Complex Nitrogen Sources Research Articles

Tierische Proteinhydrolysate als N-Quelle für die Milchsäurefermentation

Polylactic acid, a biodegradable polymer, is an ecological alternative for petroleum-derived plastics. The biotechnological production of its precursor lactic acid demands complex carbon and nitrogen sources significantly defining the fermentation costs. Whereas a variety of waste streams have already been utilized as carbon providing substrates, there is still no cheap alternative for common peptones. Protein hydrolyzates from animal by-products could be a promising solution to fill this gap.

Bioproduction of Docosahexaenoic Acid by the Marine protist Schizochytrium

The heterotrophic protist Schizochytrium sp. was batch cultivated in media containing glucose, yeast extract, peptone, dihydrogen potassium phosphate and sea salt prepared in distilled water. The increasing amounts of yeast ex-tract stimulated growth however, influenced lipid accumulation negatively. It has been observed that the sea salt concentrations above half the average seawater salinity were required for good growth and lipid accumulation. Schizochytrium sp. was able to grow on a glucose concentration as high as 60 g/l, which resulted in reduced biomass and lipid yield. Although, the culture in study, effectively utilized certain simple and complex nitrogen sources lipid yield was more in complex nitrogen source, i.e. yeast extract and pep-tone, than in simple nitrogen source. Comparison of growth at 20 and 28 ºC showed that the higher incubation temperature was more favorable for the growth. The optimum pH was optimized as 7 with a rotation speed of 220 rpm. Incubation for 120 h was required for the maximum production of bio-mass, lipid and DHA. In this period, the percentage of docosahexaenoic acid (20.0 %) was produced in the lipid (4.0 g/l).

Optimization of complex medium composition for heterotrophic cultivation of Euglena gracilis and paramylon production

Heterotrophic cultivation of Euglena gracilis was carried out on synthetic (Hutner medium) and complex cultivation media in order to optimize production of β-1,3-glucan (paramylon). For preparation of complex media, various industrial by-products (e.g., molasses, corn steep solid, yeast extract, and beef extract) were used with or without addition of pure compounds [glucose, galactose, fructose, lactose, maltose, sucrose, and (NH4)2HPO4]. Heterotrophic cultivation of E. gracilis was performed in Erlenmeyer flasks and additionally confirmed during research in the stirred tank bioreactor. The results clearly show that E. gracilis can easily metabolize glucose and fructose as carbon sources and corn steep solid as complex nitrogen and growth factors source for biomass growth and paramylon synthesis. Furthermore, it was also proved that addition of (NH4)2HPO4, beef extract, or gibberellic acid did not have positive effect on the biomass growth and paramylon synthesis. After optimization of complex medium composition and verification in the stirred tank bioreactor, it was concluded that medium composed of glucose (20 g/L) and corn steep solid (30 g/L) is the most suitable complex medium for industrial cultivation of E. gracilis and paramylon production.

Production of Indole-3-Acetic Acid by Three Species of Seimatosporium Causing Stem Canker and Dieback of Eucalyptus tereticornis

Production of Indole-3-Acetic Acid by three species of Seimatosporium (S. falcatum, S. fusisporum and S. discosioides) causing stem canker and dieback of Eucalyptus tereticornis was investigated. Though all the three species of Seimatosporium under investigation produced Indole-3-Acetic Acid, degree of production varied with the species, medium and incubation period. Indole-3-Acetic Acid production by all the three species was more in medium containing complex nitrogen source. S. discosioides produced comparatively more amount of Indole-3-Acetic Acid than other two species. No positive correlation could be observed among mycelial growth, Indole-3-Acetic Acid production and pH of the medium.

Effect of Complex Nutrients and Repeated-Batch Cultivation of Halobacterium salinarum on enhancing Bacteriorhodopsin Production

The extreme halophilic bacteria Halobacterium salinarum is known to produce bacteriorhodopsin (BR) protein in its purple membrane (PM) as a light-driven pump for the synthesis ATP. Its growth does not utilize simple carbon sources such as glucose, but relies on the complex carbon/nitrogen sources. The production yield of BR in the culture of H. salinarum also strongly depends on the complex nitrogen sources employed. From the various complex carbon/nitrogen sources employed in this work, tryptone rather than commonly used peptone was found to be the best complex nutrient for the growth of H. salinarum and its BR production. Bubble column photobioreactor operated in a repeated-batch mode was also employed to enhance the growth of H. salinarum by intermittent removing growth inhibitory metabolites. By employing 0.5% tryptone as carbon/nitrogen source in the culture medium, 201.8 mg/L of BR was obtained after 210 h repeated-batch cultivation which is about 50% higher than that obtained in shaker flask cultivation.

