Combined solvent and water activity stresses on turgor regulation and membrane adaptation in Oceanimonas baumannii ATCC 700832

Oceanimonas baumannii ATCC 700832 is a Gram negative marine bacterium capable of utilising phenol as a sole carbon source. The ability of the bacterium to tolerate low water activity when utilising either succinate or phenol as a substrate in minimal medium was studied. The membrane lipid and protein composition showed two discreet adaptive phases as salinity increased. Firstly, when NaCl concentration was increased from 0.15% (w/v), the minimum at which growth was observed, to 1% NaCl (w/v), the ratio of zwitterionic to anionic phospholipids in the membrane increased significantly. At the same time the ratio of saturated to unsaturated fatty acids and the total membrane protein decreased significantly. The second phase was observed when salinity was increased from 1% to 7% NaCl (w/v) as the ratio of zwitterionic to anionic phospholipids decreased and membrane protein increased. However, the ratio of saturated to unsaturated fatty acids was unaffected. Salinity also affected the tolerance of cultures to elevated levels of phenol. Cultures grown in 0.15% NaCl (w/v) could tolerate 12 mM phenol, whereas in the presence of 1% NaCl (w/v) cultures continued to grow in up to 20 mM phenol and in 7% NaCl (w/v) cultures 8 mM phenol could be tolerated. Changes to the composition of the membrane phospholipids and fatty acids were also observed when phenol concentrations were at the maximum that could be tolerated. Under such conditions the ratio of zwitterionic to anionic phospholipids decreased twofold compared to cultures utilising 4 mM phenol as the substrate, in all salinities except in 7% NaCl (w/v) cultures, where there was no significant effect. The ratio of saturated to unsaturated fatty acids increased significantly in all salinities compared to cultures grown with 4 mM phenol. This revised version was published online in June 2006 with corrections to the Cover Date.     

Determination of the kinetic parameters of the phenol-degrading thermophile Bacillus themoleovorans sp. A2

Phenolic compounds are pollutants in many wastewaters, e.g. from crude oil refineries, coal gasification plants or olive oil mills. Phenol removal is a key process for the biodegradation of pollutants at high temperatures because even low concentrations of phenol can inhibit microorganisms severely. Bacillus thermoleovorans sp. A2, a recently isolated thermophilic strain (temperature optimum 65 degrees C), was investigated for its capacity to degrade phenol. The experiments revealed that growth rates were about four times higher than those of mesophilic microorganisms such as Pseudomonas putida. Very high specific growth rates of 2.8 h(-1) were measured at phenol concentrations of 15 mg/l, while at phenol concentrations of 100-500 mg/l growth rates were still in the range of 1 h(-1). The growth kinetics of the thermophilic Bacillus thermoleovorans sp. A2 on phenol as sole carbon and energy source can be described using a three-parameter model developed in enzyme kinetics. The yield coefficient Yx/s of 0.8-1 g cell dry weight/g phenol was considerably higher than cell yields of mesophilic bacteria (Yx/s 0.40-0.52 g cell dry weight/g phenol). The highest growth rate was found at pH 6. Bacillus thermoleovorans sp. A2 was found to be insensitive to hydrodynamic shear stress in stirred bioreactor experiments (despite possible membrane damage caused by phenol) and flourished at an ionic strength of the medium of 0.25(-1) mol/l (equivalent to about 15-60 g NaCl/l). These exceptional properties make Bacillus thermoleovorans sp. A2 an excellent candidate for technical applications.     

Growth and nitrogen removal characteristics of Halomonas sp. B01 under high salinity

