Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
Biochemical Engineering Journal (v.26, #1)
Effect of ldhA gene deletion on the metabolism of Escherichia coli based on gene expression, enzyme activities, intracellular metabolite concentrations, and metabolic flux distribution by Md. Mohiuddin Kabir; Pei Yee Ho; Kazuyuki Shimizu (pp. 1-11).
To investigate how the central metabolism of Escherichia coli changes with the deletion of ldhA (lactate dehydrogenase gene), an integrated study was made by combining the information of cultivation characteristics, gene expressions, enzyme activities, intracellular metabolite concentrations, and metabolic flux distribution. The deletion of ldhA gene reduced the specific growth rate and the specific glucose consumption rate. The production rates of acetate, ethanol, and formate were all higher for the ldhA mutant as compared with those of the parent strain. The anaplerotic enzyme phosphoenol pyruvate carboxylase (Ppc), which is also known to be contributed to catabolic succinate formation under anaerobic condition, was downregulated in the ldhA mutant compared to the parent strain. On the other hand, phosphoenol pyruvate carboxykinase (Pck) that catalyzes the opposite reaction to Ppc was upregulated significantly in the mutant. This was mainly due to the shortage of phosphoenol pyruvate (PEP) in the ldhA mutant as was evidenced by the measurement of intracellular PEP concentration. Interestingly, two other catabolic enzymes, NAD+- and NADP+-specific malic enzymes, were both shown to be present in the parent E. coli but regulated differently in ldhA mutant. The activity of NADP+-specific malic enzyme encoded by maeB remained unchanged, whereas NAD+-specific malic enzyme encoded by sfcA was found to be upregulated significantly in the ldhA mutant compared to those in the parent strain implying that the inactivation of ldhA gene leads to the production of malate from pyruvate. Moreover, it was found that ldhA gene deletion induced a stress response as was evidenced by the significant upregulation of the genes dnaJ, grpE, groS, and fkpA involved in the heat shock response.
Keywords: Microbial growth; ldhA; mutant; E. coli; Anaerobic processes; Bioreactors; Gene expression; Enzyme activity; Metabolic flux distribution
Adsorption of serum albumin and γ-globulin from single and binary mixture and characterization of pHEMA-based affinity membrane surface by contact angle measurements by Gülay Bayramoğlu; Emine Yalçin; M. Yakup Arica (pp. 12-21).
In this study, an affinity membrane was synthesized using 2-hydroxyethylmethacrylate (HEMA) via UV-initiated photopolymerization. A dye-ligand (i.e., Procion Red HE-3B; Red-120) was covalently immobilized onto membrane. Human serum albumin (HSA) and human γ-globulin (HIgG) adsorption onto pHEMA-Red-120 membrane were studied using bare poly(hydroxyethylmethacrylate) (pHEMA) membrane as a control system. The information about surface energy, hydrophobicity and chemical heterogeneity of the affinity membrane was obtained by contact angle measurements. The contact angle values of the affinity membrane were determined by sessile drop method using water, glycerol and diiodomethane as test liquids. Component and parameters of the surface free energy of all the investigated samples were calculated from measured contact angle values using the acid–base method of the van Oss. The adsorption of HSA and HIgG significantly changed both the contact angles and component of surface free energies of the affinity membrane. The reversible HSA and HIgG adsorption on the pHEMA-Red-120 followed the Freundlich and Langmuir–Freundlich isotherm models. Selectivity of the affinity membrane was tested at different pH values to HSA and HIgG and the protein concentration of in the binary system was determined by HPLC. The affinity membrane was stable when subjected to sanitization with sodium hydroxide after repeated adsorption–elution cycles.
Keywords: Microporous membrane; Affinity chromatography; Dye-ligand; Adsorption; Separation; Albumin; γ-Globulins; Contact angle
Anaerobic co-digestion of a simulated organic fraction of municipal solid wastes and fats of animal and vegetable origin by Anna Fernández; Antoni Sánchez; Xavier Font (pp. 22-28).
The potential of mesophilic anaerobic digestion for the treatment of fats of different origin through co-digestion with the organic fraction of municipal solid wastes (OFMSW) has been evaluated. Co-digestion process was conducted in a pilot plant working in semi-continuous regime in the mesophilic range (37°C) and the hydraulic retention time (HRT) was 17 days. During the start-up period the digester was fed with increasing quantities of a simulated OFMSW (diluted dry pet food). When the designed organic loading was reached, co-digestion process was initiated. The fat used consisted of waste from the food industry (animal fat), its composition was very similar to that of the simulated OFMSW in relation to the long-chain fatty acid (LCFA) profile. Fat content in the feedstock was gradually increased up to 28%, and hence the organic loading, with stable operation of the digester. Animal fat was suddenly substituted by vegetable fat maintaining the organic loading. No accumulation of LCFA or volatile fatty acids (VFA) was detected in either case. After a short adaptation period, total fat removal throughout the experiment was over 88%, whereas biogas and methane yields were very similar to those of simulated OFMSW. In conclusion, anaerobic co-digestion of OFMSW and fat wastes appears to be a suitable technology to treat such wastes, obtaining a renewable source of energy from biogas.
