scholarly journals Protamylasse, a Residual Compound of Industrial Starch Production, Provides a Suitable Medium for Large-Scale Cyanophycin Production

2005 ◽  
Vol 71 (12) ◽  
pp. 7759-7767 ◽  
Author(s):  
Yasser Elbahloul ◽  
Kay Frey ◽  
Johan Sanders ◽  
Alexander Steinbüchel
Keyword(s):  
Recombinant Strain ◽  
Large Scale ◽  
Luria Bertani ◽  
Acid Extraction ◽  
Bacterial Strains ◽  
Biotechnological Production ◽  
Mineral Salts ◽  
Stirred Tank Reactors ◽  
Initial Ph ◽  
Optimized Conditions

ABSTRACT Protamylasse is a residual compound occurring during the industrial production of starch from potatoes. It contains a variety of nutrients and all necessary minerals and could be used as a carbon, nitrogen, and energy source for the growth of bacteria and also for cyanophycin (CGP) biosynthesis. Media containing protamylasse as the sole compound diluted only in water were therefore examined for their suitability in CGP production. Among various bacterial strains investigated in this study, a recombinant strain of Escherichia coli DH1 harboring plasmid pMa/c5-914::cphA 6803, which carries the cyanophycin synthetase structural gene (cphA) from Synechocystis sp. strain PCC6803, was found to be most suitable. Various cultivation conditions for high CGP contents were first optimized in shake flask cultures. The optimized conditions were then successfully applied to 30- and 500-liter fermentation scales in stirred tank reactors. A maximum CGP content of 28% (wt/wt) CGP per cell dry matter was obtained in 6% (vol/vol) protamylasse medium at an initial pH of 7.0 within a cultivation period of only 24 h. The CGP contents obtained with this recombinant strain employing protamylasse medium were higher than those obtained with the same strain cultivated in mineral salts medium or in expensive commercial complex media such as Luria-Bertani or Terrific broth. It was shown that most amino acids present in the protamylasse medium were almost completely utilized by the cells during cultivation. Exceptions were alanine, tryptophan, tyrosine, and most interestingly, arginine. Furthermore, CGP was easily isolated from protamylasse-grown cells by applying the acid extraction method. The CGP exhibited a molecular mass of about 26 to 30 kDa and was composed of 50% (mol/mol) aspartate, 46% (mol/mol) arginine, and 4% (mol/mol) lysine. The use of cheap residual protamylasse could contribute in establishing an economically and also ecologically feasible process for the biotechnological production of CGP.

scholarly journals Technical-Scale Production of Cyanophycin with Recombinant Strains of Escherichia coli

2002 ◽  
Vol 68 (7) ◽  
pp. 3377-3384 ◽  
Author(s):  
Kay M. Frey ◽  
Fred B. Oppermann-Sanio ◽  
Holger Schmidt ◽  
Alexander Steinbüchel
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Cell Mass ◽  
Extraction Procedure ◽  
Temperature Shift ◽  
Exponential Growth Phase ◽  
Acid Extraction ◽  
Scale Production ◽  
Mineral Salts ◽  
Cell Content

ABSTRACT By the use of Escherichia coli DH1 harboring cphA from Synechocystis sp. strain PCC6803, large-scale production of cyanophycin at 30- and 500-liter culture volumes was established. Transcription of cphA was controlled by the thermosensitive cI857 repressor, which enabled induction of cphA by a simple temperature shift in the culture fluid. Maximum cyanophycin cell content of up to 24% (wt/wt) of cellular dry matter was obtained by induction in the early exponential growth phase and cultivation of the cells in terrific broth complex medium. Synthesis of cyanophycin was found to be strongly dependent on the presence of complex components, and in mineral salts medium the cells synthesized and accumulated cyanophycin only if Casamino Acids were added. Cultivations were done at the 500-liter scale, allowing the provision of cell mass for the preparation of cyanophycin at the kilogram scale. Isolation of cyanophycin was achieved by a new acid extraction procedure which allowed large-scale purification of the polyamide from whole cells.

scholarly journals Application of the BPEC Pathway for Large-Scale Biotechnological Production of Poly(3-Mercaptopropionate) by Recombinant Escherichia coli, Including a Novel In Situ Isolation Method

