Submerged culture of chromium-enriched Termitomyces albuminosus

A species of mushroom, Termitomyces albuminosus, was cultured in liquid medium for production of chromium-enriched mycelium. The influence of chromium （Ⅲ） on mycelial growth of T. albuminosus was investigated. An optimum medium composed of 5.6g/L yeast extract, 51.6g/L hydrolyzed rice, 2g/L KH2PO4, and 20mg/L chromium（Ⅲ） with initial pH of 4.5 was obtained by using method of central composite design （CCD）. After incubation of 84h, the maximal biomass of chromium-enriched mycelia reached 24.23g DMW（dried mycelial weight）/L with 272μg/g DMW chromium content in 500mL flasks containing 100mL medium with an inoculum of 8% on a shaker of 100r/min under an optimized cultivation condition at 28 ℃.     

Approaches to valuing plant medicines: the economics of culture or the culture of economics?

This paper explores the scope and limitations of the economic valuation of biodiversity. How and why is this a useful exercise, but in what way is its usefulness constrained? These issues are discussed with particular reference to cultural values within the context of medicinal plants and phytopharmaceutical development. It is argued that whilst economic valuation is an extremely important and necessary aid to formulating conservation policy, it probably is not that informative about the motivations of people in their use of certain natural resources. This is especially true for the case of medicinal plants where belief systems about the causes of disease and therefore strategies for healing are important. The paper reviews studies which have attempted to put monetary values on medicinal plants and the option values of pharmaceuticals developed from plants, and discusses the applicability of the various approaches. The implications for conservation policy, and for likely collaboration between social and natural scientists is discussed.     

Behaviour in culture of isolated protoplasts from “Paul's scarlet” rose suspension culture cells

Summary Protoplasts were enzymatically isolated from Paul's scarlet rose suspension culture cells. They were cultured in medium similar to that used to culture the cells from which they were isolated with the addition of sucrose as an osmotic stabiliser. They were studied by light and electron microscopy and their changes in size and number per culture were recorded. Expansion was greater when the protoplasts were cultured in medium plus 12% sucrose than with 24% sucrose. Budding was observed. In medium plus 12% sucrose about 45% of the protoplasts divided but in medium plus 24% sucrose far fewer divided. Cytokinesis was abnormal: the phragmoplast disappeared soon after cytokinesis began and the cell plate became a groove and then a fibril-lined or filled tongue which progressed across the vacuole, unconnected by strands to other parts of the protoplast. The wall regenerated after several days culture in medium plus 12% sucrose fluoresced with calcofluor. The wall regenerated in medium with 24% sucrose fluoresced usually only after several weeks culture. Cytokinesis hastened formation of a wall fluorescing with calcofluor. In the electron microscope the wall was seen to contain fibrils and non-fibrillar material. The latter was the minor component in medium plus 12% sucrose but was usually the major component in medium plus 24% sucrose. The growth in plasmolysing and nonplasmolysing medium of the cells from which protoplasts are isolated was also studied.It appears that loss of the wall alters the potential of protoplasts to expand and possibly also to regenerate a wall and to divide. Wall regeneration is initially linked with expansion and cytokinesis. Osmotic pressure of the external medium is also an important factor.This work formed part of a thesis by one of us (R.S.P.) approved for the degree of Ph. D. in the University of Nottingham. The work was supported by the Agricultural Research Council.     

Production of poly-β-hydroxybutyrate by fed-batch culture of recombinantEscherichia coli

Summary A recombinantEscherichia coli strain harboring the PHB biosynthesis genes fromAlcaligenes eutrophus was used to produce poly--hydroxybutyrate (PHB) by pH-stat fedbatch culture. Initial glucose concentration for optimal growth was found to be 20g/L from a series of flask cultures. A final PHB concentration of 88.8 g/L could be obtained after 42 hrs of cultivation.     

