Extracellular protease as a reversible adhesion regulator in Pseudomonas fluorescens

The investigation of growth dynamics and protein content in a batch Pseudomonas fluorescens culture grown in a synthetic medium with glucose as the sole source of carbon and energy showed that cells reversibly adhere to the walls of the cultivation flask during the first 2-3 h of growth. Over this time period, the total protein content of free and bound cells increased exponentially at a rate of 0.25 h-1, the fraction of proteins in cells being almost the same (60-70%). The protein content in the medium increased from 3 to 50 mg/l, reaching about 30% of the total protein of the culture. The addition of the exponential culture liquid filtrate to the medium together with the inoculum led to the complete inhibition of cell adhesion and a drastic activation of proteolysis, with a concurrent release of more than 80% of cellular proteins into the medium. After 3-5 h of growth, the concentration of extracellular proteins decreased to the control level. Exogenously added proteinase K inhibited cell adhesion, the effect being more pronounced for R-type than for S-type cells. The hypothesis is discussed that the short-term reversible adhesion of cells is regulated with the involvement of a mixture of hydrocarbons, which inactivate the functional activity of bacterial adhesins, and proteases, which digest these adhesins.     

Mass cultivation of the nitrogen-fixing cyanobacteriumGloeotrichia natans,indigenous to rice-fields

Gloeotrichia natans, a nitrogen fixing cyanobacterium common in rice fields in the Philippines, was used for studies to establish key features of its physiology and potential production in outdoor cultures. Under optimal growth conditions (38 °C, pH 8.0, no carbon enrichment) the specific growth rate of rice-field isolate was 0.076 h^–1. The pH of the medium (between 6.5 and 9.0) did not influence the growth rate, but it did affect phycobiliprotein content, as reflected by a change in colour. At pH 7.0 the culture was green-brown, with phycobiliproteins constituting up to 10% of the total protein, while at pH 9.0 the culture was brownish-black and the pigment content was as high as 28% of the total protein. In outdoor cultures the specific growth rate was related directly to cell density in the range of 0.7–1.5 g dry weight 1^–1 at a rate of stirring of 30 rpm, and inversely related to cell density at half this rate. At a stirring of 30 rpm, daily production of outdoor cultures harvested to maintain cell densities of 0.7, 1.15 andw 1.5 g 1^–1 were 14.7, 17.1 and 18.1 g m^–2 d^t, respectively. This rate of production was maintained for more than 45 days. Phycobiliprotein content in the culture kept at a density of 1.5 g 1^–1 reached 14% of the total biomass.     

Effect of β-alanyl-L-histidinato zinc on protein components in osteoblastic MC3T3-El cells: Increase in osteocalcin,insulin-like growth factor-I and transforming growth factor-β

The effect of -alanyl-L-histidinato zinc (AHZ) on protein components in osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 3 days at 37°C in CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ or other reagents, and the cells were cultured further 3 or 6 days. The homgenate of cells was analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The presence of AHZ (10^–7 to 10^–5 M) caused an appreciable increase of many protein components in cells. Especially, the 67 killo-dalton (kDa) and 44 kDa proteins which are the major components from control cells were clearly increased by the presence of AHZ. Furthermore, the concentrations of osteocalcin, insulin-like growth factor-I and transforming growth factor- in the culture medium secreted from osteoblastic cells were markedly increased by the presence of AHZ (10^–6 and 10^–5 M). The effect of AHZ was a greater than that of zinc sulfate (10^–6 and 10^–5 M). The present findings suggest that AHZ can increase many proteins which are involved in the stimulation of bone formation and cell proliferation in osteoblastic cells.     

High frequency of shoot multiplication and bulblet formation of garlic in liquid cultures

In vitro shoot proliferation and bulblet production of garlic (Allium sativum L.) was studied in liquid cultures. Shoots grown in vitro were used as explants and were cultured in MS medium supplemented with 2% (w/v) sucrose and 0.5 mg l^–1 2-iP. Three culture methods (semi-solid, liquid-immersion and raft) were compared for shoot proliferation. Explants in liquid (immersion) culture exhibited an increased multiplication rate and fresh weight of shoots after 3 weeks of culture as compared with the other treatments. Bulblet formation and growth were studied in liquid medium with different concentrations of sucrose (2–13%). MS medium containing 11% (w/v) sucrose was optimal for bulblet development and bulblets developed in this medium within 9 weeks in culture. The highest multiplication rate was (135 bulblets/explant) found when explants were cultured in bulbing medium (MS medium containing 0.1 mg l^–1 NAA+11% (w/v) sucrose) supplemented with 10 M JA. Growth retardants CCC, B-9, ABA also promoted induction and growth of bulblets. Darkness promoted the bulblet induction and growth compared to light conditions (16-h photoperiod of 50 mol m^–2 s^–1). The dormancy of bulblets was broken by cold treatment at 4 °C for 8 weeks.     