Keywords: Anaerobic processes; Biogas; Fat; Long-chain fatty acids; Organic fraction of municipal solid wastes; Waste treatment
Partitioning of water in organic systems with lipase immobilized in polyurethane foams by P. Pires-Cabral; E. Dubreucq; M.M.R. da Fonseca; S. Ferreira-Dias (pp. 29-37).
Polyurethane foams are interesting enzyme supports for reactions in organic media. This study investigated the effects of: (i) support hydrophilicity; (ii) presence of immobilized lipase within the foams; and (iii) hydrophilic substrate concentration on water activity and on the partitioning of reactants between the microenvironment of the biocatalyst and the bulk organic phase. Two foams were used with different hydrophilicities. The organic phase was ethanol and butyric acid in n-hexane. The system contained water remaining from the polymerisation reaction.Experiments were carried out following a central composite rotatable design as a function of butyric acid concentration and ethanol/butyric acid molar ratio. Water activity was estimated from global medium composition by the UNIFAC-LLE group contribution method. UNIFAC calculations were also used to compare experimental bulk medium compositions with the theoretical composition of a monophasic or a biphasic system. For most experimental conditions, the organic phase composition was consistent with the presence of a water phase with no influence of the presence of enzyme in the foams. The influence of foam hydrophilicity was only significant for low water content systems (<0.05%, v/v).The system behaved as a reverse emulsion with hexane as the continuous phase and water droplets trapped within the foam matrix.
Keywords: Immobilisation; Lipase; Microenvironment; Modelling; UNIFAC; Water activity
Dynamic responses of the intracellular metabolite concentrations of the wild type and pykA mutant Escherichia coli against pulse addition of glucose or NH3 under those limiting continuous cultures by Md. Aminul Hoque; Haruo Ushiyama; Masaru Tomita; Kazuyuki Shimizu (pp. 38-49).
The dynamics of the intracellular metabolite concentrations were investigated for the wild type and pykA gene knockout mutant Escherichia coli in responses to the glucose pulse addition during glucose-limited continuous culture and in responses to the ammonia pulse addition during ammonia-limited continuous culture. For this, we developed a new automated rapid sampling device, which enables us to take samples rapidly within a second. The intracellular concentrations of G6P, F6P, 2PG, PEP, OAA, 6PG, Ribu5P, E4P and NADPH were higher for pykA mutant as compared with the wild type under both limited continuous cultures, and the concentrations of PYR, ATP and acetate were much lower for pykA mutant than those of the wild type. These phenomena reflected the fact that the accumulation of PEP caused the increased flux from PEP to OAA and that the accumulated PEP inhibited pfk, which caused the accumulation of G6P and F6P, which in turn increased the flux toward pentose phosphate (PP) pathway and increased the PP pathway metabolite concentrations. Oxygen uptake rate (OUR) was a little higher for pykA mutant as compared with that of its wild type, while CO2 production rate (CER) shows the reverse trend. OUR and CER were much less for NH3-limited condition than that of NH3-rich condition. The intracellular concentrations of PEP, ATP and PYR decreased rapidly within several seconds, whereas the concentrations of G6P, F6P FBP, 6PG, ADP, NADH and NADPH increased after glucose pulse addition during glucose-limited condition for both wild type and pykA knockout mutant. Initial decrease in PEP concentration was considered to be due to PTS system. The intracellular concentration of NADPH decreased after NH3 pulse addition under NH3-limited condition for both strains, which is due to the fact that NADPH is utilized through glutamate production under NH3 addition.
Keywords: Rapid sampling; Intracellular metabolite concentrations; Continuous culture; Transient behavior; pyk; A gene-knockout; Pulse addition
Kinetic studies of recombinant human interferon-alpha (rhIFN-α) expression in transient state continuous cultures by Poonam Srivastava; K.J. Mukherjee (pp. 50-58).