2005 ◽  
Vol 71 (2) ◽  
pp. 835-841 ◽  
Author(s):  
Nehal Thakor ◽  
Tina Lütke-Eversloh ◽  
Alexander Steinbüchel
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Sodium Dodecyl ◽  
Pha Synthase ◽  
Exponential Growth Phase ◽  
Biotechnological Production ◽  
Mineral Salts ◽  
Fed Batch Fermentation ◽  
Cell Densities

ABSTRACT Metabolically engineered Escherichia coli JM109 harboring plasmid pBPP1 and expressing the nonnatural BPEC pathway for synthesis of thermoplastic polyhydroxyalkanoates (PHA) and novel polythioesters (PTE) to provide suitable substrates of PHA synthase was investigated with respect to biotechnological production of poly(3-mercaptopropionate) [poly(3MP)]. Fed-batch fermentation processes were established at the 30- and 500-liter scales in stirred tank bioreactors to produce kilogram amounts of poly(3MP). Cultivation was done in a modified M9 mineral salts medium containing glucose or glycerol as the carbon and energy source and with 3-mercaptopropionic acid (3MP) as the precursor substrate for poly(3MP) biosynthesis provided from the late exponential growth phase. Approximately 23 g of cell dry matter (CDM) per liter and poly(3MP) cell contents of up to 45% (wt/wt) were the highest cell densities and polymer contents obtained, respectively. At best, 69.1% (wt/wt) of 3MP was converted into poly(3MP), indicating that 3MP was mostly used for poly(3MP) biosynthesis. Furthermore, a novel in situ process for rapid and convenient isolation of poly(3MP) from the cells in the bioreactor was developed. This was achieved by addition of sodium dodecyl sulfate to the cultivation broth immediately after the fermentation, heating to 90°C for 20 min with intensive stirring, and subsequent washing steps. The purity of such in situ isolated poly(3MP) was more than 98%, as revealed by gas chromatographic and elemental sulfur analyses of the material isolated.

scholarly journals Biotechnological Process for Production of β-Dipeptides from Cyanophycin on a Technical Scale and Its Optimization

2008 ◽  
Vol 75 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Ahmed Sallam ◽  
Alene Kast ◽  
Simon Przybilla ◽  
Tobias Meiswinkel ◽  
Alexander Steinb�chel
Keyword(s):  
High Performance ◽  
Large Scale ◽  
Specific Binding ◽  
Maximum Activity ◽  
Phase Iii ◽  
Acid Extraction ◽  
Mineral Salts ◽  
Sustained Activity ◽  
Fermentative Production ◽  
Layer Chromatography

ABSTRACT A triphasic process was developed for the production of β dipeptides from cyanophycin (CGP) on a large scale. Phase I comprises an optimized acid extraction method for technical isolation of CGP from biomass. It yielded highly purified CGP consisting of aspartate, arginine, and a little lysine. Phase II comprises the fermentative production of an extracellular CGPase (CphEal) from Pseudomonas alcaligenes strain DIP1 on a 500-liter scale in mineral salts medium, with citrate as the sole carbon source and CGP as an inductor. During optimization, it was shown that 2 g liter−1 citrate, pH 6.5, and 37�C are ideal parameters for CphEal production. Maximum enzyme yields were obtained after induction in the presence of 50 mg liter−1 CGP or CGP dipeptides for 5 or 3 h, respectively. Aspartate at a concentration of 4 g liter−1 induced CphEal production with only about 30% efficiency in comparison to that with CGP. CphEal was purified utilizing its affinity for the substrate and its specific binding to CGP. CphEal turned out to be a serine protease with maximum activity at 50�C and at pH 7 to 8.5. Phase III comprises degradation of CGP to β-aspartate-arginine and β-aspartate-lysine dipeptides with a purity of over 99% (by thin-layer chromatography and high-performance liquid chromatography), employing a crude CphEal preparation. Optimum degradation parameters were 100 g liter−1 CGP, 10 g liter−1 crude CphEal powder, and 4 h of incubation at 50�C. The overall efficiency of phase III was 91%, while 78% (wt/wt) of the used CphEal powder with sustained activity toward CGP was recovered. The optimized process was performed with industrial materials and equipment and is applicable to any desired scale.