Chemostat optimization of biomass production of a mixed bacterial culture utilizing methanol

Summary A continuous culture technique was used to optimize the medium composition and growth conditions of a mixed bacterial culture utilizing methanol. The improved medium resulted in satisfactory growth, high-yield coefficients and gave a product containing reduced polysaccharide concentrations. Optimal growth and biomass yields occurred at pH 6.8 a temperature of 37° C and dissolved oxygen at >20% saturation. The maximum growth rate was 0.58 h^–1 and maximum biomass yield 0.48 g g^–1. The protein content of the product ranged between 81%–83%, and nucleic acid content between 10%–12%, increasing with growth rate. The amino acid profile of the mixed culture product met and, in some cases, exceeded the UN Food and Agricultural Organization standard, indicating a good source of feed protein.Offprint requests to: A. S. Abu-Ruwaida     

Initiation of and solasodine production in hairy root cultures of Solanum mauritianum Scop.

Initiation and establishment of hairy root cultures from leaf or seedling hypocotyl explants of Solanum mauritianum Scop., using six strains of Agrobacterium rhizogenes was attempted. Success was only achieved following hypocotyl inoculation with strain LBA 9402. Transformation frequency was very low, with only one instance out of a possible 90 being recorded. Resultant hairy root cultures grew rapidly and could be maintained using a Murashige and Skoog (1962) medium supplemented with 0.1 g L^–1 myo-inositol and 3% sucrose, either as a solid or liquid culture. Under these conditions, the roots had a solasodine content of 126 g g^–1 DW. Lower levels of solasodine and decreased root growth rates were recorded when the medium strength was reduced by half or 3% glucose substituted for the 3% sucrose.Abbreviations MS Murashige and Skoog's (1962) medium     

Production of extracellular lactase fromFusarium moniliforme

Summary A strain ofFusarium moniliforme, previously used for microbial protein production, excreted lactase (-D-galactosidase, EC.3.2.1 23) when cultivated either in a whey liquid medium or on a wheat bran solid medium. The enzyme produced in both media had pH and temperature optima of 4–5 and 50–60°C respectively and was particularly suitable for processing acid whey.In the whey culture, maximum lactase yield was observed after 95 h of growth at 30°C and whey lactose concentration of 9%. The addition of ammonium, potassium and sodium ions to the growth medium considerably enhanced lactase production. A maximum enzyme yield corresponding to hydrolysis of 3 nmoles o-nitrophenyl--D-galactopyranoside sec^–1 ml^–1 of growth medium, at pH 5 and 60°C, was obtained.In the wheat bran culture, the maximum enzyme yield was obtained after 140 h of growth at 28–30°C. A marked increase in the enzyme production was observed when nitrate or phosphate was added to the growth medium. Also, the addition of certain agricultural by-products (molasses, whey) enhanced lactase production. The observed maximum yield corresponding to the hydrolysis of 182 nmoles of ONPG sec^–1 g^–1 of wheat bran, at pH 5 and 60°C, is comparable to that reported for certain microorganisms used commercially for lactase production.     

Biosynthesis and secretion of recombinant human growth hormone in Pichia pastoris

Mature human growth hormone (hGH) cDNA was cloned by homologous recombination into the yeast Pichia pastoris genome. The hGH gene expression was placed under the control of the methanol-inducible alcohol oxidase 1 (AOX1) gene promoter and the Saccharomyces cerevisiae -factor signal sequence to direct the secretion of recombinant human growth hormone (rhGH) into the growth medium. O₂-limited induction of recombinant yeast strains in shake tubes with 3 ml of culture medium produced up to 11 mg rhGH l^–1, while high cell density cultures using a 2-l bioreactor produced about 49 mg rhGH l^–1 achieving 40% of total protein of the culture medium supernatant.     

