Identification of a newly isolated Rhodotorula mucilaginosa NQ1 and its development for the synthesis of bulky carbonyl compounds by whole-cell bioreduction

A strain NQ1, which showed efficient asymmetric reduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) to enantiopure (S)-[3,5-bis(trifluoromethyl)phenyl]ethanol ((S)-BTPE), which is the key intermediate for the synthesis of a receptor antagonist and antidepressant, was isolated from a soil sample. Based on its morphological and internal transcribed spacer sequence, the strain NQ1 was identified to be Rhodotorula mucilaginosa NQ1. Some key reaction parameters involved in the bioreduction catalyzed by whole cells of R. mucilaginosa NQ1 were subsequently optimized, and the optimized conditions for the synthesis of (S)-BTPE were determined to be as follows: 5·0 ml phosphate buffer (200 mmol l⁻¹, pH 7·0), 80 mmol l⁻¹ of BTAP, 250 g (wet weight) l⁻¹ of resting cell, 35 g l⁻¹ of glucose and a reaction for 18 h at 30°C and 180 rev min⁻¹. The strain NQ1 exhibited a best yield of 99% and an excellent enantiomeric excess of 99% for the preparation of (S)-BTPE under the above optimal conditions, and could also asymmetrically reduce a variety of bulky prochiral carbonyl compounds to their corresponding optical hydroxyl compound with excellent enantioselectivity. These results indicated that R. mucilaginosa NQ1 had a good capacity to reduce BTAP to its corresponding (S)-BTPE, and might be a new potential biocatalyst for the production of valuable chiral hydroxyl compounds in industry.     

Optimization of the copy number of dibenzothiophene desulfurizing genes to increase the desulfurization activity of recombinant Rhodococcus sp.

Dibenzothiophene (DBT) degradation activity of recombinant Rhodococcus sp. T09/pRKPP was increased by about 3.5-fold by introduction of the NAD(P)H/FMN oxidoreductase gene (dszD), while DBT desulfurization activity remained the same with production of dibenzo[1,2]oxathiin-6-oxide, which was caused by insufficient activity of the last desulfurization step involving a desulfinase. Introduction of an additional dsz operon resulted in a 3.3-fold increase DBT desulfurization activity (31 mol g dry cell^–1 h^–1) compared with that of T09/pRKPP (9.5 mol g dry cell^–1 h^–1). Furthermore, optimization of DBT at 25 mg l^–1 and glucose at 10 g l^–1, increased the total DBT desulfurization activity 2- to 3-fold due to increases in the DBT desulfurizing specific activity and the final cell concentration.     

Biodesulfurization of dibenzothiophene by a newly isolated Rhodococcus erythropolis strain

A new dibenzothiophene (DBT) desulfurizing bacterium was isolated from oil-contaminated soils in Iran. HPLC analysis and PCR-based detection of the presence of the DBT desulfurization genes (dszA, dszB and dszC) indicate that this strain converts DBT to 2-hydroxybiphenyl (2-HBP) via the 4S pathway. The strain, identified as Rhodococcus erythropolis SHT87, can utilize DBT, dibenzothiophene sulfone, thiophene, 2-methylthiophene and dimethylsulfoxide as a sole sulfur source for growth at 30 °C.The maximum specific desulfurization activity of strain SHT87 resting cells in aqueous and biphasic organic–aqueous systems at 30 °C was determined to be 0.36 and 0.47 μmol 2-HBP min⁻¹ (g dry cell)⁻¹, respectively. Three mM DBT was completely metabolized by SHT87 resting cells in the aqueous and biphasic systems within 10 h. The rate and the extent of the desulfurization reaction by strain SHT87 suggest that this strain can be used for the biodesulfurization of diesel oils.     

