Macromolecular and solution properties of Cepacian: the exopolysaccharide produced by a strain of Burkholderia cepacia isolated from a cystic fibrosis patient

Light scattering and viscosity measurements were carried out on the previously chemically characterised exopolysaccharide produced by a strain of Burkholderia cepacia isolated from a cystic fibrosis patient. The same exopolysaccharide was also produced by other clinical strains in different laboratories. Therefore, the name Cepacian is now proposed for this exopolysaccharide. Experiments performed as a function of the ionic strength on the native polymer revealed a change in the overall shape of the polymer at low ionic strength. This behaviour was absent in the de-acetylated sample. Potentiometric titrations and light scattering experiments carried out on the acidic form of the native polymer revealed the formation of macromolecular aggregates with a stoichiometry n and 2n stabilised by interactions involving the uronic acid residues.     

Kinetics of production and characterization of the fucose-containing exopolysaccharide from Enterobacter A47

A fucose-containing exopolysaccharide (EPS) was produced by the bacterium Enterobacter A47 using glycerol byproduct from the biodiesel industry. The analysis of kinetic data suggested a partially growth associated EPS synthesis model. Although the EPS was composed of fucose, galactose and glucose at all cultivation stages, their relative proportion has varied considerably during the run. At the beginning (24 h), glucose was the main component (82.4 wt.%), being fucose and galactose minor components (5.0 wt.% and 10.9 wt.%, respectively), while at the end (96 h) it was composed of 26.0 wt.% fucose, 28.9 wt.% galactose and 43.7 wt.% glucose. The acyl groups content and composition have also changed, reaching their maximum content (19.2 wt.%) at the end of the run. Moreover, the molecular weight has increased linearly during the run (from 8 × 10⁵ to 5 × 10⁶). The changes observed in EPS composition and molecular weight have also had an impact upon the polymer's intrinsic viscosity, as shown by its linear increase from 3.95 to 10.72 dL g⁻¹. The results suggest that the culture might have synthesized at least two distinct EPS, with different sugar composition and average molecular weight, which predominated at different cultivation stages.     

Production of a novel biomacromolecule for nanodevices from glycerol as carbon source in different conditions

The aim of this study was the investigation of producing cruxrhodopsin as a biomacromolecule with nanofunction from glycerol as carbon source using several process parameters. The optimum medium composition for cruxrhodopsin production was found to contain glycerol 1%, yeast extract 0.05% and K₂HPO₄ 0.001%. The production of cruxrhodopsin in optimal conditions was 139.86 mg/l. In conclusion, halophilic microorganism Haloarcula sp. IRU1 could be a potential microorganism for production of cruxrhodopsin from glycerol in different conditions.     

Enhanced biological phosphorus removal in a sequencing batch reactor using propionate as the sole carbon source

An enhanced biological phosphorus removal (EBPR) system was developed in a sequencing batch reactor (SBR) using propionate as the sole carbon source. The microbial community was followed using fluorescence in situ hybridization (FISH) techniques and Candidatus 'Accumulibacter phosphatis' were quantified from the start up of the reactor until steady state. A series of SBR cycle studies was performed when 55% of the SBR biomass was Accumulibacter, a confirmed polyphosphate accumulating organism (PAO) and when Candidatus 'Competibacter phosphatis', a confirmed glycogen-accumulating organism (GAO), was essentially undetectable. These experiments evaluated two different carbon sources (propionate and acetate), and in every case, two different P-release rates were detected. The highest rate took place while there was volatile fatty acid (VFA) in the mixed liquor, and after the VFA was depleted a second P-release rate was observed. This second rate was very similar to the one detected in experiments performed without added VFA.A kinetic and stoichiometric model developed as a modification of Activated Sludge Model 2 (ASM2) including glycogen economy, was fitted to the experimental profiles. The validation and calibration of this model was carried out with the cycle study experiments performed using both VFAs. The effect of pH from 6.5 to 8.0 on anaerobic P-release and VFA-uptake and aerobic P-uptake was also studied using propionate. The optimal overall working pH was around 7.5. This is the first study of the microbial community involved in EBPR developed with propionate as a sole carbon source along with detailed process performance investigations of the propionate-utilizing PAOs.     

