Trichoderma harzianum MTCC 5179 impacts the population and functional dynamics of microbial community in the rhizosphere of black pepper (Piper nigrum L )

Abstract Employing Illumina Hiseq whole genome metagenome sequencing approach, we studied the impact of Trichoderma harzianum on altering the microbial community and its functional dynamics in the rhizhosphere soil of black pepper (Piper nigrum L.). The metagenomic datasets from the rhizosphere with (treatment) and without (control) T. harzianum inoculation were annotated using dual approach, i.e., stand alone and MG-RAST. The probiotic application of T. harzianum in the rhizhosphere soil of black pepper impacted the population dynamics of rhizosphere bacteria, archae, eukaryote as reflected through the selective recruitment of bacteria [Acidobacteriaceae bacterium (p = 1.24e-12), Candidatus koribacter versatilis (p = 2.66e-10)] and fungi [(Fusarium oxysporum (p = 0.013), Talaromyces stipitatus (p = 0.219) and Pestalotiopsis fici (p = 0.443)] in terms of abundance in population and bacterial chemotaxis (p = 0.012), iron metabolism (p = 2.97e-5) with the reduction in abundance for pathogenicity islands (p = 7.30e-3), phages and prophages (p = 7.30e-3) with regard to functional abundance. Interestingly, it was found that the enriched functional metagenomic signatures on phytoremediation such as benzoate transport and degradation (p = 2.34e-4), and degradation of heterocyclic aromatic compounds (p = 3.59e-13) in the treatment influenced the rhizosphere micro ecosystem favoring growth and health of pepper plant. The population dynamics and functional richness of rhizosphere ecosystem in black pepper influenced by the treatment with T. harzianum provides the ecological importance of T. harzianum in the cultivation of black pepper.

Trichoderma harzianum MTCC 5179 impacts the population and functional dynamics of microbial community in the rhizosphere of black pepper (Piper nigrum L.).

Employing Illumina Hiseq whole genome metagenome sequencing approach, we studied the impact of Trichoderma harzianum on altering the microbial community and its functional dynamics in the rhizhosphere soil of black pepper (Piper nigrum L.). The metagenomic datasets from the rhizosphere with (treatment) and without (control) T. harzianum inoculation were annotated using dual approach, i.e., stand alone and MG-RAST. The probiotic application of T. harzianum in the rhizhosphere soil of black pepper impacted the population dynamics of rhizosphere bacteria, archae, eukaryote as reflected through the selective recruitment of bacteria [Acidobacteriaceae bacterium (p=1.24e-12), Candidatus koribacter versatilis (p=2.66e-10)] and fungi [(Fusarium oxysporum (p=0.013), Talaromyces stipitatus (p=0.219) and Pestalotiopsis fici (p=0.443)] in terms of abundance in population and bacterial chemotaxis (p=0.012), iron metabolism (p=2.97e-5) with the reduction in abundance for pathogenicity islands (p=7.30e-3), phages and prophages (p=7.30e-3) with regard to functional abundance. Interestingly, it was found that the enriched functional metagenomic signatures on phytoremediation such as benzoate transport and degradation (p=2.34e-4), and degradation of heterocyclic aromatic compounds (p=3.59e-13) in the treatment influenced the rhizosphere micro ecosystem favoring growth and health of pepper plant. The population dynamics and functional richness of rhizosphere ecosystem in black pepper influenced by the treatment with T. harzianum provides the ecological importance of T. harzianum in the cultivation of black pepper.

Pseudomonas sp. BUP6 produces a thermotolerant alkaline lipase with trans-esterification efficiency in producing biodiesel.

