Effects of Corn Steep Liquor and Thiamine on l-Glutamic Acid Fermentation of Hydrocarbons: IV. Utilization of Hydrocarbons by Microorganisms

The effect of nutrients of natural source, such as corn steep liquor, peptone, and yeast extract, on the fermentative production of L-glutamic acid from hydrocarbons by a Corynebacterium was studied. Corn steep liquor and meat extract were found to be remarkably stimulatory to L-glutamic acid production; about 5 g per liter of L-glutamic acid were accumulated in a culture broth containing 3% n-paraffins, 0.01% corn steep liquor, and mineral salts. Among nutritional factors contained in corn steep liquor, biotin had very little effect on the accumulation of L-glutamic acid, but thiamine was highly stimulatory to L-glutamic acid production. The optimal concentration of thiamine for L-glutamic acid production was 3 to 5 μg per liter, and for cell growth, 50 μg per liter. L-Glutamic acid was accumulated in negligible quantity when the amount of thiamine in the culture broth was sufficient to support abundant growth of bacterial cells.     

Nitrogen nutrition and regulation of cephalosporin production in Streptomyces clavuligerus.

When used as sole nitrogen source, certain amino acids (e.g., proline, asparagine) supported both growth and sporulation by Streptomyces clavuligerus streaked onto solid defined medium. Ammonium supported growth but suppressed sporulation. Amino nitrogen was best for cephalosporin production in liquid defined medium, although urea was almost as useful. A comparison of amino acids showed asparagine and glutamine to be the best nitrogen sources and arginine to be almost as good. Ammonium salts supported a somewhat lower growth rate than asparagine, but antibiotic production was very poor on these inorganic nitrogen sources. Addition of ammonium to asparagine did not affect growth rate but increased mycelial mass; cephalosporin production was reduced by about 75%. Antibiotic production was more closely associated with growth in the absence of ammonium than in its presence, indicating a strong inhibitory and (or) repressive effect of NH4+ on antibiotic production. Ammonium exerted its negative effect when added at 24h or earlier, i.e. before antibiotic formation began.     

Physiology of,an enzyme production by,plant cell cultures

Red kidney bean (Phascolus vulgaris) cells, derived from roof, callus, were grown in suspension culture in shake flasks and in laboratory fermentors using batch and continuous batch culture techniques. The medium contained casein hydrolysate, sucrose, inorganic salts, vitamins, and growth hormones. In continuous batch culture yields of up to 171 g wet weight, (8.5 g dry weight) per liter were obtained in 7 days. Organic nitrogen was used preferentially. Growth on nitrate was considerably slower than on organic nitrogen sources. Indole acetic and naphthalene acetic acids were not essential for good growth of the cells whereas kinetin and 2, 4-D were. The optimum pH for growth was about p11 4.5. The presence of amylase and peroxidase was detected in culture filtrates. Amylase activity was low in either the presence or the absence of starch in the medium. Peroxidase production could be related directly with growth of the culture. Maximum peroxidase yield, as measured by the guaiacol method and expressed as horse radish peroxidase, was 1.25 × 10^?8 M.     

Formation of Short-Chain Fatty Acids from H(2) and CO(2) by a Mixed Culture of Bacteria

The biological utilization of CO(2) and H(2) for the formation of short-chain fatty acids was studied by using a mixed culture of bacteria. Optimization of a medium was carried out in continuous culture to identify limiting factors which controlled growth and production of organic acids. The optimal pH for growth and acid production was 7.0 at 37 degrees C; the maximal cell concentration obtained was 5.9 g of cells per liter (dry weight), and the maximal amount of volatile acids formed was 4.7 g/liter, with acetic acid as the predominant acid. With the optimized medium, it was found that the rate of transfer of hydrogen or carbon dioxide, or both, from gas to liquid was the limiting factor which controlled growth and production of acids.     

