Bacterial strain characterizing by EDTA requirement

A novel strain of bacteria (LPM-4) was isolated that is characterized by a unique EDTA requirement for cell growth. Suspensions of washed cells of strain LPM-4 degrated EDTA complexes with Ba2+, Mg 2+, Ca2+, and Mn2+ at constant rates (0.310-0.486 mmol EDTA/(g h)) and Zn-EDTA at an initial rate of 0.137 +/- 0.016 mmol EDTA/(g h). The temperature optima for cell growth and EDTA degradation were determined under pH-auxostat cultivation. As compared with the known EDTA-degrating bacteria, strain LPM-4 exhibited a higher specific growth rate (0.095 h(-1)) and lower mass cell yield (0.219 g cells/g EDTA) that is promising for its practical applications for EDTA removal in wastewater treatment plants.     

A novel EDTA-degrading Pseudomonas sp.

A bacterial strain LPM-410 capable of utilizing ethylenediaminetetraacetate (EDTA) as the sole source of energy, carbon, and nitrogen was isolated from sewage sludge and identified as a Pseudomonas sp. on the basis of its phenotypic characteristics. Suspensions of exponential-phase cells degraded EDTA, Mg–, Ca–, Ba–, and Mn–EDTA at constant specific rates ranging from 0.363 to 0.525 mmol EDTA/(g cells h). The more stable chelate, Zn–EDTA, was degraded at a lower rate (0.195 ± 0.030 mmol EDTA/(g cells h)), and here was no degradation of Co–, Cu–, Pb–, and Fe(III)–EDTA.     

EDTA degradation by cells of Chelativorans oligotrophicus immobilized on a biofilter

A biofilter based on light expanded clay aggregate (LECA) and cells of the obligate ethylenediamine tetraacetate (EDTA) destructor Chelativorans oligotrophicus LPM-4 has been developed. The culture steadily maintained a high level of EDTA monooxygenase activity of 180–200 nmol/min/mg of protein during three months. EDTA was converted completely or by 80% at initial concentrations of 0.5–0.7 or 2.0 g/l, respectively, in a 2-dm² biofilter at a flow rate of 20 ml/h.     

Bacterial Degradation of EDTA

Degradation of EDTA (ethylenediaminetetraacetic acid) or metal–EDTA complexes by cell suspensions of the bacterial strain DSM 9103 was studied. The activity of EDTA degradation was the highest in the phase of active cell growth and decreased considerably in the stationary phase, after substrate depletion in the medium. Exponential-phase cells were incubated in HEPES buffer (pH 7.0) with 1 mM of uncomplexed EDTA or EDTA complexes with Mg²⁺, Ca²⁺, Mn²⁺, Pb²⁺, Co²⁺, Cd²⁺, Zn²⁺, Cu²⁺, or Fe³⁺. The metal–EDTA complexes (Me–EDTA) studied could be divided into three groups according to their degradability. EDTA complexes with stability constants K below 10¹⁶ (log K < 16), such as Mg–EDTA, Ca–EDTA, and Mn–EDTA, as well as uncomplexed EDTA, were degraded by the cell suspensions at a constant rate to completion within 5–10 h of incubation. Me–EDTA complexes with log K above 16 (Zn–EDTA, Co–EDTA, Pb–EDTA, and Cu–EDTA) were not completely degraded during a 24-h incubation, which was possibly due to the toxic effect of the metal ions released. No degradation of Cd–EDTA or Fe(III)–EDTA by cell suspensions of strain DSM 9103 was observed under the conditions studied.     

A novel malic enzyme gene, <Emphasis Type="Italic">Mime2</Emphasis>, from <Emphasis Type="Italic">Mortierella isabellina</Emphasis> M6-22 contributes to lipid accumulation

Objective

This study was aimed at cloning and characterizing a novel malic enzyme (ME) gene of Mortierella isabellina M6-22 and identifying its relation with lipid accumulation.