Kinetic modeling of rhIFN-α expression was done in continuous cultures using complex media at dilution rates varying from 0.2 to 0.5h−1. The human IFN-α gene was inserted under the T7 promoter in BL21 (DE3) codon plus (RIL) cells and induced with 1mM IPTG. Post-induction growth and production profiles were monitored over time. Wash out of the cells was observed only at a dilution rate of 0.5h−1, which gave a post-induction specific growth rate ( μ) of 0.37h−1. The rate of accumulation of rhIFN-α inside the cells as inclusion bodies was used to calculate the specific product formation rate ( qp). qp was found to be strongly correlated with the dilution rates used with the maximum qp being 0.17gg−1h−1 at the first hour of induction at a dilution rate of 0.5h−1.The decline in qp with time could be related to the build up of inclusion bodies inside the cells which reached a maximum value of 50% of the total protein in the fourth hour post-induction.
Keywords: Recombinant interferon alpha; Continuous cultures; Kinetics; Expression; E. coli; Inclusion bodies; Modeling
Kinetic analyses of trichloroethylene cometabolism by toluene-degrading bacteria harboring a tod homologous gene by Katsutoshi Hori; Jun’ichi Mii; Yuki Morono; Yasunori Tanji; Hajime Unno (pp. 59-64).
We have previously constructed a bacterial library consisting of toluene-degrading bacteria isolated from soil, activated sludge, and trichloroethylene (TCE) waste. In the present study, the library was subjected to colony hybridization with todC1C2-encoding terminal oxygenase of a multicomponent enzyme that is responsible for the first oxidation of toluene, toluene dioxygenase (TDO). Three strains, A1071, IB5, and B6122, which showed positive signals with todC1C2 by the colony hybridization, were selected on the basis of results of a preliminary degradation assay for detail analyses of degradation kinetics of toluene and TCE. Their possession of the gene homologous to todC1, a large subunit of TDO of Pseudomonas putida F1, was confirmed by dot hybridization analysis using their chromosomal DNA extracts as templates. Results of degradation experiments using resting cells revealed the variety in TCE degradation ability among these strains; A1071 demonstrated degradation kinetics similar to the strain F1 while B6122 could not degrade TCE at all. The interaction between toluene and TCE on their degradation could be simply expressed by competitive inhibition.
Keywords: Trichloroethylene; Cometabolism; Toluene-degrading bacteria; Toluene dioxygenase; Kinetic parameters; Inhibition
Biological phenol removal using suspended growth and packed bed reactors by G. Tziotzios; M. Teliou; V. Kaltsouni; G. Lyberatos; D.V. Vayenas (pp. 65-71).
Phenol removal was studied using batch cultures of free suspended cells and attached growth processes. Indigenous bacteria from olive pulp were enriched and used as inoculum for the filter and a suspended-growth flask reactor. A pilot-scale packed bed reactor was constructed and operated for biological phenol removal from industrial wastewater. The packed bed reactor was found to be more resistant to high phenol concentrations and led to significantly higher removal rates than the suspended-growth reactor. Two different operating modes were used to investigate the optimal performance of the filter, i.e., continuous and draw-fill. The latter was found to achieve removal rates up to 12.65g phenol/(ld), while the continuous operating mode achieved removal rates only up to 0.082g phenol/(ld).The low operating cost combined with the high phenol removal rates indicates that the above technology may offer a feasible solution to a serious environmental problem.
Keywords: Aerobic processes; Biofilms; Packed bed bioreactors; Waste-water treatment; Phenol; Olive pulp bacteria
Degradation of trichloroethylene in a coupled anaerobic–aerobic bioreactor: Modeling and experiment by B. Tartakovsky; M.-F. Manuel; S.R. Guiot (pp. 72-81).
This work studied trichloroethylene (TCE) degradation in a single stage coupled anaerobic–aerobic granular biofilm reactor. The co-existence of aerobic methanotrophic and anaerobic methanogenic bacteria in this reactor was expected to allow for a combination of reductive and oxidative pathways of TCE degradation and result in mineralization of TCE. First, the anaerobic–aerobic coupling was simulated using a mathematical model. Simulations of TCE degradation under different operating conditions suggested the superiority of the coupled reactor system over either aerobic or anaerobic process alone. Next, the approach of combining aerobic–anaerobic TCE degradation was validated in a reactor experiment. The experiment confirmed degradation of TCE due to the co-existence of methanogenic and methanotrophic populations in a single reactor.
Keywords: Dynamic modeling; Biofilm; TCE; Anaerobic–aerobic coupling; Biodegradation
Comments on “Study on biosorption of Cr(VI) by Mucor hiemalis� by Yuh-Shan Ho (pp. 82-83).
Quotation accuracy; Pseudo-first-order; Pseudo-second-order; Kinetics; Biosorption