Phyto-fabrication of Iron Nanoparticles: Characterization and Antibacterial Capacity

2020 ◽  
Vol 16 ◽  
Author(s):  
Asma S. Algebaly ◽  
Afrah E. Mohammed ◽  
Mudawi M. Elobeid
Keyword(s):  
Electron Microscopy ◽  
Plant Extracts ◽  
Large Scale ◽  
Iron Nanoparticles ◽  
Ethanolic Extract ◽  
Green Technology ◽  
Resistant Bacteria ◽  
Scale Production ◽  
Bacterial Strains ◽  
Plant Sources

Introduction: Fabrication of iron nanoparticles (FeNPs) has recently gained a great concern for their varied applications in remediation technologies of the environment. Objective: The current study aimed to fabricate iron nanoparticles by green technology approach using different plant sources, Azadirachta indica leaf and Calligonum comosum root following two extraction methods. Methods: Currently, a mixture of FeCl2 and FeCl3 was used to react with the plant extracts which are considered as reducing and stabilizing agents for the generation of FeNPs in one step. Different techniques were used for FeNPs identification. Results: Immediately after mixing of the two reaction components, the color changed to dark brown as an indication of safe conversion of Fe ions to FeNPs, that later confirmed by zeta sizer, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). FeNPs fabricated by C. comosum showed smaller size when compared by those fabricated by A. indica. Using both plant sources, FeNPs fabricated by the aqueous extract had smaller size in relation to those fabricated by ethanolic extract. Furthermore, antibacterial ability against two bacterial strains was approved. Conclusion: The current results indicated that, at room temperature plant extracts fabricated Fe ion to Fe nanoparticles, suggesting its probable usage for large scale production as well as its suitability against bacteria. It could also be recommended for antibiotic resistant bacteria.

Study on Optimization of Fermentation Condition for Nitrilase-Producer Escherichia Coli BL21 (DE3)/pET-Nit

2011 ◽  
Vol 175-176 ◽  
pp. 192-196 ◽  
Author(s):  
Li Li Feng ◽  
Jian Fei Zhang ◽  
Hui Luo ◽  
Zheng Li ◽  
Hong Jie Zhang
Keyword(s):  
Escherichia Coli ◽  
Recombinant Strain ◽  
Culture Medium ◽  
Culture Conditions ◽  
Fermentation Condition ◽  
Hydrogen Phosphate ◽  
Strain Bl21 ◽  
Initial Ph ◽  
Potassium Hydrogen ◽  
Optimization Of Fermentation

The paper concentrated on the optimization of the recombinant strain BL21 (DE3)-PE7-Nit. The component of culture medium and the culture conditions were optimized. The optimized medium was: yeast extract 10 g/l, L-glutamate sodium 8 g/l, MgSO4.7H2O 0.7 g/l, Isopropyl-β-D-thiogalactopyranoside 0.3 mmol/L, potassium hydrogen phosphate 0.5 g / L, phosphate Potassium 0.5 g / L and the culture condition was: initial pH 7.0, inoculum 2%. The result showed that the activity of nitrilase prepared with these conditions increased by 130.37 % through optimization.

scholarly journals Microbial Production of Glyceric Acid, an Organic Acid That Can Be Mass Produced from Glycerol

2009 ◽  
Vol 75 (24) ◽  
pp. 7760-7766 ◽  
Author(s):  
Hiroshi Habe ◽  
Yuko Shimada ◽  
Toshiharu Yakushi ◽  
Hiromi Hattori ◽  
Yoshitaka Ano ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Growth Parameters ◽  
Operating Time ◽  
Enantiomeric Excess ◽  
Gluconobacter Oxydans ◽  
Acetic Acid Bacteria ◽  
Culture Broth ◽  
Glyceric Acid ◽  
Bacterial Strains ◽  
Biotechnological Production

ABSTRACT Glyceric acid (GA), an unfamiliar biotechnological product, is currently produced as a small by-product of dihydroxyacetone production from glycerol by Gluconobacter oxydans. We developed a method for the efficient biotechnological production of GA as a target compound for new surplus glycerol applications in the biodiesel and oleochemical industries. We investigated the ability of 162 acetic acid bacterial strains to produce GA from glycerol and found that the patterns of productivity and enantiomeric GA compositions obtained from several strains differed significantly. The growth parameters of two different strain types, Gluconobacter frateurii NBRC103465 and Acetobacter tropicalis NBRC16470, were optimized using a jar fermentor. G. frateurii accumulated 136.5 g/liter of GA with a 72% d-GA enantiomeric excess (ee) in the culture broth, whereas A. tropicalis produced 101.8 g/liter of d-GA with a 99% ee. The 136.5 g/liter of glycerate in the culture broth was concentrated to 236.5 g/liter by desalting electrodialysis during the 140-min operating time, and then, from 50 ml of the concentrated solution, 9.35 g of GA calcium salt was obtained by crystallization. Gene disruption analysis using G. oxydans IFO12528 revealed that the membrane-bound alcohol dehydrogenase (mADH)-encoding gene (adhA) is required for GA production, and purified mADH from G. oxydans IFO12528 catalyzed the oxidation of glycerol. These results strongly suggest that mADH is involved in GA production by acetic acid bacteria. We propose that GA is potentially mass producible from glycerol feedstock by a biotechnological process.