The present study describes the characteristics of a thermotolerant and alkaline lipase secreted by Pseudomonas sp. BUP6, a novel rumen bacterium isolated from Malabari goat, and its trans-esterification efficiency in producing biodiesel from used cooking oil (UCO). The extracellular lipase was purified to homogeneity (35.8 times purified with 14.8% yield) employing (NH4)2SO4 salt precipitation and Sephadex G-100 chromatography. The apparent molecular weight of this lipase on SDS-PAGE was 35 kDa, the identity of which was further confirmed by MALDI-TOF/MS. The purified lipase was found stable at a pH range of 7-9 with the maximum activity (707 U/ml) at pH 8.2; and was active at the temperature ranging from 35 to 50 °C with the optimum at 45 °C (891 U/ml). Triton X-100 and EDTA had no effect on the activity of lipase; whereas SDS, Tween-80 and ß-mercaptoethanol inhibited its activity significantly. Moreover, Ca2+ (1.0 mM) enhanced the activity of lipase (1428 U/ml) by 206% vis-à-vis initial activity; while Zn2+, Fe2+ and Cu2+ decreased the activity significantly. Using para-nitrophenyl palmitate as substrate, the Km (11.6 mM) and Vmax [668.9 µmol/(min/mg)] of the purified lipase were also determined. Crude lipase was used for analyzing its trans-esterification efficiency with used cooking oil and methanol which resulted in the worthy yield of fatty acid methyl esters, FAME (45%) at 37 °C, indicating its prospects in biodiesel industry. Thus, the lipase secreted by the rumen bacterium, Pseudomonas sp. BUP6, offers great potentials to be used in various industries including the production of biodiesel by trans-esterification.

Phthalates impact human health: Epidemiological evidences and plausible mechanism of action.

Disregarding the rising alarm on the hazardous nature of various phthalates and their metabolites, ruthless usage of phthalates as plasticizer in plastics and as additives in innumerable consumer products continues due low their cost, attractive properties, and lack of suitable alternatives. Globally, in silico computational, in vitro mechanistic, in vivo preclinical and limited clinical or epidemiological human studies showed that over a dozen phthalates and their metabolites ingested passively by man from the general environment, foods, drinks, breathing air, and routine household products cause various dysfunctions. Thus, this review addresses the health hazards posed by phthalates on children and adolescents, epigenetic modulation, reproductive toxicity in women and men; insulin resistance and type II diabetes; overweight and obesity, skeletal anomalies, allergy and asthma, cancer, etc., coupled with the description of major phthalates and their general uses, phthalate exposure routes, biomonitoring and risk assessment, special account on endocrine disruption; and finally, a plausible molecular cross-talk with a unique mechanism of action. This clinically focused comprehensive review on the hazards of phthalates would benefit the general population, academia, scientists, clinicians, environmentalists, and law or policy makers to decide upon whether usage of phthalates to be continued swiftly without sufficient deceleration or regulated by law or to be phased out from earth forever.

Living in the epilepsy treatment gap in rural South India: A focused ethnography of women and problems associated with stigma.

In India, women with epilepsy face unique challenges. A focused ethnography of six women within the epilepsy treatment gap was conducted in rural South India. Women were asked to describe their day-to-day lives. Data were collected through open-ended, semistructured interview questions, participant observation, and field notes. Thematic analysis was done. The disease-related stigma contributed to the women's physical, psychological, and emotional struggles; the women and their family members made every effort to conceal the disease. Educational interventions to create awareness could help women seek effective treatments for their seizures, thereby reducing the stigma and improving the quality of their lives.

Pseudomonas sp. BUP6, a novel isolate from Malabari goat produces an efficient rhamnolipid type biosurfactant.