At present, the nitrogen (N) removal efficiency of the microbial treatment in the high-salinity nitrogenous wastewaters is relatively low. Study on the N removal behavior and properties of moderately halophilic bacteria Halomonas under high salinity is of great significance for the microbial treatment of high-salinity nitrogenous wastewater. The response mechanism of Halomonas sp. B01 to high osmotic pressure stress was investigated by measuring the compatible solute ectoine concentration and superoxide dismutase (SOD) activity. The salt tolerance during growth and N removal of the strain was evaluated by measuring the activities of growth-related and N removal–related enzymes and the mRNA expression abundance of ammonia monooxygenase-encoding gene (amoA). The process of simultaneous heterotrophic nitrification and aerobic denitrification (SND) under high salinity was described by measuring the concentration of inorganic N. Halomonas sp. B01 synthesized ectoine under NaCl stress, and the intracellular ectoine concentration increased with increased NaCl concentration in the growth medium. When the NaCl concentration of the medium reached 120 g L−1, the malondialdehyde concentration and SOD activity were significantly increased to 576.1 μg mg−1 and 1.7 U mg−1, respectively. The growth-related and N removal–related enzymes of the strain were active or most active in medium with 30–60 g L−1 NaCl. The amoA of the strain cultured in medium with 60 g L−1 NaCl had the highest mRNA expression abundance. In the N removal medium containing 60 g L−1 NaCl and 2121 mg L−1 NH4+-N, SND by Halomonas sp. B01 was performed over 96 h and the N removal rate reached 98.8%. In addition to the protective mechanism of synthetic compatible solutes, Halomonas sp. B01 had the repair mechanism of SOD for lipid peroxidation. The growth-related and N removal–related enzymes of the strain were most active at a certain salt concentration; amoA also had the highest mRNA expression abundance under high salinity. Halomonas sp. B01 could efficiently perform N removal by SND under high salinity.     

Production of α-amylase fromHalobacterium halobium

Maximum production of extracellular -amylase activity inHalobacterium halobium was at 40°C in a medium containing 25% (w/v) NaCl, 1% (w/v) soluble starch and 1% (w/v) peptone, in presence of 0.1mm ZnSO₄ after 5 days in shaking cultures. The amylase had optimal activity at pH 6.5 in the presence of 1 to 3% (w/v) NaCl at 53°C.S. Patel, N. Jain and D. Madamwar are with the Post Graduate Department of Biosciences, Sadar Patel University, Vallabh Vidyanagar-388120, India.     

Factors affecting mycelial biomass and exopolysaccharide production in submerged cultivation of Antrodia cinnamomea using complex media

Submerged cultures were used to identify growth-limiting nutrients by Antrodia cinnamomea strains. The mycelial biomass and EPS production by A. cinnamomea BCRC 35396 were markedly higher than other A. cinnamomea strains. A relatively high C/N ratio was favorable for both the mycelial growth (5.41 g/l) and EPS production (0.55 g/l); the optimum ratio was 40. The glucose was available utilized preferentially for mycelial growth, rather than for EPS production. Flushing the culture medium with nitrogen had a stimulating effect on both mycelial growth and EPS production. In addition, peptone, yeast extract and malt extract appeared to be important and significant component for EPS production. Phosphate ion, magnesium ion and thiamine were probably not essential for mycelial growth. By optimizing the effects of additional nutrition, the results showed that 5% (w/v) glucose, 0.8% (w/v) peptone, 0.8% (w/v) yeast extract, 0.8% (w/v) malt extract, 0.03% (w/v) KH2PO4, 0.1% (w/v) MgSO4 .7H2O and 0.1% (w/v) thiamine could lead to the maximum production of EPS (1.36 g/l).     

Production of beta-fructofuranosidase with transfructosylating activity for fructooligosaccharides synthesis by Aspergillus japonicus NTU-1249.

Microbial beta-fructofuranosidases with transfructosylating activity can catalyze the transfructosylation of sucrose and synthesize fructooligosaccharides. Aspergillus japonicus NTU-1249 isolated from natural habitat was found to produce a significant amount of beta-fructofuranosidase with high transfructosylating activity and to have the potential for industrial production of fructooligosaccharides. In order to improve it's enzyme productivity, the medium composition and the cultivation conditions for A. japonicus NTU-1249 were studied. A. japonicus NTU-1249 can produce 83.5 units of transfructosylating activity per ml broth when cultivated in a shaking flask at 28 degrees C for 72 hours with a modified medium containing 80 g/l sucrose, 15 g/l soybean flour, 5 g/l yeast extract and 5 g/l NaCl at an initial pH of 6.0. The enzyme productivity was also optimized by submerged cultivation in a 5-litre jar fermentor with aeration at 1.5 vvm and agitation at 500 rpm. Under these operating conditions, the productivity of transfructosylating activity increased to 185.6 U/ml. Furthermore, the transfructosylating activity was improved to 256.1 U/ml in 1,000-litre pilot-scale fermentor. Enzymatic synthesis of fructooligosaccharides by beta-fructofuranosidase from A. japonicus NTU-1249 was performed in batch type by adding 5.6 units of transfructosylating activity per gram of sucrose to a 50% (w/v) sucrose solution at pH 5.0 and 50 degrees C. The yield of fructooligosaccharides was about 60% after reaction for 24 hours, and the syrup produced contained 29.8% (w/v) fructooligosaccharides, 15.2% (w/v) glucose and 5.0% (w/v) sucrose.     

