Growth of Saccharomycopsis fibuliger and Candida utilis in mixed culture on pectic materials

Hydrolysis of pectin by Saccharomycopsis fibuliger and cell growth on the products of hydrolysis by Candida utilis require different incubation conditions. A three-stage sequential culture is described in which S. fibuliger was first grown under aerobic conditions to generate cell mass. The concentration of dissolved oxygen was then reduced to promote pectolytic activity and reduce the number of viable cells in the culture. Finally culture conditions were adjusted to promote the growth of C. utilis in mixed culture with S. fibuliger. The presence of C. utilis increased the rate of pectolytic activity by S. fibuliger. A yeast product, containing 98% C. utilis cells, was obtained from the mixed culture grown on 10 g l⁻¹ pectin. Cell yields using starch or an equal mixture of starch and pectin were similar to those reported in the Symba process, although lower cell yields were recorded using pectin alone.     

Studies on the growth of Saccharomycopsis fibuliger on pectic materials

Growth experiments have indicated that the yeast Saccharomycopsis fibuliger is able to utilize pectic materials as a carbon source for cell growth. Small quantities of yeast extract or mycological peptone and maltose are necessary to initiate growth. Cell yields of 35 g/100 g pectin were obtained after modification of the pH value, the concentration of calcium ions and pectin in the growth medium. The optimum pH for cell growth was 4.8, which is similar to the reported optimum for cell growth on starch. Cell yields declined at higher concentrations of pectin, as a result of the reduced rate of oxygen absorption into the growth medium. Neither the concentration of ammonium and phosphate ions nor the type of inorganic nitrogen source significantly affected cell growth within the experimental conditions employed.     

Growth of Saccharomycopsis fibuliger and Candida utilis in mixed culture on apple processing wastes

Sequential cultures of the yeasts Saccharomycopsis fibuliger and Candida utilis were grown on selected wastes from the processing of apples. Effluent from cider manufacture supported the growth of 45.4 g cells/100 g substrate and C. utilis formed 96% of the viable cells in the harvested biomass. Whole, unripe apples yielded 44 g cells/100 g substrate with a reduction in the substrate viscosity of 84%. C. utilis formed 56% of the viable cells in the harvested biomass. Effluent from pectin manufacture contained a substantial proportion of reducing compounds and supported the growth of C. utilis without prehydrolysis by S. fibuliger, to yield 33 g cells/100 g substrate.     

An investigation into the pectolytic activity of the yeast Saccharomycopsis fibuliger

Saccharomycopsis fibuliger cells produce an inducible hydrolase, tentatively characterized as a polygalacturonase [poly(1,4-α-d-galacturonide) glycanohydrolase, EC 3.2.1.15], which is associated with the yeast cells and which causes the partial hydrolysis of pectin or poly-d-galacturonic acid. No evidence of pectinesterase (pectin pectyl hydrolyase, EC 3.1.1.11) or pectate lyase [poly(1,4-α-d-galacturonide) lyase, EC 4.1.1.1] activity has been found. Enzyme production took place at an optimum temperature of 28°C, whereas optimum activity was at ～45°C. The optimum pH for pectolytic activity was similar to the optimum pH for cell growth. A reduction in the concentration of dissolved oxygen in the culture medium and an increase in cell age caused an increase in the rate of pectin decomposition within the limits employed. Products of pectin decomposition consisted of a mixture of uronides including d-galacturonic acid.     

Nitrogen Fixation Associated with Development and Localization of Mixed Populations of Cellulomonas sp. and Azospirillum brasilense Grown on Cellulose or Wheat Straw

