Influence Of Medium Components Research Articles

Influence of medium components and pH on the production of odd-carbon fatty acids by Aurantiochytrium sp. SA-96

Odd-carbon fatty acids have been utilized for anaplerotic therapy for lifestyle-related diseases such as Alzheimer’s disease and diabetes. We examined the culture condition of Aurantiochytrium for the production of pentadecanoic acid (C15) as an odd-carbon fatty acid, and demonstrated a produce method for odd-carbon fatty acid–enriched triglyceride. To determine the optimum conditions for C15 production, selection of effective components and their respective concentrations in the culture medium, choice of the suitable Aurantiochytrium strain, and determination of the optimum culture pH were performed. The optimum conditions for the production of C15 were found to be as follows: Strain: Aurantiochytrium sp. strain SA-96 Medium composition: 0.2% yeast extract 0.5% monosodium glutamate, 1.0% sea salt, 50 mM L-Val, 25 mM sodium propionate, 3.6% glucose, and 10% soy milk whey. Culture system: Airlift culture vessels (5.0 L) and pH controller were used. Sodium hydroxide (1.0 M) solution was used for pH adjustment. Other parameters were as follows: medium volume, 3.0 L; air supply, 1.2 vvm; temperature, 23.5–26.6 °C; pH, 7.40–7.74; culture period, 72 h. The yield of odd-carbon fatty acids (C15, 85% of odd FA) was 1.04 g L−1. Preparation of odd-carbon fatty acid–enriched triglyceride: Triglyceride was isolated from the extracted lipids from cultured cells and treated with ozone/hydrogen peroxide. By removing of carboxylic acid containing triglyceride, the odd-carbon fatty acid and C15 contents of the resultant triglyceride reached to 72.3 and 60.7%, respectively.

Influence of media components and pH on the production of alpha amylase from Bacillus sp. SJC B03

Influence of media components and pH on the production of alpha amylase from Bacillus sp. SJC B03

Impact of phosphate and other medium components on physiological regulation of bacterial laccase production.

Laccases are multicopper oxidases known to catalyze the transformation of a wide range of phenolic and non-phenolic substrates using oxygen as electron acceptor and forming water as the only by product. Their potential relevance in several industries requires the constant search for novel laccases. Positive outcome of the isolation of laccase producing bacteria depends on the nature and concentration of media constituents. Several attempts to isolate laccase producing bacteria failed when the phosphate-containing M9 minimal medium was used. Shift to phosphate-less M162 medium led to successful isolations. Seven bacterial isolates belonging to genera Bacillus, Lysinibacillus, Bhargavaea and Rheinheimera were used to study the effect of medium constituents on laccase production. Inorganic phosphate (≥50 mM) was found to regulate laccase synthesis negatively though no inhibitory effect of phosphate (10-500 mM) was seen on laccase activity. All isolates ceased laccase synthesis when grown in the presence of tryptone (0.2-1%), with R. tangshanensis as an exception, or yeast extract (1.5-2%) as the only C/N source in M162 medium. Supplementation upto 0.1% of glucose in basal M162 medium increased laccase production in five isolates but decreased at higher concentrations. The influence of medium components on laccase synthesis was further affirmed by zymographic studies. These observations offer possibilities of isolating promising laccase producers from diverse environmental sources. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:541-548, 2017.

Production of Fungal Laccase Using Orange Peelings as Substrate by Submerged Static Fermentation

