Complex Nitrogen Sources Research Articles

Nutritional regulation of protease production by the feather-degrading bacterium Chryseobacterium sp. kr6

The effects of nutritional conditions on growth and protease production by the feather-degrading Chryseobacterium sp. kr6 were investigated. Higher growth was observed on feather-containing or tryptone (TR) medium when compared to casein (CA) or glucose-nitrogen (GN) base medium. Protease production occurred during growth on feather-containing and TR media, whereas no protease activity was detected on CA or GN medium, indicating that protease production is not constitutive, depending on the presence of specific complex nitrogen sources. Supplementation of whole feathers (WF) medium with glucose (WFG) or NH(4)Cl (WFN) did not result in major differences in growth and protease production, whereas soluble protein was lower in supplemented media. Glucose consumption and growth were higher on WFG than on GN medium, suggesting that the absence of a specific complex nitrogen source limited bacterial growth. On WF medium, this strain grew closely attached to the feather structures, initially on the barbules and subsequently on the feather rachis. It was observed, through zymogram analysis, that strain kr6 produced diverse proteolytic enzymes in response to different growth substrates. These results were confirmed by the differential behaviors of crude proteases towards protease inhibitors.

The Plackett-Burman design in optimization of media components for biomass production ofLactobacillus rhamnosusOXY

The central composite design was developed to search for an optimal medium for the growth of Lactobacillus rhamnosus OXY. The effect of various media components, such as carbon sources, simple and complex nitrogen sources, mineral agents, and growth factors (vitamins B, amino acids) was examined. The first-order model based on Plackett-Burman design showed that glucose, sodium pyruvate, meat extract and mineral salts significantly influenced the growth of the examined bacteria. The second-order polynomial regression confirmed that maximum biomass production could be achieved by the combination of glucose (12.38 g/l), sodium pyruvate (3.15 g/l), meat extract (4.08 g/l), potassium phosphate (1.46 g/l), sodium acetate (3.65 g/l) and ammonium citrate (1.46 g/l). The validation of the predicted model carried out in bioreactor conditions confirmed the usefulness of the new medium for the culture of L. rhamnosus OXY in large scale. The optimal medium makes the culture of the probiotic bacterium L. rhamnosus OXY more cost effective.

Optimization of biomass production with enhanced glucan and dietary fibres content by Pleurotus ostreatus ATHUM 4438 under submerged culture

This work was aimed at optimizing biomass production by the edible basidiomycete Pleurotus ostreatus ATHUM 4438 in a submerged process with enhanced glucan and dietary fibres content. β-Glucan from Pleurotus sp. (pleuran) has been used as food supplements due to its immunosuppressive activity. Like other dietary fibre components, oyster mushroom polysaccharides can stimulate the growth of colon microorganisms (probiotics), i.e. act as prebiotics. We used the FF MicroPlate for substrate utilization and growth monitoring. The pattern of substrate catabolism forms a substrate assimilation fingerprint which is useful in selecting media components for media optimization of maximum biomass production. Different carbon sources (95) were used and then 8 of them were tested in shake flask cultures. The effect of various organic and complex nitrogen sources on biomass production was also examined and response surface methodology based on central composite design was applied to explore the optimal medium composition. When the optimized culture medium was tested in a 20-L stirred tank bioreactor, using 57 g L −1 xylose and 37 g L −1 corn steep liquor, high yields (39.2 g L −1) of dry biomass was obtained. The yield coefficients for total glucan and dietary fibres on mycelial biomass formed were 140 ± 4 and 625 ± 9 mg g −1 mycelium dry weight, respectively.

Effect of nitrogen sources and vitamins on ligninolytic enzyme production by some white-rot fungi. Dye decolorization by selected culture filtrates

The effect of amino acids, complex nitrogen sources and vitamin addition on Trametes trogii, Trametes villosa and Coriolus versicolor var. antarcticus ligninolytic enzyme production, was evaluated. Dye decolorization by their culture filtrates was compared. Glutamic acid followed by peptone, were the best N sources for laccase and manganese peroxidase production. The three fungi produced two laccase isoenzymes (molecular weights from 38 up to 150kDa); their pattern of production was not affected by medium composition. Although the response was not uniform, vitamin addition sometimes stimulated ligninolytic enzyme production, but never inhibited it. Thiamine induced manganese peroxidase production. T. trogii grown in glutamic acid produced culture filtrates with the highest laccase (188.3 U/ml) and manganese peroxidase activities (4.5 U/ml), rendering the best results in decolorization. These crude filtrates were able to decolorize in half hour (at pH 4.5, 30°C): 13%, 23%, 40%, 46%, 82%, 94% and 95% of Gentian Violet, Xylidine, Congo Red, Malachite Green, Remazol Brilliant Blue R, Indigo Carmine and Anthraquinone Blue, respectively.

