Cell Dry Wt Research Articles

Enhanced supply of acetyl-CoA by exogenous pantothenate kinase promotes synthesis of poly(3-hydroxybutyrate)

BackgroundCoenzyme A (CoA) is a carrier of acyl groups. This cofactor is synthesized from pantothenic acid in five steps. The phosphorylation of pantothenate is catalyzed by pantothenate kinase (CoaA), which is a key step in the CoA biosynthetic pathway. To determine whether the enhancement of the CoA biosynthetic pathway is effective for producing useful substances, the effect of elevated acetyl-CoA levels resulting from the introduction of the exogenous coaA gene on poly(3-hydroxybutyrate) [P(3HB)] synthesis was determined in Escherichia coli, which express the genes necessary for cyanobacterial polyhydroxyalkanoate synthesis (phaABEC).ResultsE. coli containing the coaA gene in addition to the pha genes accumulated more P(3HB) compared with the transformant containing the pha genes alone. P(3HB) production was enhanced by precursor addition, with P(3HB) content increasing from 18.4% (w/w) to 29.0% in the presence of 0.5 mM pantothenate and 16.3%–28.2% by adding 0.5 mM β-alanine. Strains expressing the exogenous coaA in the presence of precursors contained acetyl-CoA in excess of 1 nmol/mg of dry cell wt, which promoted the reaction toward P(3HB) formation. The amount of acetate exported into the medium was three times lower in the cells carrying exogenous coaA and pha genes than in the cells carrying pha genes alone. This was attributed to significantly enlarging the intracellular pool size of CoA, which is the recipient of acetic acid and is advantageous for microbial production of value-added materials.ConclusionsEnhancing the CoA biosynthetic pathway with exogenous CoaA was effective at increasing P(3HB) production. Supplementing the medium with pantothenate facilitated the accumulation of P(3HB). β-Alanine was able to replace the efficacy of adding pantothenate.

Metal removal capability of two cyanobacterial species in autotrophic and mixotrophic mode of nutrition

BackgroundCyanobacteria are ecologically significant prokaryotes that can be found in heavy metals contaminated environments. As their photosynthetic machinery imposes high demands for metals, homeostasis of these micronutrients has been extensively considered in cyanobacteria. Recently, most studies have been focused on different habitats using microalgae leads to a remarkable reduction of an array of organic and inorganic nutrients, but what takes place in the extracellular environment when cells are exposed to external supplementation with heavy metals remains largely unknown.MethodsHere, extracellular polymeric substances (EPS) production in strains Nostoc sp. N27P72 and Nostoc sp. FB71 was isolated from different habitats and thenthe results were compared and reported.ResultCultures of both strains, supplemented separately with either glucose, sucrose, lactose, or maltose showed that production of EPS and cell dry weight were boosted by maltose supplementation. The production of EPS (9.1 ± 0.05 μg/ml) and increase in cell dry weight (1.01 ± 0.06 g/l) were comparatively high in Nostoc sp. N27P72 which was isolated from lime stones.The cultures were evaluated for their ability to remove Cu (II), Cr (III), and Ni (II) in culture media with and without maltose. The crude EPS showed metal adsorption capacity assuming the order Ni (II) > Cu (II) > Cr (III) from the metal-binding experiments.Nickel was preferentially biosorbed with a maximal uptake of 188.8 ± 0.14 mg (g cell dry wt) −1 crude EPS. We found that using maltose as a carbon source can increase the production of EPS, protein, and carbohydrates content and it could be a significant reason for the high ability of metal absorbance. FT-IR spectroscopy revealed that the treatment with Ni can change the functional groups and glycoside linkages in both strains. Results of Gas Chromatography-Mass Spectrometry (GC–MS) were used to determine the biochemical composition of Nostoc sp. N27P72, showed that strong Ni (II) removal capability could be associated with the high silicon containing heterocyclic compound and aromatic diacid compounds content.ConclusionThe results of this studyindicatede that strains Nostoc sp. N27P72 can be a good candidate for the commercial production of EPS and might be utilized in bioremediation field as an alternative to synthetic and abiotic flocculants.

