Catalytic potential of a poly(AAc‐co‐HPMA‐cl MBAm)‐matrix‐immobilized lipase from a thermotolerant Pseudomonas aeruginosa MTCC‐4713

A purified alkaline thermo‐tolerant lipase from Pseudomonas aeruginosa MTCC‐4713 was immobilized on a series of five noble weakly hydrophilic poly(AAc‐co‐HPMA‐cl MBAm) hydrogels. The hydrogel synthesized by copolymerizing acrylic acid and 2‐hydroxy propyl methacrylate in a ratio of 5 : 1 (HG_5:1 matrix) showed maximum binding efficiency for lipase (95.3%, specific activity 1.96 IU mg^?1 of protein). The HG_5:1 immobilized lipase was evaluated for its hydrolytic potential towards p‐NPP by studying the effect of various physical parameters and salt‐ions. The immobilized lipase was highly stable and retained ～92% of its original hydrolytic activity after fifth cycle of reuse for hydrolysis of p‐nitrophenyl palmitate at pH 7.5 and temperature 55°C. However, when the effect of pH and temperature was studied on free and bound lipase, the HG_5:1 immobilized lipase exhibited a shift in optima for pH and temperature from pH 7.5 and 55°C to 8.5 and 65°C in free and immobilized lipase, respectively. At 1 mM concentration, Fe³⁺, Hg²⁺, NH₄⁺, and Al³⁺ ions promoted and Co²⁺ ions inhibited the hydrolytic activities of free as well as immobilized lipase. However, exposure of either free or immobilized lipase to any of these ions at 5 mM concentration strongly increased the hydrolysis of p‐NPP (by ～3–4 times) in comparison to the biocatalysts not exposed to any of the salt ions. The study concluded that HG_5:1 matrix efficiently immobilized lipase of P. aeruginosa MTCC‐4713, improved the stability of the immobilized biocatalyst towards a higher pH and temperature than the free enzyme and interacted with Fe³⁺, Hg²⁺, NH₄⁺, and Al³⁺ ions to promote rapid hydrolysis of the substrate (p‐NPP). © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 4252–4259, 2006     

Purification and characterization of solvent‐tolerant,thermostable, alkaline metalloprotease from alkalophilic Pseudomonas aeruginosa MTCC 7926

BACKGROUND: Microbial proteases are becoming imperative for commercial applications. The protease secreted by Pseudomonas aeruginosa MTCC 7926, isolated from solvent‐contaminated habitat was purified and characterized for activity at various edaphic conditions. The purified alkaline protease was investigated for dehairing of animal skin, anti‐staphylococcal activity and processing of X‐ray film. RESULTS: The protease was 24‐fold purified by ammonium sulfate fractionation, sephadex G‐100 gel filtration and DEAE‐cellulose, with 36% recovery. K_M and V_max, using casein were 2.94 mg mL^?1 and 1.27 µmole min^?1, respectively. The apparent molecular mass by SDS‐PAGE was 35 kDa. Alkaline protease was active at pH 6–11 and temperature 25–65 °C. Its activity was (a) 86.8% in 100 mmol L^?1 NaCl, (b) >95% in metal ions (Mn²⁺, Ca²⁺, Mg²⁺, Fe²⁺) for 1 h, (c) >90% in bleaching agents and chemical surfactants, (d) 135.4 ± 2.0% and 119.9 ± 6.2% with rhamnolipid and cyclodextrin, respectively, (e) stable in solvents for 5–30 days at 27 °C, and (f) inhibited by EDTA, indicating metalloprotein. CONCLUSION: This work showed that purified protease retained its activity in surfactants, solvents, metals, and bleaching agents. The enzyme is an alternative for detergent formulations, dehairing of animal skin, X‐ray film processing, treatment of staphylococcal infections and possibly non‐aqueous enzymatic peptide synthesis. Copyright © 2009 Society of Chemical Industry     

