Evidence of Biocontrol Activity of Bioinoculants Against a Human Pathogen, Listeria monocytogenes.

Due to rhizodeposits and various microbial interactions, the rhizosphere is an extremely dynamic system, which provides a conductive niche not only for bacteria beneficial to plants but also for those that might pose a potential threat to humans. The importance of bioinoculants as biocontrol agents to combat phytopathogens has been widely recognized. However, little information exists with respect to their role in inhibiting human pathogens in the rhizosphere. The present study is an attempt to understand the impact of an established bacterial consortium, Azotobacter chroococcum, Bacillus megaterium, and Pseudomonas fluorescens, on the survivability of Listeria monocytogenes in the rhizosphere of Cajanus cajan and Festuca arundinacea. An experiment conducted in Hoagland's medium in the presence of C. cajan demonstrated that the presence of bioinoculants impaired growth of L. monocytogenes compared to that observed in their absence. On the other hand, in the presence of F. arundinacea, no significant differences were observed in the population dynamics of L. monocytogenes in the presence or absence of the bioinoculants. Agar plate assay through cross streak method revealed the inhibition of L. monocytogenes by bioinoculants. Potential bioactive compounds were identified by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). These results suggest that agricultural amendments can act as protective agents against human pathogens while enforcing plant growth promotion.

Effect of Agricultural Amendments on Cajanus cajan (Pigeon Pea) and Its Rhizospheric Microbial Communities--A Comparison between Chemical Fertilizers and Bioinoculants.

Inoculation of leguminous seeds with bioinoculants has been practiced in agriculture for decades to ameliorate grain yield by enhanced growth parameters and soil fertility. However, effective enhancement of plant growth parameters results not only from the direct effects these bioinoculants impose on them but also from their non-target effects. The ability of bioinoculants to reduce the application of chemicals for obtaining optimum yield of legume appears to be of great ecological and economic importance. In the present study, we compared the influence of seed inoculation of Cajanus cajan with a microbial consortium, comprising Bacillus megaterium, Pseudomonas fluorescens and Trichoderma harzianum, with that of application of chemical fertilizers on plant's growth parameters and its rhizospheric microbial communities. Real-time PCR assay was carried out to target the structure (16S rRNA) and function (nitrogen cycle) of rhizospheric microbiota, using both DNA and RNA as markers. The results showed that the microbial consortium was the most efficient in increasing grain yield (2.5-fold), even better than the recommended dose of chemical fertilizers (by 1.2-fold) and showed enhancement in nifH and amoA transcripts by 2.7- and 2.0-fold, respectively. No adverse effects of bioinoculants' application were observed over the rhizospheric microbial community, rendering the consortium to be safe for release in agricultural fields.

Effect of formulated root endophytic fungus Piriformospora indica and plant growth promoting rhizobacteria fluorescent pseudomonads R62 and R81 on Vigna mungo.

In the present investigation, the effect of three beneficial organisms (root endophytic fungus Piriformospora indica (Pi) and pseudomonads strains R62 and R81) and their four different consortia (Pi+R62, Pi+R81, R62+R81, Pi+R62+R81) was investigated on the plant Vigna mungo through their inorganic carrier-based (talcum powder and vermiculite) formulations. All the treatments resulted in significant increase in growth parameters under glasshouse as well as field conditions and showed a consistency in their performance on moving from glasshouse to field conditions. In glasshouse conditions, a maximum increase of 4.5-fold in dry root weight and 3.9-fold in dry shoot weight compared to control was obtained with vermiculite-based consortium formulation of Pi+R81. In field studies using vermiculite as carrier, a maximum enhancement of 3.2-fold in dry root weight, 3.0-fold in dry shoot weight, 8.4-fold in number of nodules and 4.0-fold in number of pods in comparison to control was obtained with the bio-inoculant formulation containing consortium of Pi+R81. The same treatment also caused the highest improvement of 1.9-fold in nitrogen content and 1.7-fold in phosphorus content, while the highest increase of 1.4-fold in potassium content was obtained with Pi alone.

Shelf-life enhancement of bio-inoculant formulation by optimizing the trace metals ions in the culture medium for production of DAPG using fluorescent pseudomonad R62.

