Growth and carotenoid production of Phaffia Rhodozyma in fed-batch cultures with different feeding methods

The growth and carotenoid production of Phaffia rhodozyma in fed-batch cultures with different feeding methods and grown at specific growth rates similar to the batch culture was compared. With constant feeding, exponential feeding, DO-stat and pH-stat fed-batch cultures of Phaffia rhodozyma, the highest biomass (17.4 g/l) and lowest carotenoid content (307 g/g cell) of Phaffia rhodozyma was from the DO-stat fed-batch culture. The lowest biomass (14.7 g/l) and highest carotenoid content (412 g/g cell) was from the exponential, fed-batch culture.     

Morphology of a spectrum of parasporal endotoxin crystals from cultures of Bacillus thuringiensis ssp. kurstaki isolate A3-4

Morphology of the parasporal -endotoxin crystals of Bacillus thuringiensis subsp. kurstaki isolate A3-4, a native (Taiwan) strain was studied by scanning (SEM) and transmission (TEM) electron microscopy. The typical bipyramidal crystals and an unusual parasporal inclusion with an embedded body were observed. The parasporal -endotoxin crystal with an embedded body, which was in different size and shape was well demonstrated in thin sections by transmission electron microscopy. The sporulated cells had multiple individually separated inclusions up to four crystals in one cell, which was unique to this isolate, and has not been reported before. The -endotoxin crystals included in the cell or released from the cell after batch fermentation or fed-batch fermentation did not show any altered morphological characteristics. However, judging from thin-sections of TEM, cells and the included parasporal crystals from fed-batch fermentation appeared larger than those from batch fermentation. It was observed that release of spore and parasporal crystals from the bacterial cell produced from batch cultures was earlier than that of fed-batch cultures. Preliminary bioassay results showed that the isolate cultures from both types of culture were equally effective against Plutella xylostella larvae. Based on the morphological observations, this strain may have a multiple insecticidal activities toward different insect species.     

Fed-batch culture of<Emphasis Type="Italic"> Bacillus thuringiensis</Emphasis> based on motile intensity

The operation of a fed-batch culture is more complicated than that of batch or continuous culture. Thus, an appropriate feeding strategy for fed-batch cultures should be carefully designed. In this study, a simple feeding strategy for fed-batch culture of Bacillus thuringiensis based on motile intensity is described. The feeding strategy consisted of two steps: (1) initiating feeding at the peak of motile intensity; (2) terminating feeding at low motile intensity (or non-motility) of the cells. In addition, the motile intensity of B. thuringiensis was used to determine the optimum environmental conditions (pH, temperature, and dissolved oxygen) and optimum medium composition. Using this fed-batch strategy, the production of thuringiensin increased 34% compared with batch culture using the same environmental conditions and medium composition. The proposed strategy for fed-batch culture helps to avoid overfeeding of substrate and facilitates on-line control. A comparison of several alternative strategies for fed-batch culture demonstrated that strategies such as glucose-stat and DO-stat result in a lower productivity than that obtained using the motility intensity method.     

Profile control of the specific growth rate in fed-batch experiments

Summary The aim of this paper is to apply a computer control scheme to a laboratory scale fermentor so that the specific growth rate in a baker's yeast fed-batch culture, which cannot be measured directly, will follow as accurately as possible the desired profile specified in advance. Using an extended Kalman filter and programmed controller/feedback compensator (PF) system proposed previously, profile control of the specific growth rate () was achieved experimentally in a baker's yeast fed-batch culture. Also, bang-bang type profile control of minimized the proportion of budding cells, which have a strong correlation with the fermentative activity in bread-making.     

Stimulation of vitamin B12 formation in aerobically-grownRhodopseudomonas gelatinosa under microaerobic condition

Summary Photopigments and vitamin B12 formation ofRhodopseudomonas gelatinosa were enhanced by a stepwise change of the culture condition from aerobic (oxidation-reduction potential, ORP>+110 mV) to microaerobic condition (ORP=0 to –200 mV). During the microaerobic culture in the malateglutamate medium, -aminolevulinic acid synthetase (ALAS) increased 2 to 4 folds in 4 h with the increases in intracellular content of carotenoid, bacteriochlorophyll and vitamin B12. Effects of light illumination on vitamin B12 formation could not be observed. Further, the production of SCP enriched with vitamin B12 and photopigments from cassava starch was done by changing aerobic to microaerobic culture resulting that intracellular carotenoid, bacteriochlorophyll and vitamin B12 increased to 310, 960 and 38 g/g cell from 230, 0 and 25 g/g cell of aerobic culture, respectively.     

