Simultaneous production of polyhydroxyalkanoates and rhamnolipids by Pseudomonas aeruginosa

The feasibility of the simultaneous production of polyhydroxyalkanoates (PHAs) and rhamnolipids, as a novel approach to reduce their production costs, was demonstrated by the cultivation of Pseudomonas aeruginosa IFO3924. Fairly large amounts of PHAs and rhamnolipids were obtained from the bacterial cells and the culture supernatant, respectively. Decanoate was a more suitable carbon source than ethanol and glucose for the simultaneous production, although glucose was suitable for cell growth without an induction period under pH control. The kind of carbon source affected PHA monomer composition markedly and PHA molecular weight slightly. Monorhamnolipids and dirhamnolipids were included in the rhamnolipids extracted from the culture supernatant using decanoate, glucose, or ethanol as the carbon source. Both PHAs and rhamnolipids were synthesized after the growth phase. PHA content in the cell reached a maximum when the carbon source was exhausted. After exhaustion of the carbon source, PHA content decreased rapidly, but rhamnolipid synthesis, which followed PHA synthesis, continued. This resulted in a time lag for the attainment of maximum levels of PHAs and rhamnolipids. The reusability of the cells used in rhamnolipid production was evaluated in the repeated batch culture of P. aeruginosa IFO3924 for the simultaneous production of PHAs and rhamnolipids. High concentrations of rhamnolipids in the culture supernatant were attained at the end of both the first and second batch cultures. High PHA content was achieved in the resting cells that were finally harvested after the second batch. Simultaneous production of PHAs and rhamnolipids will enhance the availability of valuable biocatalysts of bacterial cells, and dispel the common belief that the production cost of PHAs accumulated intracellularly is almost impossible to become lower than that of cells themselves.     

Bioconversion of starch processing waste to Phellinus linteus mycelium in solid-state cultivation

The objective of the experiment was to use starch processing waste as an alternative growth medium for cultivation of mycelia of the mushroom Phellinus linteus and to find an optimum condition under solid-state cultivation. Response surface analysis along with a central composite design was successfully applied to approximate the simultaneous effects of the substrate concentration (16-36 g l(-1)), pH (4.5-6.5), and temperature (25-35 degrees C) on the mycelial growth rate. In the model, pH and temperature significantly affected the mycelial growth but substrate concentration did not. The optimal substrate concentration, pH, and temperature for maximizing growth rate of P. linteus mycelia were found to be 16.5 g l(-1), pH 6.0, and 29.7 degrees C, respectively. Subsequent verification of these levels agreed with model predictions and the maximum mycelial growth rate at these conditions was 6.1 +/- 0.8 mm day(-1). Therefore, the results of the experiments suggest that starch processing waste could be utilized as a growth substrate for the cultivation of the mushroom mycelia of P. linteus, enhancing the usefulness of this byproduct of the starch manufacturing industry. This approach is likely to be useful for establishing similar parameters for the cultivation of other fungi.     

氯氰菊酯降解菌GF31的分离鉴定及其降解特性

从受污染的土壤中分离得到1株以氯氰菊酯为唯一碳源生长的降解菌GF31, 通过形态观察、16S rDNA基因序列分析、生理生化实验, 鉴定该菌为铜绿假单胞菌(Pseudomonas aeruginosa)。菌株GF31降解氯氰菊酯的最佳pH值为7.0, 接种量为10%, 对浓度高达300 mg/L的菊酯仍可保持较高的降解活性。外加氮源对菌株的降解效能影响显著, 有机氮比无机氮更有利于农药降解。当以0.5 g/L蛋白胨作为氮源时, 降解速率明显提高, 对100 mg/L氯氰菊酯降解的平均速率为13.64 mg/(L·d), 是以硫酸铵为氮源时的2倍。初步分析认为降解产物及碱性pH环境对菌株的生长及活性具有一定的抑制作用。     

Production of cyclodextrin glycosyltransferase by alkaliphilic <Emphasis Type="Italic">Bacillus circulans</Emphasis> in submerged and solid-state cultivation

Cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19) is an industrially important enzyme, which is used to produce cyclodextrins (CDs). In this research, we report the use of experimental factorial design to find the best conditions of pH and temperature for CGTase production by Bacillus circulans var. alkalophilus. The optimized calculated values for the tested variables were, respectively, pH 9.7 and temperature 36^oC, with a CGTase activity of 615 U mL⁻¹. The CGTase production was further studied with the optimized process parameters on submerged cultivations (SC) and solid-state cultivations (SSC) using soybean industrial fibrous residue (SIFR). The maximum CGTase activity obtained on SC was 1,155 U mL⁻¹ under aerobic conditions. Cell growth and CGTase synthesis in SSC using SIFR as substrate was excellent, with CGTase activity of 32,776 U g_(SIFR) ⁻¹. These results strongly support the use of SIFR for CGTase production since it is a non-expensive residue.     