Mixed cultures of Cellulomonas sp. and Azospirillum brasilense were grown with straw or cellulose as the carbon source under conditions favoring the fixation of atmospheric nitrogen. Rapid increases in cell numbers, up to 10⁹ cells per g of substrate, were evident after 4 and 5 days of incubation at 30°C for cellulose and straw, respectively. Nitrogen fixation (detected by acetylene reduction measured on parallel cultures) commenced after 2 and 4 days of incubation for straw and cellulose, respectively, and continued for the duration of the experiment. Pure cultures of Cellulomonas sp. showed an increase in cell numbers, but CO₂ production was low, and acetylene reduction was not detected on either cellulose or straw. Pure cultures of A. brasilense on cellulose showed an initial increase in cell numbers (10⁷ cells per g of substrate) over 4 days, followed by a decline presumably caused by the exhaustion of available carbon substrate. On straw, A. brasilense increased to 10⁹ cells per g of substrate over 5 days and then declined slowly; this growth was accompanied by acetylene reduction. Scanning electron micrographs of straw incubated with a mixed culture under the above conditions for 8 days showed cells of both species in close proximity to each other. Evidence was furnished that the close spatial relationship of cells from the two species facilitated the mutually beneficial association between them and thus increased the efficiency with which the products of straw breakdown were used for nitrogen fixation.     

Enforced growth-rate fluctuation causes pectin ring formation in the cell wall of Lilium longiflorum pollen tubes

Normally growing lily (Lilium longiflorum Thunb.) pollen tubes cultured in standard sucrose medium display a relatively steady tip-growth pattern and a rather even pectin sheath in the cell wall. In an attempt to better understand pulsatory growth, observed in some species, e.g., Petunia, and its possible role in causing the formation of thickened cell wall rings, we have imposed marked fluctuations in the growth-rate of lily pollen tubes. The appropriate growth-perturbing conditions were achieved by modulating the medium osmolarity or by applying caffeine, a non-turgor inhibitor, in a specially designed incubation chamber with a controlled medium flow. The relatively non-esterified pectin deposition in the wall of the growth-interrupted pollen tubes was detected by immunofluorescence microscopy using a monoclonal antibody, JIM 5. The observations show that the periods of slow or inhibited growth correspond to the times when the thickened walls are deposited. Since the growth fluctuations were induced by both turgor- and non-turgor-related means, the proposed endogenous regulatory role of turgor pressure is questioned. Other factors, such as the tip-focused Ca²⁺ gradient which was demonstrated by ratiometric ion imaging, and the alteration in the extensibility of the cell wall, which correlated with pectin esterification/de-esterification, emerge as candidates for the regulation of growth fluctuations.     

Binding of methylmercury(II) by HeLa S3 suspension-culture cells: Intracellular methylmercury levels and their effect on DNA replication and protein synthesis

HeLa S3 cells were exposed to varied concentrations of methylmercury over varied periods of time and its binding by the cells was studied using ²⁰³Hg-labeled methylmercuric chloride as radioactive marker. Also studied was the effect of cell-bound methylmercury on DNA replication and protein synthesis and on the growth rate of the cells. The results show that methylmercury binding is a rapid process, with much of the organomercurial bound within the the first 60 min of incubation, and that considerable quantities of organic mercury become affixed to the cells. The amounts of bound methylmercury, [CH₃Hg(II)]_bound, given in mol/cell, range from 2 × 10^?16 (at 1 h of incubation and at 1 μM CH₃Hg(II) in the medium) to almost 4 × 10^?14 (at 24 h of incubation and at 100 μM CH₃Hg(II) in the medium). A [CH₃Hg(II)]_bound value of about 30 × 10^?16 mol/cell appears to be the threshold below which cells display a normal growth pattern and below which metabolic events such as DNA replication or protein synthesis are affected only to a minor degree but above which major changes in cell metabolism and cell growth take place. Methylmercury binding by the cells is tight so that only 20% of the bound material is released from the cells over a 3-h incubation period when the cells are placed into fresh, methylmercury-free growth medium. Analysis of the binding data in terms of binding to identical and completely independent sites yields an association constant K of 7.92 × 10⁴ l/mol and for the maximum concentration of cellular binding sites the value 2.40 × 10^?14 mol/cell or 1.45 × 10¹⁰ sites/cell. Evidence is presented which shows that cellular sulfhydryl groups do not suffice to provide all the sites taken up by methylmercury and that binding, in all likelihood, involves basic nitrogen, too. The levels of cell-bound methylmercury are such that binding to HeLa DNA and HeLa chromatin, for instance, can readily take place. Methylmercury binding data obtained by using the technique of particle-induced X-ray emission (PIXE) are in good agreement with the data obtained via isotope dilution.     