Effects of Culture Age, Washing and Storage Conditions on Desiccation Tolerance of Colletotrichum truncatum Conidia

Colletotrichum truncatum conidia produced from a one week-old culture in a liquid semi-defined medium with a C:N ratio of 5:1 were more tolerant of desiccation than those harvested from two or three week-old cultures. Conidia washed with 20% (w/v) sucrose germinated better than unwashed conidia or those washed in 10% (w/v) sucrose, 10 and 20% (w/v) glucose or fructose, 0.1% (w/v) soluble starch, 0.9% (w/v) NaCl or deionized water. Washing with sucrose (20% w/v) also resulted in significantly longer germ tubes than those produced by unwashed conidia or conidia washed with deionized water or NaCl (0.9% w/v). Conidia washed twice in sucrose showed greater desiccation tolerance during storage at 15% relative humidity (RH) and 15°C than at 30% RH and 15 or 25°C or at 15% RH and 25, 5 or -10°C.     

Biological coagulation of skim latex using Acinetobacter sp. isolated from natural rubber latex centrifugation effluent

An Acinetobacter sp, isolated from latex centrifugation effluent, effectively coagulated skim rubber from skim latex. After coagulation for 48 h without the addition of any nutrients, at an optimum dilution of 1:10(v/v) and with an inoculum concentration of 6.4 mg dry cell /ml, the yield of the skim rubber was 8 % (w/v) and the COD of the residual solution was only 0.4 g/l. chemical coagulation at the same dilution resulted in 7 % (w/v) yield of dry rubber content and 2.2 g COD /l.     

Plant regeneration from cell suspension-derived protoplasts of Primula malacoides and Primula obconica

Protoplasts were isolated from cell suspension cultures of Primula malacoides cv. ‘Lovely Tokyo’ and P. obconica cv. ‘Aalsmeer Giant White’. P. obconica protoplasts were embedded in 0.1% (w/v) gellan gum-solidified discs comprising MS medium supplemented with 3 mg/l of 2,4-D or picloram, 0.1 mg/l of zeatin, 0.2 M glucose and 0.2 M mannitol, and surrounded by a liquid medium of the same composition except for the addition of 0.1% (w/v) activated charcoal. The protoplasts formed visible colonies, which were transferred to the regeneration medium containing 30 g/l of sucrose, 0.1 mg/l of picloram and 2 mg/l of zeatin for shoot induction. P. malacoides protoplasts formed visible colonies when cultured in disc culture using 0.1% (w/v) gellan gum-solidified MS medium containing 5 mg/l of 2,4-D, 1 mg/l of NAA, 0.1 mg/l of zeatin and 0.4 M glucose. Small calli were transferred to MS medium supplemented with 5 mg/l of zeatin for shoot regeneration. The shoots of both species readily rooted on plant growth regulator-free 1/2 MS medium and successfully acclimatized to greenhouse conditions. The protoplast-derived plants showed some alterations in morphological characteristics from those of the in-vitro-germinated control plants.     

Biodegradation of saline phenolic wastewater in a biological contact oxidation reactor with immobilized cells of <Emphasis Type="Italic">Oceanimonas</Emphasis> sp.

Objective

To develop a method to treat saline phenolic wastewater in a biological contact oxidation reactor (BCOR) with immobilized cells of a marine microorganism, Oceanimonas sp., isolated from seawater.

Results

Cells were immobilized on fibre carriers in the BCOR. Saline wastewater with phenol at 1.5 g/l and NaCl at 6 % (w/v) was treated. In continuous assays, 99 % removal of phenol was achieved and a kinetic model for the phenol degradation is presented based on Monod’s equation.

