Showing papers in "Applied Microbiology and Biotechnology in 1988"

Production of copolyesters of 3-hydroxybutyrate and 3-hydroxyvalerate by Alcaligenes eutrophus from butyric and pentanoic acids

Abstract: Copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) have been produced by Alcaligenes eutrophus in nitrogenfree culture solutions of butyric and pentanoic acids. When pentanoic acid was used as the sole carbon source, a copolyester with an unusually high 3HV fraction of 90 mol% was produced. Copolyesters with a wide range of compositions (0–90 mol% 3HV) were obtained by using butyric and pentanoic acids together as carbon sources. The biosynthetic pathways of poly(3-hydroxybutyrate) were investigated using [1-13C]acetate and [1-13C]butyrate. It is suggested that butyric and pentanoic acids are incorporated into the copolyester as 3HB and 3HV units respectively without decomposition of the carbon skeletons in the cell.

Characteristics of Trichoderma reesei β -xylosidase and its use in the hydrolysis of solubilized xylans

Abstract: The β-xylosidase (EC 3.2.1.37) of Trichoderma reesei was purified and its characteristics and use in the hydrolysis of steamed birch xylan were studied. The enzyme was a glycoprotein with a molecular weight of 100000 as determined by SDS-gel electrophoresis and its isoelectric point was 4.7. The pH optimum was 4.0 and temperature optimum 60°C. β-Xylosidase was competitively inhibited by xylose and the inhibition constant was 2.3 mM. The purified enzyme also showed α-arabinofuranosidase activity.

Biosorption of copper by fungal melanin

Abstract: Melanin obtained from Aureobasidium pullulans and Cladosporium resinae was an efficient biosorbent for copper. Copper uptake could be expressed using various adsorption isotherms; melanin from A. pullulans obeyed Freundlich and Langmuir isotherms whereas C. resinae melanin followed the BET isotherm indicating a more complex type of adsorption than in A. pullulans. In general, uptake capacities of melanin were greater than for intact biomass and the higher uptake by pigmented rather than albino biomass could be correlated with the presence of melanin. Cu2+ was less readily desorbed from melanin by dilute mineral acids than from intact biomass and again, the relative ease of Cu2+ desorption from pre-loaded pigmented or albino biomass was correlated with the presence or absence of melanin. Mg2+ and Zn2+ appeared to be the most effective cations for desorption with Na+ and K+ the least effective. The addition of melanin to a coppercontaining culture of the albino strain of A. pullulans resulted in some reduction of toxicity.

Transport of xylose and glucose in the xylose-fermenting yeast Pichia stipitis

Abstract: A low-affinity and a high-affinity sylose proton symport operated simultaneously in both starved and non-starved cells of Pichia stipitis. Glucose competed with xylose for transport by the low-affinity system and inhibited xylose transport by the high-affinity system non-competitively. The low affinity system was subject to substrate inhibition when glucose but not when xylose was the substrate. The differences between the characteristics of monosaccharide transport by Pichia stipitis and its imperfect state, Candida shehatae, are discussed.

Effect of culture conditions on mycelial growth and production of γ-linolenic acid by the fungus Mortierella ramanniana

Abstract: Effect of culture conditions on cell growth, lipid accumulation and γ-linolenic acid production is reported for four Mortierella species. The highest concentration as well as the highest productivity of γ-linolenic acid in lipid was determined in strains of M. ramanniana. M. ramanniana CBS 112.08 was used in the studies of the influence of medium composition, concentration of carbon- and nitrogen sources and growth temperature. Several carbon sources provided good growth and a high lipid content in biomass. The highest dry weights (11–12g/l) and lipid contents (∼24%, w/w), were observed if glucose or fructose was used as carbon source, whereas the highest amount of γ-linolenic acid (∼26%) was determined in starch-grown cells. The fatty acid composition in the lipid was influenced by the cultivation time, growth temperature and, to a minor extent, by the carbon source used. In fermentor cultures, both strains of Mortierella ramanniana showed relatively poor growth and incomplete consumption of glucose. M. vinacea, on the other hand, grew well in tower reactors. M. vinacea, which has a different morphology than M. ramanniana strains, also showed higher yields of biomass and lipid and higher yield coefficients than the latter.