Emulsifying behaviour and rheological properties of the extracellular polysaccharide produced by Pseudomonas oleovorans grown on glycerol byproduct

The functional properties of a novel extracellular polysaccharide (EPS) produced by Pseudomonas oleovorans grown on glycerol byproduct, generated by the biodiesel industry, were investigated. The EPS is a high molecular weight (4.6 × 10⁶) heteropolysaccharide, composed by neutral sugars (galactose, 68%; mannose, 17%; glucose, 13%; rhamnose, 2%; fucose, 4%) and acyl groups (3.04%). This biopolymer has pseudoplastic fluid behaviour in aqueous media. The apparent viscosity was stable for the pH range 2.9–7.1 and NaCl concentrations up to 1.0 M. Though the apparent viscosity decreased at high temperatures, at alkaline conditions and at NaCl concentrations of 2.0 M, pseudoplastic fluid behaviour was retained. The EPS was capable of stabilizing water emulsions with several hydrophobic compounds, including hydrocarbons, vegetable and mineral oils. It retained its emulsifying activity during exposure to wide temperature (30–50 °C) and pH (2–12) variations, as well as to the presence of NaCl at concentrations as high as 2.0 M.     

High-level production of 3-hydroxypropionic acid from glycerol as a sole carbon source using metabolically engineered Escherichia coli

As climate change is an important environmental issue, the conventional petrochemical-based processes to produce valuable chemicals are being shifted toward eco-friendly biological-based processes. In this study, 3-hydroxypropionic acid (3-HP), an industrially important three carbon (C3) chemical, was overproduced by metabolically engineered Escherichia coli using glycerol as a sole carbon source. As the first step to construct a glycerol-dependent 3-HP biosynthetic pathway, the dhaB1234 and gdrAB genes from Klebsiella pneumoniae encoding glycerol dehydratase and glycerol reactivase, respectively, were introduced into E. coli to convert glycerol into 3-hydroxypropionaldehyde (3-HPA). In addition, the ydcW gene from K. pneumoniae encoding γ-aminobutyraldehyde dehydrogenase, among five aldehyde dehydrogenases examined, was selected to further convert 3-HPA to 3-HP. Increasing the expression level of the ydcW gene enhanced 3-HP production titer and reduced 1,3-propanediol production. To enhance 3-HP production, fed-batch fermentation conditions were optimized by controlling dissolved oxygen (DO) level and employing different feeding strategies including intermittent feeding, pH-stat feeding, and continuous feeding strategies. Fed-batch culture of the final engineered E. coli strain with DO control and continuous feeding strategy produced 76.2 g/L of 3-HP with the yield and productivity of 0.457 g/g glycerol and 1.89 g·L⁻¹·h⁻¹, respectively. To the best of our knowledge, this is the highest 3-HP productivity achieved by any microorganism reported to date.     

Structural elucidation of an exopolysaccharide from mycelial fermentation of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis

A novel polysaccharide designated EPS-1A with an average molecular weight around 40 kDa was fractionated and purified by anion-exchange and gel-filtration chromatography from the crude exopolysaccharide (EPS) isolated from fermentation broth of Cs-HK1, a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis. The structural characteristics of EPS-1A were determined with various methods (e.g. GC, GC–MS, FT-IR, ¹H NMR and ¹³C NMR) and through acid hydrolysis, methylation, periodate-oxidation and Smith degradation. The results suggested that EPS-1A was composed of glucose, mannose and galactose at 15.2:3.6:1.0 M ratio. EPS-1A was a slightly branched polysaccharide and its backbone was composed of (1 → 6)-α-d-glucose residues (77%) and (1 → 6)-α-d-mannose residues (23%). Branching occurred at O-3 position of (1 → 6)-α-d-mannose residues of the backbone with (1 → 6)-α-d-mannose residues and (1 → 6)-α-d-glucose residues, and terminated with β-d-galactose residues.     

