Majority of Actinomadura clinical isolates from sputa or bronchoalveolar lavage fluid in Japan belongs to the cluster of Actinomadura cremea and Actinomadura nitritigenes, and the description of Actinomadura chibensis sp. nov

In Japan during 1996-2004, 21 actinomycete strains that have madurose as the diagnostic cell-wall sugar and show true branching in their substrate and aerial mycelia were isolated from sputa or bronchoalveolar lavage (BAL) fluid of patients with pulmonary infections or who were suspected of having related infections. Chemotaxonomic studies showed that all the isolates belong to the genus Actinomadura. Among them, six and seven strains were classified respectively into clusters of Actinomadura nitritigenes and Actinomadura cremea based on 16S rDNA analyses because their 16S rDNA similarities to those respective species were greater than 99.5%. To our knowledge, this is first report that strains of above two species were isolated from clinical specimens. Neither Actinomadura madurae nor Actinomadura pelletieri strain was isolated, and one new species, Actinomadura chibensis, was proposed; the remaining seven strains were not assigned into any known species, suggesting the presence of another new Actinomadura species.     

利用响应面法优化固氮类芽孢杆菌Paenibacillus sp.1–49的发酵培养基（英文）北大核心CSCD

【目的】固氮类芽孢杆菌Paenibacillus sp.1–49是本实验室分离的具有潜在生物肥料价值的微生物菌剂。该菌在常见的培养基中不能高效生长(OD_(600)≤1)。本文拟优化发酵培养基成分以获得最大菌体生物量。【方法】运用响应面分析中的因素筛选实验设计和最陡爬坡实验以及中心复合设计法,对菌株的发酵培养基进行了响应面优化。【结果】利用响应面优化法最终确定了菌株最佳培养基:蔗糖36.22g/L,Tryptone 5.31 g/L,Yeast Extract 10.92 g/L,Mg SO_4 0.51 g/L,Na Cl 3.5 g/L,Na_2Mo O_4 0.02 g/L,FeS O_4 0.02 g/L。通过摇瓶发酵后菌体OD600=10.280,达到预测值的94.6%。【结论】利用响应面法成功地对Paenibacillus sp.1-49的发酵培养基进行了优化,为该菌株和其他类芽孢杆菌的大规模发酵提供了参考。     

Improved process for exopolysaccharide production by Klebsiella pneumoniae sp. pneumoniae by a fed-batch strategy

Exopolysaccharide (EPS) was produced by Klebsiella pneumoniae K63 grown in fed-batch cultures using different procedures of the supply of carbon or nitrogen (N) source, or both. Cultures grown with excess of glucose and limitation or exhaustion of N produced 54.8 and 47.4 g(EPS) l(-1), respectively. These cultures also led to an accumulation of 'overflow' metabolites representing more than 16% of carbon conversion. The consistency indexes ( K ) obtained to the end of the cultures, characteristic of the rheological property of the biopolymer, were 16.4 Pa s(n) for N deficiency and 5.2 Pa s(n) for N limitation conditions. The simultaneous limitation of glucose and N decreased the excretion of co-metabolites (6.4% of carbon conversion) and the EPS production (18.1 g(EPS) l(-1)), while improving the quality of the polysaccharide, characterized by the highest K of 126.2 Pa s(n) and the highest pseudoplasticity degree (flow behaviour index, n=0.2).     

Ethanol production from H2 and CO2 by a newly isolated thermophilic bacterium, Moorella sp. HUC22-1

The thermophilic bacterium, Moorella sp. HUC22-1, newly isolated from a mud sample, produced ethanol from H(2) and CO(2) during growth at 55 degrees C. In batch cultures in serum bottles, 1.5 mM ethanol was produced from 270 mM H(2) and 130 mM CO(2) after 156 h, whereas less than 1 mM ethanol was produced from 23 mM fructose after 33 h. Alcohol dehydrogenase and acetaldehyde dehydrogenase activities were higher in cells grown with H(2) and CO(2) than those grown with fructose. The NADH/NAD(+) and NADPH/NADP(+) ratios in cells grown with H(2) and CO(2) were also higher than those in cells grown with fructose. When the culture pH was controlled at 5 with H(2) and CO(2) in a fermenter, ethanol production was 3.7-fold higher than that in a pH-uncontrolled culture after 220 h.     