Ultrasound-assisted fermentative production of Polyhydroxybutyrate (PHB) in Cupriavidus necator

The conventional polymers (polyethylene, polypropylene, polystyrene, etc) are non-degradable and exhibit many hazardous effects on the environment. Due to their bio incompatibility and accumulation in nature, there is a need to produce environment-friendly biodegradable polymers. Polyhydroxybutyrate is one of the biodegradable biopolymers produced by a microorganism Cupriavidus necator in order to survive the stress condition. In this study, the effect of ultrasound to intensify the productivity of biopolymer was studied. Different ultrasound parameters, mainly the effect of ultrasound power, duty cycle, ultrasound irradiation time on the productivity of PHB were studied using the one-factor-at-a-time method. Intracellular PHB was extracted using chloroform as a solvent, and analysis was carried out using GC–MS, FT-IR analytical techniques for the detection of PHB. The highest productivity of 0.2155 ± 0.0049 g/100 mL (1.7 g PHB g−1 dry cell wt−1) is reported when a substrate was irradiated with ultrasound at 22 kHz frequency, 50 W power, 10% duty cycle and 10 min of irradiation time with a constant cycle time of 1 min.

Individual and combined supplementation of carbon sources for growth augmentation and enrichment of lipids in the green microalga Tetradesmus obliquus

Microalgae with high lipid productivity offer great promise as a potential source of biodiesel, an alternative to fossil diesel. Since a target of both high biomass yield and lipid content is difficult to attain with the classical photoautotrophic mode of nutrition, mixotrophy has evolved as a preferred mode for enhancement of growth in microalgae. In the present study, this strategy was explored extensively with six different exogenous carbon sources in eight different concentrations to investigate the effect of these carbon sources on the biomass and lipid production potential of the green alga Tetradesmus obliquus (SAG 276-3a). To further augment the effect of mixotrophy on the growth and lipid accumulation of the test microalga, selected carbon sources with their specific concentrations showing enhancement in both biomass yield and lipid accumulation in the first phase of the experiment were interacted with each other. Significant rise in biomass yield by twofold was seen in the case of 0.16% citrate + 0.08% bicarbonate interaction, whereas for lipid accumulation, maximum rise in lipid content (% dry cell wt.) by 2.3-fold and lipid yield (g L-1) by 4.5-fold was observed in the case of 0.16% acetate + 0.16% citrate-supplemented cultures. The biodiesel samples as analyzed using GC–MS were predominated with saturated and monounsaturated fatty acid methyl esters. Fuel property analysis of the biodiesel samples found them to be well suited to the Indian, American (ASTM), and European (EN) biodiesel standards.

Carotenoid Production by a Novel Isolate of Microbacterium paraoxydans

This study reports extraction and characterization of carotenoid pigments from Microbacterium paraoxydans, a non-photosynthetic bacterium, cultivated in Luria-Bertani (LB) medium. The isolate was identified to be moderately halo- and osmo-tolerant capable of withstanding high (~6%) salt and sugar (30% w/v sucrose, 20% w/v glucose) concentrations after a brief period of adaptation. The pigments were characterized using a combination of UV-Vis spectral analysis with the λmax at 407, 436 and 466nm and ESI-MS with an m/z value at 536.44. The absorption profile of the pigments and their nature was influenced by carbon, nitrogen source and presence of salt in the growth medium. Highest level of pigment (~16gkg dry wt cells-1) was produced in NH4Cl supplemented LB medium. The pigment displayed free radical scavenging, anticancer activity, characteristic of the plant carotenoids. Based on the accumulation of pigments under different conditions, a biochemical pathway for synthesis of neurosporene was proposed.

Combined mutagenesis of Rhodosporidium toruloides for improved production of carotenoids and lipids.