Detection, isolation, and characterization of exopolysaccharide produced by a strain of Phormidium 94a isolated from an arid zone of Mexico

Phormidium 94a, a cyanobacteria that produces extracellular polymeric substances (EPS), was isolated from arid soils of Mexico. Microscopic localization, using histochemical techniques like the Toluidine blue technique, was done in order to demonstrate the presence of EPS. Acetone was added to precipitate the EPS. In this study we characterized the EPS by GC, HPLC, and IR techniques. The highest fraction of EPS had a molecular weight of 2000 kDa. The sugar composition was galactose, mannose, galacturonic acid, arabinose, and ribose in the three main fractions, and the sugar ratio found was different in each fraction. The low EPS concentrations had a Newtonian behavior, when the concentrations were increased, the behavior changed to pseudoplastic. The EPS rheulogical behavior is similar to low viscosity arabic gum. Also, it was found that an increase in viscosity occurred at longer hydration time. More rheological and toxicological studies are required in order to analyze its possible application in food industries.     

嗜热链球菌IMAU20246胞外多糖基因簇及其表达分析北大核心

【目的】嗜热链球菌IMAU20246是一株具有良好发酵特性且高产胞外多糖(exopolysaccharides,EPS)的菌株,但其EPS基因簇及合成途径尚不清晰。因此可通过全基因组测序及生物信息学分析菌株基因组序列,探究EPS合成及调控机制。【方法】本实验对嗜热链球菌IMAU20246进行全基因组测序并进行生物信息学分析,解析EPS生物合成相关基因簇及EPS合成途径,同时采用实时荧光定量PCR技术(quantitative real-time PCR,qRT-PCR)对其不同时间点EPS基因簇的表达进行定量分析。【结果】嗜热链球菌IMAU20246基因组中有一个18.1 kb的EPS生物合成基因簇,编码15个与EPS生物合成相关的基因。嗜热链球菌IMAU20246通过转运葡萄糖、甘露糖、果糖、半乳糖、乳糖、海藻糖、纤维二糖及蔗糖合成UDP-葡萄糖、dTDP-葡萄糖、dTDP-鼠李糖、UDP-半乳糖、UDP-呋喃半乳糖、UDP-N-乙酰葡萄糖胺和UDP-N-乙酰半乳糖胺等7种糖核苷酸。qRT-PCR的结果表明,EPS基因簇中的基因在细胞生长阶段均能表达,特别是糖基转移酶基因epsE、epsF、epsH和epsJ在培养6 h时表达量最高,此时EPS产量达到最高。【结论】本研究从基因组解析了嗜热链球菌IMAU20246 EPS基因簇及其合成途径,为菌株的进一步开发提供了理论依据。     

Efficient production of polyhydroxyalkanoates (PHAs) from Pseudomonas mendocina PSU using a biodiesel liquid waste (BLW) as the sole carbon source

Conditions for the optimal production of polyhydroxyalkanoate (PHA) by Pseudomonas mendocina PSU using a biodiesel liquid waste (BLW) were determined by response surface methodology. These were an initial carbon to nitrogen ratio (C/N) of 40 (mole/mole), an initial pH of 7.0, and a temperature of 35 °C. A biomass and PHA concentration of 3.65 g/L and about 2.6 g/L (77% DCW), respectively, were achieved in a growth associated process using 20 g/L glycerol in the BLW after 36 h of exponential growth. The PHA monomer compositions were 3HB (3-hydroxybutyrate), a short-chain-length-PHA, and the medium-chain-length-PHA e.g. 3-hydroxyoctanoate and 3-hydroxydecanoate. Both the phbC and phaC genes were characterized. The phbC enzyme had not been previously detected in a Pseudomonas mendocina species. A 2.15 g/L of an exopolysaccharide, alginate, was also produced with a similar composition to that of other Pseudomonas species.     

Potential and limitations of Klebsiella pneumoniae as a microbial cell factory utilizing glycerol as the carbon source

Klebsiella pneumoniae is a Gram-negative facultative anaerobe that metabolizes glycerol efficiently under both aerobic and anaerobic conditions. This microbe is considered an outstanding biocatalyst for transforming glycerol into a variety of value-added products. Crude glycerol is a cheap carbon source and can be converted by K. pneumoniae into useful compounds such as lactic acid, 3-hydroxypropionic acid, ethanol, 1,3-propanediol, 2,3-butanediol, and succinic acid. This review summarizes glycerol metabolism in K. pneumoniae and its potential as a microbial cell factory for the production of commercially important acids and alcohols. Although many challenges remain, K. pneumoniae is a promising workhorse when glycerol is used as the carbon source.     