Geobacillus galactosidasius sp. nov., a new thermophilic galactosidase-producing bacterium isolated from compost

Two thermophilic spore-forming strains, with optimum growth temperature at 70 °C, were isolated from compost of the “Experimental System of Composting” (Teora, Avellino, Italy). A phylogenetic analysis based on 16S rRNA gene sequences showed that these organisms represented a new species of the genus Geobacillus. Based on polyphasic taxonomic data the strains represented a novel species for which the name Geobacillus galactosidasius sp. nov. is proposed. The type strain is CF1B^T (= ATCC BAA-1450^T = DSM 18751^T).     

Process optimization for production and purification of novel fibrinolytic enzyme from Stenotrophomonas sp. KG-16-3

Intravascular thrombosis is a major cardiovascular complication responsible for high mortality worldwide. Existing thrombolytic agents are expensive and have various side effects. As a consequence, researchers continue to search for better thrombolytic agents. Fibrinolytic proteases especially those of microbial origin are considered as potential therapeutic candidates for thrombosis. The current study reports fibrinolytic protease from a bacterial isolate Stenotrophomonas sp. KG-16-3, as it exhibits high fibrinolytic activity on fibrin agarose plate. Studies on fibrinolytic protease from Stenotrophomonas sp. are lacking. So, a detailed study was conducted for the production and purification of fibrinolytic protease. Optimizing process parameters using the Design of Experiments method enhanced the yield by 1.5-fold. The fibrinolytic enzyme was purified by ammonium sulfate precipitation, ion-exchange and gel-filtration chromatography resulting in 7.1-fold purification and 16.7% yield with specific activity of 383.8?U/mg. The purified enzyme exhibited higher fibrinolytic activity than plasmin and had a molecular weight of 39?kDa. Optimal activity of the enzyme was observed at 50?°C and pH 10. The enzyme exhibited stability up to 60?°C, over pH 7–10 and in the presence of different metal ions and solvents. The activity of the enzyme was significantly reduced in the presence of phenylmethyl sulfonyl fluoride, iodoacetic acid and 1,10-phenanthroline, suggesting that the enzyme belonged to the serine–cysteine metalloprotease category. The present study is the first ever report on the Design of Experiments based optimization of fermentation conditions for the production of fibrinolytic protease from Stenotrophomonas sp.     

Investigation of lipase production by a new isolate of Aspergillus sp.

Fungi isolated from soil were screened for exogenous lipolytic activity. The highest lipase activity was found in a new soil isolate of Aspergillus sp. Some optimal cultural parameters influencing the growth and production of extracellular lipase from this Aspergillus sp. were investigated. The lipase yield was maximum on day 4 of incubation of the culture at pH 5.5 and 30 °C. When the medium was prepared using olive oil as carbon source and peptone as a nitrogen source, better lipase yields were obtained. Aeration enhanced growth and lipase production.     

Characterization and optimization of extracellular alkaline lipase production by Alcaligenes sp. Using stearic acid as carbon source

The aim of this study was to improve the production of an extracellular alkaline lipase from Alcaligenes sp. (ATCC 31371) by optimization of the culture medium, for economic production of biodiesel from waste vegetable oil. A number of carbon sources including different types of starch, sugar, sugar alcohol, organic acids, and surfactants were investigated. Polyoxyethylene (20) sorbitan tristearate, whose side chain is stearic acid, was the most effective carbon source for lipase production. Box-Behnken experimental design was used for three factors (soy protein, sodium nitrate, and stearic acid) and the optimal composition for maximum lipase production (1.7-fold enhancement) was established as soy protein 4.07%, sodium nitrate 0.17%, and stearic acid 0.28% at 28°C with an agitation rate of 220 rpm for 24 h. The enzyme was purified to homogeneity and the recovery of the lipase activity was 7.8% with a 30-fold purification. The estimated molecular size of the protein determined by SDS-PAGE was 33 kDa. The optimum pH and temperature of the purified lipase was 8.5 and 40°C, respectively. The purified enzyme was stable in the pH range of 6.0 and 9.5 and in the temperature range of 20 and 50°C.     

