The effects of pH, temperature, phytate, glucose, phosphate and surfactants on the phytase production of Mitsuokella jalaludinii, a new bacterial species from the rumen of cattle, were evaluated.
The effects of different carbon and nitrogen sources on phytase production by Mitsuokella jalaludinii were evaluated and the optimization of rice bran (RB) and soybean milk (SM) concentrations in the medium for phytase production was also determined.
Rhodovulum sulfidophilum was grown in settled undiluted and nonsterilized sardine processing wastewater (SPW). The aims were to evaluate the effects of inoculum size and media on the biomass production with simultaneous reduction of chemical oxygen demand (COD).
This study was undertaken to optimize yeast extract, glucose, and vitamin concentrations; and also culture pH for maximizing the growth of a probiotic bacterium, Lactobacillus rhamnosus, and to assess the effects of these factors by using response surface methodology. A central composite design was used as an experimental design for the allocation of treatment combinations. A polynomial regression model with cubic and quartic terms was used for analysis of the experimental data. It was found that the effects involving yeast extract, glucose, vitamins and pH on the growth of L. rhamnosus were significant, and the strongest effect was given by the yeast extract concentration. Estimated optimum conditions of the factors for the growth of L. rhamnosus are as follows: pH=6.9; vitamin solution=1.28% (v/v); glucose=5.01% (w/v) and yeast extract=6.0% (w/v).
A mycological medium was developed for primary isolation and culture of lipophilic yeasts. It was initially based on published information of nutrients and trace components that would promote the growth of these yeasts. It was subsequently modified and adjusted to specifically promote the growth of lipophilic yeasts and simultaneously avoid the luxurious growth of other fungi and bacteria. With this medium, the conventional bacteriological procedures such as microbial streaking for pure culture and anti-microbial sensitivity testing could be carried out for these lipophilic yeasts.
Isolation and culture of Burkholderia pseudomallei remains the main stay in the diagnosis of melioidosis. Thus, the search for selective and differential media for B. pseudomallei has been ongoing. A number of such media have been reported with varying efficacy. Ashdown medium is the most established selective medium for the isolation of B. pseudomallei. There are no reports of differential media differentiating B. pseudomallei from Burkholderia cepacia. This report documents such a selective and differentiating medium for B. pseudomallei. Of a total of 1042 clinical specimens containing mixed flora and gram-negative isolates that were tested on this medium, 16 of the specimens yielded B. pseudomallei. The isolation rate was found to be 1.5%. This medium was found to be simple and inexpensive, can be made by small laboratories, and called as Francis medium. Based on the colony morphology and color, a preliminary report can be made within 18-24 h for the presence of B. pseudomallei. Our study showed that this medium had an overall sensitivity of 78.4% with a specificity of 92.2%. The use of this medium as an early diagnostic tool will help to reduce mortality and morbidity of melioidosis patients.
In this study, an organic solvent tolerant bacterial strain was isolated. This strain was identified as Pseudomonas sp. strain S5, and was shown to degrade BTEX (Benzene, Toluene, Ethyl-Benzene, and Xylene). Strain S5 generates an organic solvent-tolerant lipase in the late logarithmic phase of growth. Maximum lipase production was exhibited when peptone was utilized as the sole nitrogen source. Addition of any of the selected carbon sources to the medium resulted in a significant reduction of enzyme production. Lower lipase generation was noted when an inorganic nitrogen source was used as the sole nitrogen source. This bacterium hydrolyzed all tested triglycerides and the highest levels of production were observed when olive oil was used as a natural triglyceride. Basal medium containing Tween 60 enhanced lipase production to the most significant degree. The absence of magnesium ions (Mg2+) in the basal medium was also shown to stimulate lipase production. Meanwhile, an alkaline earth metal ion, Na+, was found to stimulate the production of S5 lipase.
A Na(+)/H(+) antiporter gene was isolated from alkaliphilic Bacillus sp. G1. The full-length sequence of the Na(+)/H(+) antiporter gene was obtained using a genome walking method, and designated as g1-nhaC. An ORF preceded by a promoter-like sequence and a Shine-Dalgarno sequence, and followed by a terminator-like sequence was identified. The deduced amino acid sequence consists of 535 amino acids, and a calculated molecular mass of 57 776 Da. g1-nhaC was subsequently cloned into pET22b(+) and expressed in Escherichia coli BL21 (DE3). Recombinant E. coli harboring the g1-nhaC gene was able to grow in modified L medium at various concentrations of NaCl (0.2-2.0 M) at different pH values. The recombinant bacteria grew well in the medium with concentrations of NaCl as high as 1.75 M at pH 8.0-9.0. Minimal growth was observed at 2.0 M NaCl, pH 8.0-9.0. At pH 10, the recombinant bacteria grew well in a medium with a low concentration of NaCl (0.2 M). These results suggested that the g1-NhaC antiporter from Bacillus sp. G1 plays a role in Na(+) extrusion at lower pH values and in pH homeostasis at pH 10 under Na(+)-limiting conditions.