Conclusion

The BOCR system using immobilized cells of Oceanimonas efficiently treats saline phenolic wastewaters without having decrease the salinity of the wastewater.

     

Optimization of γ-amino butyric acid production in a newly isolated Lactobacillus brevis

An isolate from kimchi, identified as Lactobacillus brevis, accumulated γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter, in the culture medium. Optimal culture conditions for growth of L. brevis and production of GABA were 6 % (w/v) l-glutamic acid, 4 % (w/v) maltose, 2 % (w/v) yeast extract, 1 % (w/v) NaCl, 1 % (w/v) CaCl₂, 2 g Tween 80/l, and 0.02 mM pyridoxal 5′-phosphate at initial pH 5.25 and 37 °C. GABA reached 44.4 g/l after 72 h cultivation with a conversion rate 99.7 %, based on the amount (6 %) of l-glutamic acid added. GABA was purified using ion exchange column chromatography with 70 % recovery and 97 % purity.     

Rhizogenesis in callus from Conference pear (Pyrus communis L.) protoplasts

Large numbers (ca 6×10⁶ protoplasts/g f.wt) of viable (80%) protoplasts were isolated from embryo-callus tissues of Conference pear using an enzyme mixture which contained 2.0% (w/v) Meicelase, 2.0% (w/v) Rhozyme HP-150 and 0.03% (w/v) Macerozyme R-10. A medium based on ammonium-free MS salts and supplemented with 2.0 mg/l NAA, 0.5 mg/l BAP and 9% (w/v) mannitol supported protoplast division and the proliferation of multicellular colonies. Colonies were taken to the callus stage on a medium which contained MS salts plus 0.1 mg/l 2,4-D and 0.1 mg/l BAP. Roots were regenerated from these protoplastderived calli on MS medium with 0.1 mg/l NAA, 5.0 mg/l BAP and 50 mg/l casein hydrolysate.Abbreviations BAP 6-benzylaminopurine - CPW13M CPW salts medium [15] with 13% (w/v) mannitol - FDA fluorescein diacetate, f. wt-fresh weight - MS Murashige and Skoog [14] - NAA -naphthaleneacetic acid - PE plating efficiency (%) - 2,4-D 2,4-dichlorophenoxyacetic acid     

Halomonas aidingensis sp. nov., a moderately halophilic bacterium isolated from Aiding salt lake in Xinjiang, China

Two Gram-negative moderately halophilic bacterial strains, designated Ad-1(T) and C-12, were isolated from Aiding salt lake of Xinjiang in China. The novel isolates were subjected to a polyphasic taxonomic study. Cells of these strains were cocci or short rods and motile with polar flagella. Colonies produced brown-red pigment. The isolates grew in the range of 0.5-25% (w/v) NaCl, pH 5.5-10.5 and 4-45°C. Analysis of their 16S rRNA gene sequences indicated that strains Ad-1(T) and C-12 belonged to the genus Halomonas showing 92.7-98.4% similarity with the type species. The isoprenoid quinones of the isolates were Q-9 and Q-8. The major cellular fatty acids were C18:1ω7c, C16:1ω7c/6c, C16:0, C12:0-3OH and C10:0. The DNA G + C contents of strains Ad-1(T) and C-12 were 64.6 and 63.9 mol%, respectively. The DNA relatedness between the two isolates was 89.2%. The similarities of these newly isolated strains with closely related type strains were lower than 35% at the genetic level. Based on phenotypic, chemotaxonomic and genetic characteristics, the representative strain Ad-1(T) is considered to be a novel species of the genus Halomonas, for which the name Halomonas aidingensis sp. nov. is proposed, with Ad-1(T) (= CGMCC 1.10191(T) = NBRC 106173(T)) as the type strain.     