Methods

Mime2 was cloned from strain M6-22. Plasmid pET32aMIME2 was constructed to express ME of MIME2 in Escherichia coli BL21. After purification, the optimal pH and temperature of MIME2, as well as K_m and V_max for NADP⁺ were determined. The effects of EDTA or metal ions (Mn²⁺, Mg²⁺, Co²⁺, Cu²⁺, Ca²⁺, or Zn²⁺) on the enzymatic activity of MIME2 were evaluated. Besides, plasmid pRHMIME2 was created to express MIME2 in Rhodosporidium kratochvilovae YM25235, and its cell lipid content was measured by the acid-heating method. The optimal pH and temperature of MIME2 are 5.8 and 30 °C, respectively.

Results

The act ivity of MIME2 was significantly increased by Mg²⁺, Ca²⁺, or Mn²⁺ at 0.5 mM but inhibited by Cu²⁺ or Zn²⁺ (p?<?0.05). The optimal enzymatic activity of MIME2 is 177.46 U/mg, and the K_m and V_max for NADP⁺ are 0.703 mM and 156.25 μg/min, respectively. Besides, Mime2 transformation significantly increased the cell lipid content in strain YM25235 (3.15?±?0.24 vs. 2.17?±?0.31 g/L, p?<?0.01).

Conclusions

The novel ME gene Mime2 isolated from strain M6-22 contributes to lipid accumulation in strain YM25235.

     

Isolation,selection, and biological characterization research of highly effective electricigens from MFCs for phenol degradation

The microbial fuel cells (MFCs) are recognized to be highly effective for the biodegradation of phenol. For isolating the phenol-degrading bacteria, the sample containing 500 mg/L phenol was collected from the MFCs. The strain (WL027) was identified basing on the 16S rRNA gene analysis and phylogenetic analysis as Bacillus cereus. The effects of pH, temperature, concentrations of phenol, heavy metal ions, and salt on the growth of strain as well as the degradation of phenol have been carefully studied. The WL027-strain exhibited favorable tolerance for the metal cations including Cr²⁺, Co²⁺, Pb²⁺, and Cu²⁺ with the concentration of 0.2 mg/L and NaCl solution with a high concentration of 30 g/L. In 41 h, 86.44% of 500 mg/L phenol has been degraded at the initial pH at 6 and the temperature of 30 °C. The strain was highly active electrogenesis bacteria and the coulombic efficiency reached 64.25%, which showed significant advantage on the efficient energy conversion. Therefore, due to the highly efficient degradation of phenol, WL027-strain could be used in the treatment of phenol-containing wastewater.     

Lipid metabolism in large T47D human breast cancer spheroids: 31P- and 13C-NMR studies of choline and ethanolamine uptake

³¹P- and ¹³C-NMR were used to determine the kinetics of choline and ethanolamine incorporation in T47D clone 11 human breast cancer cells grown as large (300 μm) spheroids. Spheroids were perfused inside the spectrometer with 1,2-¹³C-labelled choline or ethanolamine (0.028 mM) and the buildup of labeled phosphorylcholine (PC) or phosphorylethanolamine (PE) was monitored. To analyze the NMR kinetic data, it was assumed that each signal represents a weighted average of signal from the proliferating and non-proliferating compartments of the large spheroid. The average ATP pool size was 4±1 fmol/cell compared to 8±1 fmol/cell in small (150 μm) proliferating spheroids (P < 0.0002). The average PC pool size at steady state was reduced to 11±6 fmol/cell compared to 22±8 (P < 0.007). This could be correlated with an overall reduction of choline uptake in the non-proliferating spheroid fraction. The rate of the enzyme choline kinase was 0.3 fmol/(cell h) compared to 1.0 fmol/(cell h) (P < 0.0001) for proliferating cells. The rate constant of CTP: phosphocholine cytidyltransferase (0.05 h^?1) was not significantly altered, but the rate of the enzyme was reduced from 1.3 to 0.2–0.5 fmol/(cell h). The pool size of PE in medium containing serum ethanolamine (1.7 μM) was approximately the same (15 fmol/cell) in small and large spheroids. In the presence of high ethanolamine (0.028 mM) the average PE level decreased slightly (11 fmol/cell) and the rate of the enzyme ethanolamine kinase in the non-proliferating fraction was 0.7 fmol/(cell h) versus 1.0 fmol/(cell h) in the proliferating cells (P < 0.07). The rate constant of CTP: phosphoethanolamine cytidyltransferase (0.07 h^?) was not significantly altered but the corresponding reaction rate was reduced from 1.4 to 0.2–0.8 fmol/(cell h). The kinetics of choline incorporation did not alter in the presence of 0.028 mM ethanolamine.     