To improve production of lipids and carotenoids by the oleaginous yeast Rhodosporidium toruloides by screening mutant strains. Upon physical mutagenesis of the haploid strain R. toruloides np11 with an atmospheric and room temperature plasma method followed by chemical mutagenesis with nitrosoguanidine, a mutant strain, R. toruloides XR-2, formed dark-red colonies on a screening plate. When cultivated in nitrogen-limited media, XR-2 cells grew slower but accumulated 0.23g lipids/g cell dry wt and 0.75mg carotenoids/g CDW. To improve its production capacity, different amino acids and vitamins were supplemented. p-Aminobenzoic acid and tryptophan had beneficial effects on cell growth. When cultivated in nitrogen-limited media in the presence of selected vitamins, XR-2 accumulated 0.41g lipids/g CDW and 0.69mg carotenoids/g CDW. A mutant R. toruloides strain with improved production profiles for lipids and carotenoids was obtained, indicating its potential to use combined mutagenesis for a more productive phenotype.

Putative carotenoid genes expressed under the regulation of Shine-Dalgarno regions in Escherichia coli for efficient lycopene production.

Putative genes crtE, crtB, and crtI from Deinococcus wulumiqiensis R12, a novel species, were identified by genome mining and were co-expressed using the optimized Shine-Dalgarno (SD) regions to improve lycopene yield. A lycopene biosynthesis pathway was constructed by co-expressing these three genes in Escherichia coli. After optimizing the upstream SD regions and the culture medium, the recombinant strain EDW11 produced 88mg lycopene g(-1) dry cell wt (780mg lycopene l(-1)) after 40h fermentation without IPTG induction, while the strain EDW without optimized SD regions only produced 49mg lycopene g(-1) dry cell wt (417mg lycopene l(-1)). Based on the optimization of the upstream SD regions and culture medium, the yield of the strain EDW11 reached a high level during microbial lycopene production until now.

Carbon dioxide and poultry waste utilization for production of polyhydroxyalkanoate biopolymers by Nostoc muscorum Agardh: a sustainable approach

The new paradigm is to view wastes as resources for sustainable development. In this regard, the feasibility of poultry waste and CO2 utilization for cultivation of a filamentous nitrogen-fixing cyanobacterium, Nostoc muscorum Agardh, was investigated for production polyhydroxyalkanoates, the biodegradable polymers. This cyanobacterium showed profound rise in biomass yield with up to 10 % CO2 supply in airstream with an aeration rate of 0.1 vvm. Maximum biomass yield of 1.12 g L−1 was recorded for 8 days incubation period, thus demonstrating a CO2 biofixation rate of 0.263 g L−1 day−1 at 10 % (v/v) CO2-enriched air. Poultry litter (PL) supplementation also had a positive impact on the biomass yield. The nutrient removal efficiency of N. muscorum was reflected in the significant reduction in nutrient load of PL over the experimental period. A maximum poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) [P(3HB-co-3HV)] copolymer yield of 774 mg L−1 (65 % of dry cell wt.), the value almost 11-fold higher than the control, was recorded in 10 g L−1 PL-supplemented cultures with 10 % CO2 supply under the optimized condition, thus demonstrating that N. muscorum has good potential for CO2 biomitigation and poultry waste remediation while simultaneously producing eco-friendly polymers.

Effects of calcium, magnesium and sodium chloride in enhancing lipid accumulation in two green microalgae

Biodiesel from microalgae has the potential as a sustainable fuel, since some species show exceptionally high lipid accumulation potential under various stresses. Effects of different concentrations of Ca, Mg and NaCl in the growth medium on biomass yield and lipid accumulation of Chlorella vulgaris and Scenedesmus obliquus grown under batch culture mode were investigated. Starvation of Mg showed a marginal rise in lipid content for a short period of time. Ca-starved cultures, however, demonstrated a profound rise in lipid content, i.e. 40% of dry cell wt. (dcw) was recorded against 11.9% control for C. vulgaris and 37% (dcw) against 11.3% for S. obliquus. Under increased concentration of Mg, significant rise in biomass and lipid yield was recorded. Effect of NaCl-induced osmotic stress showed lipid accumulation of ∼ 40% (dcw) in both the test algae, whereas the biomass yield was severely affected. The fatty acid profiles under the above stresses were analysed and discussed.