An efficient biosurfactant-producing bacterium Selenomonas ruminantium CT2, isolated from mangrove sediment in south of Thailand

Biosurfactant-producing bacteria, isolate CT2, was isolated from mangrove sediment in the south of Thailand. The sequence of the 16S rRNA gene from isolate CT2 showed 100?% similarity with Selenomonas ruminantium. The highest biosurfactant production (5.02?g/l) was obtained when the cells were grown on minimal salt medium containing 15?g/l molasses and 1?g/l commercial monosodium glutamate supplemented with 1?g/l NaCl, 0.1?g/l leucine, 5?% (v/v) inoculum size at 30?°C and 150?rpm after 54?h of cultivation. The biosurfactant obtained by extraction with ethyl acetate showed high surface tension reduction (25.5?mN/m), a small CMC value (8?mg/l), thermal and pH stability with respect to surface tension reduction and emulsification activity and a high level of salt tolerance. The biosurfactant obtained was confirmed as a lipopeptide by using a biochemical test, FT-IR, MNR and mass spectrometry. The crude biosurfactant showed a broad spectrum of antimicrobial activity and also had the ability to emulsify oil and enhance PAHs solubility.     

Production of raw cassava starch-degrading enzyme by Penicillium and its use in conversion of raw cassava flour to ethanol

A newly isolated strain Penicillium sp. GXU20 produced a raw starch-degrading enzyme which showed optimum activity towards raw cassava starch at pH 4.5 and 50 °C. Maximum raw cassava starch-degrading enzyme (RCSDE) activity of 20 U/ml was achieved when GXU20 was cultivated under optimized conditions using wheat bran (3.0% w/v) and soybean meal (2.5% w/v) as carbon and nitrogen sources at pH 5.0 and 28 °C. This represented about a sixfold increment as compared with the activity obtained under basal conditions. Starch hydrolysis degree of 95% of raw cassava flour (150 g/l) was achieved after 72 h of digestion by crude RCSDE (30 U/g flour). Ethanol yield reached 53.3 g/l with fermentation efficiency of 92% after 48 h of simultaneous saccharification and fermentation of raw cassava flour at 150 g/l using the RCSDE (30 U/g flour), carried out at pH 4.0 and 40 °C. This strain and its RCSDE have potential applications in processing of raw cassava starch to ethanol.     

渗透压冲击下中度嗜盐菌Halomonas sp. Y四氢嘧啶类相容性溶质的合成与释放

【背景】四氢嘧啶类物质在高温、冷冻和干燥等逆境条件下,对酶、蛋白质、核酸及整个细胞具有良好的保护作用,已经应用于酶制剂、生物医药及护肤品等相关领域。目前此类物质只能依赖中度嗜盐菌采用细菌泌乳工艺进行商业化生产,因此四氢嘧啶类高产菌株及其发酵技术的研究日益受到国内外研究者关注。【目的】分离获得高产合成四氢嘧啶类相容性溶质的中度嗜盐细菌,研究渗透压冲击对其胞内四氢嘧啶合成与释放的影响,探索细菌泌乳法制备四氢嘧啶的可行性。【方法】采用涂布平板法分离中度嗜盐菌,对分离菌株进行形态、生理生化和16S rRNA基因序列分析,鉴定其种属;采用高效液相色谱法(HPLC)和质谱法(MS)分析四氢嘧啶类物质,细菌泌乳法制备四氢嘧啶类物质。【结果】从盐池土样中分离到一株以四氢嘧啶类物质为主要相容性溶质的中度嗜盐菌Y,鉴定为盐单胞菌(Halomonas sp.)Y。盐单胞菌Y能在NaCl质量浓度为10-250 g/L的培养基中生长,最适生长的NaCl浓度为100 g/L;HPLC-MS测试结果证明盐单胞菌Y可同时合成四氢嘧啶和羟基四氢嘧啶2种相容性溶质,在最适生长的盐浓度下其合成量分别达175.5 mg/g和47.9 mg/g;在NaCl质量浓度为0-30 g/L的低渗溶液中胞内四氢嘧啶类物质经5 min即可达到最大释放率,而细菌泌乳工艺中最适合诱导四氢嘧啶释放的低渗溶液为质量浓度为10 g/L的NaCl溶液;采用细菌泌乳工艺制备四氢嘧啶,经连续11轮的高渗/低渗冲击,四氢嘧啶总合成量为6.0 g/L,总释放量为5.7 g/L,平均释放率为64.5%,底物转化率为128.9 mg/g。【结论】盐单胞菌Y是一株较高产合成四氢嘧啶类的中度嗜盐菌,能够耐受反复的渗透压冲击,采用细菌泌乳工艺显著提高了四氢嘧啶的制备效率。     

