A prototype neural network supervised control system for Bacillus thuringiensis fermentations

This article discusses the development of a prototype neural network-based supervisory control system for Bacillus thuringiensis fermentations. The input pattern to the neural network included the type of inoculum, operation temperature, pH value, accumulated process time, optical density in fermentation medium, and change in optical density. The output from the neural network was the predicted optical density for the next sampling time. The control system has been implemented in both a computer simulation and a laboratory fermentation experiment with promising results. (c) 1994 John Wiley & Sons, Inc.     

The Continuous Culture of Luminous Bacteria: a Luminostat

A method has been designed for the continuous culture of luminous bacteria. The control system for the culture uses a combination of luminescence and optical density as a light signal received by a photomultiplier. This combined signal operates pumps which exchange the growth medium. Using this method, a culture of brightly luminescing bacteria was maintained for periods up to 3 weeks.     

Studies on the Growth in Culture of Plant Cells: XII. A VERSATILE SYSTEM FOR THE LARGE SCALE BATCH OR CONTINUOUS CULTURE OF PLANT CELL SUSPENSIONS

A versatile large-scale batch culture unit has been developedfor the growth of plant cell suspension cultures. This unithas been modified to permit of intermittent or continuous renewalof culture medium and, in a modified form, incorporated intoopen continuous culture systems of the chemostat and turbidostattype. A fully automatic culture sampler has been incorporatedinto the basic culture unit. The culture systems described havebeen successfully operated using a cell suspension derived fromAcer pseudoplatanus and results are presented demonstratingsynchronous growth in batch culture, prolonged logarithmic increassein cell number under conditions of high aeration and culturemedium renewal, and steady states of growth resulting from automaticregulation of the optical density of the cell suspension andfrom fixed rates of displacement of cell suspension by new medium.The potentialities of the culture systems are discussed in thelight of the experimental results presented.     

含无机盐沉淀菌悬液中微生物生长量的快速测定

目的:消除菌悬液中无机盐沉淀对比浊法测定生长量的干扰。方法:以嗜有机甲基杆菌ME25为材料,采用比浊法,研究了EDTA对菌悬液中无机沉淀物的清除效应以及对菌体生物量测定的影响。结果:在室温、pH 4~11,1.25×10^-2 mol/L EDTA与菌悬液样品作用1min,即可去除样品中无机盐沉淀,样品稳定,在1h内不影响样品中菌体吸光度的测定;实际样品和理论样品测定,相对误差小于3.0%,回收率为98%~100%,RSD均小于0.5%。结论:采用螯合剂EDTA可快速去除菌悬液中的无机盐沉淀,有效地消除沉淀物的干扰,明显提高了比浊法测定生长量的准确度,简便易行,具有较高的实用价值。     

Optimization of a cultivation process for recombinant protein production by Escherichia coli.

A single-stage fed-batch bioprocess for the production of a recombinant protein beta-galactosidase, by E. coli has been developed. The XL1-blue strain of E. coli which harbors a multi-number foreign plasmid PT was cultured in a reformulated medium. Critical medium components were selected and their respective concentrations were optimized with the Orthogonal Table method. An exponential substrate feeding schedule was used to maintain optimum conditions. Inhibition of growth and protein expression caused by excessive concentrations of glucose and acetate was investigated and subsequently minimized with an incremental nutrient feeding schedule which limited the specific growth rate of a culture. The program necessary to facilitate the control of substrate addition is fully described. This program has been used with a 2.5 l bioreactor and a commercially available software package for optimization without on-line or off-line measurement of optical density (OD), CO2, glucose or acetate. The optimized fed-batch process limited the acetate concentration to less than 20 mM; maintained an exponential growth phase for 50 h; and produced a cell density of 51 g l-1 dry cell weight (DCW) or 154 OD600 with a beta-galactosidase activity of 990 U ml-1.     

In situ fermentation monitoring with recombinant firefly luciferase

A novel method is described for the on-line determination of viable cell number. It has been tested in fermentations of Escherichia coli. The cells are transfected with the gene for firefly luciferase and fed low levels of luciferin in the medium. The reaction requires ATP, so the nonviable cells cannot produce light. Thus, light production is linear with viable cell density from innoculation through most of exponential growth. The light emitted by these cells is then conducted from the reaction vessel to the light detection equipment by an optical fiber. With the equipment described below, as few as a 10(6) cells/mL, or an OD(600) of 0.004, are easily detectable and concentrations greater than 10(10) cells/mL are well within range. The data are collected by a computer, so adaptation to on-line control applications is straightforward. During lag phase, this method is much more accurate then optical density measurements. At the end of exponential growth, rapid changes in light production mark carbon source depletion and the onset of cell lysis. A simple model accounts for the luciferin used during the fermentation and corrects the light detected to the proper cell density. (c) 1993 John Wiley & Sons, Inc.     