Statistical experimental design was used to optimize the concentration of trace elements for production of antifungal compound, 2,4-diacetylphloroglucinol (DAPG), from fluorescent pseudomonad R62 in shake-flask cultivation. The selection of the trace metal ions, influencing DAPG production, was done using Plackett-Burman design (PBD). Only Zn(2+), Mn(2+) and MoO(4)(2-) were the most significant components (p<0.05). A quadratic model was used to fit the response. Application of response surface methodology (RSM) revealed that the optimum values of the salts of the trace elements Zn(2+) (ZnSO(4)·7H(2)O), Mn(2+) (MnCl(2)·4H(2)O), and MoO(4)(2-) (Na(2)MoO(4)·2H(2)O) were 83, 42 and 135µM, respectively, to achieve 125 mg/L of DAPG, which was nearly 13-fold more compared to its production in basal synthetic medium in shake flask. The studies in 14L bioreactor resulted in 135 mg/L of DAPG at the end of 36 h of cultivation. The culture broth containing 125 mg/L of DAPG was found to be sufficient for keeping the bio-inoculant viable in non-sterile talcum powder-based formulations (which contained 25µg DAPG/g carrier) when stored at 28°C for 6 months. The structure of the purified DAPG was confirmed using (1)H NMR and mass spectrometry.

Application of inorganic carrier-based formulations of fluorescent pseudomonads and Piriformospora indica on tomato plants and evaluation of their efficacy.

AIMS: Fluorescent pseudomonads are widely used as bioinoculants for improving plant growth and controlling phytopathogenic fungi. Piriformospora indica (Pi), a symbiotic root endophyte, also has beneficial effects on a number of plants. The present study focuses on the improvement of growth yields of tomato plants and control of Fusarium wilt using inorganic carrier-based formulations of two fluorescent pseudomonad strains (R62 and R81) and Pi. METHODS AND RESULTS: The inorganic carrier-based formulations of pseudomonad strains and Pi were tested for plant growth promotion of tomato plants under glass house and field conditions. In controlled glass house experiments, 8·8-fold increase in dry root weight and 8·6-fold increase in dry shoot weight were observed with talcum powder-based consortium formulation of R81 and Pi. Field trial experiments ascertained the glfass house results with a considerable amount of increase in plant growth responses, and amongst all the treatments, R81 + Pi treatment performed consistently well in field conditions with an increase of 2·6-, 3·1- and 3·9-fold increase in dry root weight, shoot weight and fruit yield, respectively. The fluorescent pseudomonad R81 and Pi also acted as biocontrol agents, as their treatments could control the incidence of wilt disease caused by Fusarium oxysporum f.sp. lycopersici in tomato plants under glass house conditions. CONCLUSIONS: The culture broths of pseudomonads R62, R81 and Pi were successfully used for development of talcum- and vermiculite-based bioinoculant formulations. In controlled glasshouse experiments, the talcum-based bioinoculant formulations performed significantly better over vermiculite-based formulations. In field experiments the talcum-based consortium formulation of pseudomonad R81 and Pi was most effective. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the formulations of pseudomonad strains (R62 and R81) and Pi can be used as bioinoculants for improving the productivity of tomato plants. The application of such formulations is a step forward towards sustainable agriculture.

High-density spore production of Piriformospora indica, a plant growth-promoting endophyte, by optimization of nutritional and cultural parameters.

Piriformospora indica is an axenically cultivable root endophytic fungus which exerts plant growth promoting effects on its host plants. To enable commercial production of its spores, the medium composition and culture conditions have been optimized in a 14 L bioreactor such that they result in maximum biomass during growth phase and in maximum spore yield during subsequent sporulation phase. Maximum spore yields were obtained with modified Kaefer medium using a glucose deprivation strategy. An enhancement of 100% in overall biomass productivity (0.18 g L(-1) h(-1)) and reduction of about 70% in the time (60 h) required to achieve the maximum spore yield (9.25×10(7) spores/mL) was achieved in comparison to the original Kaefer medium. The high spore yield obtained in the present study seems to be economical for commercial production of P. indica.

Co-culture of arbuscular mycorrhiza-like fungi (Piriformospora indica and Sebacina vermifera) with plant cells of Linum album for enhanced production of podophyllotoxins: a first report.

