Reciprocal Shaker Research Articles

High-level expression of the methanol-inducible β-galactosidase gene by perfusion culture of recombinant Pichia pastoris using a shaken ceramic membrane flask

The perfusion culture technique using a shaken ceramic membrane flask (SCM flask) was applied to achieve high-level expression of recombinant gene. A recombinant methylotrophic yeast strain, Pichia pastoris, was cultured aerobically with head space ventilation on a reciprocal shaker using an SCM flask. High-level production of β-galactosidase was attempted by increasing both the cell concentration and the intracellular content of β-galactosidase. The productivity and yield of β-galactosidase were compared between two-stage culture and growth-associated production methods. In the two-stage culture method, the cell concentration was first raised to 57 g/ l at 121 h by feeding glycerol and, thereafter, expression of β-galactosidase was induced by feeding methanol. The β-galactosidase activity in the culture broth quickly increased up to 96 kU/ml within 48 h of initiating the production phase, while the cell concentration continued to increase, reaching 106 g/ l after 167 h culture. On the other hand, in the growth-associated production method, β-galactosidase was produced from an early stage of the culture by the feeding of methanol. The β-galactosidase activity reached 152 kU/ml at 168 h, while the cell concentration was depressed to 49.7 g/ l. The results showed that the growth-associated production method with the feeding of methanol was highly effective for high-level expression of the methanol-inducible recombinant gene of Pichia pastoris. A β-galactosidase productivity level 10 times higher than that obtained in an ordinary fed-batch culture using a shake flask was readily achieved by continuous replenishment of the culture supernatant.

Studies on hydrogen production by continuous culture system of hydrogen-producing anaerobic bacteria

Characteristics of continuous hydrogen production and fatty acid formation by an active hydrogen-producing anaerobic bacterium, Clostridium butyricum strain SC-E1, was examined under vacuum and non-vacuum culture systems. The continuous cultures were performed using 1040 ml anaerobic glass bottles containing 600 ml of medium including glucose and polypeptone at a concentration of 0.5 or 1.0% as substrate, and were conducted at pH 6.7, hydraulic retention time (HRT) 8h, and 30°C on a reciprocal shaker. The non-vacuum cultures at 16 days of incubation showed 2.0 to 2.3 mol-H2/mol-glucose and 1.4 to 2.0 mol-H2/mol-glucose of hydrogen productivity at 0.5 and 1.0% of substrate concentration, respectively. The vacuum cultures conducted at 0.28 atm gave 1.8 to 2.3 mol-H2/mol-glucose and 1.3 to 2.2 mol-H2/mol-glucose of hydrogen productivity at 0.5 and 1.0% of substrate concentration, respectively. The fatty acid production from the vacuum cultures exhibited approximately the same yield of fatty acids as those of the non-vacuum cultures. It was concluded that the maximal hydrogen production potential by anaerobic bacteria is 1.3 to 2.2 mol-H2/mol-glucose, which is less than 50% of theoretical. In addition, the total hydrogen production rate by a two-stage bioreactor consisting of a 1-litre anaerobic fermenter (HRT 10h) and a 4-litre photobioreactor (HRT 36h) feeding at 2.4-litre of 1.0% glucose per day was estimated at 1.4 to 5.6 mol-H2/mol-glucose, which is 12 to 47% theoretical.

New method for RNA isolation from actinomycetes: application to the transcriptional analysis of the alcohol oxidoreductase gene thcE in Rhodococcus and Mycobacterium.

A new protocol for the isolation of RNA from Rhodococcus and other actinomycetes such as Mycobacterium and Amycolatopsis was developed. The method is based on rapid lysis of cells in a high-speed reciprocal shaker using small abrasive particles followed by spin column purification of the lysate. This quick procedure yields RNA preparations suitable for functional studies. This was shown for the thcE gene of R. erythropolis NI86/21, which encodes a N,N'-dimethyl-4-nitrosoaniline-dependent alcohol oxidoreductase. The thcE transcript was detected by Northern hybridization in R. erythropolis, R. fascians, Mycobacterium chlorophenolicum and Mycobacterium smegmatis. The transcriptional start point of the gene was determined by primer extension of the R. erythropolis mRNA.