This study describes the characteristics of a biosurfactant produced by Pseudomonas sp. BUP6, a rumen bacterium, and optimization of parameters required for its production. Initial screening of five parameters (pH, temperature, agitation, incubation, and substrate concentration) was carried out employing Plackett-Burman design, which reduced the number of parameters to 3 (pH, temperature, and incubation) according to their significance on the yield of biosurfactant. A suitable statistical model for the production of biosurfactant by Pseudomonas sp. BUP6 was established according to Box-Behnken design, which resulted in 11% increase (at pH 7, 35 °C, incubation 75 h) in the yield (2070 mg L-1 ) of biosurfactant. The biosurfactant was found stable at a wide range of pH (3-9) with 48 mg L-1 critical micelle concentration; and maintained over 90% of its emulsification ability even after boiling and in presence of sodium chloride (0.5%). The highest cell hydrophobicity (37%) and emulsification (69%) indices were determined with groundnut oil and kerosene, respectively. The biosurfactant was found to inhibit the growth and adhesion of E. coli and S. aureus significantly. From the phytotoxicity studies, the biosurfactant did not show any adverse effect on the germinating seeds of rice and green gram. The structural characterization of biosurfactant employing orcinol method, thin layer chromatography and FT-IR indicated that it is a rhamnolipid (glycolipid). Thus, Pseudomonas sp. BUP6, a novel isolate from Malabari goat is demonstrated as a producer of an efficient rhamnolipid type biosurfactant suitable for application in various industries.

Type 2 pyoverdine from Pseudomonas aeruginosa strain BUP2 as turn-off biosensor for the rapid detection of iron and copper ions in contaminated water.

Employing fluorescent quenching mechanism, type 2 pyoverdine (PVD) purified from Pseudomonas aeruginosa strain BUP2 (new strain isolated from the rumen of Malabari goat) was used as a simple, convenient and inexpensive tool for the rapid detection of Fe and Cu ions in contaminated drinking water samples. The fluorescence emitted at λ460 by PVD (in sterile water), mounted on a glass slide was efficiently quenched by the ions of heavy metals like Fe and Cu. The fluorescence quenching effect of PVD was monitored using UV trans-illuminator, and subsequently quantified and confirmed by spectrofluorimetry. Upon exposure for about 50 sec at 25 °C, this quenching efficiency could directly be assessed by naked eye with the aid of a UV trans-illuminator. The linear range of detection for Fe was 1 to 60 µM, while that of Cu was 1 to 20 µM. The limits of detection at µM concentration for Fe3+, Fe2+ and Cu2+ were 0.23, 0.24 and 0.38, respectively. The quenching of fluorescence was more pronounced in Fe-PVD system than Cu-PVD, and this observation was in corroboration with the Pearson acid base concept; being a hard acid, Fe3+ effectively bound with the O-ligands and this ability was less in Cu2+, a border line acid. Briefly, this study proposed the use of type 2 PVD as a turn-off biosensor for the rapid screening of heavy metals like Fe and Cu in drinking water, at ppm levels only with the aid of UV trans-illuminator at 25 °C in 50 sec.

Achromobacter denitrificans SP1 efficiently utilizes 16 phthalate diesters and their downstream products through protocatechuate 3,4-cleavage pathway.

This study provides physical and analytical evidences for the efficient utilization of most of the commercially available phthalate diesters by Achromobacter denitrificans SP1, coupled with the demonstration of a plausible degradation pathway. We tested 17 phthalate diesters [viz., ditridecyl phthalate, diisodecyl phthalate (DIDP), di(2-ethylhexyl)phthalate (DEHP), di-n-octyl phthalate (DOP), bis(2-ethylhexyl)isophthalate (BEIP), dihexyl phthalate (DHP), dibutyl phthalate (DBP), dicyclohexyl phthalate (DCHP), diphenyl phthalate (DPP), benzyl butyl phthalate (BBP), diamyl phthalate (DAmP), diisobutyl phthalate, dipropyl phthalate, dially phthalate (DAlP), diethyl phthalate, diethyl terephthalate and dimethyl phthalate (DMP)], and their major degradation products for the degradation efficiency of A. denitrificans SP1 in Wx medium. It efficiently utilized 16 phthalate diesters (except DAlP), and showed general preference toward phthalate diesters with longer side chains (utilized ~10mM in 48h) than those with shorter side chains and with cyclic structures (utilized ~5mM in 48h) accompanied by a sharp decline of pH to ~5 from initial 7. In a detailed study, about 37mM (~15g/L) DEHP was utilized in 48h. Moreover, A. denitrificans SP1 produced reddish-pink pigment when DIDP, DEHP, DOP, DHP, DBP, DIBP, BBP, DAmP, DCHP, DPP or DMP was supplied in the medium. From the available evidences, it seems that its putative phthalate diester degradation pathway contains the following check points: phthalate diesters, phthalate monoesters, phthalate (4,5-dioxygenase), protocatechuate (3,4-dioxygenase), and TCA cycle. Nonspecificity toward utilization of phthalate diesters, especially with greater specificity to phthalate diesters having longer side chain, and the characteristic sticky reddish-pink (or colorless) cell clump formation in the presence of phthalate diesters makes A. denitrificans SP1 a very attractive candidate to be employed as an efficient biofactory in waste water treatment processes.