scholarly journals Production, characterization, and antitumor efficiency of l-glutaminase from halophilic bacteria

2022 ◽  
Vol 46 (1) ◽  
Author(s):  
Eman Zakaria Gomaa
Keyword(s):  
Cell Lines ◽  
Halophilic Bacteria ◽  
Temperature Stability ◽  
Inoculum Size ◽  
Dependent Manner ◽  
Bacterial Strains ◽  
Carcinoma Cell Lines ◽  
Initial Ph ◽  
Hct 116 ◽  
Optimization Of Fermentation

Abstract Background Halophiles are an excellent source of enzymes that are not only salt stable, but also can withstand and carry out reaction efficiently under extreme conditions. l-glutaminase has attracted much attention with respect to proposed applications in several fields such as pharmaceuticals and food industries. The aim of the present study was to investigate the anticancer activity of l-glutaminase produced by halophilic bacteria. Various halophilic bacterial strains were screened for extracellular l-glutaminase production. An attempt was made to study the optimization, purification, and characterization of l-glutaminase from Bacillus sp. DV2-37. The antitumor activity of the produced enzyme was also investigated. Results The potentiality of 15 halophilic bacterial strains isolated from the marine environment that produced extracellular l-glutaminase was investigated. Bacillus sp. DV2-37 was selected as the most potent strain and optimized for enzyme production. The optimization of fermentation process revealed that the highest enzyme activity (47.12 U/ml) was observed in a medium supplemented with 1% (w/v) glucose as a carbon source, 1% (w/v) peptone as a nitrogen source, 5% (w/v) NaCl, the initial pH was 7.0, at 37 °C, using 20% (v/v) inoculum size after 96 h of incubation. The produced crude enzyme was partially purified by ammonium sulfate precipitation and dialysis. Of the various parameters tested, pH 7, 40 °C, and 5% NaCl were found to be the best for l-glutaminase activity. The enzyme also exhibited high salt and temperature stability. The antitumor effect against human breast (MCF-7), hepatocellular (HepG-2), and colon (HCT-116) carcinoma cell lines revealed that l-glutaminase produced by Bacillus sp. DV2-37 showed potent cytotoxic activity of all the tested cell lines in a dose-dependent manner with an IC50 value of 3.5, 3.4, and 3.8 µg/ml, respectively. Conclusions The present study proved that l-glutaminase produced by marine bacteria holds proper features and it has a high potential to be useful for many therapeutic applications.

scholarly journals Contrasting pH buffering patterns in neutral-alkaline soils along a 3600 km transect in northern China

2015 ◽  
Vol 12 (16) ◽  
pp. 13215-13240 ◽  
Author(s):  
W. Luo ◽  
P. N. Nelson ◽  
M.-H. Li ◽  
J. Cai ◽  
Y. Zhang ◽  
...  
Keyword(s):  
Soil Ph ◽  
Large Scale ◽  
Aridity Index ◽  
Clay Content ◽  
Northern China ◽  
Buffering Capacity ◽  
Carbonate Content ◽  
Alkaline Soils ◽  
Ph Buffering ◽  
Initial Ph

Abstract. Soil pH buffering capacity (pHBC) plays a crucial role in predicting acidification rates, yet its large-scale patterns and controls are poorly understood, especially for neutral-alkaline soils. Here, we evaluated the spatial patterns and drivers of pHBC along a 3600 km long transect (1900 km sub-transect with carbonate containing soils and 1700 km sub-transect with non-carbonate containing soils) across northern China. Soil pHBC was greater in the carbonate containing soils than in the non-carbonate containing soils. Acid addition decreased soil pH in the non-carbonate containing soils more markedly than in the carbonate containing soils. Within the carbonate soil sub-transect, soil pHBC was positively correlated with cation exchange capacity (CEC), carbonate content and exchangeable sodium (Na) concentration, but negatively correlated with initial pH and clay content, and not correlated with soil organic carbon (SOC) content. Within the non-carbonate sub-transect, soil pHBC was positively related to initial pH, clay content, CEC and exchangeable Na concentration, but not related to SOC content. Carbonate content was the primary determinant of pHBC in the carbonate containing soils and CEC was the main determinant of buffering capacity in the non-carbonate containing soils. Soil pHBC was positively related to aridity index and carbonate content across the carbonate containing soil sub-transect. Our results indicated that mechanisms controlling pHBC differ among neutral-alkaline soils of northern China, especially between carbonate and non-carbonate containing soils, leading to different rates, risks, and impacts of acidification. This understanding should be incorporated into the acidification risk assessment and landscape management in a changing world.