Cell suspension cultures of Linum album were developed from internode portions of in vitro germinated plant in Gamborg's B5 medium supplemented with 0.4 mg naphthalene acetic acid/l. The highest biomass was 8.5 g/l with podophyllotoxin and 6-methoxypodophyllotoxin at 29 and 1.9 mg/l, respectively after 12 d cultivation. Co-cultures of L. album cells with axenically cultivable arbuscular mycorrhiza-like fungi, Piriformospora indica and Sebacina vermifera, were established for the first time. These enhanced podophyllotoxin and 6-methoxypodophyllotoxin production by about four- and eight-fold, respectively, along with a 20% increase in biomass compared to the control cultures.

Laccase of Cyathus bulleri: structural, catalytic characterization and expression in Escherichia coli.

Cyathus bulleri, a ligninolytic fungus, produces a single laccase the internal peptides (3) of which bear similarity to laccases of several white rot fungi. Comparison of the total amino acid composition of this laccase with several fungal laccases indicated dissimilarity in the proportion of some basic and hydrophobic amino acids. Analysis of the circular dichroism spectrum of the protein indicated 37% alpha-helical, 26% beta-sheet and 38% random coil content which differed significantly from that in the solved structures of other laccases, which contain higher beta-sheet structures. The critical role of the carboxylic group containing amino acids was demonstrated by determining the kinetic parameters at different pH and this was confirmed by the observation that a critical Asp is strongly conserved in both Ascomycete and Basidiomycete laccases. The enzyme was denatured in the presence of a number of denaturing agents and refolded back to functional state with copper. In the folding experiments under alkaline conditions, zinc could replace copper in restoring 100% of laccase activity indicating the non-essential role of copper in this laccase. The laccase was expressed in Escherichia coli by a modification of the ligation-anchored PCR approach making it the first fungal laccase to be expressed in a bacterial host. The laccase sequence was confirmed by way of analysis of a 435 bp sequence of the insert.

Production and characterization of laccase from Cyathus bulleri and its use in decolourization of recalcitrant textile dyes.

Many fungi (particularly the white rot) are well suited for treatment of a broad range of textile dye effluents due to the versatility of the lignin-degrading enzymes produced by them. We have investigated decolourization of a number of recalcitrant reactive azo and acid dyes using the culture filtrate and purified laccase from the fungus Cyathus bulleri. For this, the enzyme was purified from the culture filtrate to a high specific activity of 4,022 IU mg(-1) protein, produced under optimized carbon, nitrogen and C/N ratio with induction by 2,6-dimethylaniline. The protein was characterized as a monomer of 58+/-5.0 kDa with carbohydrate content of 16% and was found to contain all three Cu(II) centres. The three internal peptide sequences showed sequence identity (80-92%) with laccases of a number of white rot fungi. Substrate specificity indicated highest catalytic efficiency (k(cat)/K(M)) on guaiacol followed by 2,2'-azino-bis(3-ethylthiazoline-6-sulfonic acid) (ABTS). Decolourization of a number of reactive azo and acid dyes was seen with the culture filtrate of the fungus containing predominantly laccase. In spite of no observable effect of purified laccase on other dyes, the ability to decolourize these was achieved in the presence of the redox mediator ABTS, with 50% decolourization in 0.5-5.4 days.

Biotechnological aspects of the production of the anticancer drug podophyllotoxin.