Liquid culture production of microsclerotia and submerged conidia by Trichoderma harzianum active against damping-off disease caused by Rhizoctonia solani

Media and culturing protocols were identified that supported the formation of submerged conidia and microsclerotia (MS) by Trichoderma harzianum Rifai strain T-22 using liquid culture fermentation. Liquid media with a higher carbon concentration (36 g L−1) promoted MS formation at all C:N ratios tested. Hyphae aggregated to form MS after 2 d growth and after 7 d MS were fully melanized. This is the first report of MS formation by T. harzianum or any species of Trichoderma. Furthermore, submerged conidia formation was induced by liquid culture media, but yields, desiccation tolerance, and storage stability varied with C:N ratio and carbon rate. Air-dried MS granules (<4 % moisture) retained excellent shelf life under cool and unrefrigerated storage conditions with no loss in conidial production. A low-cost complex nitrogen source based on cottonseed flour effectively supported high MS yields. Amending potting mix with dried MS formulations reduced or eliminated damping-off of melon seedlings caused by Rhizoctonia solani. Together, the results provide insights into the liquid culture production, stabilization process, and bioefficacy of the hitherto unreported MS of T. harzianum as a potential biofungicide for use in integrated management programs against soilborne diseases.

OPTIMIZATION OF ENNIATINS PRODUCTION BY AN ENDOPHYTIC STRAIN FUSARIUM DIMERUM

The goal of this study was to find suitable composition of cultivation media for enniatin production by isolated endophytic strain Fusarium dimerum. In order to find optimal cultivation media, mono- and di- saccharides, complex nitrogen sources and L-amino acids directed biosynthesis of enniatins were tested. Submerged cultivation experiments were carried out in cultivation flasks. Most promising medium for enniatin accumulation contained fructose, malt extract and peptone for bacteriology. Finally, quite expensive carbon source fructose was replaced by more available syrups. Optimization resulted in 4-times elevated enniatin biosynthesis by metabolites production microorganism. Moreover, this is the strain obtained from Magnolia soulangeana, which has similar metabolites spectrum as the isolated Fusarium dimerum. Comparison of these results with published ones revealed that this endophyte is a potential strain for enniatins biosynthesis in submerged cultivation in which the maximum accumulation 1.27 g.L-1 of enniatin in culture medium was reached in a short period (96 h). The results proved that the endophytic strain F. dimerum may potentially be applied for efficient production of bioactive enniatins.

Efficient Production of Laccase from &lt;i&gt;Armillariella tabescens&lt;/i&gt; by Solid State Fermentation

The production of laccase by solid-state fermentation (SSF) usingArmillariella tabescenswas studied. Wheat bran was selected to be the most suitable solid substrate. Several operational variables including nitrogen source, moisture content, copper and aromatic inducers were investigated. The results showed that the complex nitrogen sources, NH4NO3coupled with peptone was shown to be the best nitrogen source. 75% of initial moisture content was proved to be appropriate. Copper significantly influenced the laccase production and the yield of laccase was improved by addition of 1.5 mM copper sulphate in the medium. Guaiacol efficiently induced the laccase production and the enzyme yield (24500U/g) was enhanced by 32% compared with he control without any aromatic inducers. Efficient production of laccase fromA. tabescenscan be achieved by solid-state fermentation.

Application of protease-hydrolyzed whey as a complex nitrogen source to increase poly(3-hydroxybutyrate) production from oils by Cupriavidus necator

Whole whey hydrolyzed by Alcalase (WWH) was tested as a complex nitrogen source for the production of poly(3-hydroxybutyrate) (PHB) from waste frying oils by Cupriavidus necator H16. Addition of WWH (10 % (v/v) of cultivation media) supported the growth and PHB accumulation; PHB yields in Erlenmeyer flasks were more than 3.5-fold higher than in control cultivations. The positive influence of WWH on PHB production was confirmed in experiments performed in laboratory fermentor. C. necator cultivated with WWH produced 28.1 g PHB l(-1) resulting in a very high product yield coefficient of 0.94 g PHB per g oil. Since PHB yields were ~40 % higher than in the control cultivation, WWH can be considered as an excellent inexpensive nitrogen source for PHB production by C. necator.