Synthesis of hexyl α-glucoside and α-polyglucosides by a novel Microbacterium isolate

Alkyl-glucosides and alkyl-polyglucosides are the new-generation biodegradable surfactants with good emulsifying and wetting properties. The α-forms of these glucosides occur in antibiotics and also stimulate nasal absorption of many drugs. In this paper, we report the synthesis of hexyl α-glucoside and α-polyglucosides using cell-bound α-glucosidase activity of a novel strain of Microbacterium paraoxydans. A number of cell-bound glycosyl hydrolase activities were detected in the isolate with the maximum hydrolytic activity of 180IUg(-1) dry wt cells on p-nitrophenyl-α-D-glucopyranoside. In a micro-aqueous system, at a water activity of 0.69, 1.8gl(-1) of hexyl α-glucoside (corresponding to about 25% yield) was synthesized by whole cells with maltose and hexanol as substrates. The concentration was enhanced to 11gl(-1) (~60% yield) in a biphasic system at a water content of 60%. (1)H and (13)C NMR spectra of the purified compound confirmed the synthesized product to be hexyl-α-D-glucopyranoside, while the presence of hexyl di- and tri-glucosides was confirmed by electrospray ionization mass spectrometry. The cell-driven synthesis makes this an extremely attractive alternative for synthesis of such compounds.

Biosynthesis and characterization of a new copolymer, poly(3-hydroxyvalerate-co-5-hydroxydecenoate), from Pseudomonas aeruginosa

Using a new isolate of Pseudomonas aeruginosa, we obtained 7 g cell dry wt (CDW/l) using 5 % (w/v) glucose. Crude polyhydroxyalkanoates were obtained at 14.6 % of CDW. FTIR and NMR analysis confirmed that this was a new co-polymer: 3-hydroxyvalerate-co-5-hydroxydecenoate. Differential scanning calorimetry analysis showed two different melting temperatures of the copolymer and also indicated the glass transition temperature to be 4 °C. The polydispersity index of the polymer was 1.059.

Construction of Escherichia coli strains producing l-serine from glucose

L-Serine is usually produced from glycine. We have genetically engineered Escherichia coli to produce L-serine from glucose intracellularly. D-3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) in E. coli catalyzes the first committed step in L-serine formation but is inhibited by L-serine. To overcome this feedback inhibition, both the His(344) and Asn(346) residues of PGDH were converted to alanine and the mutated PGDH (PGDH(dr)) became insensitive to L-serine. However, overexpression of PGDH(dr) gave no significant increase of L-serine accumulation but, when L-serine deaminase genes (sdaA, sdaB and tdcG) were deleted, serine accumulated: (1) deletion of sdaA gave up to 0.03 mmol L-serine/g; (2) deletion of both sdaA and sdaB accumulated L-serine up to 0.09 mmol/g; and (3) deletion of sdaA, sdaB and tdcG gave up to 0.13 mmol L-serine/g cell dry wt.

Uranium and other heavy metal resistance and accumulation in bacteria isolated from uranium mine wastes

Ten bacterial strains isolated from uranium mine wastes were characterized in terms of their uranium and other metal resistance and accumulation. 16S rRNA gene sequence analysis identified the strains as members of genera Bacillus, Serratia, and Arthrobacter. Strains were able to utilize various carbon sources, particularly aromatic hydrocarbons, grow at broad pH and temperature ranges and produce non specific acid phosphatase relevant for metal phosphate precipitation in contaminated environment. The isolates exhibited high uranium and other heavy metals (Ni, Co, Cu and Cd) resistance and accumulation capacities. Particularly, Arthrobacter sp. J001 and Bacillus sp. J003 were superior in terms of U resistance at low pH (pH 4.0) along with metals and actinides (U and Th) removal with maximum cell loading of 1088 μmol U, 1293 μmol Th, 425 μmol Cu, 305 μmol Cd, 377 μmol Zn, 250 μmol Ni g−1 cell dry wt. Genes encoding P1B-type ATPases (Cu-CPx and Zn-CPx) and ABC transporters (nik) as catalytic tools for maintaining cellular metal homeostasis were detected within several Bacillus spp., with possible incidence of horizontal gene transfer for the later gene showing phylogenetic lineage to α Proteobacteria members. The study provides evidence on intrinsic abilities of indigenous bacteria from U-mine suitable for survival and cleaning up of contaminated mine sites.