Ralstonia eutropha as a biocatalyst for desulfurization of dibenzothiophene

The potential of Ralstonia eutropha as a biocatalyst for desulfurization of dibenzothiophene (DBT) was studied in growing and resting cell conditions. The results of both conditions showed that sulfur was removed from DBT which accompanied by the formation of 2-hydroxybiphenyl (2-HBP). In growing cell experiments, glucose was used as an energy supplying substrate in initial concentrations of 55 mM (energy-limited) and 111 mM (energy-sufficient). The growing cell behaviors were quantitatively described using the logistic equation and maintenance concept. The results indicated that 2-HBP production was higher for the energy-sufficient cultures, while the values of the specific growth rate and the maintenance coefficient for these media were lower than those of the energy-limited cultures. Additionally, the kinetic studies showed that the half-saturation constant for the energy-limited cultures was 2 times higher than the energy-sufficient ones where the inhibition constant (0.08 mM) and the maximum specific DBT desulfurization rate (0.002 mmol g_cell ⁻¹ h⁻¹) were almost constant. By defining desulfurizing capacity (D _DBT) including both the biomass concentration and time to reach a particular percentage of DBT conversion, the best condition for desulfurizing cell was determined at 23% g_cell L⁻¹ h⁻¹ which corresponded with the resting cells that were harvested at the mid-exponential growth phase.

     

Decolorization of Orange I under alkaline and anaerobic conditions by a newly isolated humus-reducing bacterium,Planococcus sp. MC01

Printing and dyeing wastewater (PDW) normally has a high pH of 9.0–13.0, but alkaliphilic bacteria capable of treating PDW have rarely been isolated. Here we report an alkaliphilic and halotolerant, humus-reducing facultative anaerobe, Planococcus sp. MC01 (CGMCC 4771 = KCTC 33120), which can effectively reduce AQDS (anthraquinone-2, 6-disulphonate, humus analog) and decolorize Orange I (>94.0%) under alkaline and anaerobic conditions. The decolorization process of Orange I fits a pseudo-first-order kinetics well, and the rate constants (k) were 0.12, 0.17, 0.14, and 0.12 h⁻¹ when acetate, glucose, sucrose, and lactate, respectively, served as electron donor. When 0.5 mmol l⁻¹ AQDS and 2.0 mmol l⁻¹ γ-FeOOH were added as electron shuttles, the decolorization process was stimulated by 44.4% and 32.8%, respectively. Additionally, strain MC01 showed high decolorizing activity with low initial concentrations of Orange I (0.01–0.2 mmol l⁻¹), and the optimal glucose concentration for decolorization was 10.0 mmol l⁻¹. Results of UV/vis spectra suggested the cleavage of the double azo bond during decolorization. To the best of our knowledge, this is the first report of an alkaliphilic facultative anaerobe capable of decolorizing Orange I under alkaline conditions.     

Desulfurization of dibenzothiophene by lyophilized cells of Pseudomonas delafieldii R-8 in the presence of dodecane

Several parameters that influence the dibenzothiophene (DBT) desulfurization by lyophilized cells of Pseudomonas delafieldii R-8 were studied in the presence of dodecane. The aqueous media tested with pH range in 4.6–8.5 made no obvious difference on the desulfurization activity. The rate and extent of desulfurization were strongly dependent on the volume ratio of oil-to-water, DBT concentration and the cell concentration. The specific desulfurization rate of DBT and 4,6-dimethyl DBT (4,6-DMDBT) could reach 11.4 and 9.4 mmol sulfur kg⁻¹ dry cells (DCW) h⁻¹, respectively. The desulfurization pattern of DBT was represented by the Michaelis–Menten equation. The kinetic parameters, the limiting maximal velocity (V_max) and Michaelis constant (K_m), for desulfurization of DBT were calculated.     

Cultivation of a desulfurizing bacterium, Mycobacterium strain G3

Various carbon and sulfur sources on the growth and desulfurization activity of Mycobacterium strain G3, which is a dibenzothiophene (DBT)-degrading microorganism, were studied. Ethanol, glucose or glycerol as the sole carbon source and MgSO₄, taurine or dimethyl sulfoxide (DMSO) as the sole sulfur source were suitable for the growth. In addition, desulfurization activity was expressed in medium containing taurine, MgSO₄ or DMSO at 0.1 mM, when 217 mM ethanol was used as the sole carbon source. The highest desulfurization activity was in the stationary phase cells after 5 days' growth, rather than those harvested during active growth, when Mycobacterium G3 was cultivated in medium containing 217 mM ethanol and 0.1 mM MgSO₄. Thus alternative sulfur sources to DBT can be used for the cultivation of this desulfurizing microorganism.     