Formation of Short-Chain Fatty Acids from H2 and CO2 by a Mixed Culture of Bacteria

The biological utilization of CO₂ and H₂ for the formation of short-chain fatty acids was studied by using a mixed culture of bacteria. Optimization of a medium was carried out in continuous culture to identify limiting factors which controlled growth and production of organic acids. The optimal pH for growth and acid production was 7.0 at 37°C; the maximal cell concentration obtained was 5.9 g of cells per liter (dry weight), and the maximal amount of volatile acids formed was 4.7 g/liter, with acetic acid as the predominant acid. With the optimized medium, it was found that the rate of transfer of hydrogen or carbon dioxide, or both, from gas to liquid was the limiting factor which controlled growth and production of acids.     

Control of dimorphism in a biochemical variant of Candida albicans

The cellular morphology of a biochemical variant of Candida albicans could be controlled by the ratio of carbon dioxide to oxygen in the culture system or by individual amino acids. Predominantly pseudohyphal morphology was observed (i) at a CO(2) to O(2) ratio of 2:1 and (ii) without the addition of carbon dioxide, when either glycine, d- or l-ornithine, l-serine, l-methionine, l-phenylalanine, or l-tyrosine was the sole nitrogen source in the culture medium. When ammonium chloride, ammonium sulfate, l-glutamic acid, l-glutamine, or l-proline was the nitrogen source, yeastlike growth was observed in the presence or absence of CO(2). More adenosylmethionine was present in pseudohyphal than in yeastlike cells, and pseudohyphal cell wall preparations contained less methionine than cell walls from the yeastlike form. These results suggest a correlation between sulfur amino acid metabolism and dimorphism.     

Reduced nitrogen influences somatic embryo quality and plant regeneration from suspension cultures of orchardgrass

Summary Production of somatic embryos in suspension cultures ofDactylis glomerata L. (orchardgrass) was stimulated by the addition of various compounds containing reduced nitrogen to Schenk and Hildebrandt (SH) basal medium. Equimolar concentrations of combinations of proline and either serine or threonine supported embryogenesis, whereas, these amino acids individually did not promote embryogenesis. SH medium supplemented with 6 to 25 mM ammonium ion or Murashige-Skoog basal medium also supported embryo production. Ammonium ion did not act synergistically with either proline or serine to enhance embryogenesis. Embryos produced in media containing amino acid combinations were formed singly, did not exhibit secondary embryogenesis, and had significantly higher conversion rates compared to those formed in either SH medium supplemented with ammonium ion only or in combination with amino acids.     

Carbon and nitrogen source nutrition of fumagillin biosynthesis by Aspergillus fumigatus

The carbon and nitrogen source requirements of Aspergillus fumigatus NRRL 2436 for growth and production of the angiogenesis inhibitor fumagillin were studied in chemically defined media. Both carbon and nitrogen sources strongly influenced fumagillin formation. Two out of 29 carbon sources tested interfered with fumagillin biosynthesis. The best combination of two carbon sources was 30 g L(-1) xylan and 50 g L(-1) mannose. Of fifteen nitrogen sources tested, three ammonium salts (chloride, sulfate, and dibasic phosphate) failed to support fumagillin formation, presumably due to the low pH which developed. The dosage-response study of the best nitrogen source, L-glutamic acid, revealed that 9 g L(-1) was optimal. Volumetric production of fumagillin was increased by 15-fold over that in the starting (Peterson-Goldstein) medium as a result of these findings.     

Mode of utilization of amino acids as growth substrates by Azospirillum brasilense

The study was undertaken to analyze the rate of uptake and utilization of various amino acids by Azospirillum brasilense Sp81 (RG) in a basal mineral salts solution under non-nitrogen fixing condition. These amino acids including other nitrogenous compounds were tested for both N- and C-sources. The kinetic constants (Km and Vmax) of uptake of some amino acids (e.g. lysine, arginine, proline, glutamine and glutamic acid) were exploited using a Hanes-Woolf plot, and discussed in the context of nitrogen starvation or both carbon and nitrogen starvation. To summarize all the kinetic data for these amino acids strongly suggested that the mode of these amino acids utilization in this bacterium followed the same general pattern, although the quantitative differences were there. A single amino acid was able to satisfy the nitrogen needs of this bacterium in basal mineral salts solution, and this possibility could be considered for the cost-effective growth medium for this bacterium in the biotechnological industry.     