A microtiter plate assay for the selection of 6-thioguanine-resistant mutants in Chinese hamster V79 cells in the presence of phorbol-12-myristate-13-acetate

6-Thioguanine-resistant mutants can be efficiently recovered from Chinese hamster V79 cells incubated at high cell densities in microtiter plates (10³ – 10⁴ cells/0.2 ml growth medium/0.4 cm²) when selected with 30 μM 6-thioguanine and 0.1 μg/ml phorbol-12-myristate-13-acetate, an inhibitor of metabolic cooperation among V79 cells. Mutant frequencies in the microtiter plates were calculated from a direct count of mutant colonies. After treatment of the V79 cells with the carcinogen benzo[a]pyrene in a fibroblast-mediated assay, the mutation frequencies determined with the microtiter assay system were quantitatively similar to those obtained with a conventional procedure in which selection with 6-thioguanine was performed in petri dishes. The mutagenic activities of 3 polycyclic aromatic hydrocarbons (activated in the cell-mediated assay) were assessed with the microtiter plate selection procedure. The active carcinogen benzo[a]pyrene at 1 μg/ml yielded about 100 mutants per 10⁵ colony-forming cells. The same dose of a less active carcinogen, cyclopenta-[c,d]pyrene, yielded about 20 mutants per 10⁵ colony-forming cells, and benz[a]anthracene, not an active carcinogen, was inactive as a mutagen at all doses tested. Because of the small requirements for growth medium and tissue culture vessels compared with other assays, this microtiter plate assay can serve as an inexpensive system for detecting the mutagenic activity of environmental chemicals in mammalian cells.     

Effects of light intensity on the growth rate of the red alga Porphyridium cruentum

The red alga, Porphyridium cruentum, which is one of the potential sources of arachidonic acid, was cultured in batch and continuous vessels. The growth rates in batch cultures were correlated to the mean light intensity in the vessels, and the cell concentrations in continuous cultures were estimated by those results. The yield of arachidonic acid was about 1.2 g per 10¹² cell at cell concentrations ranging from 0.5 to 1.5 × 10¹⁰ cell/l and independent of the mean light intensity.     

Development of reproducible assays for polygalacturonase and pectinase

Polygalacturonase and pectinase activities reported in the literature were measured by several different procedures. These procedures do not give comparable results, partly owing to the complexity of the substrates involved. This work was aimed at developing consistent and efficient assays for polygalacturonase and pectinase activities, using polygalacturonic acid and citrus pectin, respectively, as the substrate. Different enzyme mixtures produced by Aspergillus niger and Trichoderma reesei with different inducing carbon sources were used for the method development. A series of experiments were conducted to evaluate the incubation time, substrate concentration, and enzyme dilution. Accordingly, for both assays the recommended (optimal) hydrolysis time is 30 min and substrate concentration is 5 g/L. For polygalacturonase, the sample should be adjusted to have 0.3–0.8 U/mL polygalacturonase activity, because in this range the assay outcomes were consistent (independent of dilution factors). Such a range did not exist for the pectinase assay. The recommended procedure is to assay the sample at multiple (at least 2) dilution factors and determine, by linear interpolation, the dilution factor that would release reducing sugar equivalent to 0.4 g/L d-galacturonic acid, and then calculate the activity of the sample accordingly (dilution factor × 0.687 U/mL). Validation experiments showed consistent results using these assays. Effects of substrate preparation methods were also examined.     