Comparison of ligninolytic activities of selected white-rot fungi

Abstract: Six fast growing ligninolytic white-rot fungi were compared with Phanerochaete chrysosporium. The results showed that the fungi have similar ligninolytic systems, although minor differences exist. Like in P. chrysosporium the ligninolytic system could be induced by veratryl alcohol in Coriolus versicolor and Chrysosporium pruinosum. These three lignin peroxidase producing fungi were the fastest lignin degraders in stationary cultures, whereas in agitated cultures Bjerkandera adusta showed highest lignin degradation rates. Metabolites accumulating during the degradation of veratryl alcohol were analyzed and compared. Peroxidase production seems to be a common feature of all the tested fungi. Polyclonal antibodies against the lignin peroxidase with pl of 4.65 from P. chrysosporium reacted with the extracellular peroxidases of C. pruinosum, C. versicolor and B. adusta, but not with those of Pleurotus ostreatus.

Xylose fermentation by yeasts: 4. Purification and kinetic studies of xylose reductase fromPichia stipitis

Abstract: SummaryXylose reductase from the xylose-fermenting yeastPichia stipitis was purified to electrophoretic homogeneity via ion-exchange, gel and affinity chromatography. At physiological pH values the thermodynamic equilibrium constant was determined to be 0.575x1010 (l·mol-1). Product inhibiton studies are reported which clearly show that the kinetic mechanism of the xylose reductase is ordered-bi-bi with isomerisation of a stable enzyme form.

Optimization of lipid production in the oleaginous yeast Apiotrichum curvatum in wheypermeate

Abstract: Lipid production of the oleaginous yeastApiotrichum curvatum was studied in wheypermeate to determine optimum operation conditions in this medium. Studies on the influence of the carbon to nitrogen ratio (C/N-ratio) of the growth medium on lipid production in continuous cultures demonstrated that cellular lipid content in wheypermeate remained constant at 22% of the cell dry weight up to a C/N-ratio of about 25. The maximal dilution rate at which all lactose is consumed in wheypermeate with excess nitrogen was found to be 0.073 h-1. At C/N-ratios higher than 25–30 lipid content gradually increased to nearly 50% at C/N=70 and the maximal obtainable dilution rate decreased to 0.02 h-1 at C/N=70. From these studies it could be derived that maximal lipid production rates can be obtained at C/N-ratios of 30–35 in wheypermeate. Since the C/N-ratio of wheypermeate normally has a value between 70 and 101, some additional nitrogen is required to optimize the lipid production rate. Lipid production rates ofA. curvatum in wheypermeate were compared in four different culture modes: batch, fed-batch, continuous and partial recycling cultures. Highest lipid production rates were achieved in culture modes with high cell densities. A lipid production rate of nearly 1 g/l/h was reached in a partial recycling culture. It was calculated that by using this cultivation technique lipid production rates of even 2.9 g/l/h may be reached when the supply of oxygen can be optimized.

Immobilized growing lactic acid bacteria with κ-carrageenan — locust bean gum gel

Abstract: A cell entrapment process using κ-carrageenan — locust bean gum gel is presented. Streptococcus thermophilus, Lactobacillus bulgaricus and S. lactis were immobilized in small gel beads (0.5–1.0 mm and 1.0–2.0 mm diameter) and fermentations in bench bioreactors were conducted. Viability of entrapped cells, lactose utilization, lactic acid production and cell release rates were measured during fermentation. The procedure was effective for S. thermophilus, L. bulgaricus and S. lactis, and the viability of these bacteria remained very high throughout entrapment steps and subsequent storage. Bead diameter influenced the fermentation rate: smaller beads (0.5–1.0 mm) permitted an increase in release rates, lactose utilization and acid production by entrapped cells, approximating values attained with free cells.

Thermostable alkaline protease produced by Bacillus thermoruber — a new species of Bacillus

Abstract: The proteolytic activity produced by a new species of Bacillus isolated in our laboratory was investigated. This enzyme was purified to homogeneity from cell-free culture liquids of B. thermoruber. The purification procedure included ion-exchange chromatography on DEAE-Sephadex A-50 and α-casein agarose affinity chromatography. The protease consists of one polypeptide chain with a molecular weight of 39000±800. the isoelectric point was 5.3; the optimum pH and temperature for proteolytic activity (on casein) was found to be pH 9 and 45°C respectively. Enzyme activity was inhibited by PMSF and EDTA. The stability was considerably increased by addition of Ca2+, and the protease exhibited a relatively high thermal stability. The alkaline protease shows a preference for leucine in the carboxylic side of the peptide bond of the substrate. The Kmvalue for benzyloxycarbonyl-Ala-Ala-Leu-p-nitroanilide was 2.5 mM.