Induction and purification of a thermophilic chitinase produced byAeromonas sp. DYU-too7 using glucosamine

In the presence of chitin,Aeromonas sp. DYU-Too7 can produce extra-cellular, chitin-degrading enzymes. Chitin analogues and other carbon sources can be used to cultivate this bacterial strain. The chitinases produced by the strain were higher in the GIcN (glucosamine) medium than those in other media. The maximal chitinase activity occurred in the medium containing 0.1% GIcN. Cultivation ofAeromonas sp. DYU-Too7 in the GIcN medium sped up the chitinase production; however the same result did not appear when it was cultivated in the (Chitin+GIcN) medium. This result may indicate that GIcN can be utilized byAeromonas sp. DYU-Too7 as a carbon source and an inducer to produce chitinases. A chitinase with a molecular mass of 36 kDa was further purified and characterized to have an optimal reacting pH of 5.0 and an optimal reacting temperature of 50°C. This chitinase showed high stability in the proximity of 30°C and also high stability in the proximity of pH 7.0. The hydrolysates of colloidal chitin, with the aid of the 36-kDa chitinase, were analyzed by an HPLC and found to be chitobiose.     

Improved production of an enzyme that hydrolyses raw yam starch by Penicillium sp. S-22 using fed-batch fermentation

A newly isolated strain, Penicillium sp. S-22, was used to produce an enzyme that hydrolyses raw yam starch [raw yam starch digesting enzyme (RYSDE)]. The enzyme activity and overall enzyme productivity were respectively 16 U/ml and 0.19 U/ml h in the batch culture. The enzyme activity increased to 85 U/ml by feeding of partially hydrolyzed raw yam starch. When a mixture containing partially hydrolyzed raw yam starch and peptone was fed by a pH-stat strategy, the enzyme activity reached 366 U/ml, 23-fold of that obtained in the batch culture, and the overall productivity reached 3.4 U/ml h, which was 18-fold of that in the batch culture.     

Biotransformation of halophenols by a thermophilic Bacillus sp.

The thermophilic Bacillus sp. A2 transformed various halophenols. 2-Chlorophenol, 2-bromophenol, 3-bromophenol and 2-fluorophenol were transformed under resting cell conditions at 60°C to 3-chlorocatechol, 3-bromocatechol, 4-bromocatechol and 3-fluorocatechol, respectively. The hydroxylation of 3-bromophenol occurred at the proximal and distal position relative to the halogen substituent. In complex medium this strain completely transformed 2-chlorophenol and 2-bromophenol at concentrations up to 1 mM. Concomitantly, an accumulation of oxygen-and temperature sensitive halocatechols was observed. 3-Chlorocatechol possesses a half-life of 11.5 h at 60°C and is therefore readily decomposed during incubation. The hydroxylating system was present in phenolgrown cells but not in glucose-grown cells. The hydroxylase activity could also be induced by 2-chlorophenol. The product, 3-chlorocatechol, is not a substrate for the catechol 2,3-dioxygenase.Abbreviations 2-CP 2-chlorophenol - DCP dichlorophenol - TCP trichlorophenol - tetraCP tetrachlorophenol - MIC minimal inhibitory concentration - CF chloride-free - CFG chloride-free plus glucose - CFGY chloride-free plus glycerol - CFP chloride-free plus phenol - CAM chloramphenicol     

Statistical optimization of culture media for production of phycobiliprotein by Synechocystis sp. PCC 6701