The natural lignan podophyllotoxin, a dimerized product of two phenylpropanoid moieties which occurs in a few plant species, is a pharmacologically important compound for its anticancer activities. It is used as a precursor for the chemical synthesis of the anticancer drugs etoposide, teniposide and etopophose. The availability of this lignan is becoming increasingly limited because of the scarce occurrence of its natural sources and also because synthetic approaches for its production are still commercially unacceptable. Biotechnological production using cell culture may be considered as an alternative source. Selection of the best performing cell line, its maintenance and stabilization are necessary prerequisites for its production in bioreactors and subsequent scale-up of the cultivation process to the industrial level. Scale-up of growth and product yield depends on a multitude of factors, such as growth medium, physicochemical conditions, seed inoculum, type of reactor and processing conditions. The composition of the growth medium, elicitors and precursors, etc. can markedly influence the production. Optimum levels of parameters that facilitate high growth and product response in cell suspensions of Podophyllum hexandrum have already been determined by statistical design. P. hexandrum cells have successfully been cultivated in a 3-l stirred-tank bioreactor under low shear conditions in batch and fed-batch modes of operation. The batch kinetic data were used to identify the mathematical model which was then used to develop nutrient-feeding strategies for fed-batch cultivation to prolong the productive log phase of cultivation. An improvement in the production of podophyllotoxin to 48.8 mg l(-1) in a cell culture of P. hexandrum was achieved, with a corresponding volumetric productivity of 0.80 mg l(-1) day(-1), when the reactor was operated in continuous cell-retention mode. Efforts are being made to further enhance its production levels by the development of hairy root culture or by varying the channeling of precursors towards the desired biosynthetic pathway by molecular approaches.

Enzymatic synthesis of oligosaccharides, alkyl and terpenyl glucosides, by recombinant Escherichia coli-expressed Pichia etchellsii beta-glucosidase II.

The biosynthetic activity of yeast Pichia etchellsii beta-glucosidase II (BglII) expressed in recombinant Escherichia coli was utilized for synthesis of cellooligosaccharides, alkyl and terpene glucosides. Cellooligosaccharides with a degree of polymerization of 3 and greater were resolved by thin-layer chromatography (TLC) using an ethyl acetate:1-propanol:2-propanol:water (8:5:1:1) solvent system followed by visualization with 0.2% naphthoresorcinol reagent. Using 2M cellobiose and 15 IU of partially purified BglII, 57 mmol/L of oligosaccharides (comprising mostly cellotriose and cellopentaose) was synthesized in 16 h. Similarly, alkyl glucosides with chain lengths from 6 to 10 carbons were synthesized and products extracted to near purity by ethyl acetate extraction. The same extraction method was employed to separate, to near purity, various monoterpenyl (nerol, geraniol, citronellol) glucosides. A reliable and simple method for separation of cellooligosaccharides using a combination of Bio-Gel P-2 gel filtration and charcoal celite adsorption chromatography was developed. The cellooligosaccharides were separated to purity as confirmed by TLC. The enzyme was among the very few that could synthesize a wide variety of glycoconjugates.

Cytotoxicity of in vitro produced podophyllotoxin from Podophyllum hexandrum on human cancer cell line.

Podophyllotoxin was produced by cell culture of Podophyllum hexandrum under in vitro culture conditions. A maximum of 4.26 mg/L of podophyllotoxin was produced when P. hexandrum was cultivated in 3 L stirred tank bioreactor. The compound extracted from the cell culture was applied to the human breast cancer cell line (MCF-7) and 1 nM podophyllotoxin was able to inhibit the growth of the cancer cells by 50%. The most profound inhibitory effect of podophyllotoxin was observed when it was applied in the beginning of cell growth.

Immobilization of xylan-degrading enzymes from Melanocarpus albomyces IIS 68 on the smart polymer Eudragit L-100.

Xylanase of Melanocarpus albomyces IIS 68 was immobilized on Eudragit L-100. The latter is a copolymer of methacrylic acid and methyl methacrylate and is a pH-sensitive smart polymer. The immobilization was carried out by gentle adsorption and an immobilization efficiency of 0.82 was obtained. The enzyme did not leach off the polymer even in the presence of 1 M NaCl and 50% ethylene glycol. The K(m) of the enzyme changed from 5.9 mg ml(-1) to 9.1 mg ml(-1) upon immobilization. The V(max) of the immobilized enzyme showed an increase from 90.9 micro mol ml(-1) min(-1) (for the free enzyme) to 111.1 micro mol ml(-1) min(-1). The immobilized enzyme could be reused up to ten times without impairment of the xylanolytic activity. The immobilized enzyme was also evaluated for its application in pre-bleaching of eucalyptus kraft pulp.

Effect of major nutrients on podophyllotoxin production in Podophyllum hexandrum suspension cultures.