Molecular and structural characterization of the biosurfactant produced by Pseudomonas aeruginosa DAUPE 614

Pseudomonas aeruginosa DAUPE 614 produced rhamnolipids (3.9gL(-1)) when cultivated on a medium containing glycerol and ammonium nitrate. These rhamnolipids reduced the surface tension of water to 27.3mNm(-1), with a critical micelle concentration of 13.9mgL(-1). The maximum emulsification index against toluene was 86.4%. The structure of the carbohydrate moiety of the glycolipid was determined by gas chromatography-mass spectroscopy (GC-MS) analysis allied to electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR) 1D, 2D (13)C, (1)H spectroscopy. The hydroxyl fatty acids were analyzed by GC-MS as hydroxy-acetylated fatty acid methyl ester derivatives. The positions of the fatty acids in the lipid moiety were variable, with 6 mono-rhamnolipid homologues (Rha-C(10)-C(10); Rha-C(10)-C(8); Rha-C(8)-C(10); Rha-C(10)-C(12:1); Rha-C(12)-C(10); Rha-C(10)-C(12)) and 6 di-rhamnolipid homologues (Rha(2)-C(10)-C(10); Rha(2)-C(10)-C(8); Rha(2)-C(8)-C(10); Rha(2)-C(10)-C(12:1); Rha(2)-C(12)-C(10); Rha(2)-C(10)-C(12)). The ratio of Rha(2)-C(10)-C(10) to Rha-C(10)-C(10) was higher than has been reported in previous studies. Our methodology allowed us to distinguish between the isomeric pairs Rha-C(10)-C(8)/Rha-C(8)-C(10), Rha-C(10)-C(12)/Rha-C(12)-C(10), Rha(2)-C(10)-C(8)/Rha(2)-C(8)-C(10) and Rha(2)-C(12)-C(10)/Rha(2)-C(10)-C(12). For each isomeric pair, the congener with the shorter chain adjacent to the sugar was always more abundant than the congener with longer chain.     

Production of rhamnolipids in solid-state cultivation using a mixture of sugarcane bagasse and corn bran supplemented with glycerol and soybean oil

Rhamnolipid biosurfactants are attracting attention due to their low toxicity, high biodegradability, and good ecological acceptability. However, production in submerged culture is made difficult by severe foaming problems. Solid-state cultivation (SSC) is a promising alternative production method. In the current work, we report the optimization of rhamnolipid production by Pseudomonas aeruginosa UFPEDA 614 on a solid substrate containing sugarcane bagasse and corn bran. The best rhamnolipid production, 45 g/l of impregnating solution used, was obtained with a 50:50 (m/m) mixture of sugarcane bagasse and corn bran supplemented with an impregnating solution containing 6% (v/v) of each of glycerol and soybean oil. This level is comparable with those of previous studies undertaken in solid-state cultivation; the composition of the biosurfactant is similar, but our medium is cheaper. Our work therefore provides a suitable basis for future studies of the development of an SSC-based process for rhamnolipid production.     

Production and characterization of cellulolytic enzymes from the thermoacidophilic fungal Aspergillus terreus M11 under solid-state cultivation of corn stover

The production of extracellular cellulases by a newly isolated thermoacidophilic fungus, Aspergillus terreus M11, on the lignocellulosic materials was studied in solid-state fermentation (SSF). The results showed that the high-level cellulase activity was produced at 45 degrees C pH 3 and moisture 80% with corn stover and 0.8% yeast extract as carbon and nitrogen sources. 581 U endoglucanase activity, 243 U filter paper activity and 128 U beta-glucosidase activity per gram of carbon source were obtained in the optimal condition. Endoglucanase and beta-glucosidase exhibited their maximum activity at pH 2 and pH 3, respectively, and both of them showed remarkable stability in the range of pH 2-5. The activities of endoglucanase and beta-glucosidase were up to the maximum at 70 degrees C and maintained about 65% and 53% of their original activities after incubation at 70 degrees C for 6h. The enzyme preparations from this strain were used to hydrolyze Avicel. Higher hydrolysis yields of Avicel were up to 63% on 5% Avicel (w/v) for 72 h with 20 U FPase/g substrate.     