Diauxic growth of Penicillium camembertii on glucose and arginine

The growth of Penicillium camembertii during batch culture in a synthetic medium containing glucose and arginine was examined. The diauxic growth observed can be well characterized. Indeed, in a first phase, glucose and arginine were, respectively, assimilated as carbon and nitrogen sources, with an acidification of the medium (until 3.5), since arginine was taken up in exchange for protons. During this phase of growth, arginine, in addition to glucose, was also assimilated as an energy source, resulting in the release of the arginine carbon content as CO₂. Then, in a second phase, characterized by reduced growth rates after glucose depletion, arginine was assimilated as a carbon and nitrogen source, as well as an energy source, resulting in ammonium release which raised the pH (final pH 6.3), despite the amino acid/H⁺ exchange, since amino acids contain excess nitrogen in relation to their carbon content for fungi.     

Influence of Ammonium Salts and Cane Molasses on Growth of Alcaligenes eutrophus and Production of Polyhydroxybutyrate

The production of polyhydroxybutyrate (PHB) by Alcaligenes eutrophus DSM 545 was studied in a synthetic medium with 3% glucose at pH 7.0 supplemented with several ammonium substrates and cane molasses. Growth was measured by dry cell weight, and the PHB content was measured by gas chromatography. The effects of ammonium sources such as sulfate, nitrate, phosphate, and chloride salts and those of different ammonium sulfate concentrations were evaluated. The best growth and PHB production were obtained with ammonium sulfate; however, NH(inf4)(sup+) concentrations between 0.5 and 1.5 g/liter showed no significant difference. Ammonium sulfate was therefore used as the sole source of NH(inf4)(sup+) for experiments with cane molasses as the growth activator. Optimal growth and PHB production were obtained with 0.3% molasses. However, the yields of biomass (39 to 48%) and PHB (17 to 26%) varied significantly among the different ammonium substrates and cane molasses concentrations.     

Production of bakers' yeast in cheese whey ultrafiltrate

A process for the production of bakers' yeast in whey ultrafiltrate (WU) is described. Lactose in WU was converted to lactic acid and galactose by fermentation. Streptococcus thermophilus was selected for this purpose. Preculturing of S. thermophilus in skim milk considerably reduced its lag. Lactic fermentation in 2.3x-concentrated WU was delayed compared with that in unconcentrated whey, and fermentation could not be completed within 60 h. The growth rate of bakers' yeast in fermented WU differed among strains. The rate of galactose utilization was similar for all strains, but differences in lactic acid utilization occurred. Optimal pH ranges for galactose and lactic acid utilization were 5.5 to 6.0 and 5.0 to 5.5, respectively. The addition of 4 g of corn steep liquor per liter to fermented WU increased cell yields. Two sources of nitrogen were available for growth of Saccharomyces cerevisiae: amino acids (corn steep liquor) and ammonium (added during the lactic acid fermentation). Ammonium was mostly assimilated during growth on lactic acid. This process could permit the substitution of molasses by WU for the industrial production of bakers' yeast.     

Studies of the Cultural Physiology of the Lichen Alga Trebouxia

The pholosynthetic growth of three species of Trebouxia was so slow as to preclude the usual inorganic methods of algal cultivation. Heterotrophic growth was much stronger — as many as 0.4 doublings per day were obtained in Bold's solution with glucose and amino acid hydrolysates. Ammonium salts of inorganic acids could not be used in the culture medium because they caused excessive reductions in pH. Although the organisms showed variations in their growth response to amino acids, no specific requirement was found. T. erici produced significantly lower quantities of photosynthetic pigments in the dark. Of the nineteen carbohydrates tested, only glucose, mannitol, and galactosc stimulated growth of the algae. Preliminary studies showed that Trebouxia incorporated ¹⁴CO₂ more slowly than Chlorella pyrenoidosa. However, the carbon fixed by Trebouxia remained soluble in 80% ethanol longer than Chlorella. The carbon incorporated as CO₂ in short term experiments was greater than would be expected from the growth rates of the organisms studied.     