The effect of major medium ingredients (sugar, nitrogen source and phosphate) in Podophyllum hexandrum suspension cultures was investigated in order to increase the production of podophyllotoxin, the raw material in the synthesis of anticancer drugs. Amongst B5, Eriksson, MS, Nitsch, Street and White's medium, MS medium resulted in high growth and podophyllotoxin accumulation. The optimum level of nitrogen was found to be 60 mM, with a combination of ammonium salts and nitrate in the ratio of 1:2. The highest level of podophyllotoxin was obtained at 60 g glucose/l and at 1.25 mM phosphate after 30 days. Statistical design was adopted to determine the optimum levels of the parameters for cell growth and podophyllotoxin production.

Microbial beta-glucosidases: cloning, properties, and applications.

Beta-glucosidases constitute a major group among glycosylhydrolase enzymes. Out of the 82 families classified under glycosylhydrolase category, these belong to family 1 and family 3 and catalyze the selective cleavage of glucosidic bonds. This function is pivotal in many crucial biological pathways, such as degradation of structural and storage polysaccharides, cellular signaling, oncogenesis, host-pathogen interactions, as well as in a number of biotechnological applications. In recent years, interest in these enzymes has gained momentum owing to their biosynthetic abilities. The enzymes exhibit utility in syntheses of diverse oligosaccharides, glycoconjugates, alkyl- and aminoglucosides. Attempts are being made to understand the structure-function relationship of these versatile biocatalysts. Earlier reviews described the sources and properties of microbial beta-glucosidases, yeast beta-glucosidases, thermostable fungal beta-glucosidase, and the physiological functions, characteristics, and catalytic action of native beta-glucosidases from various plant, animal, and microbial sources. Recent efforts have been directed towards molecular cloning, sequencing, mutagenesis, and crystallography of the enzymes. The aim of the present article is to describe the sources and properties of recombinant beta-glucosidases, their classification schemes based on similarity at the structural and molecular levels, elucidation of structure-function relationships, directed evolution of existing enzymes toward enhanced thermostability, substrate range, biosynthetic properties, and applications.

Optimization of culture parameters for production of podophyllotoxin in suspension culture of Podophyllum hexandrum.

The root explants of the germinated seedlings of Podophyllum hexandrum were grown in MS medium supplemented with indole acetic acid (IAA) (2 mg/L) and activated charcoal (0.5%), and healthy callus culture was obtained after incubation for 3 wk at 20 degrees C. The cultivation of plant cells in shake flask was associated with problems such as clumping of cells and browning of media, which were solved by the addition of pectinase and polyvinylpyrrolidone. The effect of major media components and carbon source was studied on the growth and podophyllotoxin production in suspension culture. It was found that glucose was a better carbon source than sucrose and that NH4+:NO3- ratio (total nitrogen concentration of 60 mM) and PO4(3-) did not have much effect on the growth and product formation. The relative effect of culture parameters (inoculum level, pH, IAA, glucose, NH4+:NO3- ratio, and PO4(3-)) on the overall growth and product response of the plant cell suspension culture was further investigated by Plackett-Burman design. This indicated that inoculum level, glucose, IAA, and pH had significant effects on growth and production of podophyllotoxin. To identify the exact optimum concentrations of these parameters on culture growth and podophyllotoxin production, central composite design experiments were formulated. The overall response equations with respect to growth and podophyllotoxin production as a function of these culture parameters were developed and used to determine the optimum concentrations of these parameters, which were pH 6.0, 1.25 mg/L of IAA, 72 g/L of glucose, and inoculum level of 8 g/L.

Optimization of xylanase production by Melanocarpus albomyces IIS68 in solid state fermentation using response surface methodology.

Xylanase production by the thermophilic fungus, Melanocarpus albomyces IIS68, during solid state fermentation of wheat straw was studied and the effects of various variables were observed. Using the response surface methodology and the multivariant statistical approach, the optimum levels of the variables affecting xylanase production were determined. The optimum levels of the variables were 600-850 microm particle size, 43 h inoculum age, 1.37% Tween 80, 86% initial moisture content, 5.1% urea, 0.74% yeast extract and a harvest time of 96 h. Under these optimized conditions, xylanase activity of 7760 U/g initial dry substrate was obtained which was in very good agreement with the value predicted by the quadratic model (7890 U/g initial dry substrate).

Development of biotechnology in India.