Studies on hydrogen production by continuous culture system of hydrogen-producing anaerobic bacteria

Characteristics of continuous hydrogen production and fatty acid fonnation by an active hydrogen-producing anaerobic bacterium, Clostridium butyricum strain SC-E1, was examined under vacuum and non-vacuum culture systems. The continuous cultures were performed using 1040 ml anaerobic glass bottles containing 600 ml of medium including glucose and polypeptone at a concentration of 0.5 or 1.0% as substrate, and were conducted at pH 6.7, hydraulic retention time (HRT) 8h, and 30°C on a reciprocal shaker. The non-vacuum cultures at 16 days of incubation showed 2.0 to 2.3 mol-H2/mol-glucose and 1.4 to 2.0 mol-H2/mol-glucose of hydrogen productivity at 0.5 and 1.0% of substrate concentration, respectively. The vacuum cultures conducted at 0.28 atm gave 1.8 to 2.3 mol-H2/mol-glucose and 1.3 to 2.2 mol-H2/mol-glucose of hydrogen productivity at 0.5 and 1.0% of substrate concentration, respectively. The fatty acid production from the vacuum cultures exhibited approximately the same yield of fatty acids as those of the non-vacuum cultures. It was concluded that the maximal hydrogen production potential by anaerobic bacteria is 1.3 to 2.2 mol-H2/mol-glucose, which is less than 50% of theoretical. In addition, the total hydrogen production rate by a two-stage bioreactor consisting of a 1-litre anaerobic fennenter (HRT 10h) and a 4-litre photobioreactor (HRT 36h) feeding at 2.4-litre of 1.0% glucose per day was estimated at 1.4 to 5.6 mol-H2/mol-glucose, which is 12 to 47% theoretical.

Determination of oxalic acid (ethanedioic acid) in soil extracts using high performance liquid chromatograph

Abstract This paper describes a method for quantifying oxalate in soil HC1 extracts using reversed‐phase ion‐pairing high performance liquid chromatography with UV detection at 220 nm. The method was adapted from a procedure for determining urinary oxalate (6). The mobile phase was 10 percent KH2PO4 and 5 mM TBA adjusted to pH 2.0 with H3PO4. The analytical column was a totally porous, reversed‐phase silica based C‐8 column (Hibar Li‐Chrosorb™). An important step in this method was the pre‐ treatment of each soil extract with a reversed‐phase C‐18 column (SPICE™ C‐18). Sample pre‐treatment removed complex, non‐polar and low polarity compounds often present in soil extracts. The method was applied to calcareous, agricultural and organic soils materials. An oxalate accumulating fungus, Endothia Parasitica, was used as a verification of method applicability to plant‐fungal materials. Oxalate extraction was accomplished by placing 1:2 suspensions (soil: 0.1 M HCl) on a reciprocal shaker for two hours and subs...

Inhibitory effects on the growth of several bacteria by brown mustard and allyl isothiocyanate.

The inhibitory effects of mustard and its major pungent compound, allyl isothiocyanate (AIT) on the growth of five species of bacteria and the relation between their inhibitory activities were studied. Brown mustard extract was prepared as 20% mustard in 70% ethanol after myrosinase treatment. AIT was dissolved in 70% ethanol to form a concentration equivalent to that in the mustard extract. Bacteria were cultured in nutrient broth containing the mustard extract or AIT at 30°C on a reciprocal shaker, and bacterial growth was determined by turbidimetry. The duration of the lag phase of growth was proportional to the concentration of mustard or AIT in the medium, and the turbidity of the stationary phase was sometimes decreased by these inhibitors. The concentrations of mustard in the medium that inhibited bacterial growth for 24h were 0.138%, 0.104%, 0.064%, 0.043% and 0.089%, and those of AIT were 14.5, 12.3, 6.5, 3.6 and 7.2ppm for Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Pseudomonas fragi and Ps. aeruginosa, respectively. These results suggested that the inhibitory effect of mustard was mainly due to AIT. Furthermore, it was recognized that the effect of mustard on S. aureus and E. coli was bacteriostatic at 0.8% whereas that on Ps. aeruginosa was bactericidal at 0.2%.