Validation of the BacT/ALERT?3D automated culture system for the detection of microbial contamination of epithelial cell culture medium

Living tissue engineering for regenerative therapy cannot withstand the usual pharmacopoeia methods of purification and terminal sterilization. Consequently, these products must be manufactured under aseptic conditions at microbiologically controlled environment facilities. This study was proposed to validate BacT/ALERT(?)3D automated culture system for microbiological control of epithelial cell culture medium (ECCM). Suspensions of the nine microorganisms recommended by the European Pharmacopoeia (Chap. 2.6.27: "Microbiological control of cellular products"), plus one species from oral mucosa and two negative controls with no microorganisms were prepared in ECCM. They were inoculated in FA (anaerobic) and SN (aerobic) culture bottles (Biomérieux, Lyon, France) and incubated in a BacT/ALERT(?)3D automated culture system. For each species, five sets of bottles were inoculated for reproducibility testing: one sample was incubated at the French Health Products Agency laboratory (reference) and the four others at Cell and Tissue Bank of Lyon, France. The specificity of the positive culture bottles was verified by Gram staining and then subcultured to identify the microorganism grown. The BacT/ALERT(?)3D system detected all the inoculated microorganisms in less than 2 days except Propionibacterium acnes which was detected in 3 days. In conclusion, this study demonstrates that the BacT/ALERT(?)3D system can detect both aerobic and anaerobic bacterial and fungal contamination of an epithelial cell culture medium consistent with the European Pharmacopoeia chapter 2.6.27 recommendations. It showed the specificity, sensitivity, and precision of the BacT/ALERT(?)3D method, since all the microorganisms seeded were detected in both sites and the uncontaminated medium ECCM remained negative at 7 days.     

Isolated microspore culture of Canadian 6× triticale cultivars

Isolated microspore culture was conducted on nine Canadian triticale cultivars (X Triticosecale Wittmack) using two induction media developed for wheat, with or without 100 g l⁻¹ Ficoll. Significant interactions were observed for the number of embryos and calluses induced, green and albino plantlets regenerated and fertility of green plants. Ficoll was beneficial in both media to increase numbers of embryos and green plants for all cultivars. Overall, medium NPB99 supplemented with ficoll provided the most suitable condition for most cultivars. AC Alta performed slightly better on CHB3 supplemented with Ficoll. Only cv. Wapiti was not amenable to androgenesis. The cultivars AC Certa, AC Copia, AC Alta, Sandro, Ultima, Frank, Pronghorn and Banjo produced respectively, 10, 9, 6, 5, 4, 3, 3 and 1 green plants per Petri dish (35,000 microspores), on their optimum treatment. Twenty-two percent of total lines produced were fertile, and considered doubled haploids. The application of isolated microspore culture to triticale, opens new possibilities in breeding triticale, for the utilization of in vitro selection and genetic engineering.     

Abeta mediated diminution of MTT reduction--an artefact of single cell culture?

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) reduction assay is a frequently used and easily reproducible method to measure beta-amyloid (Abeta) toxicity in different types of single cell culture. To our knowledge, the influence of Abeta on MTT reduction has never been tested in more complex tissue. Initially, we reproduced the disturbed MTT reduction in neuron and astroglia primary cell cultures from rats as well as in the BV2 microglia cell line, utilizing four different Abeta species, namely freshly dissolved Abeta (25-35), fibrillar Abeta (1-40), oligomeric Abeta (1-42) and oligomeric Abeta (1-40). In contrast to the findings in single cell cultures, none of these Abeta species altered MTT reduction in rat organotypic hippocampal slice cultures (OHC). Moreover, application of Abeta to acutely isolated hippocampal slices from adult rats and in vivo intracerebroventricular injection of Abeta also did not influence the MTT reduction in the respective tissue. Failure of Abeta penetration into the tissue cannot explain the differences between single cells and the more complex brain tissue. Thus electrophysiological investigations disclosed an impairment of long-term potentiation (LTP) in the CA1 region of hippocampal slices from rat by application of oligomeric Abeta (1-40), but not by freshly dissolved Abeta (25-35) or fibrillar Abeta (1-40). In conclusion, the experiments revealed a glaring discrepancy between single cell cultures and complex brain tissue regarding the effect of different Abeta species on MTT reduction. Particularly, the differential effect of oligomeric versus other Abeta forms on LTP was not reflected in the MTT reduction assay. This may indicate that the Abeta oligomer effect on synaptic function reflected by LTP impairment precedes changes in formazane formation rate or that cells embedded in a more natural environment in the tissue are less susceptible to damage by Abeta, raising cautions against the consideration of single cell MTT reduction activity as a reliable assay in Alzheimer's drug discovery studies.     