Fast reversal of the initial reaction steps of the plasma membrane (Ca2+ + Mg2+)-ATPase

(1) Calmodulin-depleted red cell membranes catalyse a Ca²⁺, Mg²⁺-dependent ATP-[³H]ADP exchange at 37° C. The Ca²⁺, Mg²⁺-dependent exchange, measured at 20 μM CaCl₂, 1.5 mM MgCl₂, 1.5 mM ADP and 1.5 mM ATP, is comparable to the (Ca²⁺ + Mg²⁺)-ATPase activity, between 0.3 and 0.8 mmol/litre original cells per h. (2) EDTA-washed membranes present a Ca²⁺-dependent ATP-ADP exchange whose rate is not more than 7% of that found in a Mg²⁺-containing medium, while their Ca²⁺-dependent ATPase is essentially zero. Addition of 1.5 mM MgCl₂ to the medium restores both activities to the levels found with membranes not treated with EDTA. (3) Calmodulin (16 μg/ml) produces an eight-fold stimulation of the Ca²⁺-dependent ATP-ADP exchange, slightly less than it stimulates the Ca²⁺-dependent ATP hydrolysis. The effect of 1.5 mM MgCl₂ on the exchange is greater in the presence than in the absence of calmodulin. (4) It is proposed that the reversal of the initial phosphorylation of the Ca²⁺ pump, occurring at a fast rate at 37° C, involves a conformational change in the phosphoenzyme. Thus, it would be an ADP-liganded phosphoenzyme of the form EP(ADP) that would experience the fast conformational transition at 37° C. The great difficulty in producing an overall reversal of the Ca²⁺ pump should then be due to one or more reaction steps later than and including Ca²⁺ release and dephosphorylation.     

Improving EGSB reactor performance for simultaneous bioenergy and organic acid production from cheese whey via continuous biological H<Subscript>2</Subscript> production

Objectives

To evaluate the influence of hydraulic retention time (HRT) and cheese whey (CW) substrate concentration (15 and 25 g lactose l^?1) on the performance of EGSB reactors (R15 and R25, respectively) for H₂ production.

Results

A decrease in the HRT from 8 to 4 h favored the H₂ yield and H₂ production rate (HPR) in R15, with maximum values of 0.86 ± 0.11 mmol H₂ g COD^?1 and 0.23 ± 0.024 l H₂ h^?1 l^?1, respectively. H₂ production in R25 was also favored at a HRT of 4 h, with maximum yield and HPR values of 0.64 ± 0.023 mmol H₂ g COD^?1 and 0.31 ± 0.032 l H₂ h^?1 l^?1, respectively. The main metabolites produced were butyric, acetic and lactic acids.

Conclusions

The EGSB reactor was evaluated as a viable acidogenic step in the two-stage anaerobic treatment of CW for the increase of COD removal efficiency and biomethane production.

     