India has embarked upon a very ambitious program in biotechnology with a view to harnessing its available human and unlimited biodiversity resources. It has mainly been a government sponsored effort with very little private industry participation in investment. The Department of Biotechnology (DBT) established under the Ministry of Science and Technology in 1986 was the major instrument of action to bring together most talents, material resources, and budgetary provisions. It began sponsoring research in molecular biology, agricultural and medical sciences, plant and animal tissue culture, biofertilizers and biopesticides, environment, human genetics, microbial technology, and bioprocess engineering, etc. The establishment of a number of world class bioscience research institutes and provision of large research grants to some existing universities helped in developing specialized centres of biotechnology. Besides DBT, the Department of Science & Technology (DST), also under the Ministry of S&T, sponsors research at universities working in the basic areas of life sciences. Ministry of Education's most pioneering effort was instrumental in the creation of Biochemical Engineering Research Centre at IIT Delhi with substantial assistance from the Swiss Federal Institute of Technology, Zurich, Switzerland to make available state-of-the-art infrastructure for education, training, and research in biochemical engineering and biotechnology in 1974. This initiative catalysed biotechnology training and research at many institutions a few years later. With a brief introduction, the major thrust areas of biotechnology development in India have been reviewed in this India Paper which include education and training, agricultural biotechnology, biofertilizers and biopesticides, tissue culture for tree and woody species, medicinal and aromatic plants, biodiversity conservation and environment, vaccine development, animal, aquaculture, seri and food biotechnology, microbial technology, industrial biotechnology, biochemical engineering and associated activities such as creation of biotechnology information system and national repositories. Current status of intellectual property rights has also been discussed. Contribution to the India's advances in biotechnology by the industry, excepting a limited few, has been far below expectations. The review concludes with some cautious notes.

Purification, characterization and substrate specificities of xylanase isoenzymes from Melanocarpus albomyces IIS 68.

The ascomycetous fungus Melanocarpus albomyces when grown on wheat straw produced seven extracellular xylanase isoenzymes, designated as la, Ib, Ic, IIa, lib, llc, and lId. All seven xylanases were purified to homogeneity by gel filtration and ion-exchange chromatography. The molecular mass (kDa) of la, lb, Ic, Ila, lIb, lIc, and lId were estimated to be 22.9, 20.7, 18.6, 31.3, 25.4, 38.5, and 34.3, respectively by SDS-PAGE, and 23.7, 20.5, 17.1, 31.7, 25.1, 39.8, and 32.2, respectively by gel filtration. The isoelectric points of Ia, lb, Ic, Ila and IIb were found to be 6.2, 6.3, 6.4, 3.7, and 4.4, respectively. The activity of the isoenzymes was dependent on the type of the xylan substrates; Xylanases Ia, lb, and Ic showed highest specific activity toward larchwood xylan (an arabinoglucuronoxylan), IIa and Ilc toward birchwood xylan (a glucuronoxylan), and llb and IId toward beechwood xylan (a glucuronoxylan). Four isoenzymes la, lb, Ic, and Ila had an arabinose-releasing property on larchwood xylan. Application of specific isoenzymes as prebleaching agents in paper manufacture is proposed.

Simultaneous bioconversion of glucose and xylose to ethanol by Saccharomyces cerevisiae in the presence of xylose isomerase.

Simultaneous isomerisation and fermentation (SIF) of xylose and simultaneous isomerisation and cofermentation (SICF) of a glucose/xylose mixture was carried out by Saccharomyces cerevisiae in the presence of xylose isomerase. The SIF of 50 g I(-1) xylose gave an ethanol concentration and metabolic yield of 7.5 g l(-1) and 0.36 g (g xylose consumed)(-1). These parameters improved to 13.4 g l(-1) and 0.40 respectively, when borate was added to the medium. The SICF of a mixture of 50 g l(-1) glucose and 50 g l(-1) xylose gave an ethanol concentration and metabolic yield of 29.8 g l(-1) and 0.42 respectively, in the presence of borate. Temperature modulation from 30 degrees C to 35 degrees C during fermentation further enhanced the above parameters to 39 g l(-1) and 0.45 respectively. The approach was extended to the bioconversion of sugars present in a real lignocellulose hydrolysate (peanut-shell hydrolysate) to ethanol, with a fairly good yield.