Starter culture for the production of ‘soyiru’

Bacillus subtilis or licheniformis facilitated production of soyiru with the best results being given by using both together. Fermentation employing Streptococcus enterococcus was unsuccessful.H.A. Suberu is with the Department of Biological Sciences, Usmanu Danfodiyo University, Sokoto, Nigeria. J.A. Akinyanju is with the Department of Biological Sciences, University of Ilorin, P.M.B. 1515, Ilorin, Nigeria.     

Increased production of cloned β-galactosidase in two-stage culture of Bacillus amyloliquefaciens

Bacillus amyloliquefaciens harboring recombinant plasmid pHG5, which encodes B. stearothermophilus β-galactosidase, was cultured in a jar fermentor. By feeding lactose a considerable concentration of the enzyme was produced, but the cells stopped growing at an OD₆₆₀ of about 30. On the other hand, the microorganism grew to a very high cell concentration with an OD₆₆₀ of around 110 with glucose as a carbon source, but the enzyme specific activity was a half of the maximum value with lactose. Based on these facts, B. amyloliquefaciens was first grown using glucose, and the carbon source was then switched to lactose to induce β-galactosidase production. By this two-step culture method, both good cell growth and high enzyme productivity were obtained.     

Improvement of intracellular β-galactosidase production in fed-batch culture of Kluyveromyces fragilis

Four fed-batch control strategies were evaluated to improve the specific lactase activity of Kluyveromyces fragilis. Control strategies tested included DO-stat control, exponential feeding, exponential feeding with manual feedback control and corrected feed-forward control. Each was implemented with standard sensors (i.e., temperature, dissolved oxygen and pH sensors) commonly installed in fermenters. The highest specific activity was obtained using the corrected feed-forward control strategy, a strategy incorporating a novel method for on-line estimation of specific growth rate. The control strategy was able to operate effectively to a final cell density of 69 g dry wt l^–1 with a specific lactase activity of 2 U mg^–1 cell dry wt.     

Enhancement of productivity of recombinant α-amidating enzyme by low temperature culture

We have produced a recombinant C-terminal α-amidating enzyme (799BglIIα-AE) derived from Xenopus laevis by culturing a CHO cell line named 3μ-1S. Recently, we demonstrated that culturing 3μ-1S cells at a temperature below 37 °C led to the following phenomena: inhibited cell growth with high viability, enhanced cellular productivity (maximally at 32 °C), and suppressed medium consumption and release of impurities from the cells. Therefore, it is suggested that the 799BglIIα-AE production will be increased by culturing a sufficient number of the cells at a low temperature (especially at 32 °C). To assess this effect on batch and perfusion cultures, the culture temperature was shifted from 37 to 32 °C in the mid-exponential phase in the case of batch culture and from 37 to 34 °C when the cell density became high enough in the case of perfusion culture. Application of the low temperature culture to batch and perfusion cultures was effective in comparison with the culture at 37 °C: the productivity per medium and the productivity per time were increased severalfold with enhanced cellular productivity at a low culture temperature. The low temperature culture also increased the relative content of 799BglIIα-AE in the supernatant and reduced the glucose consumption. The method presented here would contribute to production of bioactive proteins using other recombinant cell lines. This revised version was published online in August 2006 with corrections to the Cover Date.     

Production of poly-γ-glutamic acid by fed-batch culture of Bacillus licheniformis

Fed-batch cultures of Bacillus licheniformis produced poly--glutamic acid (PGA), a water-soluble biodegradable polymer. PGA reached 35 g l^–1 with a productivity of 1 g l^–1 h^–1 by pulsed-feeding of citric acid (1.44 g h^–1) and l-glutamic acid (2.4 g h^–1) when citric acid was depleted from the culture medium.     