Analysis of bacterial community structure in bulk soil by in situ hybridization

In situ hybridization with rRNA-targeted, fluorescent (Cy3-labeled) oligonucleotide probes was used to analyze bacterial community structure in ethanol- or paraformaldehyde-fixed bulk soil after homogenization of soil samples in 0.1% pyrophosphate by mild ultrasonic treatment. In ethanol-fixed samples 37 ± 7%, and in paraformaldehyde 41 ± 8% of the 4′, 6-diamidino-2-phenylindole(DAPI)-stained cells were detected with the bacterial probe Eub338. The yield could not be increased by enzymatic and/or chemical pretreatments known to enhance the permeability of bacterial cells for probes. However, during storage in ethanol for 7 months, the detectability of bacteria increased in both ethanol- and paraformaldehyde-fixed samples to up to 47 ± 8% due to an increase in the detection yield of members of the α-subdivision of Proteobacteria from 2 ± 1% to 10 ± 3%. Approximately half of the bacteria detected by probe Eub338 could be affiliated to major phylogenetic groups such as the α-, β-, γ-, and δ-subdivisions of Proteobacteria, gram-positive bacteria with a high G+C DNA content, bacteria of the Cytophaga-Flavobacterium cluster of the CFB phylum, and the planctomycetes. The analysis revealed that bacteria of the α- and δ-subdivision of Proteobacteria and the planctomycetes were predominant. Here, members of the α-subdivision of Proteobacteria accounted for approximately 10 ± 3% of DAPI-stained cells, which corresponded to 44 ± 16 × 10⁸ cells (g soil, dry wt.)^–1, while members of the δ-subdivision of Proteobacteria made up 4 ± 2% of DAPI-stained cells [17 ± 9 × 10⁸ cells (g soil, dry wt.)^–1]. A large population of bacteria in bulk soil was represented by the planctomycetes, which accounted for 7 ± 3% of DAPI-stained cells [32 ± 12 × 10⁸ cells (g soil, dry wt.)^–1]. The detection of planctomycetes in soil confirms previous reports on the occurrence of planctomycetes in soil and indicates a yet unknown ecological significance of this group, which to date has never been isolated from terrestrial environments. Received: 29 March 1997 / Accepted: 28 May 1997     

Metabolism of EDTA and its metal chelates by whole cells and cell-free extracts of strain BNC1

The influence of metal ions on the metabolism of ethylenediaminetetraacetate (EDTA) by whole cells and cell-free extracts of strain BNC1 was investigated. Metal-EDTA chelates with thermodynamic stability constants below 10¹² were readily mineralized by whole cells with maximum specific turnover rates of 15 (MnEDTA) to 20 (Ca-, Mg-, and BaEDTA) μmol g protein⁻¹ min⁻¹. With the exception of ZnEDTA, chelates with stability constants greater than 10¹² were not oxidized at a significant rate. However, it was shown for Fe(III)EDTA that even strong complexes can be degraded after pretreatment by addition of calcium and magnesium salts in the pH range 9–11. The range of EDTA chelates converted by cell-free extracts of strain BNC1 did not depend on their thermodynamic stabilities. The EDTA chelates of Ba²⁺, Co²⁺, Mg²⁺, Mn²⁺, and Zn²⁺ were oxidized whereas Ca-, Cd-, Cu-, Fe-, Pb-, and SnEDTA were not. The first catabolic enzyme appears to be an EDTA monooxygenase since it requires O₂, NADH, and FMN for its activity and yields glyoxylate and ethylenediaminetriacetate as products. The latter is further degraded via N,N′-ethylenediaminediacetate. The maximum specific turnover rate with MgEDTA, the favoured EDTA species, was 50–130 μmol g protein⁻¹ min⁻¹, and the K _m value was 120 μmol/l (K _s for whole cells = 8 μmol/l). Whole cells as well as cell-free extracts of strain BNC1 also converted several structural analogues of EDTA. Received: 4 July 1997 / Received revision: 25 September 1997 / Accepted: 29 September 1997     

Efficient induction of formate hydrogen lyase of aerobically grown <Emphasis Type="Italic">Escherichia coli</Emphasis> in a three-step biohydrogen production process

A three-step biohydrogen production process characterized by efficient anaerobic induction of the formate hydrogen lyase (FHL) of aerobically grown Escherichia coli was established. Using E. coli strain SR13 (fhlA ⁺⁺, ΔhycA) at a cell density of 8.2 g/l medium in this process, a specific hydrogen productivity (28.0 ± 5.0 mmol h⁻¹ g⁻¹ dry cell) of one order of magnitude lower than we previously reported was realized after 8 h of anaerobic incubation. The reduced productivity was attributed partly to the inhibitory effects of accumulated metabolites on FHL induction. To avoid this inhibition, strain SR14 (SR13 ΔldhA ΔfrdBC) was constructed and used to the effect that specific hydrogen productivity increased 1.3-fold to 37.4 ± 6.9 mmol h⁻¹ g⁻¹. Furthermore, a maximum hydrogen production rate of 144.2 mmol h⁻¹ g⁻¹ was realized when a metabolite excretion system that achieved a dilution rate of 2.0 h⁻¹ was implemented. These results demonstrate that by avoiding anaerobic cultivation altogether, more economical harvesting of hydrogen-producing cells for use in our biohydrogen process was made possible.     

