The requirements of micronutrients for biomass and hydrocarbon production in Botryococcus braunii UTEX 572 were studied using response surface methodology. The concentrations of four micronutrients (iron, manganese, molybdenum, and nickel) were manipulated to achieve the best performance of B. braunii in laboratory conditions. The responses of algal biomass and hydrocarbon to the concentration variations of the four micronutrients were estimated by a second order quadratic regression model. Genetic algorithm calculations showed that the optimal level of micronutrients for algal biomass were 0.266 µM iron, 0.707 µM manganese, 0.624 µM molybdenum and 3.38 µM nickel. The maximum hydrocarbon content could be achieved when the culture media contained 10.43 µM iron, 6.53 µM manganese, 0.012 µM molybdenum and 1.73 µM nickel. The validation through an independent test in a photobioreactor suggests that the modified media with optimised concentrations of trace elements can increase algal biomass by 34.5% and hydrocarbon by 27.4%. This study indicates that micronutrients play significant roles in regulating algal growth and hydrocarbon production, and the response surface methodology can be used to optimise the composition of culture medium in algal culture.
The aim of this study was to develop novel anaerobic media using gellan gum for the isolation of previously uncultured rumen bacteria. Four anaerobic media, a basal liquid medium (BM) with agar (A-BM), a modified BM (MBM) with agar (A-MBM), an MBM with phytagel (P-MBM) and an MBM with gelrite (G-MBM) were used for the isolation of rumen bacteria and evaluated for the growth of previously uncultured rumen bacteria. Of the 214 isolates composed of 144 OTUs, 103 isolates (83 OTUs) were previously uncultured rumen bacteria. Most of the previously uncultured strains were obtained from A-MBM, G-MBM and P-MBM, but the predominant cultural members, isolated from each medium, differed. A-MBM and G-MBM showed significantly higher numbers of different OTUs derived from isolates than A-BM (P < 0·05). The Shannon index indicated that the isolates of A-MBM showed the highest diversity (H' = 3·89) compared with those of G-MBM, P-MBM and A-BM (H' = 3·59, 3·23 and 3·39, respectively). Although previously uncultured rumen bacteria were isolated from all media used, the ratio of previously uncultured bacteria to total isolates was increased in A-MBM, P-MBM and G-MBM.
Elements of micropropagation include establishment of shoot tip cultures, proliferation, rooting, and acclimatization of the resulting plantlets. The wide genetic variation in Pyrus makes micropropagation challenging for many genotypes. Initiation of shoots is most successful from forced dormant shoots or from scions grafted onto seedling rootstocks to impose juvenility. Clean shoots are recovered after testing for contaminants at the initiation stage on ½ strength Murashige and Skoog 1962 medium (MS), at pH 6.9 for 1 week or by streaking on nutrient agar. Although pear species and cultivars are cultured on several well-known media, MS is the most commonly used. Our studies showed that multiplication and growth of shoots are best on Pear Medium with higher concentrations of calcium chloride, potassium phosphate, and magnesium sulfate than MS medium and 4.4 μM N(6) benzyladenine. Pear shoots are often recalcitrant to rooting; however, a 5 s dip in 10 mM indole-3-butyric acid or naphthalene acetic acid before planting on basal medium without plant growth regulators is effective for many genotypes. Pear shoots store well at 1-4°C, and can hold for as long as 4 years without reculture. Cryopreservation protocols are available for long-term storage of pear shoot tips. Acclimation of in vitro-rooted or micrografted shoots in a mist bed follows standard procedures.
The large-scale production of nematophagous fungi as agents of biological control is one of the main challenges to be commercially used. In order to improve growth of microorganism in a culture medium, the addition of growth inducer is common. At the moment, the action of their addition in the mycelia growth and sporulation rate of nematophagous fungi is not known. The purpose of this trial was to evaluate the sporulation rate of Duddingtonia flagrans by adding two growth inducers, meso-inositol and Tween 80, both at 0.5 % in a traditional culture medium Sabouraud glucose agar (SGA) and also in a traditional culture medium enriched with wheat flour and milk powder. From a traditional sterile culture of D. flagrans, four groups were made: SGA; Sabouraud glucose agar-meso-inositol 0.5 %; Sabouraud glucose agar-Tween 80 0.5 %; and Sabouraud glucose agar-enriched (SGA-E). These media were placed at a constant temperature of 27 °C for 4 weeks. Following this, chlamydospores were gently rinsed off with sterile water and counted using a Neubauer haematocytometer to estimate the number of chlamydospores per millilitre of water. The addition of meso-inositol 0.5 % to SGA promoted a significant increase (p < 0.05) in chlamydospore production obtaining an average of 51,715,000 chlamydospores per Petri dish. The highest chlamydospore concentration was observed in the SGA-E in comparison with SGA (p < 0.01) obtaining an average of 208,760,000 chlamydospores. The aim of this study was to obtain basic knowledge regarding the effect of enriched culture medium and growth-inducing meso-inositol and Tween 80 on mycelial growth and production of chlamydospores.