Production of pyoverdine, the fluorescent pigment of Pseudomonas aeruginosa PAO1

Abstract The nutritional requirements for the production of pyoverdine were studied using Pseudomonas aeruginosa PAO₁ in a chemically defined medium, with shaking. Succinic acid and ammonium sulphate were found to be the best sources of carbon and nitrogen for pyoverdine production. The optimum carbon-to-nitrogen ratio was found to be 4:1. Elevated concentrations of phosphate inhibited pyoverdine production.     

铜绿假单胞菌外毒素A的生产,分离纯化和鉴定

通过对培养基组成、种子活化、接种量和培养条件进行优化,使铜绿假单胞菌外毒素Ａ（ＰＥ）产量达到每毫升５－１０μｇ和１９２小鼠ＬＤ５０,不低于国外报道水平。经二步纯化,ＰＥ蛋白回收率为３３．３３％（ＰＥ）和１６．６７％（ＬＤ５０）,提纯系数为４３８．５（ＰＥ）或２１８．５（ＬＤ５０）,ＳＤＳ－ＰＡＧＥ呈现一条带,相对分子质量为６６０００,琼脂糖扩散鉴定与兔抗ＰＥ产生一条沉淀线,小鼠半数致死量为０．１５     

Characterization of the amylovorin locus of Lactobacillus amylovorus DCE 471, producer of a bacteriocin active against Pseudomonas aeruginosa, in combination with colistin and pyocins

Lactobacillus amylovorus DCE 471 produces amylovorin L, a bacteriocin with an antibacterial activity against some strains of the Lactobacillus lineage. Based on the sequence of one active peptide, a gene encoding active amylovorin L was cloned and sequenced. Genome walking allowed us to sequence a larger fragment of 7577 bp of genomic DNA, with 12 predicted ORFs. The previously characterized amylovorin L peptide-encoding gene is preceded by another gene encoding a small polypeptide with a typical bacteriocin-processing double-glycine site, suggesting that amylovorin L is a two-component class IIb bacteriocin (amylovorin Lalpha/beta). Lalpha and Lbeta show the highest similarity to gassericin T from Lactobacillus gasseri SBT2055 and BlpN from Streptococcus pneumoniae R6, respectively, and to LafA and LafX, which form the lactacin F bacteriocin of Lactobacillus johnsonii NCC 533. As for other lactic acid bacteria bacteriocins, amylovorin L showed no activity against the Gram-negative opportunistic pathogen Pseudomonas aeruginosa on its own, but showed synergistic inhibitory activity when used in combination with the peptide antibiotic colistin, and, remarkably, with the P. aeruginosa soluble bacteriocins, pyocins S1 and S2.     

Cassava wastewater as a substrate for the simultaneous production of rhamnolipids and polyhydroxyalkanoates by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Glycerol, cassava wastewater (CW), waste cooking oil and CW with waste frying oils were evaluated as alternative low-cost carbon substrates for the production of rhamnolipids and polyhydroxyalkanoates (PHAs) by various Pseudomonas aeruginosa strains. The polymers and surfactants produced were characterized by gas chromatography–mass spectrophotometry (MS) and by high-performance liquid chromatography–MS, and their composition was found to vary with the carbon source and the strain used in the fermentation. The best overall production of rhamnolipids and PHAs was obtained with CW with frying oil as the carbon source, with PHA production corresponding to 39% of the cell dry weight and rhamnolipid production being 660 mg l⁻¹. Under these conditions, the surface tension of the culture decreased to 30 mN m⁻¹, and the critical micelle concentration was 26.5 mg l⁻¹. It would appear that CW with frying oil has the highest potential as an alternative substrate, and its use may contribute to a reduction in the overall environmental impact generated by discarding such residues.     

Adaptive evolution of Methylotuvimicrobium alcaliphilum to grow in the presence of rhamnolipids improves fatty acid and rhamnolipid production from CH4

