Optimization Of Nitrogen Sources Research Articles

Carotenoid production from nondetoxified xylose mother liquid or corncob hydrolysate with engineered Kluyveromyces marxianus

Carotenoids are widely utilized in the food, pharmaceutical and nutraceutical industries. Here, Kluyveromyces marxianus was engineered to overproduce carotenoids from corncob hydrolysate or xylose mother liquid (XML, a byproduct of commercial xylose purification). First, the toxicity of fat-soluble carotenoids to cells was reduced by employing xylose inducible promoters using with a two-temperature strategy to separate cell growth and product accumulation. Then, through further engineering and optimization of the carotenoid biosynthesis pathway, 1506.7 mg/L lycopene, 988.5 mg/L β-carotene or 142.9 mg/L astaxanthin were produced with glucose and xylose by K. marxianus. Finally, 397.7 mg/L and 279.7 mg/L lycopene, 297.3 mg/L and 108.8 mg/L β-carotene, and 86.4 mg/L and 56.8 mg/L astaxanthin were produced with nonsterilized andnondetoxified XML or corncob hydrolysate after nitrogen source optimization. To our knowledge, the produced amounts of lycopene, β-carotene and astaxanthin from lignocellulose biomass by yeast in this study were higher than those in previous reports.

Optimization of Nitrogen Sources and Carbon Source for Recombinant Omega-3 Fatty Acid-Containing Biomass Production

Optimization of Nitrogen Sources and Carbon Source for Recombinant Omega-3 Fatty Acid-Containing Biomass Production

Metabolic engineering of Pichia pastoris for malic acid production from methanol.

The application of rational design in reallocating metabolic flux to accumulate desired chemicals is always restricted by the native regulatory network. In this study, recombinant Pichia pastoris was constructed for malic acid production from sole methanol through rational redistribution of metabolic flux. Different malic acid accumulation modules were systematically evaluated and optimized in P. pastoris. The recombinant PP-CM301 could produce 8.55 g/L malic acid from glucose, which showed a 3.45-fold increase compared to the parent strain. To improve the efficiency of site-directed gene knockout, NHEJ-related protein Ku70 was destroyed, whereas leading to the silencing of heterogenous genes. Hence, genes related to by-product generation were deleted via a specially designed FRT/FLP system, which successfully reduced succinic acid and ethanol production. Furthermore, a key node in the methanol assimilation pathway, glucose-6-phosphate isomerase was knocked out to liberate metabolic fluxes trapped in the XuMP cycle, which finally enabled 2.79 g/L malic acid accumulation from sole methanol feeding with nitrogen source optimization. These results will provide guidance and reference for the metabolic engineering of P. pastoris to produce value-added chemicals from methanol.

Enhanced isobutanol production by co-production of polyhydroxybutyrate and cofactor engineering

Once cells have been used to produce biochemicals, there are only a few effective ways to utilize the residual cell mass, even though the utilization of leftover cells would aid in decreasing production costs. Here, a polyhydroxybutyrate (PHB) and isobutanol co-production system was designed to address this challenge. The addition of the PHB operon into Escherichia coli conferred a metabolic advantage for alcohol production, generating 1.14-fold more isobutanol. Furthermore, following nitrogen source optimization and cofactor engineering, the engineered E. coli strain produced 2-fold more isobutanol and 0.25 g/L PHB. Moreover, E. coli cells showed higher tolerance to isobutanol with the overexpression of PHB biosynthesis genes. This co-production system resulted in an increased biomass, higher glucose utilization, and lower acetate maintenance, leading to higher productivity regarding PHB and isobutanol yield. Thus, this novel system is applicable to future fermentation studies for the co-production of PHB and isobutanol.

Growth kinetic and nitrogen source optimization for liquid culture fermentation of Metarhizium robertsii blastospores and bioefficacy against the corn leafhopper Dalbulus maidis.