Light emission from the Fe2+–EDTA–ascorbic acid–H2O2 system strongly enhanced by plant phenolic acids

Oxidative reactions can result in the formation of electronically excited species that undergo radiative decay depending on electronic transition from the excited state to the ground state with subsequent ultra‐weak photon emission (UPE). We investigated the UPE from the Fe²⁺–EDTA (ethylenediaminetetraacetic acid)–AA (ascorbic acid)–H₂O₂ (hydrogen peroxide) system with a multitube luminometer (Peltier‐cooled photon counter, spectral range 380–630 nm). The UPE, of 92.6 μmol/L Fe²⁺, 185.2 μmol/L EDTA, 472 μmol/L AA, 2.6 mmol/L H₂O₂, reached 1217 ± 118 relative light units during 2 min measurement and was about two times higher (P < 0.001) than the UPE of incomplete systems (Fe²⁺–AA–H₂O₂, Fe²⁺–EDTA–H₂O₂, AA–H₂O₂) and medium alone. Substitution of Fe²⁺ with Cr²⁺, Co²⁺, Mn²⁺ or Cu²⁺ as well as of EDTA with EGTA (ethylene glycol‐bis(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid) or citrate powerfully inhibited UPE. Experiments with scavengers of reactive oxygen species (dimethyl sulfoxide, mannitol, sodium azide, superoxide dismutase) revealed the dependence of UPE only on hydroxyl radicals. Dimethyl sulfoxide at the concentration of 0.74 mmol/L inhibited UPE by 79 ± 4%. Plant phenolics (ferulic, chlorogenic and caffec acids) at the concentration of 870 μmol/L strongly enhanced UPE by 5‐, 13.9‐ and 46.8‐times (P < 0.001), respectively. It is suggested that augmentation of UPE from Fe²⁺–EDTA–AA–H₂O₂ system can be applied for detection of these phytochemicals.     

Isolation and growth characteristics of nitrilotriacetate-degrading bacteria

Two bacterial strains that degrade nitrilotriacetate (NTA) were isolated from NTA-acclimatized activated sludge. These bacteria grew well in NTA medium with optimal pH around 7. The growth rate constants of the bacteria, strains N-2 and N-5, were 0.046 h⁻¹ and 0.11 h⁻¹ at the concentration of 0.1% NTA (pH 7.0, 25°C), respectively.The growth of each bacterium was inhibited at high concentrations NTA. The growth rate decreased roughly linearly with increasing concentration of NTA. The strains N-2 and N-5 showed maximal cell growth at the concentrations of 0.2% and 0.25% NTA, respectively. The strain N-2 would not grow at the concentration of 0.5% NTA. On the other hand, the strain N-5 showed a little growth under the same conditions. Also, the bacterial growth was almost completely inhibited when divalent metal ions such as Mg⁺⁺, Ca⁺⁺, and Fe⁺⁺ were omitted from the culture medium, or slightly excess EDTA (1 mM) was added to the medium. These results suggest that the bacterial growth inhibition at high concentration of NTA is caused by the sequestration of metal ions in the medium.     

Performance of bioelectrochemical systems inoculated with Desmodesmus sp. A8 under different light sources

Light source can affect the stomata opening, photosynthesis process, and pigment content in microalgae cells. In this study, growth rate, chlorophyll a (chl a) content, and electrogenic capability of Desmodesmus sp. A8 were investigated under incandescent and fluorescent lamps. Growth rate, productivity, and chl a content of strain A8 exposed to incandescent light were recorded as 0.092 ± 0.010 day^?1, 0.019 ± 0.008 g L^?1 day^?1, and 15.10 ± 1.40 mg L^?1, which decreased to 0.086 ± 0.006 day^?1, 0.012 ± 0.004 g L^?1 day^?1, and 10.06 ± 1.59 mg L^?1, respectively, under fluorescent light. The stable current density of bioelectrochemical systems inculcated with strain A8 under incandescent and fluorescent lamps were 249.76 and 158.41 mA m^?2 at ?0.4 V vs. Ag/AgCl, coupling with dissolved oxygen within biofilm decreasing from 15.91 to 10.80 mg L^?1. This work demonstrated that illuminating microalgae under an incandescent lamp can improve biomass production and electrogenic capabilities.     