Production of capsular polysaccharide from Escherichia coli K4 for biotechnological applications

The production of industrially relevant microbial polysaccharides has recently gained much interest. The capsular polysaccharide of Escherichia coli K4 is almost identical to chondroitin, a commercially valuable biopolymer that is so far obtained from animal tissues entailing complex and expensive extraction procedures. In the present study, the production of capsular polysaccharide by E. coli K4 was investigated taking into consideration a potential industrial application. Strain physiology was first characterized in shake flask experiments to determine the optimal culture conditions for the growth of the microorganism and correlate it to polysaccharide production. Results show that the concentration of carbon source greatly affects polysaccharide production, while the complex nitrogen source is mainly responsible for the build up of biomass. Small-scale batch processes were performed to further evaluate the effect of the initial carbon source concentration and of growth temperatures on polysaccharide production, finally leading to the establishment of the medium to use in following fermentation experiments on a bigger scale. The fed-batch strategy next developed on a 2-L reactor resulted in a maximum cell density of 56 g(cww)/L and a titre of capsular polysaccharide equal to 1.4 g/L, approximately ten- and fivefold higher than results obtained in shake flask and 2-L batch experiments, respectively. The release kinetics of K4 polysaccharide into the medium were also explored to gain insight into the mechanisms underlying a complex aspect of the strain physiology.

Use of Hydrolysates from Yellowfin Tuna (Thunnus albacares) Heads as a Complex Nitrogen Source for Lactic Acid Bacteria

Two different peptones obtained by enzymatic hydrolysis of yellowfin tuna (Thunnus albacares) head waste have been shown to be effective in promoting the growth of lactic acid bacteria (Lactobacillus bulgaricus Persian Type Culture Collection (PTCC) 1332, Lactobacillus acidophilus PTCC 1643, Lactobacillus casei PTCC 1608, Lactobacillus delbrukii PTCC 1333, Lactobacillus plantarum PTCC 1058, Lactococcus lactis PTCC 1336, and Lactobacillus sakei PTCC 1712). Peptones obtained from the enzymatic hydrolysis with Alcalase or Protamex were used instead of the standard peptones used in commercial MRS media. Peptones produced by Alcalase and Protamex had a 34% and 19% degree of hydrolysis, respectively. The results showed that the peptones from Alcalase and Protamex were better at promoting lactic acid bacteria (LAB) growth than the commercial MRS media (P < 0.05). The choice of proteolytic enzyme used to produce the fish hydrolysate had a considerable impact on the performance of the resulting hydrolysate, both in terms of maximum growth rate and biomass production. Peptones produced using Alcalase, with a higher degree of hydrolysis, induced better growth and performed better overall as an LAB substrate than those using Protamex. Current study revealed that enzymatic-modified fish by-products can be used as low cast nitrogen source for bacterial growth.

Enhanced production of recombinant streptokinase in Escherichia coli using fed-batch culture

Fed-batch culture strategy is often used for increasing production of heterologous recombinant proteins in Escherichia coli. This study was initiated to investigate the effects of dissolved oxygen concentration (DOC), complex nitrogen sources and pH control agents on cell growth and intracellular expression of streptokinase (SK) in recombinant E. coli BL21(DE3). Increase in DOC set point from 30% to 50% did not affect SK expression in batch culture where as similar increase in fed-batch cultivation led to a significant improvement in SK expression (from 188 to 720 mg l −1). This increase in SK could be correlated with increase in plasmid segregational stability. Supplementation of production medium with yeast extract and tryptone and replacement of liquid ammonia with NaOH as pH control agent further enhanced SK expression without affecting cell growth. Overall, SK concentration of 1120 mg l −1 representing 14-fold increase in SK production on process scale-up from flask to bioreactor scale fed-batch culture is the highest reported concentration of SK to date.

Effect of Precultural and Nutritional Parameters on Compactin Production by Solid-State Fermentation

In the present study, production of compactin by Penicillium brevicompactum WA 2315 was studied. In the first step, various precultural parameters were studied by substituting one factor at a time. Subsequently, the effect of maltodextrin DE 18 on compactin production was studied. The optimized parameters gave maximum compactin production of 850 mug/gds as compared with 678 mug/gds before optimization. Statistical study was performed to further improve the production and develop a robust model. An improved yield of 950 mug/gds was obtained using the conditions proposed by the experimental model. The present study emphasizes the importance of precultural and nutritional parameters on the production of compactin, and further confirms the usefulness of solid-state fermentation for the production of industrially important secondary metabolites. It also confirms that complex nitrogen sources such as oil cakes can be used for the production of compactin.