Development of a semidefined growth medium for Pedobacter cryoconitis BG5 using statistical experimental design

Pedobacter cryoconitis BG5 are psychrophiles isolated from the cold environment and capable of proliferating and growing well at low temperature regime. Their cellular products have found a broad spectrum of applications, including in food, medicine, and bioremediation. Therefore, it is imperative to develop a high-cell density cultivation strategy coupled with optimized growth medium for P. cryoconitis BG5. To date, there has been no published report on the design and optimization of growth medium for P. cryoconitis, hence the objective of this research project. A preliminary screening of four commercially available media, namely tryptic soy broth, R2A, Luria Bertani broth, and nutrient broth, was conducted to formulate the basal medium. Based on the preliminary screening, tryptone, glucose, NaCl, and K₂HPO₄ along with three additional nutrients (yeast extract, MgSO₄, and NH₄Cl) were identified to form the basal medium which was further analyzed by Plackett–Burman experimental design. Central composite experimental design using response surface methodology was adopted to optimize tryptone, yeast extract, and NH₄Cl concentrations in the formulated growth medium. Statistical data analysis showed a high regression factor of 0.84 with a predicted optimum optical (600?nm) cell density of 7.5 using 23.7?g/L of tryptone, 8.8?g/L of yeast extract, and 0.7?g/L of NH₄Cl. The optimized medium for P. cryoconitis BG5 was tested, and the observed optical density was 7.8. The cost-effectiveness of the optimized medium was determined as 6.25 unit prices per gram of cell produced in a 250-ml Erlenmeyer flask.     

Effects of Mounting Media on Fading of Toluidine Blue and Pyronin G Staining in Epoxy Sections

Available mounting media cause fading of histological preparations over time. A study was designed to find the most suitable medium for durable mounting of Araldite embedded semithin sections of rabbit cerebral cortex stained with toluidine blue and pyronin G. Among four synthetic mounting media tested, only DePeX prevented fading of the sections during the first month. All mounting media tested helped preserve staining intensity after one month, since the fading rate after one year is only about half that in sections prepared without mounting medium. The average optical density of sections after one year was higher in preparations mounted with DePeX than in sections treated with the other mounting techniques tested in this study. After one year, the average optical density of sections mounted with DePeX had decreased approximately 20%.     

Colorimetric estimation of diamine oxidase activity.

A simple colorimetric method for estimation of DAO activity with 4-nitrobenzylamine as a substrate (9,10) was developed. Sensitivity of this method, based on conversion of the aldehyde formed in course of the enzymatic reaction into its 4-nitrophenylhydrazone with subsequent measuring of optical density at 590 nm in strongly alkaline medium, exceeded about 25-fold that of the conventional colorimetric procedure for estimation of DAO activity (14).Sensitivity of the spectrophotometric method for estimation of DAO activity with 4-dimethylaminomethylbenzylamine as a substrate (4) was increased about fivefold by conversion of the aldehyde formed in course of the enzymatic reaction into its 4-nitrophenylhydrazone with subsequent measuring of optical density at 530 nm in strongly alkaline medium.     

A method for the microinjection and culture of protoplasts at very low densities

A method has been developed which allows the recovery of calli from a high proportion of individual, injected, mesophyll protoplasts of Nicotiana tabacum c.v. Xanthi. A small drop of low melting point agarose is used both to hold protoplasts during microinjection and for their subsequent culture in feeder dishes. The feeder dishes consist of "beads" of protoplasts at a high density set in agarose to "feed" the infected protoplasts across a liquid medium.The method has been used successfully both with normal protoplasts and protoplasts from which the vacuole has been removed.Abbreviations NT medium Nagata-Takebe medium (Nagata and Takebe, 1971) - MS medium Murashige-Skoog medium (Murashige and Skoog, 1962) - NAA 1-Naphthaleneacetic acid - BAP 6-Benzylaminopurine - LMP agarose low melting point agarose     

Microgravity Alters the Physiological Characteristics of Escherichia coli O157:H7 ATCC 35150, ATCC 43889, and ATCC 43895 under Different Nutrient Conditions