Cultivation conditions for extracellular production of penicillinase by Escherichia coli carrying pEAP31 on a semi-large scale

Cultivation conditions for extracellular production of penicillinase on a semi-large scale were established by using Escherichia coli K-12 HB 101 carrying the plasmid pEAP31 with the penicillinase gene from alkalophilic Bacillus sp. no. 170. Extracellular production of the enzyme was affected by several parameters such as concentration of carbohydrates and Nacl, pH value of culture broth, culture temperature, culture volume and shaking speed of the cultivation flask. The organism produced a large amount of the enzyme in culture broth under the optimal conditions established. For example, 180 units/ml of the extracellular enzyme was produced when the organism was inoculated in 300 ml broth in a 500-ml volume cultivation flask and shaken at 30°C on a reciprocal shaker at 172 oscillations/min with 3.2-cm strokes.

Effects of glycose on NADP and NAD glutamate dehydrogenase activities and their relation to ammonium and pH levels in cultures of Bipolaris maydis race T

NADP-glutamate dehydrogenase (NADP-GDH) and NAD-glutamate dehydrogenase (NAD-GDH) activities from Bipolaris maydis race T (ATCC 36180) were determined by measuring the change in absorbance at 340 nm of either reduced NADP or NAD in a reaction mixture of NH4C1, α-ketoglutarate and a cell free extract of the fungus. NADP-GDH activity was high at 48 h, but low at 72 and 96 h when the fungus was incubated on a reciprocal shaker at 28 °C in a mineral salts medium containing 2 g/l glucose and 4 g/l Lasparagine. In contrast, in these cultures NAD-GDH activity was low at 48 h, but high at 72 and 96 h. At 72 and 96 h glucose was not detected in the culture medium. In addition, levels of ammonium and pH increased from 0.0 μmoles/ml and pH 5.8 at 48 h to 10.6 μmoles/ml and pH 7.2 at 72 h, and to 23.0 μmoles/ml and pH 8.4 at 96 h. Fungal mycelia were transferred after 48 h of incubation on media containing 2 g/l glucose and 4 g/l L-asparagine to fresh media containing 0, 2 or 5 g/l glucose with and without 4 g/l L-asparagine. Twenty-four h after transfer to fresh media containing 5 g/l glucose with L-asparagine or 2 or 5 g/l glucose without L-asparagine, NADP-GDH activity was high and NAD-GDH activity was low. Glucose was detected in the culture medium, ammonium was not detected and the pH remained unchanged or decreased. In contrast, 24 h after transfer to fresh media with 0 or 2 g/l glucose with L-asparagine and on media lacking glucose or L-asparagine, NADP-GDH activity was low and NAD-GDH activity was high. Glucose was not detected in the culture medium, ammonium levels were high and the pH increased. Thus, accumulation of ammonium and pH increases accompanying depletion of glucose in a L-asparagine medium could be related to a change in the capacity of B. maydis race T to assimilate and produce ammonium via pathways involving glutamate dehydrogenases.

Lipid and exopolysaccharide production during hydrocarbon growth of a marine bacterium from the sea surface

Lipid and exopolysaccharide production during hydrocarbon growth of a marine bacterium from the sea surface

Somatic embryogenesis and plant regeneration from suspension callus culture in melon (Ccumis melo L.).

Somatic embryos and plantlets were induced from suspension culture of cotyledon callus in melon (Cucumis melo L.). Cotyledons excised directly from bared mature seed were cultured on MS agar media with several concentrations of 2, 4-D and BA. Three weeks old calli were transferred to MS liquid medium without growth regulators, and cultured on a reciprocal and gyratory shaker (115 rpm). Somatic embryos and plantlets were observed within 2-3 weeks in the liquid media. The calli cultured on MS agar medium with 1.0mg/l 2, 4-D+0.1mg/l BA were the most suitable for embryogenesis.