English

We evaluated the bioflocculant production by&nbsp;Virgibacillus&nbsp;species isolated from a marine environment in the Eastern Cape Province of South Africa. Culture conditions for optimal production of the bioflocculant included: glucose as sole carbon source (flocculating activity 91.8%); complex nitrogen source (urea + yeast extract + (NH4)2SO4); inoculum size of 2% (v/v); initial pH 10; and calcium as mediating factor. Optimum dose of the purified bioflocculant for the clarification of 4 g/l kaolin clay suspension at neutral pH was 0.3 mg/ml. Chemical analysis of the purified bioflocculant revealed it to be mainly composed of uronic acids (87% w/w). Scanning electron microscopic observations were indicative of a porous bioflocculant structure. Fourier transform infrared (FTIR) spectrum revealed the presence of carboxyl and hydroxyl groups whilst the themogravimetric analysis (TGA) showed degradation temperature (Td) of 150&deg;C. The bioflocculant appears to hold promise in vast biotechnological applications in a more environmentally friendly and economical manner. &nbsp; Key words:&nbsp;Virgibacillus&nbsp;sp., bioflocculant, thermostable, polysaccharide.

Nutritional Studies on Production of Antibacterial Activity by the Zebra Mussel Antagonist, Pseudomonas fluorescens CL0145A

Pseudomonas fluorescens strain CL0145A was discovered at the New York State Museum Field Research Laboratory as an effective agent against the environmentally destructive zebra mussel, which has contaminated US waters. Dried cells of the microbe are being commercialized as an environmentally friendly solution to the problem. We found that antibiotic activity against the Gram-positive bacterium Bacillus subtilis is produced and excreted by this strain. We have carried out studies to optimize production of the antibiotic. Studies were begun in a complex corn meal medium. Activity was found in both cells and culture supernates and was maximal after one day of fermentation. Static fermentation conditions were found to be superior to shaken culture. Production of extracellular antibiotic in complex medium was found to be dependent on the content of sucrose and enzymehydrolyzed casein. Indeed, production was greater in sucrose plus enzyme-hydrolyzed casein than in the complex medium. Of a large number of carbon sources studied as improvements over sucrose, the best was glycerol. An examination of nitrogen sources showed that production was improved by replacement of enzymehydrolyzed casein with soy hydrolysates. Production in the simple glycerol-Hy-Soy medium was not improved by addition of an inorganic salt mixture or by complex nitrogen sources, with the exception of malt extract. In an attempt to keep the medium more defined, we studied the effect of amino acids and vitamins as replacements for malt extract. Of 21 amino acids and 7 vitamins, we found tryptophan, glutamine, biotin, and riboflavin to be stimulatory. The final medium contained glycerol, Hy- Soy, tryptophan, glutamine, biotin, and riboflavin.

Substitution of yeast extract by ammonium sulfate for mannitol production in cashew apple juice

Abstract The use of agriculture excess as substrate in industrial fermentations became an interesting alternative to reduce production costs and to reduce negative environmental impact caused by the disposal of these products. In this work, the use of cashew apple juice supplemented with ammonium sulfate as substrate for mannitol production by two Leuconostoc strains was studied. The carbohydrates of cashew apple juice are glucose and fructose. Process pH was not significant on fructose conversion into mannitol by Leuconostoc citreum B-742. However, for L. mesenteroides B-512F the process pH showed a significant effect on fructose conversion into mannitol. The use of cashew apple juice supplemented with ammonium sulfate in replacement of yeast extract enhanced the yield L. mesenteroides B-512F was the best mannitol producer. The optimal pH for mannitol production from fructose by this strain in pH-controlled fermentation was at pH 5.0 with mannitol yield of 95% and productivity of 1.6 g/L h. Lactic acid bacteria are known as nutritional exigent microorganisms. Usually, they are cultivated in yeast extract and other complex nitrogen source. Herein, the combination of an inorganic exogenous nitrogen source and the amino acids from the cashew apple juice was enough to support LAB growth in a low cost substrate.