The aim of this study is to provide understanding of microgravity effects on important food-borne bacteria, Escherichia coli O157:H7 ATCC 35150, ATCC 43889, and ATCC 43895, cultured in nutrient-rich or minimal medium. Physiological characteristics, such as growth (measured by optical density and plating), cell morphology, and pH, were monitored under low-shear modeled microgravity (LSMMG; space conditions) and normal gravity (NG; Earth conditions). In nutrient-rich medium, all strains except ATCC 35150 showed significantly higher optical density after 6 h of culture under LSMMG conditions than under NG conditions (P < 0.05). LSMMG-cultured cells were approximately 1.8 times larger than NG-cultured cells at 24 h; therefore, it was assumed that the increase in optical density was due to the size of individual cells rather than an increase in the cell population. The higher pH of the NG cultures relative to that of the LSMMG cultures suggests that nitrogen metabolism was slower in the latter. After 24 h of culturing in minimal media, LSMMG-cultured cells had an optical density 1.3 times higher than that of NG-cultured cells; thus, the higher optical density in the LSMMG cultures may be due to an increase in both cell size and number. Since bacteria actively grew under LSMMG conditions in minimal medium despite the lower pH, it is of some concern that LSMMG-cultured E. coli O157:H7 may be able to adapt well to acidic environments. These changes may be caused by changes in nutrient metabolism under LSMMG conditions, although this needs to be demonstrated in future studies.     

高比活力大肠杆菌磷酸转乙酰酶的纯化

从国内大肠杆菌菌株1.505提取磷酸转乙酰酶(PTA),经数步纯化荻得了高比活力酶制品。该酶制品比粗酶纯化了61倍,比活力达1040单位/mg,经聚丙烯酰胺凝胶电泳检查,仅有两条蛋白质区带。用经过纯化的酶测定辅酶A,其结果6.75单位/10微升酶液在光径1cm时,233nm的吸收值△E_(PTA)<0.009。     

Characterization of a spectrophotometric technique for the estimation of fowl and turkey sperm motility

An objective method of estimating the motility of fowl and turkey spermatozoa, depending on their rheotactic and light-scattering properties, has been developed. From a 1- to 2-minute recorder trace of the optical density of diluted semen before and after stopping its passage through a flow cuvette, three independent constants may be simply and graphically determined. These are: OD_m, the maximum optical density of semen flowing through the cuvette, shown to be dependent on the concentration of spermatozoa; % (ΔOD)_m, the maximal change of optical density following cessation of flow, which has been correlated with the percentage of motile spermatozoa in the sample; and t½, the time taken for the change of optical density to reach % (ΔOD)_m. This latter parameter has been correlated with forward motility of both fowl and turkey spermatozoa.     

Nondividing, Bacteroid-Like Rhizobium trifolii: In Vitro Induction Via Nutrient Enrichment

Rhizobium trifolii 0403 maintained in exponential phase via periodic dilution doubled in 210 min in mannitol-salts medium and doubled in 244 min in glycerolsalts. In both media, cell number and optical density increased in parallel. When exponentially growing cells in either medium were supplemented with a mixture of glucose, Casamino Acids, succinate, and yeast extract, optical density continued to increase but within less than the time required for one doubling, division ceased. The increase in optical density coupled with division cessation resulted in the formation of large, pleomorphic, nondividing cells. Large cells apparently increased in size as a result of swelling only at regions of most recent cell envelope synthesis. Greater than 95% of the cells in a population swelled, and commitment to swelling occurred within two doubling time equivalents. Swollen cells eventually reached a characteristic maximum size and exhibited osmotic fragility.     

A novel isotope labeling protocol for bacterially expressed proteins

Summary A novel protocol for isotopically labeling bacterially expressed proteins is presented. This method circumvents problems related to poor cell growth, commonly associated with the use of minimal labeled media, and problems with protein induction encountered, less commonly, when using enriched labeled media. The method involves initially growing the bacterial cells to high optical density in a commercially available enriched labeled medium. Following a suitable growth period, the cells are transferred to a different (minimal) labeled medium, appropriate for induction. The method is demonstrated using the protein melanoma growth stimulating activity (MGSA).     

Spectroscopic characterization of the Stentor photoreceptor.

1. On the basis of chromatographic and spectroscopic (absorption, fluorescence and its polarization, fluorescence lifetime, circular dichroism) characterization of the Stentor photoreceptor (stentorin) for photophobic response, the photoreceptor chromophore released from mild acid hydrolysis has been identified as hypericin. 2. The native chromophore is apparently linked to a protein (65 K) containing Lys and several hydrophobic residues, which is soluble in acetone and n-pentane. The peptide-linked stentorin (I) chromophore exhibits circular dichroism in the visible region due to the induced optical activity provided by the peptide. 3. The sodium dodecyl sulfate polyacrylamide gel electrophoresis of a 38% fraction of the sucrose density centrifugation has resolved stentorin II proteins having molecular weights of 13 000, 16 000, 65 000 and 130 000. These proteins, as well as the acetone-soluble peptide, have been spectroscopically characterized with particular emphasis on their primary photoreactivity as the photophobic receptor of Stentor coeruleus. 4. Irradiation of whole living Stentor in dilute buffer solutions induces a decrease in the pH of the medium. A strong dependence upon pH in the fluorescence spectra of both synthetic and native chromophores is also evident, showing a significant drop in the pKa of one or more hydroxyl groups in the excited state. A mechanism for the photophobic response, based on this lowering of the pKa as the primary photoprocess, has been discussed.     

