to cylindrical in shape and borne in chains, measured 3.0 to 5.0 µm long. The fungus was morphologically and physiologically identified as Syncephalastrum racemosum Schr. (2). For pathogenicity tests, healthy fruits (10 replicates) were surface sterilized and punctured inoculated aseptically with 1.0 × 106 conidia and incubated at 20 ± 5°C Typical symptoms of the disease appeared after 4 days. The fungus exhibited a strong level of cellulolytic activity as indicated by prolific growth on Indian gooseberry fiber waste under solid-state fermentation conditions. The level of cellulase activity (1) was 21 filter paper activity unit per ml at 72 hr in culture supernatant of basal medium having carboxymethyl cellulose as the carbon source. The fungus showed resistance to tannins (as much as 2%), since it could grow well in liquid growth medium (Czapek Dox broth) with 2% tannins and aonla juice with 1.8% tannins. Since Indian gooseberry is rich in fiber (2.5 to 3.4%) and tannins (1.5 to 2.0%), this may be an important pathogen. To our knowledge, this is the first report of the occurrence of Syncephalastrum racemosum on Indian gooseberry fruits. References: (1) T. K. Ghose. Pure Appl. Chem. 59(2):257, 1987. (2) J. I. Pitt and A. D. Hocking. Fungi and Food Spoilage. Academic Press. North Ryde, Australia, 1985.

Effect of Processing Conditions on the Microflora of Fresh-Cut Vegetables.

Surveys were made of commercial processing lines used to prepare fresh-cut vegetables such as chopped salad ingredients, carrot sticks, and cauliflower florets. Washing and chlorinated water dips only partially removed the microorganisms that were intrinsic to the vegetables. Major sources of in-plant contamination were the shredders used to prepare chopped lettuce and coleslaw. Gram-negative rods were the predominant microflora with species of Pseudomonas being most numerous; many were psychrotrophic. Only low numbers of lactic acid bacteria and fungi were recovered.