Isolation and properties of a pure bacterial strain capable of fluorobenzene degradation as sole carbon and energy source

A pure bacterial strain capable of aerobic biodegradation of fluorobenzene (FB) as the sole carbon and energy source was isolated by selective enrichment from sediments collected from a polluted site. 16S rRNA and fatty acid analyses support that strain F11 belongs to a novel genus within the alpha-2 subgroup of the Proteobacteria, possibly within a new clade related to the order Rhizobiales. In batch cultures, growth of strain F11 on FB led to stoichiometric release of fluoride ion. Maximum experimental growth rate of 0.04 h-1 was obtained at FB concentration of 0.4 mM. Growth kinetics were described by the Luong model. An inhibitory effect with increasing FB concentrations was observed, with no growth occurring at concentrations higher than 3.9 mM. Strain F11 was shown to be able to use a range of other organic compounds, including other fluorinated compounds such as 2-fluorobenzoate, 4-fluorobenzoate and 4-fluorophenol. To our knowledge, this is the first time biodegradation of FB, as the sole carbon and energy source, by a pure bacterium has been reported.     

Oxygen limitation improves glycerol production by Candida krusei in a bioreactor

An oxygen limitation strategy based on dynamic enzyme activity was applied to improve glycerol accumulation and decrease the residual sugar level in a fermentation of Candida krusei in a bioreactor. By applying oxygen limitation at 88 h when the activities of two glycerol synthetic enzymes cytosolic glycerol-3-phosphate dehydrogenase (ctGPD) and glycerol-3-phosphatase (GPP) were low and the activity of mitochondrial glycerol-3-phosphate dehydrogenase (mtGPD) which catalyzes the glycerol dissimilation was high, the glycerol dissimilation was efficiently reduced. The final glycerol concentration reached 51.8 g l⁻¹ at 96 h and 54.9 g l⁻¹ at 116 h, which was 18 and 60% higher than the control (without oxygen limitation), respectively. The residual sugar was consumed completely while it was 11.2 g l⁻¹ at the end of fermentation in the control. Under oxygen limitation, ethanol production was detected at a final concentration of 3.6 g l⁻¹. This work suggests a metabolic flux shift by oxygen limitation in the bioreactor.     

A cellulosic exopolysaccharide produced from sugarcane molasses by a Zoogloea sp.

An extracellular polysaccharide producing bacterium Zoogloea sp. was isolated from an agro-industrial environment in the north-eastern region of Brazil. The extracellular polysaccharide produced from sugarcane molasses was hydrolysed with trifluoroacetic acid (mild and strong conditions) giving 88% of soluble material. The main monosaccharides present in the soluble fraction were glucose (87.6%), xylose (8.6%), mannose (0.8%), ribose (1.7%), galactose (0.1%), arabinose (0.4%) and glucuronic acid (0.8%). Methylation analysis of the polysaccharide showed mainly 2,3,6-tri-O-methylhexitol (74.7%) and 2,3,-di-O-methylhexitol (17.7%). Enzyme hydrolysis of the polysaccharide with a cellulase confirmed the presence of (1→4)-β- -glucopyranosyl residues.     

Selective enrichment and isolation of Rhodopseudomonas palustris using trans-cinnamic acid as sole carbon source

Abstract Enrichment cultures for anoxygenic phototrophs capable of using cinnamic acid as sole organic carbon source consistently yielded the nonsulfur purple bacterium Rhodopseudomonas palustris . Pure cultures of R. palustris obtained from the enrichments grew photoheterotrophically on cinnamate and benzoate as well as on derivatives of these compounds. Photosynthetic growth on cinnamate was greatly stimulated by addition of exogenous CO₂, and resulted in breakage of the aromatic nucleus. Growth yield studies suggested that cinnamate was converted by R. palustris to intermediates that can be quantitatively assimilated into cell material.     

