Agitation effect on growth and metabolic behavior of plant cell suspension cultures of Thevetia peruviana at bench scale reactor

Plant cell suspension cultures of Thevetia peruviana has been explored for pharmaceutical compounds production. The aim of this study was to evaluate the agitation rate effect on growth and metabolism behavior of T. peruviana cells grown in a 7 L stirred tank reactor. Increases in agitation rates favored cell growth, secondary metabolites production and metabolic activity. The highest biomass concentration 11.92?±?0.25 g DW/L was reached at 550 rpm. The oxidase-reductase activity was stimulated at 550 and 800 rpm. Guaiacol peroxidase activity showed an increase for 300 and 550 rpm after day 7. High levels of extracellular Reactive Oxygen Species (ROS) were observed at day 7 for 550 and 800 rpm. Intracellular phenolic compounds (PC) showed an upward trend until day 7 with a maximum phenolic production of 57.78?±?4.70 mg EGA/100 g FW for 550 rpm. These results indicated that cells responded to ROS stress in a non-enzymatic manner during the first 7 days of culture, increasing PC production with antioxidant capacity. After 7 days, cells responded enzymatically. Intracellullar cardiac glycoside showed a relative increase of 1.7 and 2.1 times for 550 and 800 rpm, respectively. The maximum extracellular production of cardiac glycoside for 550 and 800 rpm was 770.34?±?42.84 mg EP/L. Taken together this study established reactor culture conditions for production of cardiac glycosides and PC, especially taxifolin.

     

Inhibition of byssal formation in Semimytilus algosus (Gould, 1850) by a film-forming bacterium isolated from biofouled substrata in northern Chile

Abstract

Semimytilus algosus is a small mussel species that fouls artificial culture systems of the scallop Argopecten purpuratus (Lamarck, 1819) in the north of Chile. Since biofouling organisms are a serious problem in culture, competing with the scallops for food and oxygen, environmentally- friendly methods are required to mitigate the effects of this fouling in the culture systems. The present study reports the evaluation of the inhibitory effect of biofilms and extracellular products (EP) of the bacterium Alteromonas strain Ni1-LEM on the byssal formation of S. algosus juveniles. Laboratory bioassays were carried out to determine the reattachment, exploratory behaviour and/or byssal thread production of the mussel in plastic Petri dishes containing bacterial biofilms, different dilutions of EP, and EP incorporated in a test substratum. It was concluded from the results that culture supernatants of the Alteromonas tested had an inhibitory effect on reattachment by S. algosus.     

Extracellular products from Chlorococcum ellipsoideum and Chlamydomonas globosa

Summary Extracellular products of Chlorococcum and Chlamydomonas were isolated from culture filtrates. Five groups of substances were found in both species: (i) steam-volatile acids, (ii) yellow water-soluble phenolic compounds, (iii) lipophilic substances, (iv) proteins and (v) polysaccharides. Chlorococcum had higher concentrations of all groups than Chlamydomonas; moreover, the two species differed slightly in the composition of groups (ii) and (iii). Probably, these is a great similarity between green algae in the production of extracellular compounds.     

Impact of Anatomic Site on Growth,Efflux-Pump Expression,Cell Structure,and Stress Responsiveness of <Emphasis Type="Italic">Bacteroides fragilis</Emphasis>

This study investigated whether B. fragilis from various human sites acquired stable traits enabling it to express certain efflux pumps (EPs), adopt a particular cell structure, and tolerate certain stressors. Isolates from blood, abscess, and stool (n = 11 each) were investigated. Bacteria from various sites portrayed different ultrastructres and EP expression. Blood isolates were tolerant to nutrient limitation and stool isolates to NaCl and bile salt stress. Stressors significantly increased EP expression. These data demonstrate that (1) B. fragilis acquires stable traits from various in vivo microenvironments; (2) that EPs are involved in stress responsiveness; and (3) that EP expression is tightly controlled and site dependent.     