Accessing the molecular interactions of phthalates and their primary metabolites with the human pregnane X receptor using in silico profiling.

Phthalates are known to cause endocrine disruption in humans and animals. Being lipophilic xenobiotic chemicals, phthalates from the surrounding environments can easily be absorbed into the biological system, thereby causing various health dysfunctions. This molecular docking study evaluates a variety of molecular interactions of 12 commonly used diphthalates and respective monophthalates onto the ligand binding domain (LBD) of the human pregnane X receptor (hPXR), a xenosensor, which would be beneficial for further in vitro and in vivo studies on hazardous phthalates. Out of 12 diphthalates and their monophthalates tested, diisodecyl phthalate (-9.16 kcal mol-1 ) showed more affinity toward hPXR whereas diisononyl phthalate (-8.77) and di(2-ethyhexyl)phthalate (-8.56), the predominant plasticizers found in a variety of plastics and allied products, showed comparable binding scores with that of the control ligands such as hyperforine (-9.99) and dexamethasone (-7.36). In addition to the above diphthalates, some of their monophthalates (monoisodecyl phthalate, mono-2-etheylhexyl phthalate, etc.) also established similar interactions with certain crucial amino acids in the LBD, which led to higher G scores. In fact, bisphenol A, a well-studied and proven endocrine disruptor, showed lesser G scores (-6.69) than certain phthalates. Copyright © 2016 John Wiley & Sons, Ltd.

Human ketosteroid receptors interact with hazardous phthalate plasticizers and their metabolites: an in silico study.

Phthalic acid esters or phthalates are ubiquitous environmental pollutants known for their adverse health effects in test animals and, of late, in humans. Thus, in this molecular docking study - using Glide (Schrödinger) - the molecular interactions of 31 ligands, including 12 diphthalates, their monophthalates and phthalic acid with selected human ketosteroid receptors, i.e., androgen (hAR), progesterone (hPR) and glucocorticoid (hGR) receptors were explored and their binding affinities were compared with that of corresponding natural steroids and a known endocrine disrupting xenobiotic, bisphenol A (BPA). Mostly, diphthalates and monophthalates showed the potential for antisteroidal activity by interacting with hAR, hPR and hGR. Of them, diphenyl phthalate showed the highest G score (-7.70 kcal mol(-1) ) with hAR, and the crucial amino acid (aa) residues in the ligand binding domain (LBD) of this receptor involved in the molecular interactions were Phe 764, Leu 704, Asn 705 and Thr 877. The mono-iso-decyl phthalate showed the highest G score (-8.36) with the hPR, and the crucial aa residues in the LBD interactions were Arg 766 Gln 725 and Phe 778. The mono-iso-decyl phthalate also showed more affinity (-8.44) towards hGR than the natural ligand, and the aa residues in the LBD interactions were Gln 570 and Met 604. In addition to these, some other phthalates established comparable interactions with certain aa residues located in the LBD of these receptors, which resulted in higher G scores. Contrastingly, BPA and some natural ligands tested in this study showed lower G scores with these receptors than certain phthalates reported herein, i.e., certain phthalates are more toxic than the proven toxic BPA. Copyright © 2015 John Wiley & Sons, Ltd.