Laccases are diphenol oxidases that have numerous applications in biotechnological processes. In this work, the production of fungal laccase using organic and inorganic feed substrates in submerged static fermentation was investigated. Study Design: One-factor-at-a-time strategy was adopted to optimize the cultural parameters for enhanced laccase production. Place and Duration of Study: Department of Microbiology, Faculty of Science, University of Port Harcourt, Nigeria, between October 2014 and November 2015. Methodology: A total of nine fungal isolates were obtained from wood decaying sites of University of Port Harcourt forest areas and subjected to laccase screening with 2,2-Azinobis-3- ethyl(benzthiazoline-6-sulphonate) (ABTS). The influence of medium components using the basal medium at pH 5.0 as base was evaluated and these cultural parameters include carbon sources (glycerol, rice bran, glucose and ground orange peelings), nitrogen sources (yeast extract, potassium nitrate, peptone and ammonium chloride), metal ions (copper sulfate and manganese sulfate) and inducer compounds (ABTS, Tween 80 and soya oil). Time course study was conducted with the unoptimized and optimized cultural medium. Results: Out of nine cultures tested, seven were found to be laccase-positive with isolates CF-1 and CF-2 being the best potential cultures. Isolate CF-1 which had the highest laccase activity was identified as Pleurotus ostreatus and was chosen for further studies. Ground orange peelings (0.1% w/v) and NH4Cl (0.1% w/v) were the most suitable carbon and nitrogen source for laccase production by the fungus. Maximum laccase production was obtained with Cu2+ at a concentration of 0.05%w/v among other metal ions. Soya oil at concentration of 0.05% (v/v) was the best inducer of the enzyme. The highest laccase production was achieved at an Initial pH 4.5. Under optimal culture medium, the maximum laccase activity was determined to be 7.21 U ml-1 on the 7th day of cultivation; which was approximately three times higher than that in basal medium (2.5 U ml-1). The results obtained indicate that the extracellular laccase production is dependent on various cultural parameters. Conclusion: One-factor-at-a-time strategy adopted in this study proved that the optimum conditions enhanced laccase production by three folds using orange peelings. The results obtained are very interesting since orange peelings are common agricultural wastes in several countries and imply that their re-utilization in the production of enzymes would help solve pollution problems caused by their improper disposal. In addition, Pleurotus ostreatus having shown promise for laccase production using low-cost lignocellulosic substrates could be suggested as a prospective candidate for higher laccase production for several biotechnological applications.

Secondary alcohol oxidase activity identified in genus of Pseudomonas isolated from the oil polluted soil

Since the interest for biocatalysts and their application in industry have increased significantly, enzymatic oxidation of secondary alcohols to the desired ketones is still not an issue. The aim of this study was to discover bacterium species which express enzymes with activities toward oxidation of secondary alcohols. Five species of Pseudomonas genus bacteria were isolated from the oil polluted soil. Particularly, Pseudomonas putida species have shown enhanced activity in oxidation reactions of secondary alcohols. The influence of medium components and cultivation parameters were investigated for P. putida 4 strain, which expressed enzyme with specificity only for secondary alcohols. The main finding of this study was that PVA was used as a carbon and energy source by bacteria and did not act as an inducer.

The influence of medium components on the differentiation period and the life expectancy of the mouse neuroblastoma cells NIE-115

The rate of differentiation and longevity of mouse neuroblastoma cells NIE-115 in culture media of various compositions have been investigated. In serum-free media the dependence of the differentiation period on concentrations of sodium ions, amino acids, and carbohydrates as well as on the pH values had maximum. Differentiated cells exhibited maximal longevity in those media in which the differentiation time was comparable with the duration of the cellular cycle.

Influence of medium components and fermentation conditions on the production of bacteriocin(s) by Bacillus licheniformis AnBa9

Recently, antibacterial peptides are gaining more attention as an alternative therapeutics and food and other products from spoilage and deterioration. Antibacterial peptide producing strains were isolated from sediments of slaughterhouse sewage wastes. One among them, identified as Bacillus licheniformis inhibited the growth of several gram positive bacteria. Response surface methodology with central composite rotary design was used for optimization of fermentation medium and conditions for antibacterial peptide production. Lactose, NH4NO3, yeast extract and NaCl and environmental factors such as pH, temperature and incubation period were selected as variables. Among ingredients, high concentration of yeast extract and NaCl had a positive effect on antibacterial peptide production and specific activity, respectively. Alkaline pH and high temperature favoured the production of antibacterial peptide by B. licheniformis AnBa9. Under optimized condition, B. licheniformis AnBa9 produced 25-fold higher production of antibacterial peptide than the un-optimized condition. Biochemical characteristics of the antibacterial peptides of B. licheniformis AnBa9 revealed that they are of bacteriocin type.