The cosmopolitan entomopathogenic and root endophytic fungus Metarhizium robertsii has a versatile lifestyle and during liquid fermentation undergoes a dimorphic transformation from hyphae to conidia or microsclerotia, or from hyphae to blastospores. In all cases, these processes are mediated by environmental and nutritional cues. Blastospores could be used in spray applications to control arthropod pests above ground and may serve as an attractive alternative to the traditional solid-grown aerial conidial spores of Metarhizium spp. found in commercial products. Nitrogen is a vital nutrient in cell metabolism and growth; however, it is the expensive component in liquid cultures of entomopathogenic fungi. Our goals in this study were to optimize nitrogen sources and titers for maximum production of M. robertsii blastospores cultured in shake flasks at highly aerated conditions and to furtherdetermine theirvirulence against the corn leafhopper Dalbulus maidis, an important vector of serious pathogens in maize crops worldwide. Our fermentation studies revealed that the low-cost corn steep liquor (CSL) was the most suitable nitrogen source to improve blastospore growth in M. robertsii. The growth kinetic assays determined the optimal titer of 80gL-1 and a yield up to 4.7 × 108 cells mL-1 within 5days of cultivation (3days preculture and 2days culture), at a total cost of US$0.30L-1. Moreover, the blastospore growth kinetic was strongly dependent on glucose and nitrogen consumptions accompanied by a slight drop in the culture pH. Insect bioassays evidenced a high virulence of these blastospores, either as dried or fresh cells, to D. maidis adults fed on maize plants. Our findings provide insights into the nutritional requirements for optimal and cost-efficient production of M. robertsii blastospores and elucidate the potential of blastospores as an ecofriendly tool against the corn leafhopper.

Isolation, optimization and estimation of probiotic <em>Lactobacillus</em> from different natural sources

Purpose: In the field of nutrition, probiotics are widely in use and lactic acid from probiotics acts as the best nourishing agent. Production of lactic acid from probiotics requires a large quantity of De Man, Rogosa and Sharpe agar (MRS) media that doubles the cost of production and may also affect vegan choice. So there arises a need to develop a cost-effective new media that replaces the animal sources. This study is about the optimizing and improving a costeffective growth medium by replacing animal products with high nutritional cheap materials obtained from plants to produce lactic acid by isolated Lactobacilli strains from pure sources. Method: This work is a comparative analytical study following use of different media for determination of best carbon sources (dates, pineapple, molasses, sucrose, fructose, galactose) and nitrogen sources (peanut, chickpea, wheat, lentils, Bengal gram, mung beans) among the different sources available to constitute the best media which is cost effective for the production of lactic acid. Results: In the optimization of nitrogen source, the highest growth was observed with the lentil seed powder. In the optimization of the carbon source, the highest increase was observed with molasses. Molasses a by-product of the sugar industry, incorporated as a carbon source, lentils as the nitrogen source in the media showed best results in terms of lactic acid production. Conclusion: The low-cost media is obtained by using plant sources, making it suitable for vegan choice. Animal products can be replaced with plant sources for the costeffective production of probiotic lactobacilli and lactic acid.

Metabolic engineering of Escherichia coli for 1,3-propanediol biosynthesis from glycerol

In this study, the engineered E. coli was constructed for efficient transformation of glycerol to 1,3-propanediol (1,3-PDO). To regenerate NADPH, the key bottleneck in 1,3-PDO production, heterologous NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDN, encoded by gapN) pathway was introduced, and the gapN expression level was fine-tuned with specific 5′-untranslated regions (5′-UTR) to balance the carbon flux distribution between the metabolic pathways of NADPH regeneration and 1,3-PDO biosynthesis. Additionally, glucose was added to the medium to promote glycerol utilization and cell growth. To elevate the utilization of glycerol in the presence of glucose, E. coli JA11 was constructed through destroying PEP-dependent glucose transport system while strengthening the ATP-dependent transport system. Subsequent optimization of nitrogen sources further improved 1,3-PDO production. Finally, under the optimal fermentation condition, E. coli JA11 produced 13.47 g/L 1,3-PDO, with a yield of 0.64 mol/mol, increased by 325% and 100% compared with the original engineered E. coli JA03, respectively.