Studies on toxigenic fungi in roasted foodstuff (Salted seed) and halotolerant activity of emodin-producingAspergillus wentii

Commercial roasted salted peanuts (3% NaCl), popcorn (1% NaCl), summer-squash (9% NaCl), sunflower (3% NaCl) and wild-melon (3% NaCl) seeds are polluted with fungi, mostlyAspergillus flavus, A. niger, Penicillium chrysogenum, P. corylophilum andRhizopus stolonifer. Contamination of popcorn with the fungi is about 10 times higher than in the other foods. These fungi, common also on unsalted seeds, are significantly inhibited in seeds (30% moisture content) treated with 9–21% NaCl. The halotolerantA. wentii represents the main fungus recovered from seeds treated by 15–21% NaCl. 9% NaCl stimulated emodin production byA. wentii on peanut and citrinin production byP. chrysogenum on popcorn and sunflower. Aflatoxin, citrinin and emodin production on popcorn persisted up to 15% NaCl. Popcorn is thus strongly susceptible to fungal invasion and toxin pollution. The halotolerance ofA. wentii was confirmed by its strong permanent growth in liquid medium at up to 15% NaCl. At 3% NaCl the mycelial growth and nitrogen content increased while the level of emodin and lipid production decreased. CO₂ evolution strongly increased at 9–15% NaCl as a characteristic ofA. wentii salt tolerance. Emodin inhibited seed viability and the inhibition dose for 50% reduction (LD₅₀) was 65 mg/L for popcorn and 45 mg/L for sunflower.     

High bioreactor production and emulsifying activity of an unusual exopolymer by Chromohalobacter canadensis 28

Unusual composition of an exopolymer (EP) from an obligate halophilic bacterium Chromohalobacter canadensis 28 has triggered an interest in development of an effective bioreactor process for its production. Its synthesis was investigated in 2‐L bioreactor at agitation speeds at interval 600‐1000 rpm, at a constant air flow rate of 0.5 vvm; aeration rates of 0.5, 1.0, and 1.5 vvm were tested at constant agitation rate of 900 rpm. EP production was affected by both, agitation and aeration. As a result twofold increase of EP yield was observed and additionally increased up to 3.08 mg/mL in a presence of surfactants. For effective scale‐up of bioreactors mass transfer parameters were estimated and lowest values of K_La obtained for the highest productivity fermentation was established. Emulsification activity of EP exceeded that of trade hydrocolloids xanthan, guar gum, and cellulose. A good synergism between EP and commercial cellulose proved its potential exploration as an enhancer of emulsifying properties of trade emulsions. A pronounced lipophilic effect of EP was established toward olive oil and liquid paraffin. Cultivation of human keratinocyte cells (HaCaT) with crude EP and purified γ‐polyglutamic acid (PGA) showed higher viability than control group.     

Production of surfactant and detergent-stable,halophilic, and alkalitolerant alpha-amylase by a moderately halophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. Strain <Emphasis Type="Italic">TSCVKK</Emphasis>

A moderately halophilic alkalitolerant Bacillus sp. Strain TSCVKK, with an ability to produce extracellular halophilic, alkalitolerant, surfactant, and detergent-stable alpha-amylase was isolated from soil samples obtained from a salt-manufacturing industry in Chennai. The culture conditions for higher amylase production were optimized with respect to NaCl, substrate, pH, and temperature. Maximum amylase production of 592 mU/ml was achieved in the medium at 48 h with 10% NaCl, 1% dextrin, 0.4% yeast extract, 0.2% tryptone, and 0.2% CaCl₂ at pH 8.0 at 30 °C. The enzyme activity in the culture supernatant was highest with 10% NaCl at pH 7.5 and 55 °C. The amylase that was partially purified by acetone precipitation was highly stable in various surfactants and detergents. Glucose, maltose, and maltooligosaccharides were the main end products of starch hydrolysis indicating that it is an alpha-amylase.     