Sucrose Fermentation by Brazilian Ethanol Production Yeasts in Media Containing Structurally Complex Nitrogen Sources

Four Saccharomyces cerevisiae Brazilian industrial ethanol production strains were grown, under shaken and static conditions, in media containing 22% (w/v) sucrose supplemented with nitrogen sources varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Sucrose fermentations by Brazilian industrial ethanol production yeasts strains were strongly affected by both the structural complexity of the nitrogen source and the availability of oxygen. Data suggest that yeast strains vary in their response to the nitrogen source's complex structure and to oxygen availability. In addition, the amount of trehalose produced could be correlated with the fermentation performance of the different yeasts, suggesting that efficient fuel ethanol production depends on finding conditions which are appropriate for a particular strain, considering demand and dependence on available nitrogen sources in the fermentation medium.

Production and stability analysis of yellowish pink pigments from Rhodotorula rubra MTCC 1446

Rhodotorula rubra MTCC1446 is a good source of yellowish pink pigment. Submerged fermentation, whey sugar lactose, coconut water, complex nitrogen sources viz. yeast extract and peptone have stimulatory effect on growth as well as yields of intracellular pigments of Rhodotorula rubra MTCC 1446. Physicochemical analysis of the extracted pigments indicated its stability near neutral pH, but the pigment is relatively heat sensitive.

Biosynthesis of cellulolytic enzymes by Tricothecium roseum with citric acid mediated induction

Tricothecium roseum, an unexplored fungus for the production of cellulolytic enzymes, was used in this study for carboxymethylcellulase (CMcase) and b-glucosidase production. The culture filtrate of this organism exhibited highest total cellulolytic activity and extracellular protein content on 9th day during the course of its growth on potato dextrose yeast extract (PDYE) broth medium supplemented with 1.0% (w/v) carboxymethylcellulose. Among various soluble carbon and complex nitrogen sources tested in this study, carboxymethylcellulose and peptone supported maximum production of both cellulolytic enzymes. Under all suitable growth conditions, the enzyme biosynthesis was remarkably increased when the inducer citric acid was added to the PDYE media containing carboxymethylcellulose. Maximum CMcase production (1.59 U/ml) and b-glucosidase production (1.82 U/ml) were achieved in citric acid amended PDYE media at 37°C. Both the cellulolytic enzyme production was highest at initial pH 6.5 and after 7 days incubation. The study clearly demonstrated that citric acid is a good inducer for extracellular cellulolytic enzyme production by the fungus.

Type III Secretion System Genes of Dickeya dadantii 3937 Are Induced by Plant Phenolic Acids

Background Dickeya dadantii is a broad-host range phytopathogen. D. dadantii 3937 (Ech3937) possesses a type III secretion system (T3SS), a major virulence factor secretion system in many Gram-negative pathogens of plants and animals. In Ech3937, the T3SS is regulated by two major regulatory pathways, HrpX/HrpY-HrpS-HrpL and GacS/GacA-rsmB-RsmA pathways. Although the plant apoplast environment, low pH, low temperature, and absence of complex nitrogen sources in media have been associated with the induction of T3SS genes of phytobacteria, no specific inducer has yet been identified.Methodology/Principal FindingsIn this work, we identified two novel plant phenolic compounds, o-coumaric acid (OCA) and t-cinnamic acid (TCA), that induced the expression of T3SS genes dspE (a T3SS effector), hrpA (a structural protein of the T3SS pilus), and hrpN (a T3SS harpin) in vitro. Assays by qRT-PCR showed higher amounts of mRNA of hrpL (a T3SS alternative sigma factor) and rsmB (an untranslated regulatory RNA), but not hrpS (a σ54-enhancer binding protein) of Ech3937 when these two plant compounds were supplemented into minimal medium (MM). However, promoter activity assays using flow cytometry showed similar promoter activities of hrpN in rsmB mutant Ech148 grown in MM and MM supplemented with these phenolic compounds. Compared with MM alone, only slightly higher promoter activities of hrpL were observed in bacterial cells grown in MM supplemented with OCA/TCA.Conclusion/SignificanceThe induction of T3SS expression by OCA and TCA is moderated through the rsmB-RsmA pathway. This is the first report of plant phenolic compounds that induce the expression T3SS genes of plant pathogenic bacteria.