A model equations of the volume transport of multicomponent and heterogeneous non-ionic solutions in double-membrane system

The volume flows model equation for a double-membrane system, in which two membranes separate three compartments (l,m,r) containing the heterogeneous, non-ionic n-component solutions is elaborated. In this system the solution concentrations fulfill the condition Clk > Cmk > Crk. The inter-membrane compartment (m) consists of the infinitesimal layer of solution. The volume of compartment m and external compartments (l and r) fulfill the conditions Vm 0 and Vl =Vr respectively. The linear dependences of the volume flux on concentration differences in binary solutions and nonlinear – in ternary solutions, were obtained. This model for binary and ternary non-electrolyte solutions is discussed. It is shown, that the double-membrane system has rectifying and amplifying properties for osmotic transport and mechanical pressure.     

Optimizing recombinant protein expression in the T7 system under the control of the proUp promoter

-Galactosidase and streptokinase expression was tested under the control of the T7 promoter in batch and fed-batch cultures. An Escherichia coli host GJ1158, which contained the T7 RNA polymerase gene under the osmo-responsive proUp promoter, was used for expression studies. -Galactosidase expression was enhanced from 26 mg l^–1 to 127 mg l^–1 in batch culture when a combination of sucrose and sorbitol was used instead of salt as an inducer. Similarly in fed-batch cultures 140 mg streptokinase l^–1 was formed with sucrose and sorbitol induction which was higher than that achieved with IPTG induced cultures.     

Humans and viruses determine the nature of virus vaccine production processes

Vaccine production processes result from the interaction between humans with a particular cell and virus system. The factors that control progress lie not only in the nature of the virus and animal cell but also in the history of the environment in which the process is to be developed. This latter constraint strongly influences the nature of the technical process that is chosen for the production of the vaccine rather than the achievement of efficiency based on one or other of the many possible engineering parameters of the virus production process. In addition to this it is also clear that we have much to learn about the production of viruses from animal cells in culture and that we may be aided by changing our present paradigm of the virus as a cellular enemy to that of the viruses are the cell's best friend.Paper presented at Cell Engineering III Meeting, Florida, 1992.     

Enhancement of producing catharanthine by suspension growth ofCatharanthus roseus

Summary Increased production of catharanthine (about 200 g/mL) was obtained at low light intensity under chemostat cultivation as compared with batch and fed-batch processes. Photo-inhibition was observed at high light intensity. Cells after being frozen with 5% DMSO produced more catharanthine (up to 350 g/L) in batch culture than those from conventional culture techniques.     

The structure of the incompatibility factors of Schizophyllum commune: Constitution of the three classes of B factors

Summary The B factors of Schizophyllum commune are of 3 classes: The high recombining class I has 7 alleles and 7 alleles; the low recombining classes are class II with 7 allels and probably 2 alleles and class III with probably 2 (or also 2) alleles and 7 allels. A fourth hypothetical class (-) was not found and either does not exist or is indistinguishable from class III by the tests employed. The and alleles differ from and by either (a) mutations affecting both mating specificity and recombination frequency, or (b) deletions involving most of the B region.The research was supported by a grant from the Atomic Energy Commission of the U.S. No. (30-1)-3875 and was performed at the Biological Laboratories, Harvard University, Cambridge, Mass., U.S.A.     

The internal pH of Acetobacterium wieringae and Acetobacter aceti during growth and production of acetic acid

The intracellular pH was measured in growing Acetobacterium wieringae and Acetobacter aceti with an acid equilibrium distribution method. [¹⁴C]-acetylsalicylic acid, [¹⁴C-benzoic acid and [¹⁴C]-acetic acid were used as pH-indicators. The extracellular pH of Acetobacterium wieringae decreased from 7.0 to 5.0 during growth; accordingly, the intracellular pH changed from 7.1 to 5.5, and a pH between 0.1 and 0.65 (interior more alkaline) was maintained. Corresponding results were obtained for Acetobacter aceti. The external pH and the internal pH decreased in parallel from 6.2 to 3.5 and from 5.8 to 3.9, respectively.This demonstrates that neither the anaerobic nor the aerobic acetogen was able to maintain a large pH in the presence of high concentrations of acetic acid.     