Mineralization of Surfactants by the Microbiota of Submerged Plant Detritus

In wetlands and canopied bodies of water, plant detritus is an important source of carbon and energy. Detrital materials possess a large surface area for sorption of dissolved organics and are colonized by a large and diverse microbiota. To examine the biodegradation of surfactants by these microorganisms, submerged oak leaves were obtained from a laundromat wastewater pond, its overflow, and a pristine control pond. Leaves were cut into disks and incubated in sterile water amended with 50 μg of ¹⁴C-labeled linear alkylbenzene sulfonate (LAS), linear alcohol ethoxylate, stearyltrimethyl ammonium chloride, distearyldimethyl ammonium chloride, benzoic acid, or mixed amino acids per liter. Sorption of the test compounds to the detritus and evolution of ¹⁴CO₂ were followed with time. All of the compounds sorbed to the detritus to various degrees, with LAS and stearyltrimethyl ammonium chloride the most sorptive and benzoic acid the least. All compounds were mineralized without a lag. With leaves from the laundromat wastewater pond, half-lives were 12.6 days for LAS, 8.4 days for linear alcohol ethoxylate, 14.2 days for stearyltrimethyl ammonium chloride, 1.0 days for benzoic acid, and 2.7 days for mixed amino acids. Mineralization of LAS and linear alcohol ethoxylate by control pond leaves was slower and exhibited an S-shaped rather than a typical first-order pattern. This study shows that detritus represents a significant site of surfactant removal in detritus-rich systems.     

Batch Production of Protein from Ethane and Ethane-Methane Mixtures

A culture of Graphium grows upon natural gas and a mineral salt solution. Ethane is the preferred substrate but methane is co-utilized. A stirred-tank type fermentor was used to study batch growth. Maximum production rate of biomass was 80 mg/liter.hr, at pH 4, using simple synthetic supporting medium with ammonium sulfate as a nitrogen source. This rate was observed after 40 hr of fermentation. A doubling time of 3.7 hr was observed. The corresponding specific growth rate was 0.187 per hr. A magnetic drive fermentor was used to study the effect of continuous recycle of gases in a gas-tight system. The rate of oxygen utilization is approximately 2.1 times higher than for ethane. Oxygen must not be allowed to become limiting in recycle gases. The calculated efficiency of overall biomass synthesis averages 30%. Hyphal and unicellular tissue of Graphium contains 52% protein. It compares favorably with standard FAO protein in its content of amino acids.     

Effect of acetate on glucose utilization by isolated rat thymocytes

The effects of acetate and ammonium salts on glucose metabolism, aminoisobutyric acid influx, and radioiodinated insulin binding in isolated thymocytes were studied. Acetate in the concentration range, 0.1–30 mm, was found to inhibit basal and insulin-stimulated CO₂ production whereas ammonium chloride at concentrations greater than 0.3 mm was slightly stimulatory. Ammonium salts inhibited glucose incorporation into glycogen and aminoisobutyric acid influx only at high concentration (30 mm). Neither acetate nor ammonium salts had significant effects on glucose incorporation into glycogen or aminoisobutyric acid influx at lower concentrations. No effect on insulin binding was observed. The observation that very low concentrations of acetate can perturb these biological assay systems suggests that other biological functions may be affected by trace amounts of buffer salts carried over from protein isolation steps.     

Benchmarking of commercially available CHO cell culture media for antibody production