Adenosine triphosphate pool levels and endogenous metabolism in Arthrobacter crystallopoietes during growth and starvation

The adenosine triphosphate (ATP) content of Arthrobactery crystallopoietes was measured during growth, starvation and recovery from starvation. During exponential growth of the cells as spheres in a glucose salts medium, the level of ATP per cell remained constant at 8.0×10^-10 g/cell. Morphogenesis to rodshaped cells and an increased growth rate following addition of casein hydrolysate was accompanied by an almost two-fold increase in the ATP level. As division of the rod-shaped cells proceeded, the level of ATP declined. After growing as rods for 12–14 h the cells underwent fragmentation to spheres during which time the ATP level again increased to the original value of 8.0×10^-10 g/cell. As the spherical cells resumed growth on the residual glucose, their ATP content declined for a short period and then remained relatively constant. During starvation of sphere or rod-shaped cells for one week, the ATP level declined by approximately 70% during the first 40–50 h and then remained constant. The endogenous metabolism rate of spherical cells declined during the first 10–20 h of starvation and then remained constant at approximately 0.02% of the cell carbon being utilized per h. Addition of glucose to spherical cells which had been starved for one week increased both the ATP content per cell and their rate of endogenous metabolism. The ATP content fluctuated and then remained at a level higher than maintained during starvation while endogenous metabolism quickly declined.Non-Standard Abbreviations ATP adenosine triphosphate - GS glucose mineral salts - HC casein hydrolysate - PVP polyvinylpyrrolidone - DMSO dimethylsulfoxide - MOPS morpholinopropane sulfonic acid - EDTA ethylene diaminetetraacetic acid     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

The effect of deuterium oxide on protein turnover in Lemna minor

Lemna minor fronds transferred to a sterile culture medium containing 50% (v/v) deuterium oxide (²H₂O) rapidly undergo a loss of soluble protein with a corresponding increase in free amino acids. The loss of protein is due to two factors: (i) the inhibition of protein synthesis for 4 h followed by a slower rate of synthesis than normal, (ii) a rapid 9–10 fold increase in protein degradation. In plants grown for longer periods (3–6 days) in 50% ²H₂O medium, protein synthesis is inhibited by 20% and the rate constant of degradation is 2–3 times that measured in fronds growing in normal (H₂O containing) complete medium. The initial loss of protein is not due to the breakdown of any specific protein fraction. Investigation of several enzymes indicates that all proteins are catabolised in response to ²H₂O treatment. The implications of these results with regard to the interpretation of density-labelling experiments are discussed.     

Pinoresinol and matairesinol accumulation in a Forsythia × intermedia cell suspension culture

A newly established Forsythia × intermedia cell suspension culture was shown to accumulate (+)- and (–)-pinoresinol as well as matairesinol. The influence of the sucrose content of the culture medium and of the cultivation time on pinoresinol and matairesinol accumulation was evaluated. The highest pinoresinol yield was achieved from cells grown in medium containing 6% sucrose for 12 ± 2 days with levels of 0.6–0.8 mg g^–1 dry weight and an average enantiomeric composition of 75 ± 5% (+)-pinoresinol. The highest matairesinol amount was reached in the same medium at the 14th ± 2 culture day with levels of 1.0–2.7 mg g^–1 dry weight. To our knowledge, this is the first report on pinoresinol accumulation in Forsythia × intermedia plants or cell suspension cultures.     

Formation of phenylethanoid glycosides by Cistanche deserticola callus grown on solid media

The optimal growth of Cistanche deserticola callus and formation of phenylethanoid glycosides (PeG) was at 25°C with light irradiation intensity of 24 mol m^–2 s^–1 on solidified B5 media supplemented with 0.5 mg 6-benzylaminopurine l^–1, 10 mg gibberellin l^–1, 800 mg casein hydrolysate l^–1 and 20 g sucrose l^–1. After 30 d culture, the biomass reached 15.5 g dry wt callus l^–1 medium and its PEG content was 10.7% (w/w). The PeG content was 42%–127% higher than those in explants.     

Culture ofGelidium sesquipedale (Clem.) Born.et Thur. in a chemostat system. Biomass production and metabolic responses affected by N flow

Gelidium sesquipedale is the most important raw material used for extraction of agar in Spain. Based on chemostats, a system of culture for macroalgae with a continuous flow of culture medium has been developed. A stressed morphotype from the South of Spain was cultured, and the effects of different rates of NO ₃ ^– flow on growth and internal constituents were investigated in the laboratory. Cultivation was successful after optimizing factors affecting growth, such as irradiance level, renewal rate and water movement. Mass production was dependent on N supply. With a flow of 35 mol NO₃ ^– g^–1 DW d^–1, optimal values of growth (2.1% d^–1) and biomass yield were obtained. In these conditions, biomass yield resembled the values observed in natural populations (about 500 g DW m^–2 y^–1). When the flow of N was reduced to 15 mol NO ₃ ^– g^–1 DW d^–1, growth rate and biomass yield were reduced three-fold, and were null when N was supplied as 7 mol NO ₃ ^– g^–1 DW d^–1. C:N ratio was an index of the physiological status of the tissue, remaining low when N was sufficient and raised to critical values when N supply was limited. Phycobiliproteins, kept at a constant irradiance level, were affected by N supply, acting as an internal nitrogen reserve, unlike chlorophylla. An effective phycobiliprotein synthesis took place when the flow of N was sufficient. Agar yield, on dry weight basis, was similar as a function of N flow, whereas agar yield of the culture was higher when N was sufficient as a result of growth not being limited by N.This system of culture, commonly used in microalgal studies, may have an important use in macroalgae as a system to obtain biomass of high quality as well as a good tool for physiological studies in conditions of continuous and controlled flow of nutrients.     