Tetrazolium salts (TTZ), such as tetrazolium violet (TV), have been widely used for microbiological studies. The formation of the colored formazan product due to bacterial reduction of the uncolored reagent is extensively exploited to stain cells or colonies in agar or on filters. But an important toxic effect of tetrazolium salts on bacteria exists that limits their use at high concentrations, impairing the efficient staining of the colonies. This is especially the case for Salmonella spp. where we observed, using a classic photometric approach and mathematical modeling of the growth, an important impact of tetrazolium violet on the apparent growth rate below the inhibitory concentration. In this study, we demonstrate that adding magnesium to the medium in the presence of TV leads to a significant increase in the apparent growth rate. Moreover, when higher TV concentrations are used which lead to total inhibition of Salmonella strains, magnesium addition to the culture media allows growth and TV reduction. This effect of magnesium may allow the use of higher TTZ concentrations in liquid growth media and enhance bacteria detection capabilities.
Endemic brucellosis threatens wild herds of wood bison (Bison bison athabascae) in and around Wood Buffalo National Park, the largest genetic reserve of wood bison in the world. The overall goal of our project was to produce and preserve disease-free embryos for the purpose of conserving the genetic diversity of this species. The aim of the present experiment was to determine the effectiveness of washing procedures for removing Brucella bacteria from in vivo-derived wood bison embryos exposed in vitro to the pathogen. Wood bison cows were given 300mg im of Folltropin diluted in 0.5% hyaluronan on the day of follicle wave emergence (Day 0) and 100mg im of hyaluronan on Day 2, and then given 2500IU im of hCG on Day 5 and inseminated 12 and 24h later. Embryos were collected on Day 13. The experiment was done in 6 replicates (n=4 bison/replicate) and an average of 9 embryos/replicate were collected. Zona pellucida-intact embryos were kept in holding medium (PBS+2% fetal calf serum) and transported to a Biosafety Level 3 laboratory at the International Vaccine Centre, University of Saskatchewan. Embryos were transferred through 5 aliquots of holding medium to remove any contaminant before exposure to Brucella. Embryos were divided equally into 2 Petri dishes (representing later wash groups with v. without antibiotics) containing 2.7mL of holding medium (n=2 to 7 embryos per dish/replicate). In a Class II biosafety cabinet, Brucella abortus biovar 1 (1×10(7) to 1×10(9)CFUmL(-1) in 0.3mL) was added to each Petri dish and incubated for 2h at 37°C in 8% CO2. A sample of holding medium was taken before exposure and after incubation for culture as negative and positive controls, respectively. After incubation, embryos in each Petri dish were subjected to a 10-step washing procedure (according to the IETS Manual, 2010) using wash medium (PBS+0.4% BSA) without antibiotics or with antibiotics (100IUmL(-1) of penicillin+100μgmL(-1) of streptomycin). The embryo wash medium was cultured at wash steps 1, 3, 6, and 9. After the tenth wash, the zona pellucida of each embryo was ruptured mechanically using a glass pipette and embryos were cultured individually. Culturing of samples was done on sheep blood agar and specific identification of Brucella organisms was done by PCR. Brucella abortus was detected in 3 embryos from the group washed in medium without antibiotics (3/27), whereas all embryos washed in medium with antibiotics were culture negative (0/27). Brucella abortus was not detected in wash media after the third wash in either group (with or without antibiotics). In summary, Brucella abortus was removed from 89% of in vitro-exposed wood bison embryos using the washing procedure without antibiotics, and from 100% using the washing procedure with antibiotics. Results validate the embryo washing technique for producing Brucella-free wood bison embryos.