Increased enzymatic activities and levels of superoxide anion and phenolic compounds in cultures of basidiomycetes after temperature stress

Selected strains of basidiomycetes (Abortiporus biennis, Trametes versicolor and Cerrena unicolor) were shown to produce enhanced extracellular peroxidase (EP), superoxide dismutase (SOD) and laccase activities following the exposure of 10-day-old fungal cultures to separate high and low temperature stress. The stressful conditions also caused an increase in the concentrations of phenol compounds and superoxide anion radicals in these cultures. At first, peroxidase activity was observed at 12 hours from the moment of temperature stress application. Laccase activity appeared at 96 hours after the maximum levels of superoxide anion radicals (48 h) and SOD activity (36–72 h). The concentration of phenolic substances grew steadily during the period of cultivation. These relations between laccase, SOD and EP as well as superoxide radicals and phenol levels in the environment of ligninolytic fungi seems to be important in the course of the biosynthesis or biodegradation of lignin, as the consequence of adaptation of these basidiomycetes to environmental temperature conditions.     

The role of extracellular polysaccharides produced by the terrestrial cyanobacterium Nostoc sp. strain HK-01 in NaCl tolerance

The terrestrial cyanobacterium Nostoc sp. HK-01 was more tolerant to NaCl stress than the aquatic cyanobacterium Anabaena sp. PCC 7120 (also called Nostoc sp. PCC 7120) which is similar to Nostoc sp. HK-01 in phylogeny. We determined the amount of extracellular polysaccharides (capsular and released polysaccharides) from the cells of both strains cultured with or without 200 mM NaCl. The amount of capsular polysaccharides from Nostoc HK-01 reached approximately 65% of the dry weight whereas that from Anabaena PCC 7120 only occupied approximately 18% of the dry weight under NaCl stress. Anabaena PCC 7120 grew well under NaCl stress when both polysaccharides from Nostoc HK-01 were added to the culture. However, Anabaena PCC 7120 barely grew under NaCl stress when both of its polysaccharides were added. Extracellular polysaccharides from Nostoc HK-01 contained abundant fucose and glucuronic acid in comparison with those from Anabaena PCC 7120. Under NaCl stress, the composition ratios of sugars in the extracellular polysaccharides from Anabaena PCC 7120 hardly changed in comparison with those in ordinary culture conditions. By contrast, the composition ratios of sugars in the extracellular polysaccharides from Nostoc HK-01 changed under NaCl stress. These results suggest that the effect of extracellular polysaccharides from Nostoc HK-01 on NaCl tolerance comes from the increased amount of capsular polysaccharides, the sugar composition, and the change of the sugar composition ratio under NaCl stress.     

Anaerobic Production of Extracellular Polysaccharide by Butyrivibrio fibrisolvens nyx

Anaerobic production of extracellular polysaccharide (EP) was examined, using a previously uncharacterized, obligately anaerobic rumen isolate, Butyrivibrio fibrisolvens nyx, which produced an EP that was rheologically similar to xanthan gum. The main objectives were to determine the nutritional requirements and conditions which promoted EP production by strain nyx. Strain nyx was grown anaerobically in defined and semidefined media. In addition to carbohydrate and nitrogen sources, strain nyx required acetic acid, folic acid, biotin, and pyridoxamine. Strain nyx produced similar amounts of EP at 35 to 40°C. Conditions that improved growth usually improved EP production. Of the carbohydrates tested, glucose supported the fastest growth and most EP production, followed by sucrose, xylose, and lactose. Strain nyx utilized ammonium sulfate, urea, or vitamin-free casein hydrolysate as nitrogen sources for growth and EP production. At 2 and 20 g/liter, respectively, ammonium sulfate and vitamin-free casein hydrolysate supported about the same rates of growth and EP production. EP was not produced in the lag or stationary phases, and EP production was exponential during exponential cell growth. Based on the results of this work, anaerobic EP production with B. fibrisolvens nyx could reduce energy costs for industrial EP production compared with the cost of aerated systems. Finally, this work demonstrated that, under appropriate growth conditions, a gastrointestinal tract (ruminal) microorganism produced high levels of EP.     