Acetoin production as an indicator of growth and metabolic inhibition of Listeria monocytogenes

It has been shown that Listeria monocytogenes produces acetoin from glucose under aerobic conditions. A defined medium with glucose as the sole carbon source was used in an aerobic shake flask culture to reliably produce acetoin. Acetoin, the reactive compound in the Voges-Proskauer test, was assayable in the medium and was used to quantify the metabolic response when inhibitors were added to the medium. Inhibitors such as lactic, acetic, propionic and benzoic acids were used to demonstrate the utility of acetoin production as an indicator of metabolic disruption. With increasing levels of inhibitor, the metabolic and growth responses were measured by acetoin production and optical density change, respectively. Both measurements decreased in a similar manner with increasing inhibitor concentrations. The data also showed the apparent mode of action of the inhibitors. A bacteriostatic effect was observed for the protonated organic acids, acetic (4 mmol l(-1)) and propionic (4 mmol l(-1)), whereas protonated lactic (4 mmol l(-1)) and benzoic (0.16 mmol l(-1)) acids gave an irreversible (apparent bacteriocidal) effect. Lactic, acetic, and propionic acids showed stimulation of metabolic activity at low concentrations, but benzoic did not. Acetoin production is a novel method for quantifying and assessing the mode of action of inhibitors against L. monocytogenes. This system can be used to screen inhibitors for applications in food safety.     

The relationship between the light scattering properties of zymogen granules and the release of their contained proteins

The optical density of suspensions of the digestive enzyme-containing zymogen granule, a roughly spherical 1 micron diameter membrane-enclosed subcellular structure isolated from the exocrine pancreas of mammals, is reduced greatly when they are suspended in physiological media. This reduction in optical density is accompanied by the release of the granule's protein contents. It has traditionally been assumed that this property is due to granule lysis; that is, dissolution of the particle and its consequent disappearance as a strongly scattering object. Thus, lysis would decrease optical density by decreasing the number density of suspended spheres (N) according to Beer's law. However, as a general matter, changes in the optical density of suspensions of spheres may be a function of changes in the refractive index (m) or radius (r) of the objects as well. In this study, we apply Mie theory of scattering by small particles, which, in conjunction with Beer's law, allows us to evaluate whether changes in the scattering properties of granule suspensions are due to changes in N, m or r. Scattering by granule suspensions was reduced in three ways-pH, calcium ion concentration, and detergent concentration. A simple reduction in particle number did not account for decreased scattering and protein release in any of these circumstances. Instead, the changes appear attributable to decreases in particle size and refractive index.     

Automation of the Limulus amoebocyte lysate test by using the Abbott MS-2 microbiology system.

A rapid, automated method for the performance of the Limulus amoebocyte lysate endotoxin assay has been developed by using the Abbott MS-2 Microbiology System. This instrument automatically determines sequential changes in the optical density of up to 176 samples at 1- or 5-min increments during a 1-h assay period. Graphic representation of optical density changes can be viewed on a cathode-ray tube or reproduced by using a hard-copy printer. Limulus amoebocyte lysate preparations that were obtained from different commercial producers and that had similar endotoxin sensitivities by the conventional gelation method varied somewhat in reactivity when determinations were based upon rate changes in optical density. Lysates from Associates of Cape Cod, Difco Laboratories, and M. A. Bioproducts were the most readily adaptable to the MS-2 System. Use of the MS-2 system increased the sensitivity of these preparations from 60- to 250-fold, and as little as 1 pg/ml was detected. Adaptation of the MS-2 instrument for this purpose provides an objective, reproducible, automated method for the performance of Limulus amoebocyte lysate tests on a variety of fluids.     

Ribonucleic acid and protein content of eukaryotic ribosomes isolated from Artemia salina

We are studying the ribosomes from the cryptobiotic embryos of Artemia salina. Here we report on the relation between the optical density at 260 nm of a ribosome solution and its RNA and protein content. Using the original Lowry method or a modified version, it has been found that 1 A₂₆₀ unit contains 42.4 ± 1.6 μg of protein, and, from the phosphorus content, that the same solution contains 41.6 ± 1.0 μg of RNA. Analytical isodensity equilibrium centrifugation gives a value of 1.570 ± 0.005 g/cm³ for the buoyant density of these ribosomes in CsCl. This density can be related to a protein content of 51%, which is in accord with the chemical determinations. The relation between the optical density of ribosomes, RNA, and protein content and the optical density of rRNA of different systems, such as Escherichia coli, yeast, A. salina, and rat liver is discussed.