Pros and cons of CLA consumption: an insight from clinical evidences.

This comprehensive review critically evaluates whether supposed health benefits propounded upon human consumption of conjugated linoleic acids (CLAs) are clinically proven or not. With a general introduction on the chemistry of CLA, major clinical evidences pertaining to intervention strategies, body composition, cardio-vascular health, immunity, asthma, cancer and diabetes are evaluated. Supposed adverse effects such as oxidative stress, insulin resistance, irritation of intestinal tract and milk fat depression are also examined. It seems that no consistent result was observed even in similar studies conducted at different laboratories, this may be due to variations in age, gender, racial and geographical disparities, coupled with type and dose of CLA supplemented. Thus, supposed promising results reported in mechanistic and pre-clinical studies cannot be extrapolated with humans, mainly due to the lack of inconsistency in analyses, prolonged intervention studies, follow-up studies and international co-ordination of concerted studies. Briefly, clinical evidences accumulated thus far show that CLA is not eliciting significantly promising and consistent health effects so as to uphold it as neither a functional nor a medical food.

A monograph on the remediation of hazardous phthalates.

Phthalates or phthalic acid esters are a group of xenobiotic and hazardous compounds blended in plastics to enhance their plasticity and versatility. Enormous quantities of phthalates are produced globally for the production of plastic goods, whose disposal and leaching out into the surroundings cause serious concerns to the environment, biota and human health. Though in silico computational, in vitro mechanistic, pre-clinical animal and clinical human studies showed endocrine disruption, hepatotoxic, teratogenic and carcinogenic properties, usage of phthalates continues due to their cuteness, attractive chemical properties, low production cost and lack of suitable alternatives. Studies revealed that microbes isolated from phthalate-contaminated environmental niches efficiently bioremediate various phthalates. Based upon this background, this review addresses the enumeration of major phthalates used in industry, routes of environmental contamination, evidences for health hazards, routes for in situ and ex situ microbial degradation, bacterial pathways involved in the degradation, major enzymes involved in the degradation process, half-lives of phthalates in environments, etc. Briefly, this handy module would enable the readers, environmentalists and policy makers to understand the impact of phthalates on the environment and the biota, coupled with the concerted microbial efforts to alleviate the burden of ever increasing load posed by phthalates.

Achromobacter denitrificans strain SP1 efficiently remediates di(2-ethylhexyl)phthalate.

This study describes how Achromobacter denitrificans strain SP1, a novel isolate from heavily plastics-contaminated sewage sludge efficiently consumed the hazardous plasticizer, di(2-ethylhexyl)phthalate (DEHP) as carbon source supplemented in a simple basal salt medium (BSM). Response surface methodology was employed for the statistical optimization of the process parameters such as temperature (32°C), agitation (200 rpm), DEHP concentration (10 mM), time (72 h) and pH (8.0). At these optimized conditions, experimentally observed DEHP degradation was 63%, while the predicted value was 59.2%; and the correlation coefficient between them was 0.998, i.e., highly significant and fit to the predicted model. Employing GC-MS analysis, the degradation pathway was partially deduced with intermediates such as mono(2-ethylhexyl)phthalate and 2-ethyl hexanol. Briefly, this first report describes A. denitrificans strain SP1 as a highly efficient bacterium for completely remediating the hazardous DEHP (10 mM) in 96 h in BSM (50% consumed in 60 h), which offers great potentials for efficiently cleaning the DEHP-contaminated environments such as soil, sediments and water upon its deployment.

Biphasic fermentation is an efficient strategy for the overproduction of δ-endotoxin from Bacillus thuringiensis.