Synechocystis sp. PCC 6701 has a brilliantly colored pigment, phycobiliprotein containing phycoerythrin. Culture medium was optimized by sequential designs in order to maximize phycobiliprotein production. The observed fresh weights after 6 days were 0.58 g/L in BG-11, 0.83 g/L in medium for Scenedesmus sp. and 0.03∼0.52 g/L in the other tested media. Medium for Scenedesmus sp. was selected to be optimized by fractional factorial design and central composite design since the medium maintained a more stable pH within a desirable range due to higher contents of phosphate. The fractional factorial design had seven factors with two levels: KNO₃, NaNO₃, NaH₂PO₄, Na₂HPO₄, Ca(NO₃)₂, FeEDTA, and MgSO₄. From the result of fractional factorial design, nitrate and phosphate were identified as significant factors. A central composite design was then applied with four variables at five levels each: nitrate, phosphate, pH, and light intensity. Parameters such as fresh weight and phycobiliprotein contents were used to determine the optimum value of the four variables. The proposed optimum media contains 0.88 g/L of nitrate, 0.32 g/L of phosphate under 25 μE·m⁻²·s⁻¹ of light intensity. The maximum phycobiliprotein contents have been increased over 400%, from 4.9 to 25.9 mg/L after optimization.     

Development of a process for the production of the anticancer lead compound pleurotin by fermentation of Hohenbuehelia atrocaerulea

Pleurotin is a naphthoquinone antibiotic originally isolated from Pleurotus griseus. Two pleurotin producing strains of Hohenbuehelia atrocaerulea have been identified, which, on solid substrate fermentation for 2 months yield 1-2 mg/l of the antibiotic. Described here is the lengthy developmental process which resulted in a production protocol being developed which reliably yields pleurotin from liquid fermentation at >300 mg/l. Critical to obtaining this increase in titer was inclusion in the media of an aqueous extract of alder wood.     

Statistical analysis and validation of process parameters influencing lovastatin production by Monascus purpureus MTCC 369 under solid-state fermentation

Monascus, a fermented rice (red mold rice), was found to reduce total cholesterol and triglyceride in serum due to the presence of lovastatin, a 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitor. Optimization and validation of different process parameters such as temperature, fermentation time, inoculum volume, and pH of the solid medium was done using Box-Behnken’s factorial design of response surface method for maximum production of lovastatin by Monascus purpureus MTCC 369. A maximum lovastatin production of 3.422 mg/g was predicted by day 14.43 of fermentation in a rice based solid medium of pH 6 when fermented at a temperature of 29.46°C, an inoculum volume of 5.11 mL, and using response surface plots and the point prediction tool of Design Expert 7.1.3 (Statease Inc., USA) software.     

Herminiimonas aquatilis sp. nov., a new species from drinking water

A bacterial strain (CCUG 36956T) isolated from drinking water was taxonomically studied in detail. Phylogenetic analyses using the 16S rRNA gene sequence of the isolate indicated that it belongs to family Oxalobacteraceae of the beta-subclass of the Proteobacteria, with the highest sequence similarity of 99.3% to the type strain of Herminiimonas fonticola. In the polyamine pattern putrescine and 2-hydroxyputrescine were the predominant compounds. In the polar lipid profile major compounds were phosphatidyl ethanolamine and diphosphatidyl glycerol. Phosphatidyl glycerol and an unknown phospholipid were detected in moderate proportions. The major respiratory quinone was a ubiquinone Q-8 and the major whole cell fatty acids were 16:1 omega7c, 17:1 omega6c, and 16:0. The strain also contained 10:0 3-OH and other fatty acids typical for members of the genus Herminiimonas. The results of DNA-DNA hybridizations and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain CCUG 36956T from H. fonticola. For this reason, we propose that strain CCUG 36956T represents a new species of the genus Herminiimonas for which we propose the name Herminiimonas aquatilis sp. nov.     