Vector-mediated chromosomal integration of the glutamate decarboxylase gene in Streptococcus thermophilus

The integrative vector, pINTRS, was used to transfer glutamate decarboxylase (GAD) activity to Streptococcus thermophilus ST128 thereby allowing for the production of γ-aminobutyric acid (GABA). In pINTRS, the gene encoding glutamate decarboxylase, gadB, was flanked by DNA fragments homologous to a S. thermophilus pseudogene to allow for integration at a non-essential locus on the chromosome. Screening techniques confirmed the insertion of gadB with either its endogenous promoter or the S. thermophilus P2201 promoter, resulting in the generation of recombinant strains, ST128/gadB or ST128/P2201-gadB. Following the integration event unwanted plasmid DNA, specifically the erythromycin resistance gene, was eliminated from the recombinant strains. Based on the production of GABA, activities of GAD for ST128/gadB and ST128/P2201-gadB were 30.6 ± 6 and 27.9 ± 7.2 μM/mg dry cell wt, respectively.

Optimization of culture conditions and comparison of biomass productivity of three green algae

Culture conditions for the mass production of three green algae, Chlorella sp., Dunaliella salina DCCBC2 and Dunaliella sp., were optimized using a response surface methodology (RSM). A central composite design was applied to investigate the effects of initial pH, nitrogen and phosphate concentrations on the cultivation of microalgae. The optimal growth conditions estimated from the design are as follows: Chlorella sp. (initial pH 7.2, ammonium 17 mM, phosphate 1.2 mM), D. salina DCCBC2 (initial pH 8.0, nitrate 3.3 mM, phosphate 0.0375 mM) and Dunaliella sp. (initial pH 8.0, nitrate 3.7 mM, phosphate 0.17 mM). Culturing the microalgae with the optimized conditions confirmed that the maximum growth rates were attained for these parameters. The optimum CO(2) concentrations of Chlorella sp., D. salina DCCBC2 and Dunaliella sp. were 1.0, 3.0 and 1.0% (v/v), respectively. The specific growth rates (μ) of Chlorella sp., D. salina DCCBC2 and Dunaliella sp. were 0.58, 0.78 and 0.56 day(-1), respectively, and the biomass productivities were 0.28, 0.54 and 0.30 g dry cell wt l(-1) day(-1), respectively. The CO(2) fixation rates of Chlorella sp., D. salina DCCBC2 and Dunaliella sp. were 42.8, 90.9 and 45.5 mg l(-1) day(-1), respectively. Mixotrophic cultivation of Chlorella sp. with glucose increased biomass productivity from 0.28 to 0.51 g dry cell wt l(-1) day(-1). However, D. salina DCCBC2 and Dunaliella sp. were not stimulated by several organic compounds tested.

2,4,6-Trinitrophenol degradation by Bacillus cereus isolated from a firing range

Bacillus cereus strain PU, isolated from soil contaminated with explosive waste, tolerated up to 1.3 mM 2,4,6 trinitrophenol (TNP) and utilize it aerobically as sole nitrogen and carbon source. Degradation of TNP was accompanied by stoichiometric release of 2.1 ± 0.15 mol nitrite/mol TNP at 539 μmol/h g dry cell wt. Metabolism of TNP was accompanied by transient accumulation of an orange-red metabolite, hydride meisenheimer complex (H-TNP), indicating a metabolic pathway involving complete reductive removal of the nitro group as nitrite.