Evaluation of Brazilian ethanol production yeasts for maltose fermentation in media containing structurally complex nitrogen sources

Maltose and glucose fermentations are strongly affected by the structural complexity of the nitrogen source and by the presence of oxygen. In this study five industrial Saccharomyces cerevisiae strains were grown in synthetic medium, containing maltose or glucose, supplemented with different nitrogen sources, with or without agitation. All strains were able to grow and efficiently ferment glucose, but not all strains were able to grow and ferment maltose well. Peptone and ammonium sulfate induced improved fermentation for all strains and conditions. Under agitation, as expected, higher biomass accumulation was detected. Casamino acids supplementation induced efficient maltose fermentation for all of the strains under aerated conditions, but differing maltose utilization patterns were observed for the static cultures. Copyright © 2012 The Institute of Brewing & Distilling

Application of response surface methodology to understand the interaction of media components during pullulan production by Aureobasidium pullulans RBF-4A3

Abstract Response surface methodology was applied to understand the interactions amongst the components of a complex medium for pullulan production by a novel Aureobasidium pullulans strain using a second order quadratic model. A total of 20 experimental runs were carried out and three dimensional response surfaces were generated to study the interaction. Results indicated that the concentration of yeast extract plays a critical role and has significant influence in pullulan production compared with peptone. Understanding the effect of complex nitrogen sources in the media using statistical methods has resulted in 70.43 g/L pullulan production which is higher as compared to earlier published reports.

The Ustilago maydis Nit2 homolog regulates nitrogen utilization and is required for efficient induction of filamentous growth.

Nitrogen catabolite repression (NCR) is a regulatory strategy found in microorganisms that restricts the utilization of complex and unfavored nitrogen sources in the presence of favored nitrogen sources. In fungi, this concept has been best studied in yeasts and filamentous ascomycetes, where the GATA transcription factors Gln3p and Gat1p (in yeasts) and Nit2/AreA (in ascomycetes) constitute the main positive regulators of NCR. The reason why functional Nit2 homologs of some phytopathogenic fungi are required for full virulence in their hosts has remained elusive. We have identified the Nit2 homolog in the basidiomycetous phytopathogen Ustilago maydis and show that it is a major, but not the exclusive, positive regulator of nitrogen utilization. By transcriptome analysis of sporidia grown on artificial media devoid of favored nitrogen sources, we show that only a subset of nitrogen-responsive genes are regulated by Nit2, including the Gal4-like transcription factor Ton1 (a target of Nit2). Ustilagic acid biosynthesis is not under the control of Nit2, while nitrogen starvation-induced filamentous growth is largely dependent on functional Nit2. nit2 deletion mutants show the delayed initiation of filamentous growth on maize leaves and exhibit strongly compromised virulence, demonstrating that Nit2 is required to efficiently initiate the pathogenicity program of U. maydis.

Effects of different nitrogen sources on the biogas production – a lab-scale investigation

For anaerobic digestion processes nitrogen sources are poorly investigated although they are known as possible process limiting factors (in the hydrolysis phase) but also as a source for fermentations for subsequent methane production by methanogenic archaea. In the present study different complex and defined nitrogen sources were investigated in a lab-scale experiment in order to study their potential to build up methane. The outcome of the study can be summarised as follows: from complex nitrogen sources yeast extract and casamino acids showed the highest methane production with approximately 600ml methane per mole of nitrogen, whereas by the use of skim milk no methane production could be observed. From defined nitrogen sources l-arginine showed the highest methane production with almost 1400ml methane per mole of nitrogen. Moreover it could be demonstrated that the carbon content and therefore C/N-ratio has only minor influence for the methane production from the used substrates.

Improved Laccase Production by Trametes pubescens MB89 in Distillery Wastewaters

Various culture parameters were optimised for laccase synthesis by Trametes pubescens MB89, including pH, carbon source, nitrogen source, lignocellulosic supplements, and reported inducers. Glucose, in conjunction with a complex nitrogen source at pH 5.0, resulted in the highest laccase yield. Adding ethanol, copper, or 2,5-xylidine prior to inoculation further improved laccase concentrations. The addition of 2,5-xylidine was further investigated with multiple additions applied at varying times. This novel application substantially improved laccase production when applied regularly from inoculation and during the growth phase, and also countered glucose repression of laccase synthesis. Single and multiple factor changes were studied in three distillery wastewaters and a wine lees. A synergistic increase in laccase synthesis was observed with the addition of glucose, copper, and 2,5-xylidine. Single addition of 2,5-xylidine proved most beneficial with distillery wastewaters, while copper addition was most beneficial when using the wine lees as a culture medium.