The oxygen requirements of yeasts for the fermentation of d-xylose and d-glucose to ethanol

Abstract: The effect of oxygen availability on d-xylose and D-glucose metabolism by Pichia stipitis, Candida shehatae and Pachysolen tannophilus was investigated. Oxygen was not required for fermentation of d-xylose or d-glucose, but stimulated the ethanol production rate from both sugars. Under oxygen-limited conditions, the highest ethanol yield coefficient (Ye/s) of 0.47 was obtained on d-xylose with. P. stipitis, while under similar conditions C. shehatae fermented d-xylose most rapidly with a specific productivity (qpmax) of 0.32 h-1. Both of these yeasts fermented d-xylose better and produced less xylitol than. P. tannophilus. Synthesis of polyols such as xylitol, arabitol, glycerol and ribitol reduced the ethanol yield in some instances and was related to the yeast strain, carbon source and oxygen availability. In general, these yeasts fermented d-glucose more rapidly than d-xylose. By contrast Saccharomyces cerevisiae fermented d-glucose at least three-fold faster under similar conditions.

A fast estimation of biochemical oxygen demand using microbial sensors

Abstract: Microbial amperometric sensors for biochemical oxygen demand (BOD) determination using Bacillus subtilis or Trichosporon cutaneum cells immobilized in polyvinylalcohol have been developed. These sensors allow BOD measurements with very short response times (15–30s), a level of precision of ±5% and an operation stability of 30 days. A linear range was obtained for a B. subtilis-based sensor up to 20 mg/l BOD and for a T. cutaneum-based sensor up to 100 mg/l BOD using a glucose/glutamic acid standard.

Comparison of steam and ammonia pretreatment for enzymatic hydrolysis of cellulose

Abstract: Aspenwood, wheat straw, wheat chaff and alfalfa stems were treated under pressure with either steam or ammonia. The material was then water or methanol/water extracted. The extent of enzymatic hydrolysis of the cellulose portion of the treated substrates was compared using two different cellulases, a commercial preparation, Celluclast, and those from the fungus Trichoderma harzianum. Both steam and ammonia treatment enhanced the accessibility of the cellulose as measured by hydrolysis. Methanol extraction of steamed material generally reduced the access of the enzyme to the cellulose, whereas methanol extraction of ammonia-treated material increased accessibility. The optimum combinations of pretreatment and extraction method depended on the substrate and on the enzyme system; no treatment suitable for all situations could be selected.

Water and water activity in the solid state fermentation of cassava starch by Aspergillus niger

Abstract: During the solid state fermentation (SSF) of cassava starch by Aspergillus niger estimations were made of total water, consumed water and the residual water remaining in small quantities after 23 h. A theoretical calculation based on the Ross equation showed that the water activity (a w) of the substrate decreased to 0.85 towards the end of the culture. Such low values were assumed to be inhibitory to growth. The a w of the substrate was increased when sugarcane bagasse was used as a high water retention capacity support. Higher growth rates and substrate conversion to biomass were obtained with this system, confirming that water availability is a critical factor in the SSF of starch substrates.

A note on the use of dinitrosalicylic acid for determining the products of enzymatic reactions

Abstract: Factors contributing to non-linearity of enzyme assays incorporating detection of reaction products using dinitrosalicylic acid (DNS) are discussed. The common practice of diluting reaction products before quantification of reducing compounds is shown to be one cause of non-linearity. Insufficiency of substrate is also an important contributory factor in most modern versions of the method, although the original procedure was correctly designed to ensure a linear reaction. The inherent inaccuracy involved in expression of the results of non-linear reactions in units implying linearity (katals or International Units) is emphasized.