Effect of Sodium Nitrite and Sodium Chloride on Growth of Lactic Acid Bacteria

The effect of NaNO2 and NaCl on the growth of 24 lactic acid bacteria strains isolated from vacuum-packed cooked ring sausages were examined by analyzing different growth parameters with Bioscreen. NaNO2 had a very limited effect on the growth of lactic acid bacteria at 50 and 100 mg/l but at 400 mg/l a more pronounced inhibitory effect was found. Bacterial growth was enhanced by 1–2% (w/v) of added NaCl, while NaCl concentrations above 3% (w/v) had a clear inhibitory effect. Leuconostoc isolates seemed to be more sensitive to sodium nitrite and sodium chloride than homofermentative lactobacilli strains. Among homofermentative lactobacilli, the strains resembling Lactobacillus curvatus were more sensitive to NaCl than those resembling Lactobacillus sake.     

Chromium(VI) bioaccumulation capacities of adapted mixed cultures isolated from industrial saline wastewaters

Enrichment mixed cultures tolerating relatively high concentrations of chromium and salt ions were isolated and their bioaccumulation properties improved by adaptation. Mixed cultures were enriched in Nutrient Broth media containing 25-300 mg l(-1) Cr(VI) and 0%, 2%, 4%, 6% (w/v) NaCl. Bioaccumulation of Cr(VI) was studied in a batch system as a function of initial pH (7, 8 and 9), Cr(VI) and NaCl concentrations. Increasing NaCl and Cr(VI) concentrations led to significant decreases in percentage uptake and dried weight of mixed cultures but increased maximum specific chromium uptake. The maximum specific chromium uptake value at pH 8 was 58.9 mg g(-1) for 316.1 mg l(-1) Cr(VI) in the absence of NaCl, while at pH 9 it was 130.1 mg g(-1) in media including 194.5 mg l(-1) Cr(VI) and 2% NaCl concentrations. At 4% NaCl, the maximum Cr(VI) uptake of 127.0 mg g(-1) for 221.1 mg l(-1) Cr(VI) occurred at pH 9, while at 6% NaCl the maximum Cr(VI) uptake of 114.9 mg g(-1) for 278.1 mg l(-1) Cr(VI) was found at pH 7.     

Proteomic insight into phenolic adaptation of a moderately halophilic Halomonas sp. strain AAD12

A gram-negative, moderately halophilic bacterium was isolated from ?amalt? Saltern area, located in the Aegean Region of Turkey. Analysis of its 16S rRNA gene sequence and physiological characteristics showed that this strain belonged to the genus Halomonas ; hence, it was designated as Halomonas sp. strain AAD12. The isolate tolerated up to 800 mg?L(-1) phenol; however, at elevated concentrations, phenol severely retarded cell growth. The increase in lag phase with increasing phenol concentrations indicated that the microorganism was undergoing serious adaptative changes. To understand the physiological responses of Halomonas sp. strain AAD12 to phenol, a 2-dimensional electrophoresis approach combined with mass spectrometric analysis was used. This approach showed that the expression of 14 protein spots were altered as phenol concentration increased from 200 to 800 mg?L(-1). Among the identified proteins were those involved in protein biosynthesis, energy, transport, and stress metabolism. So far, this is the first study on phenolic adaptation of a gram-negative, moderately halophilic bacteria using proteomic tools. The results provided new insights for understanding the general mechanism used by moderately halophilic bacteria to tolerate phenol and suggested the potential for using these microorganisms in bioremediation.     

Release of glucosylglycerol from the cyanobacterium Synechocystis spec. SAG 92.79 by hypoosmotic shock

When cells of Synechocystis, adapted to 684 mmol/l NaCl, were exposed to hypoosmotic shock by reducing NaCl concentration to more than 250 mmol/l, significant amounts of organic material were liberated which could be increased by increasing shock strength. After maximal hypoosmotic shock (684 mmol/l 2 mmol/l NaCl) 40–50% of photosynthetically labelled organic material occurred in the surrounding medium. The main compound exudates was the osmoprotective compound glucosylglycerol. Minor exudates were amino acids, organic acids and carbohydrates. In contrast a hyperosmotic shock (2 mmol/l684 mmol/l NaCl) liberated only one fourth of the amount liberated by a hypoosmotic shock.