Bradyrhizobium arachidis mediated enhancement of (oxy)matrine content in the medicinal legume Sophora flavescens

Effect of rhizobial inoculation and nitrate application on the content of bioactive compounds in legume plants is an interesting aspect for interactions among microbes, plants and chemical fertilizers, as well as for cultivated practice of legumes. In this study, nitrate (0, 5 and 20 mmol l⁻¹) and Bradyrhizobium arachidis strain CCBAU 051107^T were applied, individually or in combination, to the root rhizosphere of the medicinal legume Sophora flavescens Aiton (SFA). Then the plant growth, nodulation and active ingredients including (oxy)matrine of SFA were determined and compared. Rhizobial inoculation alone significantly increased the numbers and fresh weight of root nodules. Nodulation was significantly inhibited due to nitrate (5 and 20 mmol l⁻¹). Only oxymatrine was detected in the control plants without rhizobial inoculation and nitrate supplement, while both oxymatrine and matrine were synthesized in plants treated with inoculation of B. arachidis or supplied with nitrate. The content of oxymatrine was the highest in plants inoculated solely with rhizobia and was not significantly altered by additional application of nitrate. Combinations of B. arachidis inoculation and different concentrations of nitrate did not significantly change the concentrations of (oxy)matrine in the plant. In conclusion, sole rhizobial inoculation was the best approach to increase the contents of key active ingredients oxymatrine and matrine in the medicinal legume SFA.     

Competitive exclusion as a mode of action of a novel Bacillus cereus aquaculture biological agent

Aims: To determine the contribution of potential modes of action of a Bacillus cereus aquaculture biological control agent in inhibition of the fish pathogen, Aeromonas hydrophila. Methods and Results: When B. cereus was tested in plate well inhibition studies, no production of antimicrobial compounds was detected. Bacillus cereus had a high growth rate (0·96 h^?1), whereas Aer. hydrophila concentration decreased by c. 70% in co‐culture experiments. In nutrient limitation studies, B. cereus had a significantly higher growth rate when cultured under glucose (P < 0·05) and iron (P < 0·01) limitation in comparison with Aer. hydrophila. Bacillus cereus glucose (0·30 g l^?1 h^?1) and iron (0·60 mg l^?1 h^?1) uptake rates were also significantly higher (P < 0·01) than the Aer. hydrophila glucose (0·14 g l^?1 h^?1) and iron (0·43 mg l^?1 h^?1) uptake rates. Iron uptake was facilitated by siderophore production shown in time profile studies where relative siderophore production was c. 60% through the late exponential and sporulation phases. Conclusions: Competitive exclusion by higher growth rate, competition for organic carbon and iron, facilitated by siderophore production, could be identified as mechanisms of pathogen growth inhibition by B. cereus. Significance and Impact of the Study: This study is the first elucidation of the mechanism of action of our novel B. cereus biological agent in growth attenuation of pathogenic Aer. hydrophila. This study enhances the application knowledge and attractiveness for adoption of B. cereus NRRL 100132 for exploitation in aquaculture.     

Recombinant Pseudomonas putida carrying both the dsz and hcu genes can desulfurize dibenzothiophene in n-tetradecane

Pseudomonas putida IFO13696, a recombinant strain with dsz desulfurization genes, desulfurized dibenzothiophene (DBT) in water but not in n-tetradecane. By introducing into this recombinant strain the hcuABC genes that take part in the uptake of DBT in the oil phase into the cell, 82% of 1 mM DBT in n-tetradecane was degraded in 24 h by resting cells. The products of hcuABC genes thus acted in the uptake of DBT in n-tetradecane into the cells and were effective in desulfurization of DBT in the hydrocarbon phase.     