The uptake of silver ions by Escherichia coli K12: toxic effects and interaction with copper ions

Abstract: Growth of Escherichia coli in chloridefree medium in batch culture is inhibited completely at concentrations of AgNO3 greater than 2.5x10-6 M. Incubation of non-growing cells in HEPES buffer (pH 7.4) at increasing levels of Ag+ results in the progressive saturation of two types of binding site. At one site, the Ag+ is not released by washing with 0.1 M nitric acid, and is probably intracellular. Silver bound to the second site is released by acid-washing, but not by buffer washing, and is assumed to be surface-bound. The amounts of Ag+ taken up from solution at the two sites is 1.6x10-7 and 4.6x10-7 mol (mg dry weight)-1, respectively. Total accumulation of silver is 67 mg (g dry weight)-1, similar to literature values found for silver-resistant bacteria. Binding of Ag+ at intracellular sites (observed at low [Ag+]) appears to be independent of pH. Addition of AgNO3 to growing cells in mid-exponential phase of growth in concentrations that will inhibit growth results in substantially decreased accumulation of silver. Growth yield in chemostat culture is diminished in the presence of added Ag+, but this effect is moderated by added Cu2+, which may protect copper sites from Ag+ or compete with Ag+ for other sites at which Ag+ exerts toxic effects. Very small amounts of Cu2+ are found in cell samples from the chemostat compared to the substantial amounts of Ag+ taken up, but uptake of Cu2+ is decreased at higher [Ag+]/[Cu2+]ratios.

An acetyl esterase of Trichoderma reesei and its role in the hydrolysis of acetyl xylans

Abstract: An acetyl esterase was purified from Trichoderma reesei by cation and anion exchange chromatography. The enzyme had a molecular weight of 45 000 as determined by SDS-electrophoresis, or 67 000 as determined by gel filtration. In chromatofocusing the enzyme was shown to consist of two isoenzymes with isoelectric points of 6.8 and 6.0. The enzyme showed activity towards naphthyl acetate, triacetin and glucose-and xylose acetates. However, it liberated acetic acid from acetylated xylo-oligomers only to a small extent. The liberation of acetic acid from the oligomeric substrate was enhanced by addition of endoxylanase and β-xylosidase.

Adsorbents for extractive bioconversion applied to the acetone-butanol fermentation

Abstract: Four different polymeric resins were tested as adsorbents in extractive bioconversion applied to the fermentative production of acetone and butanol by Clostridium acetobutylicum. The polymers were tested for their ability to adsorb butanol from pure solutions, and fermentation broths. Furthermore, the effect on the fermentability of the media was tested. The pH was increased to prevent adsorption of intermediates such as acetic and butyric acids. Bonopore, the polymer giving the best adsorption pattern with no undesirable effects, was tested in repeated batch cultures with C. acetobutylicum.

Improvement in stability of an immobilized fungal laccase

Abstract: A laccase of the basidiomyceteTrametes versicolor was immobilized on porous glass beads that were activated with 3-aminopropyltriethoxysilane and glutaraldehyde. The support immobilized 100% of the enzyme, whereupon 90% of the original activity was retained. After immobilization, the enzyme was active in a wider pH and temperature range, and its heat stability and reuse were greatly improved compared to those of the free laccase. The immobilized enzyme was found reusable in treating different substrates, either recycled alone or in a sequential order.

Ammonia inhibition of the maximum growth rate (µm) of hydrogenotrophic methanogens at various pH-levels and temperatures.

Abstract: A method for the quantitative measurement of the maximum growth rate (μm) of hydrogen-consuming methanogenic populations was applied to assess the toxicity of ammonia1 under various pH and temperature conditions. The maximum uninhibited growth rate of the hydrogenotrophic population present in sludge from an industrial anaerobic wastewater treatment system appeared to be 0.126 h-1 at pH=7 and 37°C. At 350 mM ammonia the maximum growth rate had decreased to almost half that value. At a temperature of 29°C the maximum growth rates in the ammonia range tested appeared to be approximately 60% of that at 37°C, while increasing ammonia concentrations caused a similar maximum growth rate decline. At 37°C an increase of the pH to 7.8 appeared to enhance ammonia inhibition of the maximum growth rate. Increased propionate concentrations (tested up to 60 mM) appeared to have no effect on ammonia inhibition.

Soluble phosphate accumulation by Aspergillus niger from fluorapatite

Abstract: In order to determine conditions that may provide greater solubilization of insouluble phosphate, the fungus Aspergillus niger was grown in a stationary culture containing modified citrate medium supplemented with 800 mg fluorapatite per litre. Solubilization of insouluble phosphate increased with fungal growth, reaching a maximum after 11 days of culture. Soluble phosphate levels were correlated with pH of the culture medium but not with titratable acidity values, probably due to the metabolic activity of the fungus resulting from consumption of sugar in the culture medium. Fructose, glucose, xylose, and sucrose were the carbohydrates that favoured fluorapatite solubilization the most when compared with galactose and maltose. Although increasing fructose concentrations in the culture medium favoured mycelial growth, increased total acidity and a fall in pH, soluble phosphate levels were reduced, probably owing to consumption by the rapidly growing fungus. Among the nitrogen sources tested, ammonium salts favoured the production of larger amounts of soluble phosphate than organic nitrogen (peptone or urea) or nitrate, corresponding to the lowest pH and highest titratable acidity values obtained.