Increasing lipid content from biomass of microalgae to produce biofuels with optimization of nitrogen source

The aim of this research is to analyze the effect of urea fertilizer as a nitrogen source to lipid productivity of mixed culture of green microalgae consisting of Monoraphidium sp., Chlorella sorokiniana, and Scenedesmus obliquus as lipid sources. In research, cultures were cultivated in a 1.5 L glass photobioreactor with batch culture system. The operational conditions of this research were carried out at pH 6.0, 27 °C, aeration with air flow 150 mL/sec, and 2400 lux with vary amount of urea as a source of nitrogen as much (grams) 0.0; 0.5; 1.0. The Bligh and Dyer extraction is performed to produce biofuels after harvesting process and to analyze lipid content. Analysis of fatty acids using Gas Chromatography Mass Spectrometry (GCMS) Method, analysis of dry weight using Gravimetric Method, and analysis of cell density using Spectrofotometry Method. This research concludes that with 0.5 grams of urea fertilizer can produce dry weight and total lipid content optimally that were 0.26% (w/w) and 36,35% (w/w). This research concludes that increasing amount of nitrogen source could be increasing green microalgae biomass but is not for increased lipid content. The high lipid content can be produced by decreasing 50% nitrogen source.

Pigment Production of Monascus sp. Isolated from Angkak in Semarang Region, Central Java, Indonesia

Angkak (red fungi rice, red yeast rice) is a traditional fermentation by fungi Monascus in a lot of Asia Countries, which is useful as a natural food colorant. The aim of this research was to obtain pure isolate of red fungus from angkak in Semarang. The growth and the red pigment production of the selected isolate in the different initial medium pH and nitrogen sources were observed. The treatment was done by growing the isolate in PDB (Potato Dextrose Broth) medium at initial pH 3, 5, 7 and 9 and as well as optimization of nitrogen sources with 1% ammonium chloride, 1% ammonium nitrate, and 1% peptone. Analysis of the fungi growth was performed by weighing mycelia biomass using dry weight method and the red pigments were analysis using spectrophotometer at a wavelength (λmax) of 500 nm. The results showed that the highest absorbance value of pigment (0.81) was obtained at pH 7, and the highest value of the cell dry weight was also obtained at pH 7 (1.23 g/L). The results showed that 1% ammonium chloride was the best nitrogen source for absorbance of red pigment production (0.82), but the highest of dry weight (2.56 g/L) was obtained by using medium with 1% ammonium nitrate as a nitrogen source.

Applying the strategy of light environment control to improve the biomass and polysaccharide production of Nostoc flagelliforme

The strategy of light environment control was applied to improve biomass and extracellular polysaccharides (EPS) production of the cyanobacterium Nostoc flagelliforme by adjusting multiple wavelengths (red 660 nm, blue 460 nm, green 520 nm) and light intensity assisted by nitrogen source optimization. A mixed wavelength with low light intensity was more suitable for cell growth. Wavelength shift approach, i.e., manipulation of light wavelength at appropriate culture stages, increased both biomass and EPS production, and optimum shift time was at 9 days. The effects of four nitrogen sources under different light conditions were subsequently evaluated, and urea showed the best performance. The optimized wavelength shift approach (9-day illumination with white light followed by 9-day culture with mixed wavelengths of red/blue/green = 12:5:5) with urea as nitrogen source improved the biomass from 0.72 ± 0.02 to 1.20 ± 0.02 g L−1 (i.e., by 66 %) and EPS production from 27.31 ± 1.00 to 86.65 ± 2.56 mg L−1 (i.e., by 217.3 %). These results provide information on novel culture strategies for microalgal biotechnology by applying light environment control.