Partial characterisation of two bacteriocins produced byLactobacillus paracasei subsp.paracasei ST242BZ and ST284BZ and the effect of medium components on their production

The influence of medium components on production of bacteriocins ST242BZ (10.0 kDa) and ST284BZ (3.5 kDa) byLactobacillus paracasei subsp.paracasei ST242BZ and ST284BZ have been studied. Growth in MRS broth (pH of 6.5) yielded bacteriocin levels of 12800 AU/ml. Modified MRS with tryptone as the only nitrogen source, MRS supplemented with KH2PO4 (10–100 g/l), or MRS supplemented with thiamine increased bacteriocin ST242BZ production to 25600 AU/ml. Tryptone, combinations of tryptone, meat extract and yeast extract, or thiamine did not increase bacteriocin ST284BZ production. However, MRS supplemented with K2HPO4 (50–100 g/l) increased bacteriocin ST284BZ production up to 25600 AU/ml. Our results suggest that production of bacteriocins ST242BZ and ST284BZ are stimulated by potassium ions.

Influence of Media Components and pH on Somatic Embryo Induction in Three Genotypes of Soybean

The influence of media components on the initiation of somatic embryogenesis in three genotypes of soybean was investigated. The following genotypes were used: Iroquois, Macon, and Savoy. Media modifications included sucrose concentration, type and concentration of auxin at two pH levels, and pH level independently. Immature cotyledons were used as the source of explant. Cotyledons were placed on a medium containing MS salts, B5 vitamins, sucrose, and auxin. Gelrite (0.2%) was used as the solidifying agent. Sucrose concentrations of 1, 2, 3, 4.5, or 6% were used. The auxins used included 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthaleneacetic acid (NAA) with each at concentrations of 45.2, 90.4, 135.7, 180.9, and 226.2 μM. The pH of each the media was adjusted to either 5.7 or 7.0 with 1 N NaOH. In an additional experiment, the effect of the two pH levels, 5.7 and 7.0, was investigated independently. Overall, the frequency of somatic embryogenesis significantly varied among the different genotypes used in this study, with Iroquois showing the highest response. Frequency of somatic embryogenesis also varied in response to the different treatments used, including sucrose and auxin. The highest initiation (91.7%) and mean number of somatic embryos per responding explant (14.9) of Iroquois was observed in a medium containing 2% sucrose. The highest initiation (97.1%) and mean number of somatic embryos per responding explant (19.5) was observed in Iroquois on 135.7 μM 2,4-D and Savoy on 135.7 μM 2,4-D, respectively, for the auxin by pH level experiment. No significant differences were observed among the two pH treatments used.

Influence of medium components on ex vivo megakaryocyte expansion.