Introducing Alternaria tenuissima SBUp1, as an endophytic fungus of Ferula assa-foetida from Iran,which is a rich source of rosmarinic acid

Endophytic fungi are the endogenous micro-organisms to interacting with the plant cells, which do not exhibit any symptoms on the host plant and may produce some of the main secondary metabolites of the host plant cells. Ferula assa-foetida is a perennial and endemic medicinal plant of Iran, which is a rich source of sesquiterpene, coumarins, polysulfides and phenolic acids. In this study, 28 endophytic fungi isolates including Fusarium (60·7%), Aspergillus (7·1%), Alternaria (17·9%) and Plectosphaerella (7·1%) were isolated from F. assa-foetida root (57·1%), stem (32·1%) and leaf (10·8%) collected from Parvand protected area. Subsequently, their ability to produce phenolic acids was evaluated. The high amounts of total phenol (326·09 mg g⁻¹ of dry weight, DW), total flavonoid (901·11 mg g⁻¹ DW) and antioxidant activity (247·96 mg l⁻¹) were found in the supernatant fluid of SBUp1 isolate. The high-performance liquid chromatography analysis of 14 phenolic acids showed that rosmarinic acid (RA) is the main phenolic acid in the supernatant fluid of SBUp1 by 64·11 mg g⁻¹ DW confirmed by the liquid chromatography coupled with mass spectrometric analysis. According to morphological identification followed by phylogenetic study based on internal transcribed spacer (ITS) sequencing (ITS1-5.8S-ITS2) analysis, the SBUp1 isolate was identified as Alternaria tenuissima. Eventually, to our knowledge, it is the first document confirming A. tenuissima as an endophytic fungus of F. assa-foetida, which is a rich source of RA.     

Synergistic effect on anti-methicillin-resistant Staphylococcus aureus among combinations of α-mangostin-rich extract,lawsone methyl ether and ampicillin

α-Mangostin-rich extract (AME) exhibited satisfactory inhibitory activities against all tested MRSA strains, with minimum inhibitory concentrations (MICs) of 7·8–31·25 µg ml⁻¹, whereas lawsone methyl ether (LME) and ampicillin revealed weak antibacterial activity with MICs of 62·5–125 µg ml⁻¹. However, the combination of AME and LME showed synergistic effects against all tested MRSA strains with fractional inhibitory concentration index (FICI) values of 0·008–0·009, while the combination of AME and ampicillin, as well as LME and ampicillin produced synergistic effects with FICIs of 0·016–0·257. A time-kill assay against MRSA (DMST 20654 strain) revealed a 6-log reduction in CFU per ml, which completely inhibited bacterial growth for the combinations of AME and LME, AME and ampicillin, and LME and ampicillin at a 8-h incubation, while those against MRSA (2468 strain) were at 10-h incubation. The combination of α-mangostin and LME as well as the combinations of each compound with ampicillin synergized the alteration of membrane permeability. In addition, α-mangostin, LME and ampicillin inhibited the biofilm formation of MRSA. These findings indicated that the combinations of AME and LME or each of them in combination with ampicillin had enhanced antibacterial activity against MRSA. Therefore, these compounds might be used as the antibacterial cocktails for treatment of MRSA.     

Desulfurization of 2,4,6,8-tetraethyl dibenzothiophene by recombinant Mycobacterium sp. strain MR65

Recombinant Mycobacterium sp. strain MR65 harboring dszABCD genes was used to desulfurize alkyl dibenzothiophenes (C_x-DBTs) in n-hexadecane. The specific desulfurization activity for 2,4,6,8-tetraethyl DBT (C₈-DBT) by DszC enzyme was about twice that for 4,6-dipropyl DBT (C₆-DBT). However, the degradation rate of 2,4,6,8-tetraethyl DBT in n-hexadecane by resting cells of strain MR65 was only about 40% of that of 4,6-dipropyl DBT. These results indicated that the desulfurization ability for Cx-DBTs by resting cells depends on carbon number substituted at positions 4 and 6 and that the rate-limiting step in the desulfurization reaction of highly alkylated C_x-DBTs is the transfer process from the oil phase into the cell.     