27] Biosynthetic matrices from cells in culture

The molecular mechanisms involved in cell adhesion have prompted considerable interest over the years. The study of interactions between cells and matrices in vitro is aimed at shedding some light on the extremely complicated system of interactions that must occur in vivo. The microexudate carpet as evidenced by scanning electron microscope observations promotes cell attachment and spreading of the cells. Thus biochemical studies of the mechanism of this phenomenon are feasible utilizing a modification of a quantitative adhesion assay. The assay is started by adding [ 3 H]leucine-labeled singlecell suspension to each well of the multiple-well dish. The dish is then placed in a reciprocal shaker water bath at 37 ° and 50 strokes per minute. Adhesion kinetics are measured by removing by aspiration the nonadherent cells from wells at various times. The rate of adhesion is expressed as percentage of cells adhered by comparing the radioactivity in each well with that of a 1-ml aliquot of the original cell suspension to which 0.5 ml of 1.5 N NH 4 OH is added. Data obtained in this system indicates that active metabolism is required for adhesion, as evidenced by the inhibition by KCN and iodoacetate combined.

Technological problems in cultivation of plant cells at high density

AbstractUsing Cudrania tricuspidata cells as model plant cells which have high sensitivity to hydrodynamic stress, technology problems in the cultivation of the plant cells at high density were investigated. Using “shake” flasks on a reciprocal shaker and Erlenmeyer flasks on a rotary shaker and with a high supply of oxygen on order to obtain high cell densities in shaken cultures, particles breakdown and damage to the largest cell aggregate group (above 1981 μm in diameter) occurred and normal cell growth became impeded. The mass‐transfer coefficient (K)for a model solid–liquid system (β‐naphthol particles and water) in place of a system of plant cells and a liquid medium was proposed as an intensity index of hydrodynamic stress effects on plant cells in subsequent cultures under various conditions in the bioreactor systems. Normal cell growth was obtained under culture conditions for K values less than about 4.4 × 10−3 cm/sec. The characteristics of various bioreactors used until now were investigated by considering the three main technological factors (capacity of oxygen supply, intensity of hydrodynamic stress effects on plant cells, and intensity of culture broth mixing and air‐bubble desperation). The most suitable bioreactor for culturing plant cells at high density was ajar fermentor with a modified paddle‐type impeller (J‐M). The yield of cell mass in the 10‐liter J‐M (working volume 5 liter) was about 30 g dry weight per liter of medium.

A method of extraction of fungitoxic compounds from leaves infected with virus

Cowpea leaves, inoculated with cucumber mosaic virus (CMV), were cut from their petioles several hours after inoculation, and the intercellular spaces of the leaves were filled with water by vacuum infiltration. Immediately, the leaves were immersed in water and incubated at 27°G on a reciprocal shaker. After incubation for 30 h, the leaf ambient fluids were collected and extracted by ethyl ether. The ether fraction was concentrated by evaporation of the ether and analysed by thin layer chromatography (t.l.c). The main substances separated by t.l.c. had antifungal activity. By using this method, fungitoxic compounds accumulated in the infected tissue following multiplication of the virus were extracted efficiently without killing the tissues. The incubated leaves, prior to extraction, retained high virus infectivity. Such fungitoxic compounds were absent in comparable fluid from virusuninoculated leaves.

A new soil test for simultaneous extraction of macro‐ and micro‐nutrients in alkaline soils

Abstract A new soil test was developed for simultaneous extraction of NO3, P, K, Zn, Fe, Cu and Mn from alkaline soils. The new extraction solution is 1 M in ammonium bicarbonate (NH4HCO3), 0.005 M in Diethylene Triamine Pentaacetic Acid (DTPA) and has a pH of 7.6. The new extracting solution should be stored under mineral oil. Ten grams of soil are weighed out into 125 ml Erlenmeyer flasks. Two 2.5 ml scoops of Fisher G carbon black (to remove color for colorimetric determination of nitrates) are added to each soil, followed by 20 ml extracting solution. The soil mixture is then shaken on an Eberbach reciprocal shaker for 15 minutes at 180 cycles per minute. The extract is then filtered through a Whatman 42 filter paper or its equivalent for NO3, P, K, Zn, Fe, Cu and Mn determinations. The results obtained with the new procedure are highly correlated with results obtained with Olsen's P test, ammonium acetate K test, and Lindsay and Norvell's DTPA‐Zn, Fe, Cu and Mn test. Regression equations between the ...