On the mechanism of salt tolerance

As glycerol was suggested as an osmotic agent in the salt tolerantDebaryomyces hansenii the concentrations of total, intracellular, and extracellular glycerol produced by this yeast was followed during growth in 4 mM, 0.68 M, and 2.7 M NaCl media. The total amount of glycerol was not directly proportional to biomass production but to the cultural salinity with maximum concentrations just prior to or at the beginning of the stationary phase. In all cultures the cells lost some glycerol to the media, at 2.7 M NaCl the extracellular glycerol even amounted maximally to 80% of the total. A distinct maximum of intracellular glycerol, related to dry weight or cell number, appeared during the log phase at all NaCl concentrations. As the intracellular calculated glycerol concentrations amounted to 0.2 M, 0.8 M, and 2.6 M in late log phase cells at 4 mM, 0.68 M, and 2.7 M NaCl, respectively, whereas the corresponding analysed values for the glycerol concentrations of the media were 0.7 mM, 2.5 mM, and 3.0 mM, glycerol contributes to the osmotic balance of the cells.During the course of growth all cultures showed a decreasing heat production related to cell substance produced, most pronounced at 2.7 M NaCl. At 2.7 M NaCl the total heat production amounted to-1690 kJ per mole glucose consumed in contrast to-1200 and-1130 kJ at 4 mM and 0.68 M NaCl, respectively. TheY _m -values were of an inverse order, being 129, 120, and 93 at 4 mM, 0.68 M, and 2.7 M NaCl, respectively.     

Water relations of polyol accumulation by Zygosaccharomyces rouxii in continuous culture

Summary Glycerol and arabitol were the main polyols accumulated by Zygosaccharomyces rouxii in continuous culture but the intracellular and extracellular concentrations of the polyols varied with the dilution rate and osmoticum used to adjust the water activity (a_w) to 0.960. When the a_w was adjusted with NaCl, glycerol was the main polyol accumulated intracellularly whereas glycerol and arabitol were accumulated when polyethylene glycol (PEG) 400 was used. The extracellular glycerol and arabitol concentrations at 0.960 a_w (NaCl or PEG 400) were similar or decreased relative to cultures at 0.998 a_w. Compared to steady-state cultivation at 0.998 a_w, the yeast retained at 0.960 a_w (NaCl or PEG 400) a greater proportion of the total glycerol intracellularly against an increased concentration ratio without significantly greater production of glycerol. Arabitol was only significant in osmoregulation when cultivated at 0.960 a_w (PEG 400). The intracellular glycerol concentration was insufficient to balance the a_w across the membrane, but an equilibrium could be achieved under certain conditions if arabitol was also osmotically active. Offprint requests to: P. J. van Zyl     

Extracellular production of a hybrid β-glucanase from Bacillus by Escherichia coli under different cultivation conditions in shaking cultures and bioreactors

Cultivation conditions for the extracellular production of a hybrid β-glucanase from Bacillus were established by using Escherichiacoli JM109 carrying the plasmid pLF3. This plasmid contained a novel secretion system consisting of the kil gene (killing protein) of plasmid ColE1 under the stationary-phase promoter of either the fic or the bolA gene, an omega interposon (Prentki and Krisch 1984) located upstream of the promoters and a hybrid β-glucanase gene of Bacillus. When controlled by the fic promoter, the kil gene led to a higher total production of β-glucanase and a higher protein secretion than when it was under control of the bolA promoter. When the effect of different distances between the stationary-phase promoters and the kil gene was investigated, a shorter distance was generally found to result in a higher secretion. With a complex growth medium, the kinetics of extracellular production of the enzyme depended on several operating variables, such as the salt concentration (NaCl) and the oxygen supply, which were varied by changing the culture volume and the shaking speed. In defined media the secretion of β-glucanase into the medium was increased significantly by the addition of glycerol as a carbon source and by prolonged cultivation. The strain with the highest production and secretion yield of β-glucanase [E. coli JM109(pLF3)] was tested on the fermenter scale. Received: 1 July 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996     

Optimization of culture conditions for the production of halothermophilic protease from halophilic bacterium <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TVSP101

An extremely halophilic Chromohalobacter sp. TVSP101 was isolated from solar salterns and screened for the production of extracellular halothermophilic protease. Identification of the bacterium was done based upon biochemical tests and the 16S rRNA sequence. The partially purified enzyme displayed maximum activity at pH 8 and required 4.5 M of NaCl for optimum proteolytic activity. In addition, this enzyme was thermophilic and active in broad range of temperature 60–80°C with 80°C as optimum. The Chromohalobacter sp. required 4 M NaCl for its optimum growth and protease secretion and no growth was observed below 1 M of NaCl. The initial pH of the medium for growth and enzyme production was in the range 7.0–8.0 with optimum at pH 7.2. Various cations at 1 mM concentration in the growth medium had no significant effect in enhancing the growth and enzyme production but 0.5 M MgCl₂ concentration enhanced enzyme production. Casein or skim milk powder 1% (w/v) along with 1% peptone proved to be the best nitrogen sources for maximum biomass and enzyme production. The carbon sources glucose and glycerol repressed the protease secretion. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of halophilic protease.     