This study illustrates a biphasic solid-state fermentation (SSF) strategy for the overproduction of δ-endotoxin from Bacillus thuringiensis subsp. kurstaki (Btk) and also purification of δ-endotoxin from the solid-fermented medium. The fermentation strategy had two phases (biphasic); i.e., the first short phase was semisolid state (12 h), and the remaining long phase was strict SSF. To achieve the biphasic SSF, after 12 h (150 rpm, 37 °C) fermentation of the medium [Luria-Bertani (LB) supplemented with 30 % (w/v) raw soybean flour (phase I)], the supernatant in it was completely centrifuged out (1,000 × g, 10 min) aseptically for harvesting the extracellular enzymes as by-product. The resultant wet solid matter without free-flowing liquid but with embedded Btk was incubated 60 h more (phase II) for enhancing δ-endotoxin production at static condition (37 °C). Coupled with this, δ-endotoxin was purified by the modified phase separation method, and its purity was physically confirmed by both staining and microscopic techniques. The maximum δ-endotoxin yield from solid medium (48 h) was 15.8 mg/mL (recovery was 55-59 %) LB-equivalent, while that of LB control (recovery was 95 %) was only 0.43 mg/mL (72 h), i.e., thus, in comparison, 36.74-fold more yield in solid medium obtained by 24 h less gestation period. The purified crystal proteins showed apparent molecular weights (MWs) of 45, 35, and 6 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, this unique study physically demonstrates how Btk δ-endotoxin is purified (95-99 % purity) from solid-fermented matter for the first time, coupled with its overproduction at the expense of only 21.5 % higher production cost.

Production, optimization, and partial purification of lipase from Pseudomonas sp. strain BUP6, a novel rumen bacterium characterized from Malabari goat.

This study introduces a novel bacterium-Pseudomonas sp. strain BUP6-isolated from the rumen of the Malabari goat with efficiency for producing lipase. It showed significant production of lipase when grown in a newly designed basal medium, supplemented with vegetable oil. Suitability of five vegetable oils such as groundnut oil, coconut oil, olive oil, sunflower oil, and palm oil as inducer for the production of lipase was examined, and groundnut oil supported the highest production of lipase (96.15 U/mL). Various physical parameters required for the maximum production of lipase were statistically optimized. Plackett-Burmann design was employed to study the interactive effects of physical parameters and found that temperature, agitation, and pH effected the production of lipase significantly. The optimum conditions for lipase production (37 °C, 200 rpm, and pH 6.9) were detected by Box-Behnken design and response surface methodology, which resulted in the 0.3-fold increase (i.e., 126 U/mL) of the lipase activity over the unoptimized condition. The apparent molecular mass of partially purified lipase was 35 kDa, as judged by SDS-PAGE; the activity of lipase was also confirmed by native PAGE. Thus, this study focuses on the need for the exploitation of rumen microbes for the production of industrially significant and human-friendly biomolecules to meet the future needs.

Achromobacter denitrificans SP1 produces pharmaceutically active 25C prodigiosin upon utilizing hazardous di(2-ethylhexyl)phthalate.

This first report describes the purification and identification of an orange-red pigment produced by Achromobacter denitrificans strain SP1 (isolated from sewage sludge heavily contaminated with plastics) during its growth in a simple basal salt medium supplemented with the hazardous di(2-ethylhexyl)phthalate (DEHP) blended in PVC blood bag (in situ) or free DEHP (ex situ) as carbon source. The cell-bound pigment was elucidated, characterized at molecular level, and described as an unusual 25C prodigiosin analog for the first time. At laboratory conditions (in flasks), the dry cell mass was 75.2mg/g blood bag, which upon extraction yielded 7.1mg prodigiosin; at this stage the pH of the medium was dropped from 7.2 to 3.5. Considering its pharmaceutical importance, taking 10 known prodigiosins as controls, this 25C prodigiosin was subjected to molecular docking studies, showed comparable and promising binding efficiencies with the crucial molecular human targets like cycloxygenase-2, ZAP-70 kinase and Jak-3 kinase.