Reinfusion of ex vivo-expanded autologous megakaryocytes together with a stem cell transplantation may be useful to prevent or reduce the period of chemotherapy-induced thrombocytopenia. In this study, we analyzed several serum-containing and serum-free media to identify the most suitable medium for megakaryocyte expansion. Moreover, two thrombopoietin (Tpo)-mimetic peptides were tested to evaluate whether they could replace Tpo in an expansion protocol. To analyze the effects of different media on megakaryocyte expansion, we used an in vitro liquid culture system. For this purpose, CD34(+) cells were isolated from peripheral blood and cultured for 8 days in the presence of Tpo and interleukin-3 (IL-3). The presence of megakaryocytes was analyzed by flow cytometric analysis after staining for CD41 expression. For our standard culture procedure, megakaryocyte medium (MK medium) supplemented with 10% AB plasma was used. Addition of 5% or 2.5% AB plasma yielded higher numbers of megakaryocytes, implying the presence of inhibitory factors in plasma. However, some plasma components are required for optimal megakaryocyte expansion because addition of less than 1% AB plasma or addition of human serum albumin instead of AB plasma resulted in the formation of lower numbers of megakaryocytes. Two commercially available serum-free media were also tested: Cellgro and Stemspan. If CD34(+) cells were cultured in Cellgro medium similar numbers of megakaryocytes were obtained as when CD34(+) cells were cultured in MK medium supplemented with 10% AB plasma. In MK medium with 2.5% AB plasma, higher numbers of megakaryocytes were cultured than in MK medium supplemented with 10% AB plasma. Therefore, Cellgro medium is not the best alternative medium. In cultures with Stemspan medium, higher numbers of megakaryocytes were obtained compared to MK medium with 10% AB plasma. Stemspan is thus a good alternative for MK medium. Two Tpo-mimetic peptides, AF13948 and PK1M, were tested for their ability to replace Tpo. In cultures with AF13948, comparable numbers of megakaryocytes were obtained as in the presence of Tpo, but in cultures with PK1M the number of megakaryocytes was lower. This study shows that high concentrations of plasma in medium inhibits megakaryocyte formation, but some plasma components are required for optimal megakaryocyte expansion. For an ex vivo expansion protocol, it is worthwhile to test several media, because the number of megakaryocytes differs widely with the medium used.

Influence of Media Components on Efficacy of Paclobutrazol in Inhibiting Growth of Broccoli and Petunia

Three experiments were conducted to evaluate media component effects on paclobutrazol activity. In Expts. 1 and 2, a broccoli (Brassica oleracea var. botrytis L.) seedling bioassay was used to compare the activity of paclobutrazol at six concentrations (0-0.32 mg·L-1). Results from Expt. 1 indicated that an average of 4-, 5-, and 10-fold higher concentrations were required in old composted pine bark, fresh pine bark, and composted pine bark samples, respectively, to achieve the same activity observed in sphagnum peatmoss (peat) samples. Activity in coir was similar to that in peat while activity in vermiculite and perlite was greater than that in peat. Activity in a fibrous peat sample was greater than in two less-fibrous peat samples. Results from Expt. 2 indicated that paclobutrazol activity was reduced more in the fine (<2 mm) fraction of fresh and composted bark samples than in medium (2-4 mm) or coarse (>4 mm) fractions. In Expt. 3, petunia {Petunia hybrida Vilm. `Madness Red') was grown in a mixture of either 60% composted pine bark: 0% peat or 0% composted bark: 60% peat. The paclobutrazol concentration required to achieve the same size control was 14 times higher in the former mixture than in the latter. Thus, media components differ greatly in their influence on paclobutrazol activity and the bioassay procedure may serve as a useful tool for predicting media-paclobutrazol interactions. Chemical name used: (±)-(R*,R*)-β-[(4-chlorophenyl)methyl]-α-(l,l-dimethyl)-lH-l,2,4-triazole-l-ethanol (paclobutrazol).

Influence of medium components on astaxanthin content and production of Haematococcus pluvialis

Response surface methodology was used to examine the effects of five components of the medium on astaxanthin content and astaxanthin production of Haematococcus pluvialis in batch culture based on the central composite rotatable design. The medium components considered included sodium acetate, potassium nitrate, major elements, trace elements and vitamins. Within the range of the concentrations tested, all the medium components, except the vitamins exhibited significant effects on the astaxanthin content and astaxanthin production. The optimal nutrient concentrations for astaxanthin content (mg g −1 dry cells) were 1·64 g litre −1 sodium acetate, 0·03 g litre −1 potassium nitrate, 40 ml litre −1 of the major element stock solution and 0·14 ml litre −1 of the trace element stock solution. The optimal nutrient concentrations for astaxanthin production (mg litre −1) were similar to those for the astaxanthin content (mg g −1 dry cells); the higher concentrations of sodium acetate and the major element stock solution plus a lower concentration of potassium nitrate could significantly enhance the astaxanthin production.