Extraction of petroleum hydrocarbons from oil-contaminated sediments.

Several methods and solvents for extraction of petroleum hydrocarbons from estuarine sediments were examined. Four methods of extraction were used. Shaking the sediment using a reciprocal shaker proved to be the most efficient method in terms of greater yeild of extracted material. Of the three solvents compared, hexane, benzene and chloroform, benzene was most effective.

Morphological Observations of Diplodia maydis on Synthetic and Natural Substrates as Revealed by Scanning Electron Microscopy

Mycelial and spore morphology of Diplodia maydis were investigated by using scanning electron microscopy after growth on various media and natural substrates (oat and corn kernels, and corn husks). Of several specimen preparation methods studied, Parducz fixation followed by critical-point or freeze-drying gave adequate preservation for pycnidia, mycelia, and spores. Morphological characteristics were similar in rotary and reciprocal shaker cultures and differed from that found in stationary cultures in the amount of slime-like material produced and precipitated matter on the mycelial surfaces. In general, mycelial surfaces were smooth. Large areas of coalesced material were present in all samples examined. Slime-like material produced in liquid media appeared as a finely laced net, randomly appearing throughout the mycelia with bead-like structures present along the net. A fine netting also was observed interspersed among the spores inside the pycnidia obtained from oats. Slime-like material was observed to cover the pycnidia produced on oat and corn kernels. In the latter case, the spores were less protected by the outer slime-like covering. Thickened node-like structures were observed in mycelial mats produced in modified Fries 2 medium, on potato dextrose agar plates, and on infected oats. Round and ovate thickened node-like structures were observed in mycelium produced on corn kernels. In general, node-like structures were less abundant in mycelia from naturally infected substrates. Conidia were commonly rounded to tapered and two celled, with a distinctive ridged septum at the middle. Dried spores were collapsed in a characteristic flask-like fashion.

Morphological Observations of Diplodia maydis on Synthetic and Natural Substrates as Revealed by Scanning Electron Microscopy

Mycelial and spore morphology of Diplodia maydis were investigated by using scanning electron microscopy after growth on various media and natural substrates (oat and corn kernels, and corn husks). Of several specimen preparation methods studied, Parducz fixation followed by critical-point or freeze-drying gave adequate preservation for pycnidia, mycelia, and spores. Morphological characteristics were similar in rotary and reciprocal shaker cultures and differed from that found in stationary cultures in the amount of slime-like material produced and precipitated matter on the mycelial surfaces. In general, mycelial surfaces were smooth. Large areas of coalesced material were present in all samples examined. Slime-like material produced in liquid media appeared as a finely laced net, randomly appearing throughout the mycelia with bead-like structures present along the net. A fine netting also was observed interspersed among the spores inside the pycnidia obtained from oats. Slime-like material was observed to cover the pycnidia produced on oat and corn kernels. In the latter case, the spores were less protected by the outer slime-like covering. Thickened node-like structures were observed in mycelial mats produced in modified Fries 2 medium, on potato dextrose agar plates, and on infected oats. Round and ovate thickened node-like structures were observed in mycelium produced on corn kernels. In general, node-like structures were less abundant in mycelia from naturally infected substrates. Conidia were commonly rounded to tapered and two celled, with a distinctive ridged septum at the middle. Dried spores were collapsed in a characteristic flask-like fashion.

Ultrastructure and lipid changes in Pyrenochaeta terrestris during aging.

