60h Of Incubation Research Articles

Exploring the Probiotic and Plant Growth Promoting Potential of Endophytic Bacteria Isolated from the Vegetable Crop Bhendi Abelmoschus esculentus

Endophytes are microbes that reside within the plant tissues and facilitate the growth of the host plant. Inthe present study, occurrence of endophytic bacteria in the most common vegetable plant of India, Bhendi(Abelmoschus esculentus) was screened and analysed for its plant growth promoting (PGP) potential. Presenceof a rod shaped bacterium within the tissues of the host plant was observed. The isolate was identified asBacillus firmus by its biochemical profile. Phosphate solubilization and IAA production by the isolate at 60h of incubation was found to be 37.3 g ml–1 and 39.47 g ml–1 respectively. B. firmus was found to producetrihydroxamate type siderophore with hexadentate nature. The isolate exhibited extracellular pectinolytic,proteolytic, cellulolytic, chitinolytic enzyme activity. Further, the isolate exhibited probiotic properties suchas tolerance to acid (pH2), bile salt (2%), gastric juice, auto-aggregation potential, antibiotic resistance andthe absence of haemolytic activity. Seed biopriming and scanning electron microscope (SEM) imagingconfirmed the endophytic and plant growth promoting potential of B. firmus. The results of this studyillustrate the possible utilization of the endophytic bacterium B. firmus as a bioinoculant for plant growthpromotion.

Transcriptome analysis provides new insights into the tolerance and reduction of Lysinibacillus fusiformis 15-4 to hexavalent chromium.

Microbial bioremediation of Cr(VI)-contaminated environments has drawn extensive concern. However, the molecular processes underlying the microbial Cr(VI) tolerance and reduction remain unclear. We isolated a Cr(VI)-reducing Lysinibacillus fusiformis strain 15-4 from soil on the Qinghai-Tibet Plateau. When grown in 1mM and 2mM Cr(VI)-containing medium, strain 15-4 could reduce 100% and 93.7% of Cr(VI) to Cr(III) after 36h and 60h of incubation, respectively. To know the molecular processes in response to Cr(VI), transcriptome sequencing was carried out using RNA-Seq technology. The results annotated a total of 3913 expressed genes in the strain. One thousand ninety-eight genes (28.1%) were significantly (fold change ≥ 2, false discovery rate ≤ 0.05) expressed in response to Cr(VI), of which 605 (55.1%) were upregulated and 493 (44.9%) were downregulated. The enrichment analysis showed that a total of 630 differentially expressed genes (DEGs) were enriched to 122 KEGG pathways, of which 8 pathways were significantly (p < 0.05) enriched in Cr(VI)-treated sample, including ATP-binding cassette (ABC) transporters (97 DEGs), ribosome (40), sulfur metabolism (16), aminoacyl-tRNA biosynthesis (19), porphyrin metabolism (20), quorum sensing (44), oxidative phosphorylation (17), and histidine metabolism (10), suggesting that these pathways play key roles to cope with Cr(VI) in the strain. The highly upregulated DEGs consisted of 29 oxidoreductase, 18 dehydrogenase, 14 cell redox homeostasis and stress response protein, and 10 DNA damage and repair protein genes. However, seven Na+:H+ antiporter complex-coding DEGs and most of transcriptional regulator-coding DEGs were significantly downregulated in the Cr-treated sample. Many of FMN/NAD(P)H-dependent reductase-encoding genes were greatly induced by Cr, suggesting the involvement of these genes in Cr(VI) reduction in strain 15-4. Sulfur and iron ions as well as the thiol-disulfide exchange reactions might play synergistic roles in Cr reduction.Key points• Lysinibacillus fusiformis 15-4 was able to tolerate and reduce Cr(VI) to Cr(III).• Transcriptome analysis revealed that 1098 DEGs and 8 key KEGG pathways significantly responded to Cr(VI).• Sulfur metabolism, protein biosynthesis, and porphyrin metabolism were the key pathways associated with the survival of strain 15-4 in response to Cr(VI).

Bioleaching characteristics, influencing factors of Cu solubilization and survival of Herbaspirillum sp. GW103 in Cu contaminated mine soil

This study was aimed at assess the potential of diazotrophic bacteria, Herbaspirillum sp. GW103, for bioleaching of Cu in mine soil. The strain exhibited resistance to As (550mgL−1), Cu (350mgL−1), Zn (300mgL−1) and Pb (200mgL−1). The copper resistance was further confirmed by locating copA and copB genes. The survival of the isolate GW103 during bioleaching was analyzed using green fluorescent protein tagged GW103. Response surface methodology based Box–Behnken design was used to optimize the physical and chemical conditions for Cu bioleaching. Five significant variables (temperature, incubation time, CaCO3, coconut oil cake (COC), agitation rate) were selected for the optimization. Second-order polynomials were established to identify the relationship between Cu bioleaching and variables. The optimal conditions for maximum Cu bioleaching (66%) were 30°C, 60h of incubation with 1.75% of CaCO3 and 3% COC at 140rpm. The results of Cu sequential extraction studies indicated that the isolate GW103 leached Cu from ion-exchangeable, reducible, strong organic and residual fractions. Obtained results point out that the isolate GW103 could be used for bioleaching of Cu from mine soils.