SODIUM: SOME EFFECTS ON BLUEGREEN ALGAL GROWTH1

The growth of heterocystous bluegreen algae in various concentrations of sodium, was examined in axenic culture as well as in situ studies. Anabaena cylindrica Lemm. with no Na⁺ added, suffered from decreased rates of acetylene reduction, ¹⁴C, assimilation, excretion of organic C as well as lower concentrations of chlorophyll a and particulate organic C compared to cultures supplied with 5, 10, and 50 mg Na⁺·l⁻¹ Sodium deficient algae released, extracellularly a higher percentage of previously fixed C as organic C. No differences in any parameter measured were demonstrable among cultures grown with 5, 10, and 50 mg Na⁺·l⁻¹ High nitrate concentrations (20 mg NO₃⁻·l⁻¹) resulted in decreased rates of acetylene reduction and heterocyst numbers in. Na sufficient, and Na deficient cultures: however, decreased, cellular Na content at high NO₃⁻ levels occurred only in N deficient, cultures. Higher percentages of excreted organic C occurred with increasing NO₃⁻ concentrations in Na deficient cultures. Sodium enrichment of natural bluegreen populations with the addition of 50, 100, and 200 mg Na⁺·l⁻¹ elicited neither a stimulatory nor an inhibitory response in photosynthetic C fixation. In contrast, the addition of small amounts of Na⁺ (5 mg·l⁻) resulted in increased C fixation. However, since the Na. concentration of the lake water, at ca. 5 mg Na⁺·l⁻¹, was sufficient for growth of the bluegreens present, sodium, is not assumed to be limiting under most natural conditions. No increase in in situ acetylene reduction rates occurred with additions of sodium.     

Disruption of glpF gene encoding the glycerol facilitator improves 1,3-propanediol production from glucose via glycerol in Escherichia coli

Engineered Escherichia coli has recently been applied to produce 1,3-propanediol (1,3-PDO) from glucose. A metabolic intermediate in the production pathway, glycerol, is partially secreted into the extracellular of E. coli through a glycerol facilitator encoded by glpF, and this secretion consequently decreases 1,3-PDO production. Therefore, we aimed to determine whether disrupting the glpF gene would improve 1,3-PDO production in E. coli. The intracellular glycerol concentration in a glpF-disruptant was 7·5 times higher than in a non-disruptant. The glpF-disrupted and non-disrupted E. coli strains produced 0·26 and 0·09 g l⁻¹ of 1,3-PDO, respectively, from 1% glucose after 72 h of cultivation. The specific growth rate (μ) and the 1,3-PDO yield from glucose (Y_P/S) in the disruptant were higher than those in the non-disruptant (ΔglpF, μ = 0·08 ± 0·00 h⁻¹, Y_P/S = 0·06 mol mol-glucose⁻¹; BW25113, μ = 0·06 ± 0·00 h⁻¹, Y_P/S = 0·02 mol mol-glucose⁻¹). Disruption of the glpF gene decreased the production of the by-product, acetic acid. These results indicated that disruption of glpF increased the intracellular concentration of glycerol and consequently increased 1,3-PDO production in E. coli.     

Biological response of protists Haematococcus lacustris and Euglena gracilis to conductive polymer poly (3,4-ethylenedioxythiophene) polystyrene sulfonate

Improving the growth and pigment accumulation of microalgae by electrochemical approaches was considered a novel and promising method. In this research, we investigated the effect of conductive polymer poly (3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) dispersible in water on growth and pigment accumulation of Haematococcus lacustris and Euglena gracilis. The results revealed that effect of PEDOT:PSS was strongly cell-dependent and each cell type has its own peculiar response. For H. lacustris, the cell density in the 50 mg·l⁻¹ treatment group increased by 50·27%, and the astaxanthin yield in the 10 mg·l⁻¹ treatment group increased by 37·08%. However, under the high concentrations of PEDOT:PSS treatment, cell growth was significantly inhibited, and meanwhile, the smaller and more active zoospores were observed, which reflected the changes in cell life cycle and growth mode. Cell growth of E. gracilis in all the PEDOT:PSS treatment groups were notably inhibited. Chlorophyll a content in E. gracilis decreased while chlorophyll b content increased in response to the PEDOT:PSS treatment. The results laid a foundation for further development of electrochemical methods to promote microalgae growth and explore the interactions between conductive polymers and microalgae cells.     