Rhamnolipids (RLs) are well-studied biosurfactants naturally produced by pathogenic strains of Pseudomonas aeruginosa. Current methods to produce RLs in native and heterologous hosts have focused on carbohydrates as production substrate; however, methane (CH₄) provides an intriguing alternative as a substrate for RL production because it is low cost and may mitigate greenhouse gas emissions. Here, we demonstrate RL production from CH₄ by Methylotuvimicrobium alcaliphilum DSM19304. RLs are inhibitory to M. alcaliphilum growth (<0.05 g/l). Adaptive laboratory evolution was performed by growing M. alcaliphilum in increasing concentrations of RLs, producing a strain that grew in the presence of 5 g/l of RLs. Metabolomics and proteomics of the adapted strain grown on CH₄ in the absence of RLs revealed metabolic changes, increase in fatty acid production and secretion, alterations in gluconeogenesis, and increased secretion of lactate and osmolyte products compared with the parent strain. Expression of plasmid-borne RL production genes in the parent M. alcaliphilum strain resulted in cessation of growth and cell death. In contrast, the adapted strain transformed with the RL production genes showed no growth inhibition and produced up to 1 μM of RLs, a 600-fold increase compared with the parent strain, solely from CH₄. This work has promise for developing technologies to produce fatty acid-derived bioproducts, including biosurfactants, from CH₄.     

高效氯氰菊酯降解菌CH7的分离鉴定及降解条件的优化

从农药厂活性污泥中,分离到一株能以高效氯氰菊酯为唯一碳源生长的细菌CH7。经生理生化试验和16S rD-NA分析,将菌株CH7鉴定为铜绿假单胞菌(Pseudomonas aeruginosa)。采用Box-behnken设计试验、响应面法(response surfacemethodology)优化菌株CH7的降解条件。在最优条件下(29.4°C,pH7.0,接种量0.15g/L),菌株CH7在12d内对100mg/L高效氯氰菊酯的降解率为90%。     

Pilot testing of a bioremediation system for water and soils contaminated with heavy metals: vegetable depuration module

Abstract

We present a novel constructed wetland called a vegetable depuration module (VDM) as a pilot test of a bioremediation system (BS) for decontaminating water and soil polluted with heavy metals. The VDM consisted of a pool filled with stones of different granulometry and a substrate top layer composed of a mixture of soil and volcanic ash (50:50, v/v) supplemented with 350?ppm Zn. The BS of sunflower plants colonized by the arbuscular mycorrhizal fungus Rhizophagus intraradices was planted in the VDM. Initially, the substrate registered high concentrations of Zn, Cr, Mn, Cu, and Sr, and had Eh > +500?mV and pH 8.4. Irrigation with a Cu solution by vertical flow was carried out. After 3?months, bioaccumulation factors ranged from 1.00 to 8.90, and translocation rates were >1 for Sr and Cu. Total metals extracted by the BS and percolation were 31%, 34%, 50%, 45%, and 57% for Zn, Cu, Mn, Cr, and Sr, respectively. Only the BS was capable of extracting 94% of Cu and 38% of Zn. VDM allowed us to calibrate the extractive performance of the studied elements in BS. This biotechnological development holds great potential for phytoremediation of polluted areas.     

Characterization of Pseudomonas aeruginosa isolates from dogs and cats in Japan: current status of antimicrobial resistance and prevailing resistance mechanisms

Seventy-three Pseudomonas aeruginosa isolates were collected from dogs and cats in Japan to investigate antimicrobial susceptibility and resistance mechanisms to anti-pseudomonal agents. Resistance rates against orbifloxacin, enrofloxacin, ciprofloxacin, cefotaxime, aztreonam and gentamicin were 34.2, 31.5, 20.5, 17.8, 12.3 and 4.1%, respectively. The degree of resistance to cefotaxime, orbifloxacin, and enrofloxacin was greatly affected by efflux pump inhibitors, indicating overexpression of efflux pump contributes to these resistances. Notably, orbifloxacin and enrofloxacin resistance was observed even in isolates without mutations in the target sites. This is the first report on cephalosporin- and fluoroquinolone-resistant isolates of P. aeruginosa from Japanese companion animals.     

Focus: Antimicrobial Resistance: The Intriguing Carbapenemases of Pseudomonas aeruginosa: Current Status,Genetic Profile,and Global Epidemiology