Osmotic stability of the cell membrane of Escherichia coli from a temperature-limited fed-batch process

The temperature-limited fed-batch (TLFB) process is a technique where the oxygen consumption rate is controlled by a gradually declining temperature profile rather than a growth-limiting glucose-feeding profile. In Escherichia coli cultures, it has been proven to prevent an extensive release of endotoxins, i.e. lipopolysaccharides, that occurs in the glucose-limited fed-batch (GLFB) processes at specific growth rates below 0.1 h^–1. The TLFB and the GLFB process were compared to each other when applied to produce the periplasmic, constitutively expressed, enzyme -lactamase. The extraction of the enzyme was performed by osmotic shock. A higher production of -lactamase was achieved with the TLFB technique while no difference in the endotoxin release was found during the extraction procedure. Furthermore, it was found that growth at declining temperature, generated by the TLFB technique, gradually stabilizes the cytoplasmic membrane, resulting in a significantly increased product quality in the extract from the TLFB cultures in the osmotic shock treatment.     

Postnatal development of the intrinsic nervous system in the circumvallate papilla-vonEbner gland complex

We have studied the postnatal development of the intrinsic nervous system in the circumvallate papilla–vonEbner gland complex using NADPH-diaphorase cytochemistry, immunocytochemistry (for nitric oxide synthase-1 and -internexin) and electron microscopy. In rats sacrificed in their first day post partum (1p.p.), only isolated NADPH-diaphorase positive neurons were visible in the organ. At 2p.p., a small group of neurons was visible at the base of the papillae and positive neurons formed short chains close to the developing glandular tubules. In the following days, the NADPH-diapharase positive cells increased in number and nerve fibres were associated to small ganglia located at the base of the papilla or in the gland. After the first week of extrauterine life, the intrinsic nervous system was similar to the intrinsic system of adult animals. An immunocytochemical positivity for nitric oxide synthase-1 appeared at 4p.p. in neurons located in the gland and at 7p.p. in cells located at the base of the papilla. Immunocytochemical staining for -internexin showed that at 1p.p. developing nerve fibres were present in the connective tissue of the tongue's muscle layer. At 2–3p.p., developing nerve fibres were also present at the bases and in the core of the papilla. In the following days, the positivity for -internexin was reduced and one week after birth was virtually absent. Ultrastructural examination revealed that since 1p.p. isolated neurons can be found at the base of the papilla. In conclusion, the intrinsic nervous system originates from neurons present in the organ at the birth which, in the first days, undergo a biochemical and morphological maturation while the nerve fibres rapidly grow. These findings support the hypothesis that the intrinsic nervous system of the circumvallate papilla has a role in the maturation of the vonEbner gland.     

Partitioning and separation of α-lactalbumin and β-lactoglobulin in polyethylene glycol/ammonium sulphate aqueous two-phase systems

The partition behaviour of -lactalbumin (la) and -lactoglobulin (lg) on PEG/(NH₄)₂SO₄ system was studied. For purified proteins, a partition coefficient of 12.8 for la and 0.34 for lg, with mass recovery yields of 96.7% for la in the upper phase and 83.8% for lg in the lower phase was obtained, in 18% (w/w) PEG 900/14% (w/w) (NH₄)₂SO₄ system, at pH 7. PEG/(NH₄)₂SO₄ system was an economical alternative for the recovery and separation of the two proteins in cheese whey, allowing a 50% reduction in costs. An efficient and inexpensive separation of both proteins in cheese whey could be achieved, by using 16% (w/w) PEG 900/15% (w/w) (NH₄)₂SO₄, at pH 7.5.     

Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus-infected insect cells

The baculovirus infection process ofSpodoptera frugiperda (Sf9) insect cells in oxygen-controlled bioreactors in serum-free medium was investigated using a recombinantAutographa californica (AcNPV) virus expressing -galactosidase enzyme as a model system. A variety of monitoring techniques including trypan blue exclusion, fluorescent dye staining, oxygen uptake rate (OUR) measurements, and glucose consumption were applied to infected cells to determine the best way of evaluating cell integrity and assessing the course of baculovirus infection. The metabolism of newly-infected cells increased 90% during the first 24 hours, but as infection proceeded, and cells gradually succumbed to the baculovirus infection, the cytopathic effect of the baculovirus on the cells became evident. Oxygen and glucose uptake rate measurements appeared to more accurately assess the condition of infected cells than conventional trypan blue staining, which tended to overestimate cell viability in the mid stages of infection. The optimal harvest time varied, depending on which technique — SDS-PAGE, chromogenic (ONPG) or fluorometric (C₁₂FDG) — was used to monitor -galactosidase production. Specific -galactosidase production was found to be insensitive to a wide range of culture dissolved oxygen tensions, whereas resuspending cells in fresh medium prior to infection increased volumetric productivity approximately two-fold (800,000 units -galactosidase/ml) compared to cultures infected in batch mode and allowed successful infections to occur at higher cell densities.Abbreviations ONPG ortho-phenyl 2--D-galactopyranoside - OUR oxygen uptake rate (-mol O₂/liter/hour) - q_glucose specific glucose uptake rate (mg glucose/10⁶cell/hour) - q_glutamine specific glutamine uptake rate (mg glutamine/10⁶cell/hour) - qO₂ specific oxygen uptake rate (-mol O₂/10⁶cell/hour) - MOI virus multiplicity of infection (viral plaque forming units/cell)     