scholarly journals Conversion of Fructose to HMF in a Continuous Fixed Bed Reactor with Outstanding Selectivity

Molecules ◽  
2018 ◽  
Vol 23 (7) ◽  
pp. 1802 ◽  
Author(s):  
Eric Weingart ◽  
Sarah Tschirner ◽  
Linda Teevs ◽  
Ulf Prüße
Keyword(s):  
Large Scale ◽  
Continuous Process ◽  
Fixed Bed ◽  
Fixed Bed Reactor ◽  
Water Addition ◽  
Batch Systems ◽  
Formation Conditions ◽  
High Yields ◽  
Optimized Conditions ◽  
By Products

5-Hydroxymethylfurfural (HMF) is a very promising component for bio-based plastics. Efficient synthesis of HMF from biomass is still challenging because of fast degradation of HMF to by-products under formation conditions. Therefore, different studies, conducted mainly in monophasic and biphasic batch systems with and without water addition have been published and are still under investigation. However, to produce HMF at a large scale, a continuous process is preferable. Until now, only a few studies have been published in this context. In this work, it is shown that fluorous alcohol hexafluoroisopropanol (HFIP) can act as superior reaction solvent for HMF synthesis from fructose in a fixed bed reactor. Very high yields of 76% HMF can be achieved in this system under optimized conditions, whilst the catalyst is very stable over several days. Such high yields are only described elsewhere with high boiling reaction solvents like dimethylsulfoxide (DMSO), whereas HFIP with a boiling point of 58 °C is very easy to separate from HMF.

scholarly journals Investigation of Malic Acid Production in Aspergillus oryzae under Nitrogen Starvation Conditions

2013 ◽  
Vol 79 (19) ◽  
pp. 6050-6058 ◽  
Author(s):  
Christoph Knuf ◽  
Intawat Nookaew ◽  
Stephen H. Brown ◽  
Michael McCulloch ◽  
Alan Berry ◽  
...  
Keyword(s):  
Malic Acid ◽  
Acid Production ◽  
Large Scale ◽  
High Capacity ◽  
Nitrogen Sources ◽  
Building Blocks ◽  
Close Relative ◽  
Biotechnological Production ◽  
Content Type ◽  
The One

ABSTRACTMalic acid has great potential for replacing petrochemical building blocks in the future. For this application, high yields, rates, and titers are essential in order to sustain a viable biotechnological production process. Natural high-capacity malic acid producers like the malic acid producerAspergillus flavushave so far been disqualified because of special growth requirements or the production of mycotoxins. AsA. oryzaeis a very close relative or even an ecotype ofA. flavus, it is likely that its high malic acid production capabilities with a generally regarded as safe (GRAS) status may be combined with already existing large-scale fermentation experience. In order to verify the malic acid production potential, two wild-type strains, NRRL3485 and NRRL3488, were compared in shake flasks. As NRRL3488 showed a volumetric production rate twice as high as that of NRRL3485, this strain was selected for further investigation of the influence of two different nitrogen sources on malic acid secretion. The cultivation in lab-scale fermentors resulted in a higher final titer, 30.27 ± 1.05 g liter−1, using peptone than the one of 22.27 ± 0.46 g liter−1obtained when ammonium was used. Through transcriptome analysis, a binding site similar to the one of theSaccharomyces cerevisiaeyeast transcription factor Msn2/4 was identified in the upstream regions of glycolytic genes and the cytosolic malic acid production pathway from pyruvate via oxaloacetate to malate, which suggests that malic acid production is a stress response. Furthermore, the pyruvate carboxylase reaction was identified as a target for metabolic engineering, after it was confirmed to be transcriptionally regulated through the correlation of intracellular fluxes and transcriptional changes.

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