The simultaneous production of sorbitol from fructose and gluconic acid from glucose using an oxidoreductase of Zymomonas mobilis

Abstract: The production of sorbitol and gluconic acid by toluene-treated, permeabilized cells of Zymomonas mobilis has been evaluated. From a 60% total sugar solution (300 g/l glucose and 300 g/l fructose), a sorbitol concentration of 290 g/l and a gluconic acid concentration of 283 g/l were achieved after 15 h in a batch process using free toluene-treated cells. A continuous process with immobilized cells was developed and only a small loss of enzyme activity (less than 5%) was evident after 120 h. With a strongly basic anion exchange resin and an eluent of 0.11 M Na2B4O7/0.11 M H3BO3, good separation of sorbitol and gluconic acid was achieved.

Degradation of 1,4-dichlorobenzene by enriched and constructed bacteria

Abstract: Three strains, RHO1, R3 and B1, tentatively identified as a Pseudomonas sp., an Alcaligenes sp. and a Pseudomonas sp. which were able to use 1,4-dichlorobenzene as the sole carbon and energy source were isolated from water of the Rhine river and from the sewage plant at Leverkusen-Burrig. A hybrid strain, WR1313, which uses chlorobenzene as the growth substrate, was obtained by mating the benzene-growing Pseudomonas putida strain F1 with strain B13, a Pseudomonas sp. degrading chlorocatechols. Further selection of this strain for growth on 1,4-dichlorobenzene allowed the isolation of strain WR1323. During growth on 1,4-dichlorobenzene the strains released stoichiometric amounts of chloride. The affinity of the organisms to 1,4-dichlorobenzene was measured with strain R3 showing a Ks value of 1.2 mg/l. Respiration data and enzyme activities in cell extracts as well as the isolation of 3,6-dichlorocatechol from the culture fluid are consistent with the degradation of 1,4-dichlorobenzene via 3,6-dichlorocatechol, 2,5-dichloro-cis,cis-muconate, 2-chloro-4-carboxymethylenebut-2-en-4-olide.

Biological tertiary treatment of urban wastewaters with chitosan-immobilized Phormidium

Abstract: Chitosan:Phormidium aggregates (chitosan: algae=1:2, dry weight basis) were used as a biological tertiary treatment to remove the nitrogen (NH4+, NO2-, NO3-) and phosphorus (PO43-) from a secondary effluent. In a batch system, 71 and 92% of P−PO43-were removed after 6 and 24 h, respectively. The orthophosphate removal rate was identical for all three concentrations of algae-chitosan tested (3.3, 4.6, 5.9 g d. wt.·l-1), and was 90 μg±2 μg P−PO43-·l-1·h-1, for a 90% removal. Under control conditions (chitosan flakes only added to the effluent) 73 and 78% of PO43-were removed after 6 and 24 h respectively. A 95% removal of inorganic nitrogen (NH4+, NO2-, NO3-) was attained after 4–6 h withPhormidium immobilized on chitosan flakes, as compared to 30% with chitosan flakes alone (5 g d. wt.·l-1). The system gave a similar performance when operated semi-continuously over 5 days at a daily retention time of 1.0. In the presence of chitosan-immobilized algae, medium P−PO43-levels were reduced by 87.3%±6.4% after 24 h (61.1 μg±7.0 μg P·l-1·h-1). The reduction of inorganic nitrogen in the medium was 98% after 24 h (370 μg±50 μg N·l-1·h-1). In the presence of chitosan alone, some 60% orthophosphate removal was recorded, whereas no reduction of nitrogen was observed. Disappearance of orthophosphate was attributed to its co-precipitation with calcium released from the chitosan by abrasion. The presence of the algae protected the chitosan from abrasion andPhormidium directly assimilated the orthophosphate and inorganic nitrogen, thus reducing their levels in the effluent.