Influence of ε-caprolactam on growth and physiology of environmental bacteria

ε-Caprolactam was found to have an effect on ecologically important soil bacteria. It inhibited the growth of several Bacillus sp. and Rhizobium sp. but cells of Arthrobacter sp. were able to grow in the presence of caprolactam. Sphingomonas sp. lost its inherent capacity to produce extracellular polymer (EPS) if grown in medium containing caprolactam. In the case of raw domestic sewage, the diversity of native bacteria was diminished in presence of caprolactam. Polluted sea water yielded predominantly one type of caprolactam-degrading bacteria of the genus Achromobacter. These cells efficiently utilized up to 10 g caprolactam/L as the sole source of carbon and nitrogen in synthetic medium even in the presence of 20 g NaCl/L. Compared to cells of Arthrobacter sp., cells of Achromobacter sp. accumulated high amount of 6-aminocaproic acid due to degradation of caprolactam. When using caprolactam as sole source of carbon and nitrogen, Achromobacter cells showed unique physiological ability to produce EPS upon prolonged incubation in solid medium and in broth with low phosphate (C:N:P ratio 100:20:0.05). Hydrolyzed cell-free EPS had glucose as its major component though the only substrate provided in the medium for growth was caprolactam.     

Large-Scale Production of Rhizobium meliloti on Whey

Whey, a by-product of the cheese industry, can sustain the growth of fast-growing rhizobia. To avoid any latency of growth, rhizobial inoculum must be prepared under inducing conditions. In unsupplemented whey, the number of cells of Rhizobium meliloti Balsac reached 5 × 10⁹ CFU/ml in 48 h of incubation. This is comparable to the yield obtained with yeast-mannitol broth, the standard medium for the growth of rhizobia. In raw whey supplemented with yeast extract (1.0 g/liter) and phosphate (0.5 g/liter), the number of cells reached 10¹⁰ CFU/ml in 48 h of incubation. This is a twofold increase compared with the population normally obtained in industrial production. Whey represents a relatively inexpensive and efficient substrate medium for the large-scale production of fast-growing rhizobia.     

Ethanol Inhibition Kinetics of Kluyveromyces marxianus Grown on Jerusalem Artichoke Juice

The kinetics of ethanol inhibition on cell growth and ethanol production by Kluyveromyces marxianus UCD (FST) 55-82 were studied during batch growth. The liquid medium contained 10% (wt/vol) inulin-type sugars derived from an extract of Jerusalem artichoke (Helianthus tuberosus) tubers, supplemented with small amounts of Tween 80, oleic acid, and corn steep liquor. Initial ethanol concentrations ranging from 0 to 80 g/liter in the liquid medium were used to study the inhibitory effect of ethanol on the following parameters: maximum specific growth rate (μ_max), cell and ethanol yields, and sugar utilization. It was found that as the initial ethanol concentration increased from 0 to 80 g/liter, and maximum specific growth rate of K. marxianus cells decreased from 0.42 to 0.09 h⁻¹, whereas the ethanol and cell yields and sugar utilization remained almost constant. A simple kinetic model was used to correlate the μ_max results and the rates of cell and ethanol production, and the appropriate constants were evaluated.     

Single-Cell Protein Production by the Acid-Tolerant Fungus Scytalidium acidophilum from Acid Hydrolysates of Waste Paper

The bioconversion of waste paper to single-cell protein at pH <1 by Scytalidium acidophilum is described. Waste paper pretreated with 72% H₂SO₄ at 4°C was diluted with water to a pH of <0.1 and hydrolyzed. This yielded an adequate sugar-containing substrate for the growth of the fungus. A total of 97% of the sugars (glucose, galactose, mannose, xylose, arabinose) in the hydrolysates were converted to cell biomass. Microbial contamination was not observed. Based on the sugars consumed, S. acidophilum produced higher yields in shake cultures than many other Fungi Imperfecti. In aerated cultures, productivity increased, and yields of 43 to 46% containing 44 to 47% crude protein were obtained. This compares favorably with Candida utilis, a yeast used commercially to produce single-cell protein. The chemical constituents and the essential amino acids of the fungal cells were similar to those of other fungi. The nucleic acid content was characteristic of microbes containing low levels of nucleic acid. The advantages of using S. acidophilum for single-cell protein production are discussed.     