Microbiologists have been using agar growth medium for over 120 years. It revolutionized microbiology in the 1890’s when microbiologists were seeking effective methods to isolate microorganisms, which led to the successful cultivation of microorganisms as single clones. But there has been a disparity between total cell counts and cultivable cell counts on plates, often referred to as the “great plate count anomaly”, that has long been a phenomenon that still remains unsolved. Here we report that a common practice microbiologists have been employing to prepare agar medium has a hidden pitfall: when phosphate was autoclaved together with agar to prepare solid growth media (PT medium), total colony counts were remarkably lower when compared with those grown on agar plates in which phosphate and agar were separately autoclaved and mixed right before solidification (PS medium). We used a pure culture Gemmatimonas aurantiaca T-27(T) and three representative sources of environmental samples, soil, sediment and water, as inocula and compared colony counts between PT and PS agar plates. There were higher numbers of colony forming units (CFUs) on PS medium compared to PT medium using G. aurantiaca or any of the environmental samples. Chemical analysis of PT agar plates suggested that hydrogen peroxide was contributing to growth inhibition. Comparison of 454 pyrosequences of the environmental samples to the isolates revealed that taxa grown on PS medium were more reflective of the original community structure than those grown on PT medium. Moreover, more hitherto-uncultivated microbes grew on PS than on PT medium.
Living color: Fluorescent pH-sensitive nanoparticles 12 nm in diameter were prepared and incorporated into agarose gel in a Petri dish to image pH changes during bacterial growth and metabolism.
Extraction of phycobiliproteins (R-phycoerythrin, R-PE and R-phycocyanin, R-PC) from macro-algae is difficult due to the presence of large polysaccharides (agar, cellulose etc.) present in the cell wall which offer major hindrance for cell disruption. The present study is aimed at developing most suitable methodology for the primary extraction of R-PE and R-PC from marine macro-algae, Gelidium pusillum(Stackhouse) Le Jolis. Such extraction of phycobiliproteins by using ultrasonication and other conventional methods such as maceration, maceration in presence of liquid nitrogen, homogenization, and freezing and thawing (alone and in combinations) is reported for the first time. Standardization of ultrasonication for different parameters such as ultrasonication amplitude (60, 90 and 120µm) and ultrasonication time (1, 2, 4, 6, 8 and 10mins) at different temperatures (30, 35 and 40°C) was carried out. Kinetic parameters were estimated for extraction of phycobiliproteins by ultrasonication based on second order mass transfer kinetics. Based on calorimetric measurements, power, ultrasound intensity and acoustic power density were estimated to be 41.97W, 14.81W/cm(2) and 0.419W/cm(3), respectively. Synergistic effect of ultrasonication was observed when employed in combination with other conventional primary extraction methods. Homogenization in combination with ultrasonication resulted in an enhancement in efficiency by 9.3% over homogenization alone. Similarly, maceration in combination with ultrasonication resulted in an enhancement in efficiency by 31% over maceration alone. Among all the methods employed, maceration in combination with ultrasonication resulted in the highest extraction efficiency of 77 and 93% for R-PE and R-PC, respectively followed by homogenization in combination with ultrasonication (69.6% for R-PE and 74.1% for R-PC). HPLC analysis was carried out in order to ensure that R-PE was present in the extract and remained intact even after processing. Microscopic studies indicated a clear relation between the extraction efficiency of phycobiliproteins and degree of cell disruption in a given primary extraction method. These combination methods were found to be effective for extraction of phycobiliproteins from rigid biomass of Gelidium pusillum macro-algae and can be employed for downstream processing of biomolecules also from other macro-algae.
A Gram-reaction-negative, aerobic, nonmotile, nonspore-forming, and rod-shaped bacterial strain designated Gsoil 3017(T) was isolated from soil of ginseng field and investigated by phenotypic and phylogenetic analyses. Strain Gsoil 3017(T) grew at 10-37 °C (optimal growth at 30 °C) and at pH 5.5-8.0 (optimal growth at pH 7) on R2A and nutrient agar without additional NaCl as a supplement. Strain Gsoil 3017(T) possessed β-glucosidase activity, which was responsible for its ability to transform ginsenosides Rb1, Rc, and Rd (the three dominant active components of ginseng) to F2 and C-K, respectively. Based on 16S rRNA gene phylogeny, the novel strain represents a new branch within the genus Mucilaginibacter family Sphingobacteriaceae, and clusters with Mucilaginibacter frigoritolerans FT22(T) (95.6%) and Mucilaginibacter gotjawali SA3-7(T) (95.6%). The G+C content of the genomic DNA was 48.7%. The predominant respiratory quinone was MK-7, and the major fatty acids were iso-C15:0, iso-C17:0 3-OH, and summed feature 3 (comprising C16:1 ω6c and/or C16:1 ω7c). The major polar lipid was phosphatidylethanolamine. Strain Gsoil 3017(T) could be differentiated genotypically and phenotypically from other type strains of the genus Mucilaginibacter. The isolate therefore represents a novel species, for which the name Mucilaginibacter ginsenosidivorans sp. nov. is proposed, with the type strain Gsoil 3017(T) (=KACC 14954(T) = JCM 17081(T)).