The production of extracellular and intracellular free amino acids during aerated fermentation of glucose by Baker’s yeast (Saccharomyces cerevisiae)

During a study of the effects of a high level of NaCl on the content of free intracellular amino acids in baker’s yeast grown in aerated fermentation of glucose it was found (Malaneyet al. 1988, 1989; Malaney and Tanner 1988) that 0.6 mol/L exogenous NaCl significantly increased the content of free intracellular citrulline, glutamine, ornithine, arginine and lysine (all basic amino acids) over that observed at zero mol/L exogenous NaCl. (Exogenous is defined as salt added beyond that present in the mineral salts in the culture medium.) This paper describes the production and relative relationships of both extracellular and free intracellular amino acids byS. cerevisiae under conditions of high NaCl content in the growth medium at pH 5 and 32 °C. For early culture times (6 h), the production of glutamine, citrulline, valine, isoleucine, ornithine, lysine and histidine were all enhanced by the addition of NaCl. For late times (24 h), except for ornithine, the early-time-enhanced amino acids continued to be enhanced by the addition of NaCl. In addition, the yields of several other amino acids also were increased by exogenous salt at this late time. These include aspartic acid, threonine, glutamic acid, cystine, methionine, tyrosine, phenylalanine and arginine. Deceased, August 12, 1988. This study was supported by theNational Science Foundation (Grant No. CPE8209945) in conjunction with the NSF United States — Taiwan Cooperative Science Program.     

Galanthamine production by Leucojum aestivum L. shoot culture in a modified bubble column bioreactor with internal sections

Shoot culture of summer snowflake (Leucojum aestivum L.) was successfully cultivated in an advanced modified glass‐column bioreactor with internal sections for production of Amaryllidaceae alkaloids. The highest amounts of dry biomass (20.8 g/L) and galanthamine (1.7 mg/L) were achieved when shoots were cultured at 22°C and 18 L/(L·h) flow rate of inlet air. At these conditions, the L. aestivum shoot culture possessed mixotrophic‐type nutrition, synthesizing the highest amounts of chlorophyll (0.24 mg/g DW (dry weight) chlorophyll A and 0.13 mg/g DW chlorophyll B). The alkaloids extract of shoot biomass showed high acetylcholinesterase inhibitory activity (IC₅₀ = 4.6 mg). The gas chromatography–mass spectrometry (GC/MS) profiling of biosynthesized alkaloids revealed that galanthamine and related compounds were presented in higher extracellular proportions while lycorine and hemanthamine‐type compounds had higher intracellular proportions. The developed modified bubble‐column bioreactor with internal sections provided conditions ensuring the growth and galanthamine production by L. aestivum shoot culture.     

Production of Extracellular Halo-alkaline Protease from a Newly Isolated Haloalkaliphilic Bacillus sp. Isolated from Seawater in Western India

Summary Haloalkaliphilic, gram positive, aerobic, coccoid Bacillus sp. Po2 was isolated from a seawater sample in Gujarat, India. On the basis of 16s rRNA gene homology, Po2 was 95% related to Bacillus pseudofirmus. A substantial level of extracellular alkaline protease was produced by Po2, which corresponded with the growth and reached a maximum level (264 U/ml) during the stationary phase at 24 h. The production thereafter remained nearly static at optimal level till 36 h. Po2 could grow in the range of 0–20% NaCl (w/v) and pH 7–9, optimally at 10% NaCl (w/v) and pH 8. The protease production was salt-dependent and optimum production required 15% NaCl (w/v) and pH 8. Among the organic nitrogen sources, optimum growth and protease production (260 U/ml) were supported by the combination of peptone and yeast extract. However, growth and protease production were highly suppressed by the inorganic nitrogen sources used; with the exception of potassium nitrate, which supported both growth and protease production to limited extent (24 U/ml). Strong inhibition of enzyme production was observed at above 1% glucose (w/v). Wheat flour served as both carbon and nitrogen source supporting growth and protease production.     