In this study, eight commercially available, chemically defined Chinese hamster ovary (CHO) cell culture media from different vendors were evaluated in batch culture using an IgG-producing CHO DG44 cell line as a model. Medium adaptation revealed that the occurrence of even small aggregates might be a good indicator of cell growth performance in subsequent high cell density cultures. Batch experiments confirmed that the culture medium has a significant impact on bioprocess performance, but high amino acid concentrations alone were not sufficient to ensure superior cell growth and high antibody production. However, some key amino acids that were limiting in most media could be identified. Unbalanced glucose and amino acids led to high cell-specific lactate and ammonium production rates. In some media, persistently high glucose concentrations probably induced the suppression of respiration and oxidative phosphorylation, known as Crabtree effect, which resulted in high cell-specific glycolysis rates along with a continuous and high lactate production. In additional experiments, two of the eight basal media were supplemented with feeds from two different manufacturers in six combinations, in order to understand the combined impact of media and feeds on cell metabolism in a CHO fed-batch process. Cell growth, nutrient consumption and metabolite production rates, antibody production, and IgG quality were evaluated in detail. Concentrated feed supplements boosted cell concentrations almost threefold and antibody titers up to sevenfold. Depending on the fed-batch strategy, fourfold higher peak cell concentrations and eightfold increased IgG titers (up to 5.8 g/L) were achieved. The glycolytic flux was remarkably similar among the fed-batches; however, substantially different specific lactate production rates were observed in the different media and feed combinations. Further analysis revealed that in addition to the feed additives, the basal medium can make a considerable contribution to the ammonium metabolism of the cells. The glycosylation of the recombinant antibody was influenced by the selection of basal medium and feeds. Differences of up to 50 % in the monogalacto-fucosylated (G1F) and high mannose fraction of the IgG were observed.     

A basal-defined medium for the study of proteolytic activity of Serratia marcescens.

A simple defined basal medium is presented for the study of proteolytic activity, induction and repression, and protease purification with Serratia marcescens. Since the medium contains no protein, it does not interfere with or present artifact to protein assays, column chromatography, or electrophoresis. The medium consists of the basal salts and buffer medium of Bromke and Hammel (1979) plus a carbon-energy source such as glycerol, calcium chloride for the cation requirement for protease activity, and an amino acid, preferably leucine. Growth parameters and proteolytic activities are presented for unsupplemented medium and for the medium supplemented with each of 18 amino acids. Unsupplemented medium completes the logarithmic phase in 12.5 h of incubation and has a constitutive level of proteolytic activity. Supplementation with any amino acid, except cysteine and tryptophan, increases significantly the proteolytic activity, but has a varied effect on growth parameters.     

Growth characteristics of a new methylomonad.

A methylomonad culture was isolated from pond water and examined as a potential source of single-cell protein. A medium containing magnesium sulfate, ammonium hydroxide, sodium phosphate, tap water, and methanol supported the growth of the isolate. Optimal growth conditions in batch cultures for the organism were: temperature, 30 to 33 degrees C; pH 7.1; and phosphate concentration, 0.015 M. The minimum doubling time obtained was 1.6 h. The specific growth rate in batch culture was dependent on the methanol concentration, reaching a maximum around 0.2% (wt/vol). Growth inhibition was apparent above 0.3% (wt/vol), and growth was completely inhibited above 4.6% (wt/vol) methanol. Although the inhibitory effect of formaldehyde on the specific growth rate was much greater than that of formate, the organism utilized formaldehyde, but not formate, as a sole carbon and energy source in batch cultures. The isolate was identified primarily by its inability to utilize any carbon source other than methanol and formaldehyde for growth. Although it is capable of rapid growth on methanol, the organism showed a very weak catalase activity. The amino acid content of the cells compared favorably with the reference levels for the essential amino acids specific by the Food and Agricultural Organization of the United Nations.     

Growth characteristics of a new methylomonad.

A methylomonad culture was isolated from pond water and examined as a potential source of single-cell protein. A medium containing magnesium sulfate, ammonium hydroxide, sodium phosphate, tap water, and methanol supported the growth of the isolate. Optimal growth conditions in batch cultures for the organism were: temperature, 30 to 33 degrees C; pH 7.1; and phosphate concentration, 0.015 M. The minimum doubling time obtained was 1.6 h. The specific growth rate in batch culture was dependent on the methanol concentration, reaching a maximum around 0.2% (wt/vol). Growth inhibition was apparent above 0.3% (wt/vol), and growth was completely inhibited above 4.6% (wt/vol) methanol. Although the inhibitory effect of formaldehyde on the specific growth rate was much greater than that of formate, the organism utilized formaldehyde, but not formate, as a sole carbon and energy source in batch cultures. The isolate was identified primarily by its inability to utilize any carbon source other than methanol and formaldehyde for growth. Although it is capable of rapid growth on methanol, the organism showed a very weak catalase activity. The amino acid content of the cells compared favorably with the reference levels for the essential amino acids specific by the Food and Agricultural Organization of the United Nations.