Phthalates efficiently bind to human peroxisome proliferator activated receptor and retinoid X receptor α, ß, γ subtypes: an in silico approach.

This exhaustive in silico study looks into the molecular interactions of phthalates and their metabolites with human peroxisome proliferator-activated receptor (hPPAR) and retinoid X receptor (hRXR) α, ß and γ subtypes--the nuclear receptor proteins function as transcription factors by regulating the expression of downstream genes. Apart from the much discussed plasticizer bisphenol A, we examined the binding affinities of 15 common diphthalates and their monophthalates, natural (linoleic acid, conjugated linoleic acid) and synthetic (bezafibrate, pioglitazone, GW 50156) ligands with hPPARs. In addition to these phthalates, specific natural (retinoic and phytanic acids) and synthetic (bexarotene, rosiglitazone) ligands were examined with hRXRs. The Maestro, Schrödinger Suite 2012 was used for the molecular docking study. In general, natural ligands of hPPAR showed less binding efficiencies than phthalic acid esters and drugs. The diphthalate di-iso-decyl phthalate showed the highest G score (-9.99) with hPPAR (γ), while its monophthalate (mono-iso-decyl phthalate) showed a comparatively less G score (-9.56). Though the PPAR modulator GW 50156 showed strong affinity with all hPPAR subtypes, its highest G score (-12.43) was with hPPARß. Hazardous di(2-ethylhexyl)phthalate generally showed a greater preference to hRXRs than hPPARs, but its highest G score (-10.87) was with hRXRα; while its monophthalate (Mono(2-ethylhexyl)phthalate) showed a lesser G score (-8.59). The drug bexarotene showed the highest G score (-13.32) with hRXRß. Moreover, bisphenol A showed more affinity towards hRXR. Briefly, this study gives an overview on the preference of phthalic acid esters, natural and synthetic ligands on to hPPAR and hRXR subtypes, which would lead to further in vitro mechanistic as well as in vivo preclinical and clinical studies.

Potato flour mediated solid-state fermentation for the enhanced production of Bacillus thuringiensis-toxin.

In this study, we explored the efficacy of raw potato flour (PF) as supplement to the conventional LB medium (LB control, designated as M1) for enhancing the concomitant production of endospores and δ-endotoxin from Bacillus thuringiensis subsp. kurstaki by solid-state fermentation (SSF). Of different concentrations and combinations of media tested, 10% (w/v) PF supplemented LB medium (M2) was found as the best source for the maximum yield of toxin. After 12 h submerged fermentation (SmF) at 37°C and 125 rpm, M2 was made into a wet-solid matter for SSF by removing the supernatant (1000 ×g, 10 min); the resultant pellet subsequently incubated statically (37°C) for the production of B. thuringiensis subsp. kurstaki toxin (Btk-toxin). In comparison to M1, yield of δ-endotoxin purified by sucrose density gradient centrifugation method from M2 was about 6-fold higher (53% recovery). This maximum yield from M2 was obtained at 48 h (as against 72 h from M1), thus the gestation period of M2 was reduced by 24 h with higher yield. In addition to the quantitative data, qualitative photomicrographs taken by image analyzer, scanning electron and fluorescent microscopes and digital camera showed physical evidences for the upper hand of SSF over conventional SmF for the enhanced production of Btk-toxin. SDS-PAGE image of the purified δ-endotoxin showed three major fractions with apparent MWs 66, 45 and 30 kDa. Briefly, if low-cost agricultural products like PF is used as supplement to LB, by SSF strategy, production of Btk-toxin could be enhanced to 6-fold in short gestation time without losing its entomotoxicity efficiency.

Candida tropicalis BPU1, a novel isolate from the rumen of the Malabari goat, is a dual producer of biosurfactant and polyhydroxybutyrate.