Pullulanases of alkaline and broad pH range from a newly isolated alkalophilicBacillus sp. S-1 and aMicrococcus sp. Y-1

Summary Two highly alkalophilic bacteria, and potent producers of alkaline pullulanase, were isolated from Korean soils. The two isolates, identified asBacillus sp. S-1 andMicrococcus sp. Y-1, grow on starch under alkaline conditions and effectively secrete extracellular pullulanases. The two isolates were extremely alkalophilic since bacterial growth and enzyme production occurred at pH values ranging from pH 6.0 to 12.0 forMicrococcus sp. Y-1 and pH 6.0 to 10.0 forBacillus sp. S-1. Both strains secrete enzymes that possess amylolytic and pullulanolytic acitivities. Extracellular crude enzymes of both isolates gave maltotriose as the major product formed from soluble starch and pullulan hydrolysis. Compared to other alkalophilic microbes such asMicrococcus sp. (0.57 units ml^–1),Bacillus sp. KSM-1876 (0.56 units ml^–1) andBacillus No. 202-1 (1.89 units ml^–1) these isolates secreted extremely high concentrations (7.0 units ml^–1 forBacillus sp. S-1 and 7.6 units ml^–1 forMicrococcus sp. Y-1) of pullulanases in batch culture. The pullulanase activities from both strains were mostly found in the culture medium (85–90%). The extracellular enzymes of both bacteria were alkalophilic and moderately thermoactive; optimal activity was detected at pH 8.0–10.0 and between 50 and 60°C. Even at pH 12.0, 65% of original Y-1 pullulanase activity and 10% of S-1 pullulanase activity remained. The two newly isolated strains had broad pH ranges and moderate thermostability for their enzyme activities. These result strongly indicate that these new bacterial isolates have potential as producers of pullulanases for use in the starch industry.     

An acidic and thermostable carboxymethyl cellulase from the yeast Cryptococcus sp. S-2: purification, characterization and improvement of its recombinant enzyme production by high cell-density fermentation of Pichia pastoris

The extracellular carboxymethyl cellulase (CSCMCase) from the yeast, Cryptococcus sp. S-2, was produced when grown on cellobiose. It was purified to homogeneity from the supernatant by ultrafiltration, DEAE-5PW anion exchange column and TSK-Gel G3000SW gel filtration. The purified enzyme was monomeric protein with molecular mass of approximately 34 kDa. The optimum temperature and pH for the action of the enzyme were at 40–50 °C and 3.5, respectively. It was stable at pH range of 5.5–7.5 and retained approximately 50% of its maximum activity after incubating at 90 °C for 1 h. Moreover, it could able to hydrolyze carboxymethyl cellulose sodium salt higher than insoluble cellulose substrate such as Avicel, SIGMACELL^® and CM cellulose. Due to its action at acidic pH and moderately stable at high temperature, the gene encoding carboxymethyl cellulase (CSCMCase) was isolated and improved the enzyme yield by high cell-density fermentation of Pichia pastoris. The CSCMCase cDNA contains 1023 nucleotides and encodes a 341-amino acid. It was successfully expressed under the control of alcohol oxidase I promoter using methanol induction of P. pastoris fermentation in a 2L ABLE bioreactor. The production of the recombinant carboxymethyl cellulases was higher than that from Cryptococcus sp. S-2 of 657-fold (2.75 and 4.2 × 10⁻³ mg protein L⁻¹, respectively) indicating that the leader sequence of CSCMCase has been recognized and processed as efficiently by P. pastoris. Furthermore, the recombinant enzyme was purified in two-step of ultrafiltration and hydrophobic interaction chromatography which would be much more convenient for large-scale purification for successful industrial application.     

Polyhydroxybutyrate accumulation by a Serratia sp.

A strain of Serratia sp. showed intracellular electron-transparent inclusion bodies when incubated in the presence of citrate and glycerol 2-phosphate without nitrogen source following pre-growth under carbon-limitation in continuous culture. About 1.3 mmol citrate were consumed per 450 mg biomass, giving a calculated yield of maximally 55% of stored material per g of biomass dry wt. The inclusion bodies were stained with Sudan Black and Nile Red (NR), suggesting a lipid material, which was confirmed as polyhydroxybutyrate (PHB) by analysis of molecular fragments by GC and by FTIR spectroscopy of isolated bio-PHB in comparison with reference material. Multi-parameter flow cytometry in conjunction with NR fluorescence, and electron microscopy, showed that not all cells contained heavy PHB bodies, suggesting the potential for increasing the overall yield. The economic attractiveness is enhanced by the co-production of nanoscale hydroxyapatite (HA), a possible high-value precursor for bone replacement materials.