Predicting mab product yields from cultivation media components, using near‐infrared and 2D‐fluorescence spectroscopies

The yield of monoclonal antibody (Mab) production processes depends on media formulation, inocula quality, and process conditions. As in industrial processes tight cultivation conditions are used, and inocula quality and viable cell densities are controlled to reasonable levels, media formulation and raw materials lot-to-lot variability in quality will have, in those circumstances, the highest impact on process performance. In the particular Mab process studied, two different raw materials were used: a complex carbon and nitrogen source made of specific peptones and defined chemical media containing multiple components. Using different spectroscopy techniques for each of the raw material types, it was concluded that for the complex peptone-based ingredient, near-infrared (NIR) spectroscopy was more capable of capturing lot-to-lot variability. For the chemically defined media containing fluorophores, two-dimensional (2D)-fluorescence spectroscopy was more capable of capturing lot-to-lot variability. Because in Mab cultivation processes both types of raw materials are used, combining the NIR and 2D-fluorescence spectra for each of the media components enabled predictive models for yield to be developed that out-performed any other model involving either one raw material alone, or only one type of spectroscopic tool for both raw materials. For each particular raw material, the capability of each spectroscopy to detect lot-to-lot differences was demonstrated after spectra preprocessing and specific wavelength regions selection. The work described and the findings reported here open up several possibilities that could be used to feed-forward control the process. These include, for example, enabling specific actions to be taken regarding media formulation with particular lots, and all types of predictive control actions aimed at increasing batch-to-batch yield and product quality consistency at harvest.

Design of an efficient medium for heterologous protein production in Yarrowia lipolytica: case of human interferon alpha 2b

BackgroundThe non conventional yeast Yarrowia lipolytica has aroused a strong industrial interest for heterologous protein production. However most of the studies describing recombinant protein production by this yeast rely on the use of complex media, such media are not convenient for large scale production particularly for products intended for pharmaceutical applications. In addition medium composition can also affect the production yield. Hence it is necessary to design an efficient medium for therapeutic protein expression by this host.ResultsFive different media, including four minimal media and a complex medium, were assessed in shake flasks for the production of human interferon alpha 2b (hIFN α2b) by Y. lipolytica under the control of POX2 promoter inducible with oleic acid. The chemically defined medium SM4 formulated by Invitrogen for Pichia pastoris growth was the most suitable. Using statistical experimental design this medium was further optimized. The selected minimal medium consisting in SM4 supplemented with 10 mg/l FeCl3, 1 g/l glutamate, 5 ml/l PTM1 (Pichia Trace Metals) solution and a vitamin solution composed of myo-inositol, thiamin and biotin was called GNY medium. Compared to shake flask, bioreactor culture in GNY medium resulted in 416-fold increase of hIFN α2b production and 2-fold increase of the biological activity.Furthermore, SM4 enrichment with 5 ml/l PTM1 solution contributed to protect hIFN α2b against the degradation by the 28 kDa protease identified by zymography gel in culture supernatant. The screening of the inhibitory effect of the trace elements present in PTM1 solution on the activity of this protease was achieved using a Box-Behnken design. Statistical data analysis showed that FeCl3 and MnSO4 had the most inhibitory effect.ConclusionWe have designed an efficient medium for large scale production of heterologous proteins by Y. lipolytica. The optimized medium GNY is suitable for the production of hIFN α2b with the advantage that no complex nitrogen sources with non-defined composition were required.

Optimization of cold-active chitinase production from the Antarctic bacterium, Sanguibacter antarcticus KOPRI 21702

In the present study, cultivation conditions and medium components were optimized using statistical design and analysis to enhance the production of Chi21702, a cold-active extracellular chitinase from the Antarctic bacterium Sanguibacter antarcticus KOPRI 21702. Identification of significant carbon sources and other key elements was performed using a statistical design technique. Chitin and glycerol were selected as main carbon sources, and the ratio of complex nitrogen sources to carbon sources was determined to be 0.5. Among 15 mineral components included in basal medium, NaCl, Fe(C₆H₅O₇), and MgCl₂ were found to have the most influence on Chi21702 production. The optimal parameters of temperature, initial pH, and dissolved oxygen level were found to be 25°C, 6.5, and above 30% of air saturation, respectively. The maximum Chi21702 activity obtained under the optimized conditions was 90 U/L. Through statistical optimization methods, a 7.5-fold increase in Chi21702 production was achieved over unoptimized conditions. Chi21702 showed relatively high activity, even at low temperatures close to 0°C. The information obtained in the present study could be applied to the production of cold-active endochitinase on a large scale, suitable for a process at low temperature in industry.