以摄氧率为基准的放大技术

以菌体最大摄氧率（ＯＵＲ）ｍａｘ为基准进行发酵放大，考察了ＯＵＲ与菌体产生量（ｘ）之间的关系。得出在啤酒酵母、苏云金杆菌的发酵过程中，ＯＵＲ与ｘ密切相关，ＯＵＲ增加，ｘ增加；同时得出进出口流量改变对求得的ＯＵＲ值差异影响很大，但不管以什么基准求ＯＵＲ，ｘ与所得ＯＵＲ均成正比关系     

Structure–Activity Relationships of 2‐Sufonylpyrimidines as Quorum‐Sensing Inhibitors to Tackle Biofilm Formation and eDNA Release of Pseudomonas aeruginosa

Drug‐resistant Pseudomonas aeruginosa (PA) strains are on the rise, making treatment with current antibiotics ineffective. Hence, circumventing resistance or restoring the activity of antibiotics by novel approaches is of high demand. Targeting the Pseudomonas quinolone signal quorum sensing (PQS‐QS) system is an intriguing strategy to abolish PA pathogenicity without affecting the viability of the pathogen. Herein we report the structure–activity relationships of 2‐sulfonylpyrimidines, which were previously identified as dual‐target inhibitors of the PQS receptor PqsR and the PQS synthase PqsD. The SAR elucidation was guided by a combined approach using ligand efficiency and ligand lipophilicity efficiency to select the most promising compounds. In addition, the most effective inhibitors were rationally modified by the guidance of QSAR using Hansch analyses. Finally, these inhibitors showed the capacity to decrease biofilm mass and extracellular DNA, which are important determinants for antibiotic resistance.     

Fermentation and oxygen transfer characteristics in recombinant human growth hormone production by Pichia pastoris in sorbitol batch and methanol fed‐batch operation

BACKGROUND: The influence of methanol feed rate on recombinant human growth hormone (rhGH) production by Pichia pastoris hGH‐Mut⁺ in medium containing sorbitol was investigated at three different specific growth rates (µ), namely, 0.02 (MS‐0.02), 0.03 (MS‐0.03), and 0.04 (MS‐0.04). RESULTS: Increasing methanol feed rate above MS‐0.03 did not affect sorbitol consumption, showing that µ = 0.03 h^?1 is a threshold limiting value, above which sorbitol utilization became independent of methanol feed rate. Moreover, when sorbitol was consumed, no further cell growth was observed. Increase in methanol feed rate triggered cell synthesis and the highest cell concentration was obtained at MS‐0.04 as 48 g L^?1 (t = 18 h); whereas, the highest rhGH production, 270 mg L^?1, was obtained at MS‐0.03 as a consequence of lower extracellular protease production and higher AOX activity (41 U g^?1 CDW). Oxygen uptake rate increased with increasing µ, having the maximum value, 76.6 mmol m^?3 s^?1, at MS‐0.04. K_La had a tendency to increase with µ, having a maximum value of 0.15 s^?1 at MS‐0.04 (t = 15 h). CONCLUSION: By considering rhGH concentration and oxygen transfer characteristics, the bioprocess can be improved by a two‐stage feeding strategy, operating at MS‐0.04 at the beginning of fermentation, and thereafter shifting to MS‐0.03. Copyright © 2009 Society of Chemical Industry     

Design and characterization of a one‐compartment scale‐down system for simulating dissolved oxygen tension gradients

BACKGROUND: A laboratory scale one‐compartment scale‐down system (1‐CSDS), used to generate dissolved oxygen tension (DOT) gradients was designed and characterized. The system consists of a 1.5‐L stirred‐tank bioreactor coupled to an automatic DOT controller that changes the oxygen partial pressure in the inlet gas through a feedback proportional–integral–derivative algorithm, while maintaining the hydrodynamic conditions constant. Oscillatory control of DOT was achieved by employing time‐dependent square wave or sinusoidal setpoints. RESULTS: The 1‐CSDS can be modeled as a first‐order dynamic system, but showing a permanent lag between the system response and the setpoint. The 1‐CSDS had a faster response rate for generating oscillating DOT when a square wave setpoint was used rather than a sinusoidal setpoint. The 1‐CSDS generated symmetric DOT oscillations at periods above of 100 s. CONCLUSION: The 1‐CSDS is suited to investigate the responses of microorganisms and cells, of biotechnological importance, to oscillatory DOT conditions. It was found that the response of the 1‐CSDS was limited by the k_La. Copyright © 2010 Society of Chemical Industry     