Saccharomyces cerevisiae TFS9, a novel isolated yeast capable of high caffeine-tolerant and its application in biodecaffeination approach

There is a great call for using microbial bio-decaffeination approach to remove caffeinefrom caffeinated products and industrial wastes. We aimed in this study to screen strainsof yeasts which exhibit high caffeine tolerance and to investigate the bio-degradation ofcaffeine under growth conditions. Sixteen yeast strains were isolated from the cultivatedtea soils collected from sites of northern Iran and evaluated for the caffeine tolerance bythe agar dilution method. Based on the tolerance efficiency, strain TFS9 was selected andidentified as Saccharomyces cerevisiae TFS9 (GenBank accession number KF414526)on the morphological and bioochemical characteristics as well as molecular phylogeneticstudies based on amplification the ITS1–5.8S–ITS2 rDNA sequences. The time course ofcaffeine removal by growing cells of the strain TFS9 in the minimal salt medium containingcaffeine as the sole source of carbon was estimated by a decrease in caffeine absorbanceusing UV-visible spectrophotometer. The concentration of caffeine in the supernatantof the yeast culture medium decreased by 84.8% (from 3.5g/l to 0.53 g/l) after 60h ofincubation by using of S. cerevisiae TFS9, without additional optimization process. Resultsof experimental studies suggest a simple and cost-effective process for the microbialdecaffeination of caffeine-containing solutions, and provide a promising approach fordeveloping safe processes that can be used effectively for decaffeination of industrialeffluents. The present study provides the first evidence on the caffeine bio-degradationusing yeast species of S. cerevisiae.

Disturbance of SHH signalling pathway during early embryogenesis in the cadmium-induced omphalocele chick model

Administration of cadmium (Cd) after 60h (H) incubation induces ventral body wall defect (VBWD) similar to the omphalocele phenotype in the chick embryo. In this model, the earliest histological changes have been observed in somites commencing at 4-h post-treatment (4H). The molecular mechanism by which Cd acts in this critical period of embryogenesis still remains unclear. Sonic hedgehog (SHH) signalling plays an important role in vertebrate development, including somitogenesis and thus ventral body wall formation. Patched (PTCH), a cell membrane receptor for SHH, is expressed in somites and Patched knockout mice display somite dysfunction. Another transmembrane receptor, Smoothened (SMO), is also expressed in somites and transduces the SHH signal regulated by PTCH. We designed this study to test the hypothesis that SHH signalling is downregulated during the critical period of early embryogenesis in the Cd-induced omphalocele chick model. After 60h of incubation, chicks were exposed to either chick saline or 50μL of 50μM cadmium acetate and divided into two groups: control and Cd (n=24 for each group). Chicks were harvested 1, 4, and 8h post-treatment. Real-time RT-PCR was performed to evaluate the relative mRNA expression level of SHH, PTCH and SMO. Immunofluorescence confocal microscopy was then performed to evaluate protein expression/distribution of SHH, PTCH and SMO. The relative mRNA expression levels of SHH, PTCH and SMO were significantly downregulated in the Cd group compared to controls at 4H post treatment, whereas, there were no significant differences at the other time points. Immunohistochemistry revealed that the intensity of SHH, PTCH and SMO was markedly diminished at 4h in Cd-treated embryos compared to controls. Disturbance of the SHH signalling pathway as evidenced by SHH, PTCH and SMO downregulation during the narrow window of early embryogenesis may result in somite maldevelopment, contributing to the omphalocele phenotype in the Cd chick model.