Desulfurization of dibenzothiophene by Bacillus subtilis recombinants carrying dszABC and dszD genes

The desulfurization (dsz) genes from Rhodococcus erythropolis DS-3 were successfully integrated into the chromosomes of Bacillus subtilis ATCC 21332 and UV1 using an integration vector pDGSDN, yielding two recombinant strains, B. subtilis M29 and M28 in which the integrated dsz genes were expressed efficiently under the promoter, Pspac. The dibenzothiophene (DBT) desulfurization efficiency of M29 was 16.2 mg DBT l⁻¹ h⁻¹ at 36 h, significantly higher than that of R. erythropolis DS−3 and B. subtilis M28 and also showed no product inhibition. The interfacial tension of the supernatant fermented by M29 varied from 48 mN m⁻¹ to 4.2 mN m⁻¹, lower than that of the recombinant strain, M28, reveals that the biosurfactant secreted from M29 may have an important function in the DBT desulfurization process.     

Lead,cadmium and nickel removal efficiency of white-rot fungus Phlebia brevispora

Widespread of heavy metals contamination has led to several environmental problems. Some biological methods to remove heavy metals from contaminated wastewater are being widely explored. In the present study, the efficiency of a white-rot fungus, Phlebia brevispora to remove different metals (Pb, Cd and Ni) has been evaluated. Atomic absorption spectroscopy of treated and untreated metal containing water revealed that all the metals were efficiently removed by the fungus. Among all the used metals, cadmium was the most toxic metal for fungal growth. Phlebia brevispora removed maximum Pb (97·5%) from 100 mmol l⁻¹ Pb solution, which was closely followed by Cd (91·6%) and Ni (72·7%). Scanning electron microscopic images revealed that the presence of metal altered the morphology and fine texture of fungal hyphae. However, the attachment of metal on mycelia surface was not observed during energy-dispersive X-ray analysis, which points towards the intracellular compartmentation of metals in vacuoles. Thus, the study demonstrated an application of P. brevispora for efficient removal of Pb, Cd and Ni from the metal contaminated water, which can further be applied for bioremediation of heavy metals present in the industrial effluent.     

Rhamnolipid production by Pseudomonas aeruginosa engineered with the Vitreoscilla hemoglobin gene

The potential of Pseudomonas aeruginosa expressing the Vitreoscilla hemoglobin gene (vgb) for rhamnolipid production was studied. P. aeruginosa (NRRL B-771) and its transposon mediated vgb transferred recombinant strain, PaJC, were used in the research. The optimization of rhamnolipid production was carried out in the different conditions of cultivation (agitation rate, the composition of culture medium and temperature) in a time-course manner. The nutrient source, especially the carbon type, had a dramatic effect on rhamnolipid production. The PaJC strain and the wild type cells of P. aeruginosa started producing biosurfactant at the stationary phase and its concentration reached maximum at 24 h (838 mg/l⁻¹) and at 72 h (751 mg l⁻¹) of the incubation respectively. Rhamnolipid production was optimal in batch cultures when the temperature and agitation rate were controlled at 30°C and 100 rpm. It reached 8373 mg l⁻¹ when the PaJC cells were grown in 1.0% glucose supplemented minimal media. Genetic engineering of biosurfactant producing strains with vgb may be an effective method to increase its production.     

Ethanol fermentation by an efficient strain,NRRL B-4286, of Zymomonas mobilis

We studied the effect of the initial substrate concentration over the range of 100–250 g·l⁻¹ on the fermentation kinetics in batch cultures of Zymomonas mobilis NRRL B-4286 on glucose, fructose, and sucrose, using an adapted initial inoculum. With increasing concentrations of substrate, parameters related to growth were more rapidly and strongly affected than those related to ethanol production. This strain produced 94.0 g·l⁻¹, 76.9 g·l⁻¹, and 66.5 g·l⁻¹ of ethanol at glucose, fructose, and sucrose concentrations of 200 g·l⁻¹, respectively, more than the amount produced by the efficient strain ZM4 (NRRL B-14023).