Effects of glucose and nitrogen source concentration on batch fermentation kinetics ofLactococcus lactisunder hemin-stimulated respirative condition

Analytical solutions to the ordinary differential equations governing the kinetics of cell growth, substrate utilization, and product formation of batch fermentation processes were derived and used to study the kinetics of the hemin-stimulated respiratory cultivation of Lactococcus lactis at varied initial glucose concentrations and nitrogen source concentrations. Studies revealed that initial glucose concentration varying in the range of 60 to 90 g/L had no significant substrate inhibitive effect. Furthermore, elevating the concentration of complex nitrogen sources while maintaining glucose concentration at 60% led to a high final biomass concentration of 6.6 g/L, substantially higher than that obtained with the basic medium, which was 4.1 g/L.

Screening of Bioflocculant-Producing Strain by Ion Implantation and Flocculating Characteristics of Bioflocculants

A bioflocculant-producing mutator strain, NIM-192, was screened out through nitrogen ion implanting into FJ-7 strain. The results showed that NIM-192 had good genetic stability and high flocculating activity, and the flocculating rate increased by 34.26% than that of the original. Sucrose, complex nitrogen source contained yeast extract, urea and pH 7.0 ∼ 9.0 were chosen as the best carbon source, nitrogen source and initial solution pH for bioflocculant production, respectively. The bioflocculant kept high and stable flocculating activity at alkalinous reaction mixture with a pH beyond 7.0, while the flocculating activity was remarkably reduced when the reaction pH was lower than 7.0. Addition of many cations could obviously increase the flocculating rate, among which Ca2+ demonstrated the best effect. The bioflocculant had very strong acid-base stability and thermo-stability. The flocculating rate kept over 86% when pH of the bioflocculant was in a range of 3.0 ∼ 12.0, and the change of flocculating activity was not great when heated at 100°C for 60 min.

A low-sodium-salt formulation for the fermentation of salinosporamides by Salinispora tropica strain NPS21184

In this paper, we described the development of a potassium-chloride-based-salt formulation containing low sodium concentrations (5.0 to 11 mM) to support the growth of Salinispora tropica strain NPS21184 and its production of salinosporamide A (NPI-0052). The sodium present in the media was essentially derived from the complex nitrogen sources Hy Soy, yeast extract, and peptone used in the media. We demonstrated that good growth rate and yield of S. tropica strain NPS21184 were detected in both agar and liquid media containing the potassium-chloride-based-salt formulation with sodium concentration as low as 5.0 mM, significantly less than the critical seawater-growth requirement concentration of 50 mM sodium for a marine microorganism. We also observed good production of NPI-0052 (176 to 243 mg/l) by S. tropica strain NPS21184 grown in production media containing the potassium chloride-based-salt formulation. The production of deschloro analog, salinosporamide B (NPI-0047), was significantly lower in the low-sodium-salt-formulation medium than in the high-sodium-salt-formulation media. We demonstrated that while S. tropica strain NPS21184 is a novel marine actinomycete that requires high salt content for growth, it does not require sodium-chloride-based seawater-type media for growth and production of NPI-0052.

Medium optimization for enhanced production of cytosine-substituted mildiomycin analogue (MIL-C) by Streptoverticillium rimofaciens ZJU 5119.

Cytosine-substituted mildiomycin analogue (MIL-C) was produced effectively by supplementing cytosine into the culture of Streptoverticillium rimofaciens. In order to improve the yield of MIL-C, statistically-based experimental designs were applied to optimize the fermentation medium for S. rimofaciens ZJU 5119. Fifteen culture conditions were examined for their significances on MIL-C production using Plackett-Burman design. The Plackett-Burman design and one-variable-at-a-time design indicated that glucose and rice meal as the complex carbon sources, and peanut cake meal and NH4NO3 as the complex nitrogen sources were beneficial for MIL-C production in S. rimofaciens ZJU 5119. The results of further central composition design (CCD) showed that the optimal concentration of glucose, rice meal and peanut cake meal were 18.7 g/L, 64.8 g/L and 65.1 g/L, respectively. By using this optimal fermentation medium, the MIL-C concentration was increased up to 1336.5 mg/L, an approximate 3.8-fold improvement over the previous concentration (350.0 mg/L) with un-optimized medium. This work will be very helpful to the large-scale production of MIL-C in the future.