Optimization of lab scale methanol production by Methylosinus trichosporium OB3b

Methylosinus trichosporium OB3b is a methanotrophic bacterium containing particulate methane monooxygenase (MMO), which catalyzes the hydroxylation of methane to methanol. The methanol is further oxidized to formaldehyde by methanol dehydrogenase (MDH). We developed a novel compulsory circulation diffusion system for cell cultivation. A methane/air mixture (1:1, v/v) was prepared in a tightly sealed gas reservoir and pumped into a nitrate mineral salt culture medium under optimal conditions (5 μM CuSO4, pH 7.0, 30°C). Cells were harvested, washed, and resuspended (0.6 mg dry cells/mL) in a 500 mL flask in 100 mL of 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl and 1 mM EDTA as MDH inhibitors, and 20 mM sodium formate. A single 12 h batch reaction at 25°C yielded a final concentration of 13.2 mM methanol. The use of a repeated batch mode, in which the accumulated methanol was removed after each of three 8 h cycles over a 24 h period, showed a productivity of 2.17 μmol methanol/h/mg dry cell wt. Finally, a lab-scale reaction performed using a 3 L cylindrical reactor with a working volume of 1 L produced 13.7 mM methanol after 16 h. Our results identify a simple process for improving the productivity of biologically derived methanol and, therefore the utility of methane as an energy source.

Cell Surface of Lactococcus lactis Is Covered by a Protective Polysaccharide Pellicle

In Gram-positive bacteria, the functional role of surface polysaccharides (PS) that are not of capsular nature remains poorly understood. Here, we report the presence of a novel cell wall PS pellicle on the surface of Lactococcus lactis. Spontaneous PS-negative mutants were selected using semi-liquid growth conditions, and all mutations were mapped in a single chromosomal locus coding for PS biosynthesis. PS molecules were shown to be composed of hexasaccharide phosphate repeating units that are distinct from other bacterial PS. Using complementary atomic force and transmission electron microscopy techniques, we showed that the PS layer forms an outer pellicle surrounding the cell. Notably, we found that this cell wall layer confers a protective barrier against host phagocytosis by murine macrophages. Altogether, our results suggest that the PS pellicle could represent a new cell envelope structural component of Gram-positive bacteria.

Relationship between glucose catabolism and xanthan production in Xanthomonas oryzae pv. oryzae

Two genes involved in central carbon metabolism were inactivated to modulate intracellular glucose 6-phosphate and to evaluate its effects on xanthan production in wild-type Xanthomonas oryzae pv. oryzae. Upon the inactivation of the phosphogluconate dehydratase gene (edd), intracellular glucose 6-phosphate increased from 0.05 to 1.17 mmol/g (dry cell wt). This was accompanied by increased xanthan production of up to 2.55 g/l (culture medium). In contrast, inactivation of 6-phosphogluconate dehydrogenase gene (gndA) did not influence intracellular glucose 6-phosphate nor xanthan production. The intracellular availability of glucose 6-phosphate is proposed as a rate-limiting factor in xanthan production, and it may be possible to increases production of xanthan by modulating the activities of enzymes in central carbon metabolism.

Altered composition of Ralstonia eutropha poly(hydroxyalkanoate) through expression of PHA synthase from Allochromatium vinosum ATCC 35206

The class III poly(hydroxyalkanoate) synthase (PHAS) genes (phaC and phaE) of a photosynthetic bacterium, Allochromatium vinosum ATCC 35206, were cloned, sequenced and expressed in a heterologous host. PCR coupled with a chromosomal gene-walking method was used to clone and subsequently sequence the contiguous phaC (1,068 bps) and phaE (1,065 bps) genes of A. vinosum ATCC 35206. BLASTP search of protein databases showed that the gene-products of phaC and phaE are different (<66% identities) from the previously reported class III PHASs such as those of A. vinosum DSM180. Domain analysis revealed the presence of a conserved alpha/beta-hydrolase fold in PhaC, the putative gene-product of phaC. Upon electroporation of a poly(hydroxybutanoate) (PHB)-negative mutant of Ralstonia eutropha PHB(-)4 with a shuttle plasmid pBHR1 containing the newly cloned phaC and phaE genes, the bacteria resumed the synthesis of PHB, albeit at a low level (4-5% of the cell dry wt) due to kanamycin selection pressure. We further showed that the recombinant strain grown in kanamycin-containing culture medium synthesized a blend of PHA that also contains a high content of 3-hydroxyoctanoate and 3-hydroxydecanoate as its repeat-unit monomers. Genomic analysis suggested the existence of two PHA synthase genes in R. eutropha. The results of this study not only make available a phylogenetically diverse type III phaC and phaE genes, but also confirm through kanamycin selection pressure the existence of multiple PHA biosynthesis systems in R. eutropha.