Direct recovery of hepatitis B core antigen (HBcAg) from unclarified Escherichia coli homogenates via expanded bed adsorption chromatography (EBA) has been explored in this study. Streamline DEAE was selected as the anion exchanger to recover HBcAg from heat-treated and non-heat-treated unclarified feedstocks. The use of anion-exchanger for direct extraction of proteins from unclarified feedstock is not preferred due to lack of specificity of its ligand. In this study, thermal treatment of the unclarified feedstock at 60 degrees C has resulted in 1.2- and 1.8-fold increases in yield and purity of HBcAg, respectively, compared with that purified from non-heat-treated feedstock. Heating the crude feedstock has resulted in denaturation and precipitation of contaminants in the feedstock, hence reducing non-specific interactions between the cell debris and adsorbent. The selectivity of the anion-exchanger has also been increased as shown in the breakthrough curve obtained. Enzyme-linked immunosorbent assay showed that the antigenicity of the HBcAg from heat-treated unclarified feedstock is still preserved.
In the present study, various explants of Murraya paniculata (Jack) Linn., such as cotyledons, shoots and young stems were cultured on MS medium supplemented with various concentrations of Benzyl Amino Purine (BAP) under 25 +/- 1 degree C with 16 h light and 8 h dark and also 8 h light and 16 h dark to obtain complete plant regeneration. In vitro flowering was observed from shoot explants cultured on MS supplemented with 0.5-2.0 mg L(-1) Naphthalene Acetic Acid (NAA) and also on MS basal medium under similar conditions. The leaves and flowers obtained from both in vivo and in vitro conditions were examined and compared. Morphological studies such as leaf clearing, epidermal peeling were studied using light and scanning electron microscope. Macromorphological studies of the flowers produced from in vivo and in vitro conditions were also examined. Morphologically, there were no differences between in vivo and in vitro flowers except the flowers produced from tissue culture systems were smaller in size with protruding stigmas. Differences were also found in the number of layers of palisade cells and the presence or absence of epicuticle layer of the leaves. Leaves produced from tissue culture system were smaller in size with membranous texture. Stomata were present only on the abaxial surfaces of both in vivo and in vitro leaves but the stomata were raised above the epidermis in the latter.
Cryptococcus albidus and C. laurentii were the predominant non-neoformans cryptococci isolated during an environmental sampling study for C. gattii at Klang Valley, Malaysia. Cryptococcus gattii was not isolated from any of the environmental samples. Cryptococcus albidus and C. laurentii were isolated mainly from vegetative samples of Eucalyptus trees and bird droppings. Upon testing on canavanine-glycine-bromothymol blue (CGB) agar, all the C. albidus isolates remained unchanged. Interestingly, a total of 29 (76.3%) C. laurentii isolates formed blue colours on the CGB agar. Sequence analysis of ITS1-5.8rDNA-ITS2 gene sequences (468 bp) of four CGB-blue C. laurentii isolates demonstrated the closest match (99%) with that of C. laurentii CBS 7140. This study demonstrated the diverse environmental niche of C. albidus and C. laurentii in Malaysia.
Malignant prostate tissues have markedly reduced zinc (Zn(2+)) contents in comparison to non-malignant tissues. In this study, we restored a high intracellular Zn(2+) level to LNCaP prostate cancer cells by culturing the cells in a growth medium supplemented with a supraphysiological concentration of Zn(2+) (10 microg/ml) over 5 weeks. The intracellular Zn(2+) level increased in the Zn(2+)-treated cells, and there was a marked increase in the presence of zincosomes, a Zn(2+)-specific intracellular organelle. The proliferation rate of the Zn(2+)-treated cells was markedly reduced. There was also a significant increase (36.6% +/- 6.4%) in the total tyrosine phosphorylated proteins. Vaccinia H1-related (VHR) phosphatase, zeta chain-associated protein-70 (ZAP-70) kinase and phosphorylated extracellular signal-regulated protein kinase 1 and 2 (p-ERK 1 and 2) were also present in higher abundance. Treatment with TPEN, which chelates Zn(2+), reduced the abundance of VHR phosphatase and ZAP-70 kinase, but increased the abundance of p-ERK 1. However, the TPEN treatment restored the Zn(2+)-treated LNCaP cell proliferation to a rate comparable to that of the non Zn(2+)-treated cells. These results highlight the importance of a high intracellular Zn(2+) content and the VHR/ZAP-70-associated pathways in the modulation of LNCaP prostate cancer cell growth.