Plant regeneration from stem cortex protoplasts of Brassica napus

Cell layer strips composed of the epidermis and 7–9 layers of subepidermal cells were isolated from the 3–4 terminal internodes of Brassica napus cv Westar plants at the early flowering stage. The strips were precultured for one day in modified liquid MS [11] medium and subsequently incubated for 17–18 h in a 0.4 M mannitol solution containing 1% Macerozyme and 1% Cellulase Onozuka R-10. Protoplast yield was 2–2.8×10⁶ per 1.0g of tissue. Protoplasts were cultured at 1×10⁵/ml in three different media: S₁ [13], B [12] and L[8]. The first cell divisions occurred after 2–8 days of culture at frequencies of 20–54%. The highest growth rate of colonies was obtained in L medium containing 0.4 M sucrose and 2% Ficoll. After 4 weeks, green calli, 1–2 mm in diameter were transferred onto B₅ [2] medium with 3 mgl^-1 zeatin, 1% sucrose, 0.1 M mannitol and 0.5% agarose for shoot regeneration. Up to 20% of the calli regenerated shoots which subsequently were rooted and established in soil in the greenhouse.     

Production of laccase byBotrytis cinerea and fermentation studies with strain F226

After induction, seven strains ofBotrytis cinerea released into the culture broth considerable amounts of laccase in a brief production time. The set-up of a suitable production process was studied with a selected strain in a 10-L fermenter. The optimum fermentation conditions were a 3% inoculum with a high degree of sporulation, a simple medium containing 20 g L^–1 of glucose and 2 g L^–1 of yeast extract at pH 3.5, 2 g L^–1 gallic acid as inducer, added after 2 days of growth, an agitation speed of 300 rpm, an aeration rate of 1.2 vvm and a temperature of 24°C. By optimizing the culture conditions, the enzyme activity reached 28 U ml^–1 in 5 days with a specific activity of 560 U mg^–1 protein. The best procedure to obtain a suitable crude enzyme preparation was concentration of the supernatant medium to 10% of the initial volume by ultrafiltration, followed by a fractional precipitation with ethanol. The optimum pH and temperature for laccase activity were 5.5 and 40°C, respectively, with syringaldazine as the substrate.     

Fermentative production of P(3HB-co-3HV) from propionic acid by Alcaligenes eutrophus in fed-batch culture with pH-stat continuous substrate feeding method

The feeding of propionic acid for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] by Alcaligenes eutrophus ATCC17697 was optimized using a fed-batch culture system. The concentration of propionic acid was maintained at 3 g l^–1 as growth was inhibited by propionic acid in the broth. A pH-stat substrate feeding system was used in which propionic acid was fed automatically to maintain a pH of the culture broth at 7.0. By feeding a substrate solution containing 20% (w/v) propionic acid, 4.9% (w/v) ammonia water [at a molar ratio of carbon to nitrogen (C/N molar ratio) of 10] in cell growth phase, the concentration of propionic acid in the broth was maintained at 3 g l^–1 giving a specific growth rate of 0.4 h^–1. To promote P(3HB-co-3HV) production, two stage fed-batch culture which consisted of the stage for the cell growth and the stage for the P(3HB-co-3HV) accumulation was carried out. When the substrate solution whose C/N molar ratio was 50 was fed in P(3HB-co-3HV) accumulation phase, the cell concentration and the P(3HB-co-3HV) content in the cells reached 64 g l^–1 and 58% (w/w) in 55.5 h, respectively.     