Aerobic and anaerobic de-epoxydation of mycotoxin deoxynivalenol by bacteria originating from agricultural soil

One hundred and fifty soil samples collected from different crop fields in southern Ontario, Canada were screened to obtain microorganisms capable of transforming deoxynivalenol (DON) to de-epoxy DON (dE-DON). Microbial DON to dE-DON transformation (i.e. de-epoxydation) was monitored by using liquid chromatography-ultraviolet-mass spectrometry (LC-UV-MS). The effects of growth substrates, temperature, pH, incubation time and aerobic versus anaerobic conditions on the ability of the microbes to de-epoxydize DON were evaluated. A mixed microbial culture from one composite soil sample showed 100% DON to dE-DON biotransformation in mineral salts broth (MSB) after 144h of incubation. Treatments of the culture with selective antibiotics followed an elevated temperature (50°C) for 1.5h considerably reduced the microbial diversity. Partial 16S-rRNA gene sequence analysis of the bacteria in the enriched culture indicated the presence of at least six bacterial genera, namely Serratia, Clostridium, Citrobacter, Enterococcus, Stenotrophomonas and Streptomyces. The enriched culture completely de-epoxydized DON after 60h of incubation. Bacterial de-epoxydation of DON occurred at pH 6.0-7.5, and a wide array of temperatures (12-40°C). The culture showed rapid de-epoxydation activity under aerobic conditions compared to anaerobic conditions. This is the first report on microbial DON to dE-DON transformation under aerobic conditions and moderate temperatures. The culture could be used to detoxify DON contaminated feed and might be a potential source for gene(s) for DON de-epoxydation.

Production of extracellular exoinulinase from Kluyveromyces marxianus YS-1 using root tubers of Asparagus officinalis

Root tubers of Asparagus officinalis were used as a source of raw inulin for the production of exoinulinase (EC 3.2.1.7) from Kluyveromyces marxianus YS-1. Root extract prepared at 10kg/cm2 pressure for 10min showed maximum inulinase production. Medium components and process parameters were standardized to improve the enzyme production. Inulinase yield of 40.2IU/mL in a medium containing raw inulin (3.5%), beef extract (2%), SDS (0.001%), Mn2+ (2.0mM), Mg2+ (1.5mM), Co2+ (2mM) and pH 6.5 has been obtained under agitation (150rpm) after 60h of incubation at 30°C at shake flask level. After optimization, the enzyme production was 4.8 times more than the basal medium. To test the feasibility of raw inulin from A. officinalis for the production of inulinase, trials were also made in a bioreactor (1.5L). Inulinase activity of 50.2IU/mL was obtained from raw inulin (4.0%) under agitation (200rpm) and aeration (0.75vvm) at 30°C after 60h of fermentation. Inulinase yield in bioreactor was almost six times higher than the basal medium used initially in shake flask.

Restriction of Germline Proliferation by Soft X-ray Irradiation of Chicken Embryos and its Application to Chimera Production

Primordial germ cells (PGCs) are the progenitor cells of gametes. Avian PGCs are located in the central region of the area pellucida at the blastoderm stage. PGCs enter the circulation soon after the formation of blood vessels in incubating eggs and eventually settle in the gonadal primordium. We have now examined exposure of chicken embryos to soft (low-energy) x-rays as a means of depleting endogenous PGCs and thereby improving the efficiency of chimera production. The blastoderm of White Leghorn eggs was exposed to soft x-rays for 0, 20, 40 or 60s before incubation. The irradiated embryos manifested delayed development at 60h of incubation. They also showed reduced numbers of circulating PGCs at stages 14 and 15 and of gonadal PGCs at stage 30. The hatchability of irradiated embryos was lower than that of nonirradiated controls. Irradiation for 20s was found to provide the best outcome taking into consideration both the restriction of PGC proliferation and hatchability. Dispersed blastoderm cells of quail (black plumage) embryos were introduced into the blastoderm of chicken embryos irradiated for 20 s or of nonirradiated embryos. The number of donor-derived PGCs was higher in the irradiated embryos than in the nonirradiated controls at stage 30. These results suggest that soft x-irradiation of chicken embryos is a feasible approach to depletion of endogenous germ cells and consequent improvement in the efficiency of incorporation of donor PGCs.

Gibberellic-acid-induced synthesis and release of cell-wall-degrading endoxylanase by isolated aleurone layers of barley

When aleurone layers of barley (Hordeum vulgare L.) are incubated with gibberellic acid (GA3) xylose and arabinose-both as free sugars and bound to larger molecules-are released into the medium. Release begins 10-12h after the start of incubation and continues for at least 60h. At the same time there is a GA3-induced breakdown of the cell wall resulting in a loss of 2/3 of the cell-wall pentose during 60h of incubation. GA3 causes the appearance in the medium of an enzyme (or enzymes) which hydrolyze larchwood xylan and aleurone-layer arabinoxylan. Release of the enzyme(s) into the medium begins 28-32h after the start of incubation. Enzyme activity does not accumulate to any large extent in the tissue prior to release into the medium, and is present in very low levels only in the absence of GA3. Xylanase activity is associated with a protein (or proteins) with a molecular weight of 29,000. The hydrolysis of the xylans is largely caused by endoxylanase activity, indicating the importance of endoglycosidases in the GA3-induced breakdown of the aleurone cell wall.