This unique study reports a new strain (BPU1) of Candida tropicalis isolated from the rumen of the Malabari goat, showing dual production of biosurfactant and polyhydroxybutyrate. C. tropicalis strain BPU1, a facultative anaerobe, was tuned to become an aerobe in specially designed flask, the Benjamin flask. The puffy circular colonies were smooth, white-to-cream in colour, with pseudo-filaments. The strain fermented glucose, sucrose, maltose and dextrose, but not lactose and cellulose. It assimilated (NH4 )2 SO4 , peptone, glycine and arginine, but not NaNO3 , as the nitrogen source. Interestingly, it utilized groundnut oil (up to 0.3%) in a specially designed basal mineral salt medium (BSM). Its capability for dual production of a biosurfactant and a polyhydroxybutyarate (PHB) was explored by various methods from the BSM-oil medium. Extracted biosurfactant from 6 day-old culture was biochemically characterized as a complex of lipid and carbohydrate with an Rf value of 0.88 by thin layer chromatography. Its PHB production was confirmed by specific staining methods with Nile blue sulphate, Sudan black B and Sudan 3. Briefly, this first-ever report gives ample physical evidence for the dual production of a glycolipid (biosurfactant) and PHB by C. tropicalis strain BPU1 on a specially designed medium, which would open up elaborate research on this yeast.

Conjugated linoleic acid isomers and their precursor fatty acids regulate peroxisome proliferator-activated receptor subtypes and major peroxisome proliferator responsive element-bearing target genes in HepG2 cell model.

The purpose of this study was to examine the induction profiles (as judged by quantitative reverse transcription polymerase chain reaction (qRT-PCR)) of peroxisome proliferator-activated receptor (PPAR) α, ß, γ subtypes and major PPAR-target genes bearing a functional peroxisome proliferator responsive element (PPRE) in HepG2 cell model upon feeding with cis-9,trans-11-octadecadienoic acid (9-CLA) or trans-10,cis-12-octadecadienoic acid (10-CLA) or their precursor fatty acids (FAs). HepG2 cells were treated with 100 µmol/L 9-CLA or 10-CLA or their precursor FAs, viz., oleic, linoleic, and trans-11-vaccenic acids against bezafibrate control to evaluate the induction/expression profiles of PPAR α, ß, γ subtypes and major PPAR-target genes bearing a functional PPRE, i.e., fatty acid transporter (FAT), glucose transporter-2 (GLUT-2), liver-type FA binding protein (L-FABP), acyl CoA oxidase-1 (ACOX-1), and peroxisomal bifunctional enzyme (PBE) with reference to ß-actin as house keeping gene. Of the three housekeeping genes (glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ß-actin, and ubiquitin), ß-actin was found to be stable. Dimethyl sulfoxide (DMSO), the common solubilizer of agonists, showed a significantly higher induction of genes analyzed. qRT-PCR profiles of CLAs and their precursor FAs clearly showed upregulation of FAT, GLUT-2, and L-FABP (~0.5-2.0-fold). Compared to 10-CLA, 9-CLA decreased the induction of the FA metabolizing gene ACOX-1 less than did PBE, while 10-CLA decreased the induction of PBE less than did ACOX-1. Both CLAs and precursor FAs upregulated PPRE-bearing genes, but with comparatively less or marginal activation of PPAR subtypes. This indicates that the binding of CLAs and their precursor FAs to PPAR subtypes results in PPAR activation, thereby induction of the target transporter genes coupled with downstream lipid metabolising genes such as ACOX-1 and PBE. To sum up, the expression profiles of these candidate genes showed that CLAs and their precursor FAs are involved in lipid signalling by modulating the PPAR α, ß, or γ subtype for the indirect activation of the PPAR-target genes, which may in turn be responsible for the supposed health effects of CLA, and that care should be taken while calculating the actual fold induction values of candidate genes with reference to housekeeping gene and DMSO as they may impart false positive results.