Properties of poly(AAc‐co‐HPMA‐cl‐EGDMA) hydrogel‐bound lipase of Pseudomonas aeruginosa MTCC‐4713 and its use in synthesis of methyl acrylate

Microbial lipases (E.C. 3.1.1.3) are preferred biocatalysts for the synthesis of esters in organic solvents. Various extracellular thermoalkaliphilic lipases have been reported from Pseudomonas sp. In the present study, a purified alkaline thermoalkalophilic extracellular lipase of Pseudomonas aeruginosa MTCC‐4713 was efficiently immobilized onto a synthetic poly(AAc‐co‐HPMA‐cl‐EGDMA) hydrogel by adsorption and the bound lipase was evaluated for its hydrolytic potential towards various p‐nitrophenyl acyl esters varying in their C‐chain lengths. The bound lipase showed optimal hydrolytic activity towards p‐nitrophenyl palmitate (p‐NPP) at pH 8.5 and temperature 45°C. The hydrolytic activity of the hydrogel‐bound lipase was markedly enhanced by the presence of Hg²⁺, Fe³⁺, and NH salt ions in that order. The hydrogel‐immobilized lipase (25 mg) was used to perform esterification in various n‐alkane(s) that resulted in ～ 84.9 mM of methyl acrylate at 45°C in n‐heptane under shaking (120 rpm) after 6 h, when methanol and acrylic acid were used in a ratio of 100 mM:100 mM, respectively. Addition of a molecular sieve (3Å × 1.5 mm) to the reaction system at a concentration of 100 mg/reaction vol (1 mL) resulted in a moderate enhancement in conversion of reactants into methyl acrylate (85.6 mM). During the repetitive esterification under optimum conditions, the hydrogel‐bound lipase produced 71.3 mM of ester after 10th cycle of reuse. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 183–191, 2007     

Multiobjective optimization of microalgae (Chlorella sp.) growth in a photobioreactor using Box‐Behnken design approach

This study investigates a parameter optimization approach to maximize the specific growth rate of the Chlorella vulgaris microalgae species, its biomass productivity, and CO₂ capture rate. For this purpose, the Box‐Behnken experimental design technique is applied with temperature, nitrogen to phosphorus ratio, and light‐dark cycle per day, as the growth controlling parameters. For each response, a quadratic model is developed separately describing the algal specific growth rate, biomass productivity, and CO₂ capture rate, respectively. The maximum specific growth rate of 0.84 d^?1 is obtained at 25 °C, with a nitrogen to phosphorus ratio of 3.4:1, and light‐dark cycles of 24/0 h. Maximum biomass productivity of 147.3 mg L^?1 d^?1 is found at 30 °C, with a nitrogen to phosphorus ratio of 3:1, and light‐dark cycles of 12/12 h. In addition, the maximum CO₂ capture rate of 159.5 mg L^?1 d^?1 is also obtained at 30 °C, with a nitrogen to phosphorus ratio of 4:1, and light‐dark cycles of 23/1 h. Finally, a multi‐response optimization method is applied to maximize the specific growth rate, biomass productivity, and CO₂ capture rate, simultaneously. The optimal set of 30 °C, a nitrogen to phosphorus ratio 3:1, and light‐dark cycles 16/8 h, provide the maximum specific growth rate of 0.66 per day, biomass productivity of 147.6 mg L^?1 d^?1, and CO₂ capture rate of 141.7 mg L^?1 d^?1.