Production of alkaline serine protease subtilisin Carlsberg by Bacillus licheniformis on complex medium in a stirred tank reactor

Bacillus licheniformis (DSM 641) was cultivated on complex medium in batch and fed-batch operations in a 20-l working volume stirred tank reactor. The medium composition (maltose, glucose, sucrose, fructose, ammonia, phosphate) and O2 and CO2 in the off-gas were monitored on-line; pH, pO2, turbidity, culture fluorescence were monitored in situ; optical density, concentrations of sugars, amino acids, phosphate, proteins, DNA, protease activity and total solids content were monitored off-line. Problems of on-line sampling, cell concentration monitoring, and culture fluorescence measurements and the influence of medium components on the enzyme productivity are discussed. Close relationships between variations of pH, pO2, O2 transfer rate and CO2 production rate on the one hand and cell mass and fluorescence intensity on the other were demonstrated in batch and in fed-batch cultures. Using suitable cultivation conditions, alkaline protease with high volume activity [15300 units (U)/ml] and specific activity (510 U/mg) was produced. By replacing the complex medium with a semisynthetic one, the volumetric activity was reduced by a factor of ten (to 1650 U/ml), but the specific productivity by a factor of only two (to 210 U/ml).

The influence of medium components on Cu 2+-dependent oxidation of low-density lipoproteins and its sensitivity to superoxide dismutase

The extent of in vitro Cu 2+-dependent oxidation of low-density lipoproteins (LDL) has been reported to vary widely depending upon reaction conditions. In this study, the effect of proteins and amino acids on Cu 2+-induced LDL oxidation was examined. Treatment of LDL with 5 μM CuSO 4 for 18 h in either phosphate-buffered saline (PBS) or Ham's F-10 medium resulted in extensive oxidation as determined by the content of thiobarbituric acid reactive substances (TBARS) and by increased lipoprotein electronegativity. In PBS, oxidation was entirely blocked by histidine and the tripeptide, gly-his-lys (GHK). Oxidation was also prevented by bovine serum albumin, but superoxide dismutase (SOD) provided only 20% protection. Both proteins bound similar amounts of Cu 2+, but albumin appeared to be a more effective peroxyl radical trap as evidenced by its ability to prevent LDL oxidation induced by 2,2′-azo-bis(2-amidinopropane hydrochloride). In F-10 medium, SOD had marked inhibitory effects, in contrast to PBS. The addition of disulfides to PBS markedly enhanced the ability of SOD to inhibit oxidation. These results indicate that medium components which affect Cu 2+ availability influence LDL oxidation and suggest that albumin is ideally suited as a plasma antioxidant to prevent oxidative modification of LDL. Furthermore, in certain instances, the inhibitory effects of SOD may be attributable to effects such as Cu 2+ binding rather than dismutation of superoxide.

In vitro susceptibilities of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum to sparfloxacin and PD 127391