Glucose and Fructose Fermentation by Wine Yeasts in Media Containing Structurally Complex Nitrogen Sources

: Glucose and fructose fermentations by industrial yeasts strains are strongly affected by both the structural complexity of the nitrogen source and the availability of oxygen. In this study two Saccharomyces cerevisiae industrial wine strains were grown, under shaken and static conditions, in a media containing either a) 20% (w/v) glucose, or b) 10% (w/v) fructose and 10% (w/v) glucose or c) 20% (w/v) fructose, all supplemented with nitrogen sources varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Data suggest that a complex structured nitrogen source is not submitted to the same control mechanisms as those involved in the utilization of simpler structured nitrogen sources, and mutual interaction between carbon and nitrogen sources, including the mechanisms involved in the regulation of aerobic/anaerobic metabolism, may play an important role in defining yeast fermentation performance and the differing response to the structural complexity of the nitrogen source, with a strong impact on fermentation performance.

Characterisation of the substrate specificity of the nitrile hydrolyzing system of the acidotolerant black yeast Exophiala oligosperma R1

The `black yeast' Exophiala oligosperma R1 can utilise various organic nitriles under acidic conditions as nitrogen sources. The induction of a phenylacetonitrile converting activity was optimised by growing the strain in the presence of different nitriles and /or complex or inorganic nitrogen sources. The highest nitrile hydrolysing activity was observed with cells grown with 2-cyanopyridine and NaNO3. The cells metabolised the inducer and grew with 2-cyanopyridine as sole source of nitrogen. Cell extracts converted various (substituted) benzonitriles and phenylacetonitriles. They usually converted the isomers carrying a substituent in the meta-position with higher relative activities than the corresponding para- or ortho-substituted isomers. Aliphatic substrates such as acrylonitrile and 2-hydroxy-3-butenenitrile were also hydrolysed. The highest specific activity was detected with 4-cyanopyridine. Most nitriles were almost exclusively converted to the corresponding acids and no or only low amounts of the corresponding amides were formed. The cells hydrolysed amides only with extremely low activities. It was therefore concluded that the cells harboured a nitrilase activity. The specific activities of whole cells and cell extracts were compared for different nitriles and evidence obtained for limitation in the substrate-uptake by whole cells. The conversion of 2-hydroxy-3-butenenitrile to 2-hydroxy-3-butenoic acid at pH 4 demonstrated the unique ability of cells of E. oligosperma R1 to hydrolyse aliphatic α-hydroxynitriles under acidic conditions. The organism could grow with phenylacetonitrile as sole source of carbon, energy and nitrogen. The degradation of phenylacetonitrile presumably proceeds via phenylacetic acid, 2-hydroxyphenylacetic acid, 2,5-dihydroxyphenylacetic acid (homogentisate), maleylacetoacetate and fumarylacetoacetate.

Oligopeptide assimilation and transport by Oenococcus oeni

Oenococcus oeni is a slow-growing wine bacterium with a low growth yield. It thrives better on complex nitrogen sources than on free amino-acid medium. We aimed to characterize the oligopeptide use of this micro-organism. Several peptides of two to eight amino-acid residues were able to provide essential amino acids. The disappearance of various peptides from extracellular medium was assessed with whole cells. Initial rates of utilization varied with the peptide, and free amino acids were released into the medium. Oenococcus oeni was able to transport the oligopeptides with two to five amino-acid residues tested and to hydrolyse them further. This study has clear implications for the relationship between wine nitrogen composition and the ability of O. oeni to cope with its environment.

Saprophytic growth of inoculated Frankia sp. in soil microcosms

The potential of two Frankia strains to grow saprophytically was studied in nonsterile soil microcosms with ground leaf litter of Alnus glutinosa as the sole carbon and nitrogen sources. Strains Ag45/Mut15 and ArI3, which represent two taxonomic subgroups within the Alnus host infection group were inoculated alone, or together to investigate potential competition. Their growth was analyzed by in situ and dot-blot hybridization. A significant increase in cell numbers and filament length was observed during the first 6 weeks after inoculation for strain Ag45/Mut15, both alone and in mixed culture with strain ArI3, followed by a decrease until the end of the study after 12 weeks. The number of filaments remained unchanged. In contrast, the cell numbers and filament length of strain ArI3 were reduced significantly during the first 2 weeks and were undetectable for the remainder of the study. These results were comparable with those obtained in sterile mineral medium amended with leaf litter of A. glutinosa, although reductions in cell numbers and filament length were less pronounced than in soil microcosms. In concomitant control studies without leaf litter amendments for both experimental setups, filaments of both strains could only be detected immediately after inoculation. These results were matched in all experimental setups by concomitant shifts in the rRNA content of both strains, i.e., an immediate decline in the rRNA content for strain ArI3 after inoculation, and an increase in the rRNA content, followed by a late decline during incubation for strain Ag45/Mut15. These results demonstrated that Frankia strain Ag45/Mut15 could grow saprophytically in soil with complex carbon and nitrogen sources such as leaf litter, while the growth of strain ArI3 was not supported.