     

Simulation of heterotrophic storage and growth processes in activated sludge under aerobic conditions

In this work, an extension of the Activated Sludge Model No. 3 (ASM3) is presented which takes oxygen transfer, microbial maintenance, and biomass decay into account, in order to describe the heterotrophic storage and growth processes in activated sludge. The sensitivity of the effluent chemical oxygen demand and oxygen uptake rate to the stoichiometric and kinetic coefficients was analyzed. Model calibration was successfully performed by comparing measured and predicted values for model components. Thereafter, the model was evaluated with the experimental results of four independent case studies. Results show that the established model is able to better and mechanistically describe the heterotrophic storage and growth processes in activated sludge.     

Two types of organophosphate pesticides and their combined effects on heterotrophic growth rates in activated sludge process

BACKGROUND: Pesticides are sometimes non‐biodegradable and, moreover, toxic to microorganisms. If pesticides exceed the tolerance of microorganisms, failure of the activated sludge process (ASP) occurs. Therefore the effects of two types of organophosphate pesticides on heterotrophic growth rate constant in sludge from ASP were investigated. Oxygen uptake rate was employed to measure the rate constants. RESULTS: The results indicated that the value of heterotrophic growth rate constant decreased from 3.88 d^?1 to 1.46 d^?1 or by 62% when 0.5 mg L^?1 of glyphosate was added. When adding 0.5 mg L^?1 of malathion, the value of heterotrophic growth rate constant decreased to 1.33 d^?1 or by 66%. The value of heterotrophic growth rate constant decreased to 1.98 d^?1 or by 49% when 0.5 mg L^?1 of pesticide combination (50% for each) was added. CONCLUSIONS: The inhibitory effects of glyphosate and malathion were in good agreement with non‐competitive inhibition kinetics, but pesticide combination did not follow non‐competitive kinetics. The inhibition coefficient values for glyphosate, malathion and their combination were 0.29, 0.29 and 0.58 mg L^?1, respectively. For comparison, linear and exponential types of models were derived by regression. According to non‐competitive kinetics, and linear and exponential models, the inhibitory effects of glyphosate and malathion were almost consistent. Finally, the degree of inhibition was simulated using different types of model. It was found that the toxicity of the two pesticides agreed with the antagonism well. Copyright © 2009 Society of Chemical Industry     

Screening and identification of Pseudomonas aeruginosa AB4 for improved production,characterization and application of a glycolipid biosurfactant using low‐cost agro‐based raw materials

BACKGROUND: The study is focused on (i) screening and taxonomic identity of a bacterial strain for biosurfactant production, and (ii) evaluation of its potential for production of a biosurfactant using agro‐based feedstock(s) and characterization of it for application in the removal of heavy metals. RESULTS: The production of biosurfactant by an isolate Pseudomonas aeruginosa AB4 (identified on the basis of 16S rRNA analysis) using various cost‐effective substrates were examined at conditions 40 °C, 120 rpm for 7 days. It revealed maximum (40 gL^?1) rhamnolipids production and 46% reduction of initial surface tension. Its optimum production was achieved at (i) C:N ratio 10:0.6, (ii) pH 8.5 and (iii) 40 °C. The cell–free supernatant examined for biosurfactant activity by (i) haemolytic assay, (ii) CTAB‐ methylene blue assay, (iii) drop collapse test, (iv) oil spreading technique and (v) EI 24 assay showed its glycolipid nature and stable emulsification. Analysis of partially purified rhamnolipids by (i) thin layer chromatography (TLC), (ii) high performance thin layer chromatography (HPTLC), (iii) high performance liquid chromatography (HPLC), (iv) Fourier transform infrared (FT‐IR) and (v) gas chromatography–mass spectrometry (GC‐MS) confirmed its structure as methyl ester of 3‐hydroxy decanoic acid (a glycolipid) with two major structural congeners (Rha‐C₁₀‐C₁₀ and Rha‐C₁₀‐C₈) of mono‐rhamnolipids. Finally, it showed sequestration of Cd and Pb, suggesting its application in biosurfactant‐assisted heavy metal bioremediation. CONCLUSION: This work has screened and identified a bacterium with superior biosurfactant production capabilities, characterized the glycolipidic biosurfactants as rhamnolipid and indicated the feasibility of biosurfactant production using novel renewable, relatively inexpensive and easily available resources such as non‐edible vegetable de‐oiled seed cakes and showed its utility in remediation of heavy metals. Copyright © 2010 Society of Chemical Industry     