Worldwide, Pseudomonas aeruginosa remains a leading nosocomial pathogen that is difficult to treat and constitutes a challenging menace to healthcare systems. P. aeruginosa shows increased and alarming resistance to carbapenems, long acknowledged as last-resort antibiotics for treatment of resistant infections. Varied and recalcitrant pathways of resistance to carbapenems can simultaneously occur in P. aeruginosa, including the production of carbapenemases, broadest spectrum types of β-lactamases that hydrolyze virtually almost all β-lactams, including carbapenems. The organism can produce chromosomal, plasmid-encoded, and integron- or transposon-mediated carbapenemases from different molecular classes. These include Ambler class A (KPC and some types of GES enzymes), class B (different metallo-β-lactamases such as IMP, VIM, and NDM), and class D (oxacillinases with carbapenem-hydrolyzing capacity like OXA-198) enzymes. Additionally, derepression of chromosomal AmpC cephalosporinases in P. aeruginosa contributes to carbapenem resistance in the presence of other concomitant mechanisms such as impermeability or efflux overexpression. Epidemiologic and molecular evidence of carbapenemases in P. aeruginosa has been long accumulating, and reports of their existence in different geographical areas of the world currently exist. Such reports are continuously being updated and reveal emerging varieties of carbapenemases and/or new genetic environments. This review summarizes carbapenemases of importance in P. aeruginosa, highlights their genetic profile, and presents current knowledge about their global epidemiology.     

Production,Purification, and Antibiofilm Activity of a Novel Exopolysaccharide from Arthrobacter sp. B4

A novel exopolysaccharide (EPS), namely, B4-EPS, is produced by Arthrobacter sp. B4. Response surface methodology (RSM) was employed to optimize the fermentation medium for increasing B4-EPS production. Based on Plackett–Burman design (PBD), glucose, yeast extract, and KH₂PO₄ were selected as significant variables, which were further optimized by a central composite design (CCD). According to response surface and canonical analysis, the optimal medium was composed of 16.94 g/L glucose, 2.33 g/L yeast extract, and 5.32 g/L KH₂PO₄. Under this condition, the maximum yield of B4-EPS reached about 8.54 g/L after 72 hr of batch fermentation, which was pretty close to the predicted value (8.52 g/L). Furthermore, B4-EPS was refined by column chromatography. The main homogeneous fraction (B4-EPS1) was collected and applied to assay of antibiofilm activity. B4-EPS1 exhibited a dose-dependent inhibitory effect on biofilm formation of Pseudomonas aeruginosa PAO1 without antibacterial activity. About 86.1% of biofilm formation of P. aeruginosa PAO1 was inhibited in the presence of 50 µg/mL B4-EPS1, which was more effective than the peer published data. Moreover, B4-EPS1 could prevent biofilm formation of other strains. These data suggest B4-EPS may represent a promising strategy to combat bacterial biofilms in the future.     

Development and application of a polymicrobial, in vitro, wound biofilm model

Aims: The goal of this investigation was to develop an in vitro, polymicrobial, wound biofilm capable of supporting the growth of bacteria with variable oxygen requirements. Methods and Results: The strict anaerobe Clostridium perfringens was isolated by cultivating wound homogenates using the drip‐flow reactor (DFR), and a three‐species biofilm model was established using methicillin‐resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and Cl. perfringens in the colony‐drip‐flow reactor model. Plate counts revealed that MRSA, Ps. aeruginosa and Cl. perfringens grew to 7·39 ± 0·45, 10·22 ± 0·22 and 7·13 ± 0·77 log CFU per membrane, respectively. The three‐species model was employed to evaluate the efficacy of two antimicrobial dressings, Curity? AMD and Acticoat?, compared to sterile gauze controls. Microbial growth on Curity? AMD and gauze was not significantly different, for any species, whereas Acticoat? was found to significantly reduce growth for all three species. Conclusions: Using the colony‐DFR, a three‐species biofilm was successfully grown, and the biofilms displayed a unique structure consisting of distinct layers that appeared to be inhabited exclusively or predominantly by a single species. Significance and Impact of the Study: The primary accomplishment of this study was the isolation and growth of an obligate anaerobe in an in vitro model without establishing an artificially anaerobic environment.     

Production of recombinant RNase Ba and its application in downstream processing of plasmid DNA for pharmaceutical use

The demand for new strategies in downstream processing of biopharmaceutical plasmid DNA has increased in response to the importance of nucleic acids as active pharmaceutical ingredients (API) in gene therapy and genetic vaccination. Led by the problematic usage of animal-derived proteins for producing reagents of clinical applications, we present an opportunity of removing RNA prior to chromatographic steps by using a recombinant RNase Ba (barnase of Bacillus amyloliquefaciens) as an alternative to bovine RNase A. An expression vector for RNase Ba production was constructed enabling periplasmic localization of the recombinant protein. Cultivation of the RNase-producing clone showed stable activity (3.6 kU mL(-1) during stationary phase) throughout the cultivation process. After purification the RNase activity was tested and compared to that of commercially available RNase A. RNase Ba showed no DNase activity even after prolonged incubation with plasmid DNA. Thus, it is a suitable substitute for bovine RNase A in pharmaceutical purification processes.