Optimization of Nitrogen Source(s) for the Growth and Amylase Production from Bacillus licheniformis JAR-26 under Submerged Fermentation

Optimization of Nitrogen Source(s) for the Growth and Amylase Production from Bacillus licheniformis JAR-26 under Submerged Fermentation

Enhancement of l-phenylalanine production by engineered Escherichia coli using phased exponential l-tyrosine feeding combined with nitrogen source optimization

Nitrogen source optimization combined with phased exponential L-tyrosine feeding was employed to enhance L-phenylalanine production by a tyrosine-auxotroph strain, Escherichia coli YP1617. The absence of (NH4)2SO4, the use of corn steep powder and yeast extract as composite organic nitrogen source were more suitable for cell growth and L-phenylalanine production. Moreover, the optimal initial L-tyrosine level was 0.3 g L(-1) and exponential L-tyrosine feeding slightly improved L-phenylalanine production. Nerveless, L-phenylalanine production was greatly enhanced by a strategy of phased exponential L-tyrosine feeding, where exponential feeding was started at the set specific growth rate of 0.08, 0.05, and 0.02 h(-1) after 12, 32, and 52 h, respectively. Compared with exponential L-tyrosine feeding at the set specific growth rate of 0.08 h(-1), the developed strategy obtained a 15.33% increase in L-phenylalanine production (L-phenylalanine of 56.20 g L(-1)) and a 45.28% decrease in L-tyrosine supplementation.

Impact of Nitrogen Precursor’s N/C Ratio on ORR Activity in Fe/N/C Catalysts for PEFC Applications

Among the non-precious metal catalysts, metal-nitrogen doped carbon materials (M/N/C) have been considered a suitable candidates in recent decades due to their high activity in acid medium. Yeager and co-workers revealed that it is possible to avoid using expensive macrocycles by heat-treating, in an inert atmosphere, a cost-effective metal precursor and a nitrogen precursor impregnated on an amorphous carbon black [1]. Following this approach, many methods have been explored to prepare high active M/N/C catalysts.In our approach, to synthesize the iron based M/N/C (Fe/N/C) catalyst, a nitrogen source, a high surface area carbon source (Ketjenblack® 600JD) and an iron precursor (iron acetate) are pyrolyzed together in a closed container (quartz ampoule). This method is designed to increase the residence time of evaporated and decomposed products on the carbon under pressure, thereby increasing the density of nitrogen-based catalytic sites.In our previous nitrogen source optimization studies, the most active catalysts were obtained using melamine, having a N/C ratio of 2 [2]. This enhanced activity was attributed to the increase in bulk nitrogen content and hence increased density of active catalytic sites, without significant pore blockage via carbon deposition. In another study, it is found that the best ORR performance was obtained with 1.2 wt% nominal Fe with respect to the entire mixture [3]. As nitrogen is believed to be a part of the catalytic site, increasing the surface nitrogen density in the presence of metal could potentially increase active site density.Based on results obtained from our previous studies, however, it is concluded that when increasing the N/C ratio of nitrogen precursor, the nitrogen retention and ORR activity increases. To test this, Fe/N/C catalysts were prepared nitrogen precursors of very high N/C ratio, namely, 3-Aminotriazole (N/C=2), 3, 5-Diaminotriazole (N/C=2.5) and 5-Aminotetrazole (N/C=5 and these results are compared with melamine (N/C=2).Fig. 1 shows the comparative ORR performance curves for Fe/N/C catalysts prepared with various nitrogen sources in oxygen saturated 0.5M H2SO4. Among the nitrogen sauces, the Fe/N/C catalyst prepared with melamine shows enhanced ORR activity in relation to other azole precursors. However, there is only a marginal difference in performance was observed for Fe/N/C catalyst prepared triazole compounds. Surprisingly, in the case of tetrazole, although it possesses the high nitrogen content (N/C=5) in relation to others, it shows extremely ORR low activity. The results clearly indicate that, for nitrogen precursors of high N/C ratio, the pathway of decomposition and active site formation becomes critical. AcknowledgementWe gratefully acknowledge the financial support from the U.S. Department of Energy (EERE), under a Non PGM Catalyst development effort (Contract no EE 0000459) lead by Northeastern University ReferencesS. Gupta, D. Tryk, I. Bae, W. Aldred, and E. Yeager, J. Appl. Electrochem., 19 (1989) 19. V. Nallathambi, N. Leonard, R. Kothandaraman and S. Calabrese Barton, Electrochem. Solid-State Lett., 14 (2011) B55. S. Ganesan, N. Leonard, and S. Calabrese Barton, Electrochem. Soc. Trans., 58 (2013) 1691.