Pyrenochaeta terrestris, the onion pink root fungus, was grown on a reciprocal shaker in a synthetic medium which contained cellulose as the only carbon source. The mycelium was processed for lipid analysis and ultrastructural investigations after 8, 16, 24, and 32-day growth intervals.Hyphal cells contained membrane complexes. An electron-dense substance was present in large quantities on hyphal cell walls until after the 24-day period. As the cultures aged, organelles in some hyphal cells disintegrated and viable hyphal cells grew inside senescent cells in some cases. It was not possible to correlate positively lipid content observed at the ultrastructural level with the biochemical lipid analysis as a result of the relatively small amount of lipid observed in hyphal cells at the ultrastructural level.The lipid analysis indicated that the lipid content and mycelial weight reached a maximum at the 16-day period. The major fatty acids present were: C16:0, C18:0, C18:1, and C18:2. As aging occurred, the amount of unsaturation increased in the free fatty acids, the total fatty acids, and the fatty acids of the diglycerides and triglycerides. Only the fatty acids of the monoglycerides failed to increase in unsaturation. The sterol content increased slightly over the total growth period.

Acid and alkaline phosphatases of vibrio parahaemolyticus.

Characteristics of phosphatases of Vibrio parahaemolyticus A55 K+ 105 were investigated. Phosphatases were extracted from intact cells in a 3% NaCl solution by gentle treatment on a reciprocal shaker for 15 min. The pH dependence of phosphatase of the extracts showed two optima; one optimum was found at pH 5.1–5.7, the other at pH 10.5–11.1, and the results were identical with that of resting cells. The activities of both acid and alkaline phosphatases were maximum in the presence of 0.1 m salt (KCl or NaCl). The production of alkaline phosphatase of resting cells in the induction medium containing p-nitrophenylphosphate was largely suppressed by the addition of 0.5% phosphate into the medium. The alkaline phosphatase of the extracts was stimulated about 2.5-fold by the addition of 10−3m Mg++ or Zn++, whereas it was inhibited completely by 10−3 m ethylenediaminetetraacetic acid. On the other hand, Co++ activated acid phosphatase to 98-fold. Both acid and alkaline phosphatases were found in the culture filtrated after cultivation of the organism in a 3% NaCl-nutrient broth medium for 16 hr.

Perithecium Formation by Ceratocystis fimbriata in Response to Nitrogen and Calcium

In an earlier study a synthetic medium, the A-N medium, developed empirically with maltose (18.02 g/liter) as the carbohydrate, mineral salts, thiamine, and a mixture of asparagine and calcium nitrate (furnishing 4 and 4, respectively, of a total of 636 mg N/liter), was shown to support perithecial formation by Ceratocystis fimbriata Ell. & Halst. as abundantly as malt extract agar, a poorly defined natural medium (5). The response to a mixture of nitrogen sources was unexpected because nitrate as the sole N source resulted in a starvation type of colony, and asparagine as the sole N source resulted in a starvation type of colony, and asparagine as the sole N source resulted in a vegetative colony. The present study, to clarify the reason for this response, was briefly reported earlier (6). The materials, methods, and the fungus isolate, referred to herein as the Minnesota isolate, were the same as used previously (5). Although this isolate had been identified as Ceratocystis variospora (Davids.) Moreau, additional studies indicated that it should be referred to the species C. fimbriata because C. variospora was not valid (7), a conclusion supported by Webster and Butler (10). An additional isolate of C. fimbriata, L-35, isolated from mallet-wound canker on almond (8) is referred to herein as the California isolate. One possible explanation for the response to the A-N medium was that the fungus was utilizing nitrate nitrogen when organic nitrogen was available. To test this, the Minnesota isolate was grown in the A-N medium without agar and shaken on a reciprocal shaker. The dry weight of mycelium, pH of the medium, and the residual asparagine, nitrate, and carbohydrate (maltose and glucose) were determined at intervals. The pH of the medium increased to 7.5 from the initial 4.8 during the period of active growth, but fell to 3.3 to 3.8 after growth ceased. Nitrate determinations by the diphenylamine test (9)