Enhanced growth of Acidovorax sp. strain 2AN during nitrate-dependent Fe(II) oxidation in batch and continuous-flow systems

Microbial nitrate-dependent, Fe(II) oxidation (NDFO) is a ubiquitous biogeochemical process in anoxic sediments. Since most microorganisms that can oxidize Fe(II) with nitrate require an additional organic substrate for growth or sustained Fe(II) oxidation, the energetic benefits of NDFO are unclear. The process may also be self-limiting in batch cultures due to formation of Fe-oxide cell encrustations. We hypothesized that NDFO provides energetic benefits via a mixotrophic physiology in environments where cells encounter very low substrate concentrations, thereby minimizing cell encrustations. Acidovorax sp. strain 2AN was incubated in anoxic batch reactors in a defined medium containing 5 to 6 mM NO₃⁻, 8 to 9 mM Fe²⁺, and 1.5 mM acetate. Almost 90% of the Fe(II) was oxidized within 7 days with concomitant reduction of nitrate and complete consumption of acetate. Batch-grown cells became heavily encrusted with Fe(III) oxyhydroxides, lost motility, and formed aggregates. Encrusted cells could neither oxidize more Fe(II) nor utilize further acetate additions. In similar experiments with chelated iron (Fe(II)-EDTA), encrusted cells were not produced, and further additions of acetate and Fe(II)-EDTA could be oxidized. Experiments using a novel, continuous-flow culture system with low concentrations of substrate, e.g., 100 μM NO₃⁻, 20 μM acetate, and 50 to 250 μM Fe²⁺, showed that the growth yield of Acidovorax sp. strain 2AN was always greater in the presence of Fe(II) than in its absence, and electron microscopy showed that encrustation was minimized. Our results provide evidence that, under environmentally relevant concentrations of substrates, NDFO can enhance growth without the formation of growth-limiting cell encrustations.     

Growth of Fusarium moniliforme on n-Alkanes: Comparison of an immobilization method with conventional processes

Summary In submerged culture there was negligible growth of Fusarium moniliforme with either n-tetradecane or gasoil (C₁₃–C₁₉) as the only carbon and energy source. In surface culture the cell yield was about 0.25 g dm^–3 dry weight after four weeks incubation. Some oxidation products, mainly isomeric tetradecanones (4-one, 5-one, 6-one and 7-one), could be identified. However the cell yield in a trickle-flow column was about 3 g dm^–3 dry weight after 7 days. Only traces of oxidation products could be detected. In a fixed-bed reactor, filled with glass rings, cell yields were similar to those in the trickle-flow column and depended on the medium flow rate.After termination of growth in the fixed-bed reactor, similar amounts of gibberellic acid were produced in a nitrogen-free medium with either gasoil or glucose.     

Control of the Life Cycle of Methanosarcina mazei S-6 by Manipulation of Growth Conditions

The morphology of Methanosarcina mazei was controlled by magnesium, calcium, and substrate concentrations and by inoculum size; these factors allowed manipulation of the morphology and interconversions between pseudosarcinal aggregates and individual, coccoid cells. M. mazei grew as aggregates in medium with a low concentration of catabolic substrate (either 50 mM acetate, 50 mM methanol, or 10 mM trimethylamine) unless Ca²⁺ and Mg²⁺ concentrations were high. Growth in medium high in Ca²⁺, Mg²⁺, and substrate (i.e., 150 mM acetate, 150 mM methanol, or 40 mM trimethylamine) converted pseudosarcinal aggregates to individual cocci. In such media, aggregates separated into individual cells which continued to grow exclusively as single cells during subsequent transfers. Conversion of single cells back to aggregates was complicated, because conditions which supported the aggregated morphology (e.g., low calcium or magnesium concentration) caused lysis of coccoid inocula. We recovered aggregates from coccoid cells by inoculating serial dilutions into medium high in calcium and magnesium. Cells from very dilute inocula grew into aggregates which disaggregated on continued incubation. However, timely transfer of the aggregates to medium low in calcium, magnesium, and catabolic substrates allowed continued growth as aggregates. We demonstrated the activity of the enzyme (disaggregatase) which caused the dispersion of aggregates into individual cells; disaggregatase was produced not only during disaggregation but also in growing cultures of single cells. Uronic acids, the monomeric constituents of the Methanosarcina matrix, were also produced during disaggregation and during growth as coccoids.     