Optimization of nitrogen source for enhanced production of squalene from thraustochytrid Aurantiochytrium sp.

Nitrogen (N) sources, the critical medium component, were optimized for squalene production by microalga Aurantiochytrium sp. in heterotrophic cultures. In screening experiments monosodium glutamate, yeast extract and tryptone were found to enhance cell growth and squalene production. The optimal levels of the three nitrogen sources were further determined through central composite experimental design. The squalene content and yield were both influenced not only by monosodium glutamate, tryptone and yeast extract, but also by their interactions. The squalene content and squalene yield were described by the second-order polynomial equations with high confidence levels (>99%). The optimal concentrations of monosodium glutamate, yeast extract and tryptone were predicted to be 6.61 g/L, 6.13 g/L and 4.50 g/L for squalene content and 6.94 g/L, 6.22 g/L and 4.40 g/L for squalene yield, respectively. In the verification experiment, the squalene content and squalene yield reached 0.72 mg/g and 5.90 mg/L, respectively, which were much higher than those obtained in previous studies.

Nitrogen Source Optimization for Cellulase Production by Penicillium funiculosum, using a Sequential Experimental Design Methodology and the Desirability Function

The present study aimed at maximizing cellulase production by Penicillium funiculosum using sequential experimental design methodology for optimizing the concentrations of nitrogen sources. Three sequential experimental designs were performed. The first and the second series of experiments consisted of a 2(4) and a 2(3) factorial designs, respectively, and in the third one, a central composite rotational design was used for better visualizing the optimum conditions. The following nitrogen sources were evaluated: urea, ammonium sulfate, peptone, and yeast extract. Peptone and ammonium sulfate were removed from the medium optimization since they did not present significant statistical effect on cellulase production. The optimal concentrations of urea and yeast extract predicted by the model were 0.97 and 0.36 g/L, respectively, which were validated experimentally. By the use of the desirability function, it was possible to maximize the three main enzyme activities simultaneously, which resulted in values for FPase of 227 U/L, for CMCase of 6,917 U/L, and for beta-glucosidase of 1,375 U/L. These values corresponded to increases of 3.3-, 3.2-, and 6.7-folds, respectively, when compared to those obtained in the first experimental design. The results showed that the use of sequential experimental designs associated to the use of the desirability function can be used satisfactorily to maximize cellulase production by P. funiculosum.

Induction of cellulases and hemicellulases from Neurospora crassa under solid-state cultivation for bioconversion of sorghum bagasse into ethanol

The cellulolytic and hemicellulolytic system from the mesophilic fungus Neurospora crassa was produced under solid-state cultivation (SSC) on wheat straw and wheat bran mixtures. Following optimization of nitrogen source, pH and initial moisture of the growth medium, yields as high as 492.8, 1.08, 26.7, 297.8 and 0.132 (in U g −1 of carbon source) were obtained for endoglucanase, exoglucanase, β-glucosidase, xylanase and β-xylosidase, respectively. The potential of the multienzyme system was demonstrated for hydrolysis of sorghum bagasse (SB) into fermentable carbohydrates. N. crassa cells were found able to assimilate the majority of the released sugars and generated limited levels of other metabolic products during simultaneous saccharification and fermentation of this valuable substrate into ethanol.