Role of Cations in Accumulation and Release of Phosphate by Acinetobacter Strain 210A

Cells of the strictly aerobic Acinetobacter strain 210A, containing aerobically large amounts of polyphosphate (100 mg of phosphorus per g [dry weight] of biomass), released in the absence of oxygen 1.49 mmol of P_i, 0.77 meq of Mg²⁺, 0.48 meq of K⁺, 0.02 meq of Ca²⁺, and 0.14 meq of NH₄⁺ per g (dry weight) of biomass. The drop in pH during this anaerobic phase was caused by the release of 1.8 protons per PO₄³⁻ molecule. Cells of Acinetobacter strain 132, which do not accumulate polyphosphate aerobically, released only 0.33 mmol of P_i and 0.13 meq of Mg²⁺ per g (dry weight) of biomass but released K⁺ in amounts comparable to those released by strain 210A. Stationary-phase cultures of Acinetobacter strain 210A, in which polyphosphate could not be detected by Neisser staining, aerobically took up phosphate simultaneously with Mg²⁺, the most important counterion in polyphosphate. In the absence of dissolved phosphate in the medium, no Mg²⁺ was taken up. Cells containing polyphosphate granules were able to grow in a Mg-free medium, whereas cells without these granules were not. Mg²⁺ was not essential as a counterion because it could be replaced by Ca²⁺. The presence of small amounts of K⁺ was essential for polyphosphate formation in cells of strain 210A. During continuous cultivation under K⁺ limitation, cells of Acinetobacter strain 210A contained only 14 mg of phosphorus per g (dry weight) of biomass, whereas this element was accumulated in amounts of 59 mg/g under substrate limitation and 41 mg/g under Mg²⁺ limitation. For phosphate uptake in activated sludge, the presence of K⁺ seemed to be crucial.     

Uptake and degradation of EDTA by Escherichia coli

It was found that Escherichia coli exhibited a growth by utilization of Fe(III)EDTA as a sole nitrogen source. No significant growth was detected when Fe(III)EDTA was replaced by EDTA complexes with other metal ions such as Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺, and Zn²⁺. When EDTA uptake was measured in the presence of various ions, it was remarkable only when Fe³⁺ was present. The cell extract of E. coli exhibited a significant degradation of EDTA only in the presence of Fe³⁺. It is likely that the capability of E. coli for the growth by utilization of Fe(III)EDTA results from the Fe³⁺-dependent uptake and degradation of EDTA.     

A Strain of Pseudomonas sp. Isolated from Piggery Wastewater Treatment Systems with Heterotrophic Nitrification Capability in Taiwan

A high concentration of NH₄⁺ in piggery wastewater is major problem in Taiwan. Therefore, in our study, we isolated native heterotrophic nitrifiers for piggery wastewater treatment. Heterotrophic nitrifier AS-1 was isolated and characterized from the activated sludge of a piggery wastewater system. Sets of triplicate crimp-sealed serum bottles were used to demonstrate the heterotrophic nitrifying capability of strain AS-1 in an incubator at 30°C. All serum bottles contained 80 mL medium, and the remainder of the bottle headspace was filled with pure oxygen. The experimental results showed that 2.5 ± 0.2 mmol L⁻¹ NH₄⁺ was removed by 58 hours, and, eventually, 1.5 ± 0.5 mmol L⁻¹ N₂ and 0.2 ± 0.0 mmol L⁻¹ N₂O were produced. The removal rate of NH₄⁺ by the strain AS-1 was 1.75 mmol NH₄⁺ g cell⁻¹ h⁻¹. This strain was then identified as Pseudomonas alcaligenes (97% identity) by sequencing its 16S rDNA and comparing it with other microorganisms. Thus, strain AS-1 displays high promise for future application for in situ NH₄⁺ removal from piggery wastewater.