A study of the effect of specific growth rate and acetate on recombinant protein production of Escherichia coli JM107

The growth of Escherichia coli in fed-batch and continuous culture is examined and results show that -amylase production is strongly dependent on specific growth rate (dilution rate) of the culture and production is greatest at an intermediate rate. Using continuous culture, it has also been found that the presence of acetate above a certain concentration reduces both _max, and the production of recombinant protein.     

Adjustment,the hands and healing

Chiropractic is the largest unorthodox health profession in the United States. It is licensed in all 50 states and its services covered by governmental and private health insurance. Yet chiropractic remains, in the opinion of medical commentators, an unscientific healing cult. Chiropractic holds that much illness is caused, directly or indirectly, by derangements in homeostasis that result from subtle vertebral malalignments called subluxations. Only a minority of straight chiropracters continue to provide spinal manipulation as their only treatment, while the majority mixers have embraced physical therapy, vitamin supplements and a wide variety of drugless symptomatic treaments Clinical trials suggest that spinal manipulation has short-term benefit in back pain, but the success of chiropractic is not fully explained by that effect. The clinical art, though, as manifested in the chiropractor-patient interaction contributes greatly to chiropractic healing. This process promotes patient acceptance and validation, fulfills expectations, provides explanations and engages the patient's commitment. The same process lies at the core of the art of medicine. Seeing the clinical art as it functions in chiropractic can help us to understand its independent power in medicine.     

Effects of shoot and root application of thiamin on salt-stressed sunflower plants

Plants of sunflower (Helianthus annuus L. cv Giza2) were salt-stressed with a combination of NaCl and CaCl₂ inconcentrations having different osmotic potentials (s from 0 to –1.0MPa) and were treated with 5 and 10mg L^–1 of thiamin either sprayed on the shoot orapplied to the root. The membranes of leaf discs from salt-stressed plantsappeared to be less stable (more injured) under heat(51°C) and drought (40% polyethylene glycol6000) stresses than control plants. Salinity slowed the rate of growth (lengthand dry mass production), lowered leaf relative water content (RWC) and leafandroot water potential (w), decreased the contents of chlorophyll (Chl),soluble sugars (SS) and the K⁺/Na⁺ ratio butenhanced total free amino acids (TAA), Na⁺,Ca²⁺and Cl^– accumulation in the shoot and root system. Root orshoot application of thiamin reduced membrane injury by either heat ordehydration stress, lowered leaf w, improved uptake of K⁺,and increased leaf RWC, Chl, SS, TAA contents and dry mass production. Theeffects of salinity (s), thiamin (Thi.) and their interaction(s×Thi) on the parameters tested were significant.Salinity was dominant (as indicated by ² values) in affectingthe contents of Ca²⁺, Cl^–, TAA and membranestability to heat and leaf w. The role of thiamin was dominant forNa⁺, K⁺ and SS contents and the contribution ofinteraction was dominant for growth parameters, Chl. and root w.     

Characteristic nonlinearities of the 3/s ictal electroencephalogram identified by nonlinear autoregressive analysis

We describe a method for the characterization of electroencephalographic (EEG) signals based on a model which features nonlinear feedback. The characteristic EEG fingerprints obtained through this approach display the time-course of nonlinear interactions, rather than aspects susceptible to standard spectral analysis. Fingerprints of seizure discharges in six patients (five with typical absence seizures, one with complex partial seizures) revealed significant nonlinear interactions. The timing and pattern of these interactions correlated closely with the seizure type. Nonlinear autoregressive (NLAR) analysis is compared with other nonlinear dynamical measures that have been applied to the EEG.