Lactate increases coenzyme Q10 production by Agrobacterium tumefaciens

By the optimization of nitrogen source for coenzyme Q10 (ubiquinone, CoQ10) production in Agrobacterium tumefaciens KCCM 10413 culture, the highest CoQ10 production was achieved in medium containing corn steep powder (CSP). Components for a stimulatory effect on the production of CoQ10 in CSP were screened, and lactate was found to increase dry cell weight (DCW) and the specific CoQ10 content. In a fed-batch culture of A. tumefaciens, supplementation with 1.5 g of lactate l−1 further improved DCW, the specific CoQ10 content, and CoQ10 production by 16.0, 5.8, and 22.8%, respectively. It has been reported that lactate stimulates cell growth and acts as an accelerator driving the tricarboxylic acid (TCA) cycle (Roberto et al. 2002, Biotechnol Let 24:427–431; Matsuoka et al. 1996, Biosci Biotechnol Biochem 60:575–579). In this study, lactate supplementation increased DCW and the specific CoQ10 content in A. tumefaciens culture, probably by accelerating TCA cycle and energy production as reported previously, leading to the increase of CoQ10 production.

Studies on optimization of nitrogen sources for astaxanthin production by Phaffia rhodozyma

Fermentation of Phaffia rhodozyma is a major method for producing astaxanthin, an important pigment with industrial and pharmaceutical application. To improve astaxanthin productivity, single factor and mixture design experiments were used to investigate the effects of nitrogen source on Phaffia rhodozyma cultivation and astaxanthin production. Results of single factor experiments showed nitrogen source could significantly affect P. rhodozyma cultivation with respect to carbon source utilization, yeast growth and astaxanthin accumulation. Further studies of mixture design experiments using (NH(4))(2)SO(4), KNO(3) and beef extract as nitrogen sources indicated that the proportion of three nitrogen sources was very important to astaxanthin production. Validation experiments showed that the optimal nitrogen source was composed of 0.28 g/L (NH(4))(2)SO(4), 0.49 g/L KNO(3) and 1.19 g/L beef extract. The kinetic characteristics of batch cultivation were investigated in a 5-L pH-stat fermentor. The maximum amount of biomass and highest astaxanthin yield in terms of volume and in terms of biomass were 7.71 mg/L and 1.00 mg/g, respectively.

Optimization of nitrogen sources for heterotrophic production of eicosapentaenoic acid by the diatom Nitzschia laevis

The nitrogen (N) sources including nitrate, ammonium, urea, tryptone and yeast extract were optimized for eicosapentaenoic acid (EPA) production by the diatom Nitzschia laevis in heterotrophic cultures. First, nitrate and urea were found to be the preferred N sources for both cell growth and EPA content. The optimal concentrations of nitrate and urea for cell growth and EPA yield were 620 mg/liter and 600 mg/liter, respectively. Secondly, tryptone and yeast extract were respectively added to the medium and both of them were found to enhance EPA production compared with the control. To investigate the co-effects of tryptone and yeast extract, in the subsequent experiments, a central composite experimental design was used to optimize the two complex nitrogen sources. The results showed that the EPA production was influenced not only by tryptone or yeast extract, but also by their interactions. The cell dry weight (DW), EPA content and EPA yield could be described by the second-order polynomial equations with high confidence levels (>99%). Based on the surface plots of the responses, the optimal concentrations of tryptone and yeast extract were determined to be 1.6 g/liter and 0.8 g/liter, respectively. To verify the predicted models, the alga was grown in the medium with tryptone and yeast extract at optimal concentrations. The resulting DW, EPA content and EPA yield reached 6.48 g/liter, 2.74% (w/w), and 175 mg/liter, respectively, which agreed with the predicted values, and were much higher than those obtained in the previous studies.