A novel chemically defined medium, named KG medium, supplemented with N-3-oxo-hexanoylhomoserine lactone (3-oxo-C6-HSL), an acylhomoserine lactone (AHL) used as signalling molecules in Gram-negative bacterial cell-to-cell communication, as the sole source of carbon and nitrogen, was designed and successfully used for the enrichment and isolation of AHL-degrading bacteria. A 3-oxo-C6-HSL-degrading bacterium, 13sw7, was isolated from sewage after six enrichment transfers in the 3-oxo-C6-HSL-containing KG medium. On the basis of the almost complete 16S ribosomal DNA sequence, isolate 13sw7 was clustered with unculturable beta-proteobacteria. This study indicates that the AHL-containing KG medium is effective in isolating AHL-degrading bacteria, including those previously considered unculturable, from environmental sources. To the best of our knowledge, this is the first documentation of the isolation of an AHL-degrading proteobacterium from sewage.
Cupriavidus sp. USMAA1020, a local isolate was able to biosynthesis poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer with various 4HB precursors as the sole carbon source. Manipulation of the culture conditions such as cell concentration, phosphate ratio and culture aeration significantly affected the synthesis of P(3HB-co-4HB) copolymer and 4HB composition. P(3HB-co-4HB) copolymer with 4HB compositions ranging from 23 to 75 mol% 4HB with various mechanical and thermal properties were successfully produced by varying the medium aeration. The physical and mechanical properties of P(3HB-co-4HB) copolymers were characterized by NMR spectroscopy, gel-permeation chromatography, tensile test, and differential scanning calorimetry. The number-average molecular weights (M (n)) of copolymers ranged from 260 x 10(3) to 590 x 10(3)Da, and the polydispersities (M (w)/M (n)) were between 1.8 and 3.0. Increases in the 4HB composition lowered the molecular weight of these copolymers. In addition, the increase in 4HB composition affected the randomness of copolymer, melting temperature (T (m)), glass transition temperature (T (g)), tensile strength, and elongation to break. Enzymatic degradation of P(3HB-co-4HB) films with an extracellular depolymerase from Ochrobactrum sp. DP5 showed that the degradation rate increased proportionally with time as the 4HB fraction increased from 17 to 50 mol% but were much lower with higher 4HB fraction. Degradation of P(3HB-co-4HB) films with lipase from Chromobacterium viscosum exhibited highest degradation rate at 75 mol% 4HB. The biocompatibility of P(3HB-co-4HB) copolymers were evaluated and these copolymers have been shown to support the growth and proliferation of fibroblast cells.
The aim of this study was to evaluate Tualang honey as a supplement to fetal bovine serum in cell cultures using MTT assay, chromosome aberration test and gene expression analyses. The MTT assay showed the highest percentage of cell proliferation (105.3% increment than control) of human osteoblast cell line (CRL 1543) in 0.0195% honey in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. There was enhanced cell proliferation corresponding to the decrease in concentrations of honey as indicated by the mitotic index values when the osteoblast cell line was incubated at 37 degrees C for 48 hours. There were no chromosome aberrations both in the honey treated as well as distilled water treated (negative control) cell lines. In the case of gene expression analyses, fibroblast cell lines (CCL 171) were treated with honey (0.0195%) for 24 and 48 hours separately. Though there was over expression for the bcl-xl gene at both 24 and 48 hours, under expression for bcl-xs gene at 24 hours and over expression at 48 hours and under expression for both c-myc and p53 genes at both 24 and 48 hours, none of them were statistically significant in altering the expression of mRNA.
Consumption of probiotics has been associated with decreased risk of colon cancer and reported to have antimutagenic/ anti-carcinogenic properties. One possible mechanism for this effect involves physical binding of the mutagenic compounds, such as heterocyclic amines (HCAs), to the bacteria. Therefore, the objective of this study was to examine the binding capacity of bifidobacterial strains of human origin on mutagenic heterocyclic amines which are suspected to play a role in human cancers. In vitro binding of the mutagens Trp-p-2, IQ, MeIQx, 7,8DiMeIQx and PhIP by three bacterial strains in two media of different pH was analysed using high performance liquid chromatography. Bifidobacterium pseudocatenulatum G4 showed the highest decrease in the total HCAs content, followed by Bifidobacterium longum, and Escherichia coli. pH affects binding capacity; the highest binding was obtained at pH 6.8. Gram-positive tested strains were found to be consistently more effective than the gram-negative strain. There were significant decreases in the amount of HCAs in the presence of different cell concentrations of B. pseudocatenulatum G4; the highest decrease was detected at the concentration of 10(10) cfu/ml. The results showed that HCAs were able to bind with all bacterial strains tested in vitro, thus it may be possible to decrease their absorption by human intestine and increase their elimination via faeces.