A systematic analysis of TCA Escherichia coli mutants reveals suitable genetic backgrounds for enhanced hydrogen and ethanol production using glycerol as main carbon source

Biodiesel has emerged as an environmentally friendly alternative to fossil fuels; however, the low price of glycerol feed‐stocks generated from the biodiesel industry has become a burden to this industry. A feasible alternative is the microbial biotransformation of waste glycerol to hydrogen and ethanol. Escherichia coli, a microorganism commonly used for metabolic engineering, is able to biotransform glycerol into these products. Nevertheless, the wild type strain yields can be improved by rewiring the carbon flux to the desired products by genetic engineering. Due to the importance of the central carbon metabolism in hydrogen and ethanol synthesis, E. coli single null mutant strains for enzymes of the TCA cycle and other related reactions were studied in this work. These strains were grown anaerobically in a glycerol‐based medium and the concentrations of ethanol, glycerol, succinate and hydrogen were analysed by HPLC and GC. It was found that the reductive branch is the more relevant pathway for the aim of this work, with malate playing a central role. It was also found that the putative C4‐transporter dcuD mutant improved the target product yields. These results will contribute to reveal novel metabolic engineering strategies for improving hydrogen and ethanol production by E. coli.     

Growth of Penicillium janthinellum on glycine as sole carbon and nitrogen source

Penicillium janthinellum is able to grow on glycine as the sole carbon and nitrogen source. The amino acid is transaminated to glyoxylate which is further metabolised to pyruvate by the glycerate pathway. The reaction product of partially purified glycerate kinase from this fungus is 2-phosphoglycerate. Phosphoglycerate mutase initiates gluconeogenesis from glycine. Partially purified phosphoglycerate mutase is inhibited by fructose 6-phosphate. The possible significance of this regulation is discussed.     

Production of PHB by a Bacillus megaterium strain using sugarcane molasses and corn steep liquor as sole carbon and nitrogen sources

Poly(hydroxybutyric acid) (PHB) and other biodegradable polyesters are promising candidates for the development of environment-friendly, totally biodegradable plastics. The use of cane molasses and corn steep liquor, two of the cheapest substrates available in Egypt, may help to reduce the cost of producing such biopolyesters. In this work, the effect of different carbon sources was studied. Maximum production of PHB was obtained with cane molasses and glucose as sole carbon sources (40.8, 39.9 per mg cell dry matter, respectively). The best growth was obtained with 3% molasses, while maximum yield of PHB (46.2% per mg cell dry matter) was obtained with 2% molasses. Corn steep liquor was the best nitrogen source for PHB synthesis (32.7 mg per cell dry matter), on the other hand, best growth was observed when ammonium chloride, ammonium sulphate, ammonium oxalate or ammonium phosphate were used as nitrogen sources.     

Biosynthesis of 1,3-propanediol from recombinant E. coli by optimization process using pure and crude glycerol as a sole carbon source under two-phase fermentation system

The environmental and nutritional condition for 1,3-propanediol (1,3-PD) production by the novel recombinant E. coli BP41Y3 expressing fusion protein were first optimized using conventional approach. The optimum environmental conditions were: initial pH at 8.0, incubation at 37 °C without shaking and agitation. Among ten nutrient variables, fumarate, (NH₄)₂HPO₄ and peptone were selected to study on their interaction effect using the response surface methodology. The optimum medium contained modified Riesenberg medium (containing pure glycerol as a sole carbon source) supplemented with 63.65 mM fumarate, 3.80 g/L (NH₄)₂HPO₄ and 1.12 g/L peptone, giving the maximum 1,3-PD production of 2.43 g/L. This was 3.5-fold higher than the original medium (0.7 g/L). Two-phase cultivation system was conducted and the effect of pH control (at 6.5, 7.0 and 8.0) was investigated under anaerobic condition by comparing with the no pH control condition. The cultivation system without pH control (initial pH of 8.0) gave the maximum values of 1.65 g/L 1,3-PD, the 1,3-PD production rate of 0.13 g/L h and the yield of 0.31 mol 1,3-PD/mol crude glycerol. Hence, using crude glycerol as a sole carbon source resulted in 32 % lower 1,3-PD production from this recombinant strain that may be due to the presence of various impurities in the crude glycerol of biodiesel plant. In addition, succinic acid was found to be a major product during fermentation by giving the maximum concentration of 11.92 g/L after 24 h anaerobic cultivation.