Effect of nutrient deficiency on accumulation and relative molecular weight of poly-β-hydroxybutyric acid by methylotrophic bacterium,Pseudomonas 135

Summary Pseudomonas 135, a facultative methylotroph, was cultivated on methanol as a sole carbon and energy source for the accumulation of poly--hydroxybutyric acid (PHB). The cells grew fairly well on minimal synthetic medium containing 0.5% (v/v) of methanol at pH 7.0 and 30° C. The maximum specific growth rate was determined to be 0.26–0.28 h^–1 with a growth yield of 0.38 in the optimized growth medium. For stimulation of PHB accumulation in the cells, deficiency of nutrients such as NH _inf4 ^sup+ , Mg²⁺ and PO _inf4 ^sup3– was crucial even though cell growth was significantly suppressed. The PHB content of a 40-h culture was determined to be 37% of the total cell mass in NH _inf4 ^sup+ -limited medium, 42.5% on Mg²⁺-deficient medium, and 34.5% on PO _inf4 ^sup3– -deficient medium. The maximum content of PHB in the cells could reach 55% in NH _inf4 ^sup+ -limited fed-batch culture. The average relative molecular eight determined by gel permeation chromatography was 3.7 × 10⁵ in NH _inf4 ^sup+ -limited culture, 2.5 × 10⁵ in Mg²⁺-deficientmedium, and 3.1 × 10⁵ in PO _inf4 ^sup3– -deficient medium. Polydispersity determined in each culture was relatively high (about 10–11). The solid PHB had a melting temperature of 173° C. Correspondence to: J. M. Lebeault     

Biochemical properties of pigeon milk and its effect on growth

Summary The avian juvenile food pigeon milk was studied for its chemical composition and effect on growth in vivo and in vitro. Pigeon milk on a wet weight basis consisted of 9–13% protein, 9–11% fat, 0.9–1.5% carbohydrate, 0.8–1.1% ash, 0.10–0.12% non-protein nitrogen, energy content 5.6–6.8 kcal·g^-1. Except for proteins there was little or no decrease in pigeon milk constitutents during the first week of secretion. Pigeon milk proteins consisted of trichloroacetic acid (precipitable), trichloroacetic acid (soluble), and free amino acid components in the ranges 8.4–12.1%, 0.5–0.7% and 1.4–2.5%, respectively; whereas the level of trichloroacetic acid (precipitable) and trichloroacetic acid (soluble) components decreased by about 30%, that of the free amino acids increased by 9% in the first week. About 0.6–1.0% of pigeon milk sugar was found in the trichloroacetic acid (soluble) fraction and increased by 67% in the first week. The remainder was found in the trichloroacetic acid (precipitable) fraction and did not change during this period. Major lipids of pigeon milk were the neutral lipids (7.8–8.4%); the minor lipids were glycolipids (0.9–1.6%), phospholipids (0.5–1.4%) and cholesterol (0.5–0.6%). Squabs fed pigeon milk increased their body weight by 22-fold in the first 3 weeks after hatching, and crude extracts of pigeon milk stimulated the growth of cultured hamster ovary cells. These results reflect the ability of pigeon milk to stimulate growth both in vivo and in vitro.Abbreviations AOAC association of official analytical chemists - BRIT board of radiation and isotope technology - CHO chinese hamster ovary - DNA deoxyribonucleic acid - EDTA ethylenediaminetetra-acetic acid - FCS foetal calf serum - GF growth factor - GS goat serum - MEM minimum essential medium - NPN nonprotein nitrogen - PBS phosphate-buffered saline - PM pigeon milk - TCA(P) trichloroacetic acid precipitable fraction - TCA(S) trichloroacetic acid soluble fraction     

The culture of rat myeloma and rat hybridoma cells in a protein-free medium

Y0 is a rat x rat hybridoma cell line, which does not secrete immunoglobulin, produced using a fusion partner derived from the Y3 (Y3,Ag.1.2.3) rat myoloma cell line. Y0 and Y3 have both been widely used as fusion partners in the production of rat x rat hybridomas. Y0 has also been used in recombinant gene technology. Y0 cells grown in shake flask culture, using RPMI 1640 medium with 4mM l-glutamine and 5% foetal bovine serum, reached a maximal cell density of 1.5×10⁶ cells ml^–1 with 86% viability. Y0 cells which has been adapted to grow in ABC protein-free medium reached a maximal density, in shake flask culture, of 8.75×10⁵ cells ml^–1 with 79% viability. An improved protein-free medium, designated W38 medium, was developed. In shake flask culture, W38 medium supported Y0 cell growth to a density of 2.02×10⁶ cells ml^–1 with 96% viability. Two Y3 hybridomas, YID 13.9.4 cells and SAM 618 cells were adapted to growth in W38 medium. For both hybridomas, cell growth and product yield in shake flask culture using W38 medium was superior to that obtained with serum-containing RPMI 1640 medium.Abbreviations F12 Hams F12 medium - DMEM Dulbeccos medium - RPMI RPMI 1640 medium - FBS foetal bovine serum