Box-Wilson design (BWD) model was applied to determine the optimum values of influencing parameters in anaerobic fermentation to produce hydrogen using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564). The main focus of the study was to find the optimal relationship between the hydrogen yield and three variables including initial substrate concentration, initial medium pH and reaction temperature. Microbial growth kinetic parameters for hydrogen production under anaerobic conditions were determined using the Monod model with incorporation of a substrate inhibition term. The values of micro(max) (maximum specific growth rate) and K, (saturation constant) were 0.398 h(-1) and 5.509 g L(-1), respectively, using glucose as the substrate. The experimental substrate and biomass-concentration profiles were in good agreement with those obtained by the kinetic-model predictions. By varying the conditions of the initial substrate concentration (1-40 g L(-1)), reaction temperature (25-40 degrees C) and initial medium pH (4-8), the model predicted a maximum hydrogen yield of 3.24 mol H2 (mol glucose)(-1). The experimental data collected utilising this design was successfully fitted to a second-order polynomial model. An optimum operating condition of 10 g L(-1) initial substrate concentration, 37 degrees C reaction temperature and 6.0 +/- 0.2 initial medium pH gave 80% of the predicted maximum yield of hydrogen where as the experimental yield obtained in this study was 77.75% exhibiting a close accuracy between estimated and experimental values. This is the first report to predict bio-hydrogen yield by applying Box-Wilson Design in anaerobic fermentation while optimizing the effects of environmental factors prevailing there by investigating the effects of environmental factors.
Induction strategies for the periplasmic production of recombinant human IFN-alpha2b (interferon-alpha2b) by recombinant Escherichia coli Rosetta-gami 2(DE3) were optimized in shake-flask cultures using response surface methodology based on the central composite design. The factors included in the present study were induction point, which related to the attenuance of the cell culture, IPTG (isopropyl beta-D-thiogalactoside) concentration and induction temperature. Second-order polynomial models were used to correlate the abovementioned factors to soluble periplasmic IFN-alpha2b formation and percentage of soluble IFN-alpha2b translocated to the periplasmic space of E. coli. The models were found to be significant and subsequently validated. The proposed induction strategies consisted of induction at an attenuance of 4 (measured as D600), IPTG concentration of 0.05 mM and temperature of 25 degrees C. The optimized induction strategy reduced inclusion-body formation as evidenced by electron microscopy and yielded 323.8 ng/ml of IFN-alpha2b in the periplasmic space with translocation of 74% of the total soluble product. In comparison with the non-optimized condition, soluble periplasmic production and the percentage of soluble IFN-alpha2b translocated to the periplasmic space obtained in optimized induction strategies were increased by approx. 20-fold and 1.4-fold respectively.
An efficient in vitro plant regeneration system was established for elite, recalcitrant Malaysian indica rice, Oryza sativa L. CV. MR 219 using mature seeds as explant on Murashige and Skoog and Chu N6 media containing 2,4-dichlorophenoxy acetic acid and kinetin either alone or in different combinations. L-proline, casein hydrolysate and L-glutamine were added to callus induction media for enhancement of embryogenic callus induction. The highest frequency of friable callus induction (84%) was observed in N6 medium containing 2.5 mg l(-1) 2,4-dichlorophenoxy acetic acid, 0.2 mg l(-1) kinetin, 2.5 mg l(-1) L-proline, 300 mg l(-1) casein hydrolysate, 20 mg l(-1) L-glutamine and 30 g l(-1) sucrose under culture in continuous lighting conditions. The maximum regeneration frequency (71%) was observed, when 30-day-old N6 friable calli were cultured on MS medium supplemented with 3 mg l(-1) 6-benzyl aminopurine, 1 mg l(-1) naphthalene acetic acid, 2.5 mg l(-1) L-proline, 300 mg l(-1) casein hydrolysate and 3% maltose. Developed shoots were rooted in half strength MS medium supplemented with 2% sucrose and were successfully transplanted to soil with 95% survival. This protocol may be used for other recalcitrant indica rice genotypes and to transfer desirable genes in to Malaysian indica rice cultivar MR219 for crop improvement.