Is cell culture stressful? Effects of degradable and nondegradable culture surfaces on U937 cell function

In vitro cell culture has become one of the most widely used techniques in biological and health sciences research, with the most common culture supports being either tissue culture grade polystyrene (TCPS) or polydimethylsiloxane (PDMS). It has previously been shown that monocyte-derived macrophages (MDMs) respond to material surface chemistry, synthesizing and releasing degradative activities that could produce products, which alter the cell's response. In this study, functional parameters of differentiated U937 macrophage-like cells were compared when cultured on nondegradable standard control surfaces versus models of biomaterials (polycarbonate-based polyurethanes) used in the manufacture of medical devices previously shown to degrade and/or elicit pathways of inflammation. Although the influence of PDMS and TCPS on cell function is often underappreciated by investigators, both surfaces elicited enzyme markers of inflammation. Cells on TCPS had the highest intracellular and released esterase activities and protein levels. Cells on PDMS had the most released acid phosphatase activity and protein (P < 0.001), as well as de novo 57- and 59-kDa released proteins. The criteria for defining an activated cell phenotype become critically important when materials such as PDMS and TCPS are used as standard control surfaces whether in experiments for research in elucidating metabolic pathways or in screening drugs and materials for therapeutic uses.     

Production of cucumber fruits from the culture of ‘Marketmore-76’ plantlets

A procedure to produce fruits from cultured shoot tips of Cucumis sativus L. cultivar Marketmore-76 in vitro is described. Four-week-old shoot tips, derived from sterile germinated seedlings on a MS medium, were cultured in a 3.8-1 Mason jar using an automated plant culture system. Tips readily generated roots, leaves and flowers after another 4 to 8 weeks in culture. Administration of compressed air at a 300 ml/min flow rate for 30 min 10 or 15 times a day induced the development of parthenocarpic fruits from flowers. Fruits, up to 170 mm long by 35 mm diameter, were obtained within 30 to 45 d after flower opening.Abbreviations APCS automated plant culture system - BA N-(phenylmethyl)-1H-purin-6-amine - BM basal medium - cv cultivar - MS Murashige and Skoog - NAA 1-naphthaleneacetic acid     

Predominant growth of α-amylase regulation mutant in continuous culture of Bacillus caldolyticus

The production of α-amylase in batch and continuous cultures of the strain SP of Bacillus caldolyticus was studied using a maltose-casitone medium. The three quantitative parameters of α-amylase production (maximum values of the specific production rate, volumetric productivity and concentration of α-amylase) increased in continuous culture by 2.5, 4.6 and 3.8 times respectively in comparison with a batch culture. It was found that a mutant strain M1 of α-amylase production was predominant in every run of the continuous cultures. The strain M1 differs from the strain SP in that it can produce α-amylase constitutively in a batch culture while the cells of strain SP require maltose for α-amylase production. On the contrary, α-amylase production by the strain M1 was repressed partly by maltose. Moreover, glucose repression on α-amylase production was not observed for the M1 strain while it was remarkable for the strain SP. The above-mentioned properties of the mutant M1 concerning the regulation of α-amylase production are not only advantageous for industrial use but also interesting from the viewpoint of basic microbiology.     

Evaluation of agronomic traits in tissue culture – derived progeny of bird's-foot trefoil

Two newly selected Lotus corniculatus cultivars (Bokor and Zora), introduced recently as pasture legumes, were studied with respect to their in vitro regeneration capacity and field performance of R₁ regenerates. Multiple buds were induced in callus tissues derived from apical buds, petioles, leaf and cotyledon explants. The rooted regenerates, acclimated in a greenhouse, were fertile. Seeds from the R₁ generation were sown in the field and the plants found to be superior to controls in stem height and green mass production.     