Study on the dissolved oxygen control strategy in large‐scale vitamin B12 fermentation by Pseudomonas denitrificans

BACKGROUND: There are two different routes for vitamin B₁₂ biosynthesis, which results in discrepancies and uncertainties of the dissolved oxygen (DO) concentration for vitamin B₁₂ fermentation. In this paper, the DO control strategy was explored for industrial vitamin B₁₂ fermentation by Pesudomonas denitrificans in 120000‐L fermenter. RESULTS: A DO‐stat strategy was first successfully scaled up from a 9000 L fermenter to a 120 000 L fermenter. Then a multi‐stage DO control strategy was further established in the 120 000 L fermenter, in which the DO level was shifted from 8–10% (20–48 h) to 2–5% (49–106 h) and below 2% (107–168 h) by gradually reducing the rate of aeration and agitation. As a result, 198.80 mg L^?1 of vitamin B₁₂ was obtained, which was significantly higher than those obtained under the fermentations with one‐stage DO control. CONCLUSIONS: The comparatively low DO level was favorable for vitamin B₁₂ biosynthesis, but it would have an extremely negative effect on cell growth. Compared with the low DO level maintained at all times of the fermentation process, a multi‐stage DO control strategy could not only increase the biomass but also improve vitamin B₁₂ biosynthesis. Copyright © 2012 Society of Chemical Industry     

Three‐dimensional simulations of biofilm growth in porous media

Biofilm growth occurs in a variety of random porous media in a range of industrial processes; prediction of its growth and subsequent influence on hydrodynamics is hence desirable. In this study, we present the first numerical 3D pore‐scale model of biofilm growth in porous media, based on a lattice Boltzmann simulation platform complemented with an individual‐based biofilm model (IbM). We use it to explore the coupled interaction between nutrient mass transport, biofilm growth, and hydrodynamics. Biofilm is shown to be very effective at reducing the permeability of porous media, particularly under nutrient limited conditions. We conclude with a direct comparison of 3D and 2D biofilm growth simulations in porous media and show the necessity of performing the simulations in 3D. © 2008 American Institute of Chemical Engineers AIChE J, 2009     

Interest of Homodialkyl Neamine Derivatives against Resistant P. aeruginosa,E. coli,and β-Lactamases-Producing Bacteria—Effect of Alkyl Chain Length on the Interaction with LPS

Development of novel therapeutics to treat antibiotic-resistant infections, especially those caused by ESKAPE pathogens, is urgent. One of the most critical pathogens is P. aeruginosa, which is able to develop a large number of factors associated with antibiotic resistance, including high level of impermeability. Gram-negative bacteria are protected from the environment by an asymmetric Outer Membrane primarily composed of lipopolysaccharides (LPS) at the outer leaflet and phospholipids in the inner leaflet. Based on a large hemi-synthesis program focusing on amphiphilic aminoglycoside derivatives, we extend the antimicrobial activity of 3′,6-dinonyl neamine and its branched isomer, 3′,6-di(dimethyloctyl) neamine on clinical P. aeruginosa, ESBL, and carbapenemase strains. We also investigated the capacity of 3′,6-homodialkyl neamine derivatives carrying different alkyl chains (C7–C11) to interact with LPS and alter membrane permeability. 3′,6-Dinonyl neamine and its branched isomer, 3′,6-di(dimethyloctyl) neamine showed low MICs on clinical P. aeruginosa, ESBL, and carbapenemase strains with no MIC increase for long-duration incubation. In contrast from what was observed for membrane permeability, length of alkyl chains was critical for the capacity of 3′,6-homodialkyl neamine derivatives to bind to LPS. We demonstrated the high antibacterial potential of the amphiphilic neamine derivatives in the fight against ESKAPE pathogens and pointed out some particular characteristics making the 3′,6-dinonyl- and 3′,6-di(dimethyloctyl)-neamine derivatives the best candidates for further development.