The in vitro activities of two investigational quinolones, sparfloxacin (previously designated AT 4140) and PD 127391, were determined for 30 strains each of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum and compared with those of ciprofloxacin, tetracycline, clindamycin, and erythromycin. Erythromycin was the most active compound against M. pneumoniae (maximum MIC, less than 0.008 microgram/ml). PD 127391 (MICs, less than 0.008 to 0.031 microgram/ml), sparfloxacin (MICs, less than 0.008 to 0.25 microgram/ml), clindamycin (MICs, less than 0.008 to 0.5 microgram/ml), and tetracycline (MICs, 0.063 to 0.25 microgram/ml) were superior to ciprofloxacin (MICs, 0.5 to 2 microgram/ml). Sparfloxacin and PD 127391 were active against M. hominis (MICs, less than 0.008 to 0.031 microgram/ml for each) at concentrations comparable to those of clindamycin (MICs, less than 0.008 to 0.063 microgram/ml) and at concentrations lower than those of ciprofloxacin (MICs, 0.125 to 0.5 microgram/ml). As expected, M. hominis was resistant to erythromycin (MICs, 32 to greater than or equal to 256 micrograms/ml). For U. urealyticum, PD 127391 (MICs, 0.031 to 0.5 microgram/ml) and sparfloxacin (MICs, 0.063 to 1 microgram/ml) were superior to erythromycin (MICs, 0.25 to 4 micrograms/ml), ciprofloxacin (MICs, 0.5 to 8 micrograms/ml), and clindamycin (MICs, 0.25 to 64 micrograms/ml. Both new quinolones were equally active against tetracycline-susceptible as well as resistant strains of M. hominis and U. urealyticum. The possible influence of medium components and/or pH on MICs was evaluated by testing a Staphylococcus aureus reference strain with each antibiotic in SP-4 broth and 10-B broth and comparing the results with published MICs for this strain. MICs determined in 10-B broth for erythromycin were affected most. This study shows that the activities of sparfloxacin and PD 127391 are similar to one another and comparable or superior to those of other drugs used to treat mycoplasmal infections. The MICs of both new quinolones were consistently 2 to several dilutions lower than those of ciprofloxacin for each species.

Staphylococcus aureus growth and type 5 capsular polysaccharide production in synthetic media

The production of type 5 capsular polysaccharide by Staphylococcus aureus in synthetic media was investigated. The influence of medium components on capsular polysaccharide synthesis appeared to relate to the presence or absence of the component rather than to concentration gradient. The production of type 5 capsular polysaccharide was linked to energy availability and energy source, but not to carbohydrate concentration or carbon/nitrogen ratio. Regulation of capsular polysaccharide production by S. aureus in response to medium changes would appear to differ from that typically displayed in other organisms that produce polysaccharides.

Automated determination of glucose in fermentation broths with p-hydroxy-benzoic-acid hydrazide (p-HBAH)

The substrate concentration is usually the most important process variable for automatic control of cultivation and for product formation. Glucose is the most popular substrate. An on-line technique for glucose analysis is described which is based on the p-HBAH method. The influence of medium components and metabolites was determined. Their influence on the measurement can be taken into account based on the calibration curves presented. This method attains the sensitivity of enzymatic glucose analysis and is less expensive and easier to handle.

Nutritional Factors Influencing Growth and Lipase Production by Geotrichum Candidum

The conditions necessary for optimum microbial lipase activity vary with the source of the enzyme, the substrate, and the treatment accorded the lipase preparation. Similarly, it appears that the production of bacterial and fungal lipases is related directly to the nutritional and physical environment of the cultures under observation. Thus, it has been suggested (1) that certain bacterial tipases are adaptive in nature and that production is stimulated by the presence of specific substrates, or that the elaboration of the enzyme d,epends upon preferential utilization of certain substrates by the organisms. Peters and Nelson (3) observed with Candida lipolytica that low lipase activity was associated with conditions favorable for rapid growth, whereas high lipase activity was associated with less favorable conditions. In this connection, Tammisto (9) suggested that the failure of certain lipolytic species to hydrolize fat when they were grown on glycerol-fat agar was due to the inactivation of the lipase by acids developed in the glycerol fermentation. This viewpoint is partially substantianted by the observation (2) that the extracellular lipase of Geotrichum candidum is completely inactive at pH 4.0 and that activity rapidly diminishes between pH 5.0 and pH 4.0. Specifically, lipase production has been reported to be influenced by the presence of certain buffers (3), carbohydrates (3, 10), vitamins (4), nitrogen sources (10), agar (10), fats (7), and various combinations of nutrients (9) in the cnlture medium. However, the varied responses of different microorganisms have no t permitted a unified concept of the influence of the components of the growth medium on microbial lipase production. The characteristics of the lipase (2) and some of the factors influencing the rate of liberation of water insoluble fat ty acids in cream by G. candidum (6) have been described recently. I t is the purpose of this report to present data pertaining to the influence of media components on the growth and lipase production by G. candidum.