Effects of Some Environmental Factors on Growth Characteristics of Candida utilis on Peat Hydrolysates

Samples of peat from Pine Island and Brookston bogs in Minnesota were hydrolyzed with various concentrations of HCl or H₂SO₄ solutions, before or after debituminization (an extraction process used to remove waxy materials, bitumens, from peat), to produce peak hydrolysates as growth substrates for Candida utilis. Hydrolysates were neutralized with concentrated NaOH solution to pH 3.5, 4.5, 5.0, 5.5, 6.0, and 7.0. The precipitated humates were removed by filtration. The resulting peat hydrolysates were amended with reagent-grade K₂HPO₄, K₂SO₄, and MgSO₄, 200, 100, and 50 mg per liter of peat hydrolysate, respectively. The debituminized peat produced more total nitrogen (TN) and reducing substances (RS) than the nondebituminized peat. Peat hydrolysates produced by HCl solutions contained slightly higher RS and TN than those produced by H₂SO₄ solutions; however, the latter were better growth substrates than the former. The yield coefficients in both H₂SO₄ and HCl hydrolysates initially decreased at 12 to 24 h and then increased gradually over the remaining incubation period (24 to 96 h). As TN and RS were increased, an increase in cell density, biomass, and productivity was observed. In contrast, a decrease in specific growth rate occurred as the RS and TN were increased. The generation time of C. utilis was affected by the concentrations of RS and TN. A peak substrate yield coefficient was found at pH 5.0 in HCl hydrolysates and at pH 6.0 to 6.5 in H₂SO₄ hydrolysates. Good linear correlation coefficients were found between RS and biomass of C. utilis. The coefficients of correlation increased as the TN level in hydrolysates was increased.     

Optimization of the growth parameters of Aspergillus foetidus

The batch production of gluconic acid in the presence of glucose, sucrose and molasses was investigated using free mycelia of Aspergillus foetidus NRRL 337 in shake flasks. Eight growth parameters were chosen as independent variables. The temperature, pH, substrate type and initial concentrations, inoculum percentage and shake rate directly affected the specific microorganism growth and gluconic acid production rates. The optimum temperature and initial pH values were found to be 33°C and five to six, respectively. The maximum specific growth and gluconic acid production rates were established as 57 g/dm³ of glucose, 75 g/dm³ of sucrose and 150 g/dm³ of molasses. The optimum values of the shake rate, inoculum percentage and initial ammonium nitrate concentration were determined as 100 1/min, 0.5% and 1.5 g/dm³, respectively. The maximum gluconic acid concentrations corresponding to these initial substrate concentrations were observed to be 8.3 g/dm³, 17.4 g/dm³ 37.0 g/dm³, respectively. The optimum specific microbial growth and gluconic acid production rates were found as 0.0145 1/h and 0.0375 g/g × h, respectively, for the fermentation conditions of S_Go = 57 g/dm³, T = 28°C, initial pH = 6.5, N = 84 1/min, A = 0.5 g/dm³ and I = 0.5%.     

Production of polysaccharides by Silene vulgaris callus culture depending on carbohydrates of the medium

Sources of carbohydrate nutrition such as sucrose, glucose, and galactose, with the exception of arabinose, were shown to influence positively callus growth and polysaccharide (pectin silenan and acidic arabinogalactan) biosynthesis. Galactose was found to cause a stimulatory effect on yield and productivity of arabinogalactan. Low concentrations of sucrose failed to support the cell growth and polysaccharide biosynthesis. Increasing sucrose concentrations led to biomass accumulation but failed to enhance efficiency of the substrate utilization. The optimal medium for the campion cell culture growth was found to be one containing 30 g/liter of sucrose or a mixture of sucrose with glucose (in 15 g/liter). Increasing sucrose concentrations in the medium from 30 to 100 g/liter failed to significantly influence the polysaccharide yields while the polysaccharide productivity per liter of the medium grew due to promotion of culture productivity in biomass. Variations of the carbon sources in the nutrient media were shown to influence insignificantly the biochemical characteristics of arabinogalactan and silenan while an increase in the sucrose concentration to 50-100 g/liter led to a diminution of the galacturonic acid content in silenan and to changes in contents of the neutral monosaccharide residues in silenan and arabinogalactan.