Malic acid accumulation by Aspergillus flavus

Abstract: The accumulation and excretion of l-malic acid, and to a lesser extent succinic and fumaric acids, by Aspergillus flavus occurs under aerobic conditions in a medium containing a high glucose concentration, a limiting amount of nitrogen and a neutralizing agent (CaCO3). An overall malic, succinic, and fumaric acid molar yield of up to 68.6% (moles of acid produced per mole of glucose utilized) is obtained after incubation for 6 to 8 days, although a transient molar yield of 76% was measured. Glucose consumption and formation of acids were faster in a fermentor than in shake flasks. During the acid production stage, the activity of malate dehydrogenase increased 6 to 10-fold while that of fumarase changed only slightly. Cycloheximide greatly inhibited both l-malic acid production and the increase in malate dehydrogenase activity, without affecting fumarase activity. The results suggest that de novo protein synthesis is involved in the increase of malate dehydrogenase and that this enzyme is essential for l-malic acid production and accumulation.

Synchronous anaerobic and aerobic degradation of DDT by an immobilized mixed culture system

Abstract: For the investigation of a mixed anaerobic and aerobic degradation of xenobiotics the reductive dechlorination of 1,1,1-trichloro-2,2-bis (4-chlorophenyl)ethane (DDT) to 1,1-dichloro-2,2-bis (4-chlorophenyl)ethane (DDD) and the oxidative degradation of the DDT-conversion product 4,4′-dichlorodiphenylmethane (DDM) were studied. Enrichments from digested sewage sludge led to the isolation of an Enterobacter cloacae-strain which is able to reductive dechlorination of DDT during the fermentation of lactose. From fresh sewage sludge 11 bacterial strains were isolated in batch-culture and in continuous culture utilizing diphenylmethane, a non chlorinated structural analogon of DDM, as sole source of carbon and energy. One of these isolates, Alcaliaenes sp. cometabolizes DDM during the aerobic growth with diphenylmethane. By coimmobilization of Alcaligenes sp. and Enterobacter cloacae in Ca-alginate a system could be established, in which the reductive dechlorination of DDT and the oxidative degradation of DDM and diphenylmethane proceeds simultaneously in one reactor vessel.

Extracellular ligninase of Phanerochaete chrysosporium Burdsall has no role in the degradation of DDT

Abstract: The white rot fungus Phanerochaete chrysosporium Burdsall degraded DDT [1,1-bis(4-chlorophenyl)-2, 2,2-trichloroethane] in submerged agitated cultures. The ability of the fungus to metabolize this persistent environmental pollutant is not dependent on the formation of its extracellular lignin-degrading enzyme system.

Production of the raw-starch digesting amylase of Aspergillus sp. K-27

Abstract: Aspergillus sp. K-27, isolated from soil, produced extracellular glucoamylase and α-amylase using wheat starch as a carbon source, and its productivity was doubled by the addition of α-methyl-d-glucoside to the medium. The crude enzyme preparation, which was found to be a mixture of 70% glucoamylase and 30% α-amylase, well degraded not only cereal starches but also tuber and root starches, and the initial velocity for potato starch was 72% of that for corn starch.

Biosurfactant production by Pseudomonas fluorescens 378: growth and product characteristics

Abstract: An isolate of Pseudomonas fluorescens, strain 378 was shown to produce a novel surface active compound (code name AP-6). The compound is unique in being a high molecular weight compound but has, in some aspects, properties of a low molecular weight surfactant. The product is extracellular and its formation appeared to be partly growth-associated. Using a semisynthetic medium, fermentor cultivations were performed in the pH range 6.8–8.4. The product yield was optimal at pH 8.0 and gave a final concentration of 210 times critical micelle dilution. At higher pH, specific growth rate, final biomass and product concentration decreased. It consists mainly of carbohydrates and protein, the molecular weight is 1×106 and the isoelectric point is pH 9.1.

Permeabilization of plant cells for release of intracellularly stored products: viability studies

Abstract: The effects of various chemical substances on the permeability of plasma membranes and tonoplasts of three suspension cultures (Catharanthus roseus, Thalictrum rugosum and Chenopodium rubrum) have been studied. The permeability of the plasma membrane is monitored by measuring the activity of the cytosolic enzyme isocitrate dehydrogenase and the permeability of the tonoplast is measured by determining the release of substances stored in the vacuoles (inorganic phosphate, berberine and betanin for the three cell lines, respectively). The minimum concentration required for quantitative release of vacuolar products have been established for five different permeabilization agents. Cell viability is lost upon permeabilization except for treatment of Catharanthus roseus with DMSO and Triton X-100.