Xylanase production using inexpensive agricultural wastes and its partial characterization from a halophilic <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TPSV 101

A halophilic and alkali-tolerant Chromohalobacter sp. TPSV 101 with an ability to produce extracellular halophilic, alkali-tolerant and moderately thermostable xylanase was isolated from solar salterns. Identification of the bacterium was done based upon biochemical tests and 16S rRNA sequence. The culture conditions for higher xylanase production were optimized with respect to NaCl, pH, temperature, substrates and metal ions and additives. Maximum xylanase production was achieved in the medium with 20% NaCl, pH-9.0 at 40°C supplemented with 1% (w/v) sugarcane bagasse and 0.5% feather hydrolysate as carbon and nitrogen sources. Sugarcane bagasse (250 U/ml) and wheat bran (190 U/ml) were the best inducer of xylanase when used as carbon source as compared to xylan (61 U/ml). The xylanase that was partially purified by protein concentrator had a molecular mass of 15 kDa approximately. The xylanase from Chromohalobacter sp. TPSV 101 was active at pH 9.0 and required 20% NaCl for optimal xylanolytic activity and was active over a broad range of temperature 40–80°C with 65°C as optimum. The early stage hydrolysis products of sugarcane bagasse were xylose and xylobiose, after longer periods of incubation only xylose was detected.     

Enhancing rice callus regeneration by extracellular products of Tolypothrix tenuis (Cyanobacteria)

Cyanobacteria produce a wide array of substances with activity in many biological systems. The aim of the present research was to compare the effect of differently treated extracellular products (EP) from Tolypothrix tenuis (Cyanobacteria) on rice plantlet regeneration as well as on the pigments, protein, total free porphyrin contents, and 5-aminolevulinate dehydratase (ALA-D) activity in rice callus during differentiation. Rice embryo calli were regenerated in Murashige & Skoog medium supplemented with EP with protein (EPp) or without protein (EPnp) or autoclaved (EPa), as well as with benzyladenine (BA) and benzyladenine + naphthaleneacetic acid (BA + NAA). At day 75, calli percentage regeneration were: EPnp (84%), EPp (58%), BA + NAA (45%), BA (44%), EPa (40%). The same trend was found for chlorophyll a, b, total and carotenoid contents. Protein content in BA, BA + NAA and EPnp treatments was 35% higher than in EPp and EPa. Total free porphyrin was similar in all treatments. ALA-D activity in BA, EPp and EPa treatments was 28% higher than in BA + NAA and EPnp. The extracellular bioactive substance(s) from T. tenuis would contain a mixture of thermolabile plant growth regulators that replaced and improved the effects of synthetic plant growth regulators on rice callus organogenesis. Calli were rhizogenic in all the regeneration media tested. The pigment content of the calli was related to percentage regeneration but not to the total free porphyrin and ALA-D activity.     

Glycerol production by two filamentous fungi grown at different ionic and nonionic osmotics and cheese whey

Glycerol production by a highly glycerol-producing local isolate (Eurotium amstelodami) and a standard reference isolate (Aspergillus wentii) was markedly enhanced by high saline media. Glycerol concentration depended on the external osmotic. Thus, the highest glycerol concentration was found in the presence of NaCl, followed by KCl, with considerably lower values for MgCl₂ and CaCl₂ saline media. With glucose (5–50%) used as a nonionic osmotic, low levels of glycerol were obtained and the main pool of polyols was mannitol. Glycerol production was gradually increased with the increase of NaCl concentration of cheese whey, reaching maxima by both organisms when whey was supplemented with 8% NaCl (total of 16% NaCl). The quantity of glycerol produced byA. wentii was twice higher than that obtained byE. amstelodami on whey treated with 8% NaCl.