Tissue culture of the desert plant Anastatica hiërochuntica

Summary Callus derived from the winter annual desert plant Anastatica hiërochuntica was grown on different media, Murashige and Skoog (1962) medium giving the best results. Large amounts of lignified xylem elements were formed resulting in an extremely hard tissue. The growth responses to different auxins, cytokinins and abscisic acid were investigated. When salts (high Na⁺, Ca²⁺ and Cl^--contents) as they can be found in aqueous extracts of desert soils from a natural A. hiëerochuntica habitat were added to Abou-Mandour (1977) or MS-media, growth of callus was inhibited drastically. In the presence of abscisic acid, however, original growth was completely restored. In salt free control media on the other hand, ABA proved to be inhibitory. Drought stress caused a decrease of both cytokinins and indoleacetic acid in the callus while ABA levels were increased, but by far not as distinct as in intact plants. Proline level was not affected by stress.Abbreviations ABA abscisic acid - AM Abou-Mandour-medium - BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DHZR dihydroxyzeatinriboside - DW dry weight - ELISA enzyme linked immuno sorbent assay - FW freshweight - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - IBA indole-3-butyric acid - IPA isopentenyladenosine - Kin kinetin - MS Murashige and Skoog-medium     

Control of molecular weight of poly-β-hydroxybutyric acid produced in fet-batch culture of Protomonas extorquens

Summary To control molecular weight of poly--hydroxybutyric acid (PHB) produced in a fedbatch culture of Protomonas extorquens, the effects of cultural temperature, pH, molar ratio of methanol and ammonia, and concentration of methanol in the medium on polymerization were inverstigated. Change of methanol concentration affected average molecular weight of PHB. When the cultivation was carried out at 0.05 g/l of methanol, average molecular weight of PHB reached above 8×10⁵. On the other hand, in the case of 32 g/l of methanol average molecular weight of PHB was less than 0.5×10⁵. Although every sample had a wide molelcular weight distribution, it became possible to control voluntarily average molecular weight of PHB.     

Selective growth of a mutant in continuous culture of Bacillus caldolyticus for production of α-amylase

Summary In a continuous culture of Bacillus caldolyticus strain SP, which requires maltose as an inducer for production of -amylase in batch culture, a predominant mutant strain M1 which produced high amounts of -amylase in the absence of maltose in batch culture, developed. The change of cell population from strain SP to strain M1 in maltose-casitone medium was linear with time in the transient state after the change from batch to continuous culture at a dilution rate of 0.17 h^-1, and was completed in about 11 generations of bacterial growth. The dilution rate effect of continuous culture on -amylase activity was almost the same with both strains SP and M1. The maximum -amylase activity of 380 units/ml was observed at an intermediate dilution rate that was 11.5 times higher than -amylase activity at the end of a batch culture using the same medium. It was deduced that the enhancement of -amylase production in continuous culture was attributed partly to the predominant growth of a mutant strain with higher -amylase productivity.     

Production of interferon-β in a culture of fibroblast cells on some polymeric films

Normal human skin (NB1-RGB) cells were cultured in the presenceof polyinosinic and polycytidylic acids, diethylaminoethyldextran, cycloheximide and actinomycin D, which induced humaninterferon-. The simplest induction method, that requiredonly polyinosinic and polycytidylic acids and diethylaminoethyldextran was found to give the highest production ofinterferon- by the cells. The cell growth and productionof interferon- were investigated for NB1-RGB cellscultured on silk fibroin, poly(-methyl-L-glutamate),poly(-benzyl-L-glutamate) and collagen films prepared bythe Langmuir-Blodgett (LB) and casting methods. The cell densityof NB1-RGB cells cultured on the LB films was found to be higherthan that on the cast films made of the same polymer. Thisindicates that not only the chemical structure of the polymersused for the preparation of the films but the preparationmethods of the films, i.e., casting and LB methods, are also astrong factor affecting the cell growth. The production ofinterferon- per unit number of cells was found to behigher on the cast films than that on the LB films made of thesame polymer. This is explained by the fact that the optimalsuppressed growth of NB1-RGB cells on the cast films leads tothe enhanced production of interferon- on the cast filmscompared to those on the LB films prepared by the same polymer.