Dynamics of phenol degradation by Pseudomonas putida

Pure cultures of Pseudomonas putida (ATCC 17484) were grown in continuous culture on phenol at dilution rates of 0.074-0.085 h(-1) and subjected to step increases in phenol feed concentration. Three distinct patterns of dynamic response were obtained depending on the size of the step change used: low level, moderate level, or high level. During low level responses no accumulations of phenol or non-phenol, non-glucose-dissolved organic carbon, DOC(NGP), were observed. Moderate level responses were characterized by the transient accumulation of DOC(NGP) with a significant delay prior to phenol leakage. High level responses demonstrated a rapid onset of phenol leakage and no apparent accumulations of DOC(NGP). The addition of phenol to a continuous culture of the same organism on glucose did not result in transient DOC(NGP) accumulations, although transient phenol levels exceeded 90 mg l(-1). These results were consistent with intermediate metabolite production during phenol step tests coupled with substrate-inhibited phenol uptake and suggested that traditional kinetic models based on the Haldane equation may be inadequate for describing the dynamics of phenol degrading systems. (c) 1993 John Wiley & Sons, Inc.     

Aerobic batch degradation of phenol using immobilized Pseudomonas putida

Alginate concentrations between 2 and 4% had little effect on the degradation rate of phenol by alginate-immobilized Pseudomonas putida. Ten-degree shifts from 25°C resulted in approximately 30% slower degradation. Maximal degradation rates were favored at pH 5.5–6.0. The response of degradation rate to increased air flow in the bubble column used was almost linear and an optimal higher than 16 vol vol⁻¹ was indicated, although free cells appeared in the reaction medium above 12 vol vol⁻¹. When the initial phenol concentration was raised, degradation rate was not significantly affected until levels higher than 1200 mg ml⁻¹ where performance was markedly reduced. Increasing the ratio of total bead volume to medium volume gave progressively smaller increases in degradation rate. At a medium volume to total bead volume ratio of 5:1, the maximum degradation rate was 250 mg L⁻¹ h⁻¹. Received 24 November 1998/ Accepted in revised form 27 January 1999     

Influence of phenol on the biodegradation of pyridine by freely suspended and immobilized Pseudomonas putida MK1

AIMS: To study the effect of co-contaminants (phenol) on the biodegradation of pyridine by freely suspended and calcium alginate immobilized bacteria. METHODS AND RESULTS: Varying concentrations of phenol were added to free and calcium alginate immobilized Pseudomonas putida MK1 (KCTC 12283) to examine the effect of this pollutant on pyridine degradation. When the concentration of phenol reached 0.38 g l(-1), pyridine degradation by freely suspended bacteria was inhibited. The increased inhibition with the higher phenol levels was apparent in increased lag times. Pyridine degradation was essentially completely inhibited at 0.5 g l(-1) phenol. However, immobilized cells showed tolerance against 0.5 g l(-1) phenol and pyridine degradation by immobilized cell could be achieved. CONCLUSIONS: This works shows that calcium alginate immobilization of microbial cells can effectively increase the tolerance of P. putida MK1 to phenol and results in increased degradation of pyridine. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of wastewater stream can be negatively affected by the presence of co-pollutants. This work demonstrates the potential of calcium alginate immobilization of microbes to protect cells against compound toxicity resulting in an increase in pollutant degradation.     

Involvement of the plasmid pPGH1 in the phenol degradation of Pseudomonas putida strain H

Pseudomonas putida strain H (wildtype) was shown to harbour two plasmids with a molecular mass of about 50 kb and 200–220 kb, respectively. Evidence is presented that the larger one, pPGH1, is involved in the phenol degradation via the meta-cleavage pathway.     

蚯蚓对细菌降解土壤中菲的作用

通过30d室内培养试验,分别研究了接种蚯蚓(E)、细菌(B)以及同时接种细菌和蚯蚓(BE)对土壤中菲降解的影响.结果表明: 在土壤中菲的初始污染浓度为50 mg*kg-1的条件下,各处理间菲的降解率差异显著,其降解率的大小顺序依次为:BE》B》E》CK(对照); 在150 mg*kg-1菲的初始污染浓度下,BE处理中菲的降解率高达98.86%,显著高于CK和E处理.B处理中细菌的双加氧酶活性在3种菲初始污染浓度下没有显著差异,而BE处理中双加氧酶的活性随着土壤中菲的初始污染浓度的升高而增加.在相同菲污染浓度下BE处理中蚯蚓体内的菲含量明显高于E处理.表明蚯蚓能够通过生物富集作用降低土壤中菲的浓度,而蚯蚓与细菌的相互作用能够进一步促进土壤中菲的降解.     

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid.

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k₁), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k₁ value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k₁ value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k₁ values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.     

Biotransformation kinetics of Pseudomonas putida for cometabolism of phenol and 4-chlorophenol in the presence of sodium glutamate

A kinetic model to describe the degradation of phenol and cometabolictransformation of 4-chlorophenol (4-cp) in the presence of sodium glutamate(SG) has been developed and validated experimentally. The integrated modelaccounts for cell growth, toxicity of 4-cp, cross-inhibitions among the threesubstrates, and the different roles of the specific growth substrate (phenol)and the conventional carbon source (SG) in the cometabolism of 4-cp. In thisternary substrate system, the overall phenol degradation and 4-cp transformation rates are greatly enhanced by the addition of SG since SG is able to attenuate the toxicity of 4-cp and therefore increase the cell growth rate. Model analysis indicates that the maximum specific degradation rate of phenol (0.819 mg (mg.h)^-1) is lowered by SG by up to 46% whereas the specific transformation rate of 4-cp is notdirectly affected by the presence of SG. The competitive inhibition coefficient of 4-cp to phenol degradation (K_i,cp) and that of phenol to 4-cp transformation (K_i,ph) were determined to be 6.49 mg l^-1 and 0.193 mg l^-1, respectively, indicatingthat phenol imposes much larger competitive inhibition to 4-cp transformation than the converse. The model developed can simultaneously predict phenol degradation and 4-cp transformation, and is useful for dealing with cometabolism involving multiple substrates.     

微生物酶法转化生产L-半胱氨酸的工艺研究

L-半胱氨酸(Cys)侧链巯基是构成蛋白质活性基团的重要氨基酸,在生物化学、医药、食品、饲料、化妆品等行业具有广泛的用途,国内外的需求量逐年增长.然而,Cys难以通过单纯的微生物发酵来进行生产;由于化学合成的步骤繁多,也很难进行化学合成.传统生产方法沿用毛发酸解制取L-半胱氨酸,收率低,能耗高,水解过程产生难闻气体及大量废酸,环境污染严重.     

Solvent-impregnated resins as an in situ product recovery tool for phenol recovery from Pseudomonas putida S12TPL fermentations

The sustainable production of fine/bulk chemicals is often hampered by product toxicity and inhibition to the producing micro-organisms. Consequently, the product must be removed from the micro-organisms' environment. To achieve this, so-called solvent-impregnated resins (SIRs) as well as commercial resins have been added to a Pseudomonas putida S12TPL fermentation that produces phenol as a model compound from glucose. The SIRs contained an ionic liquid which extracts phenol effectively. It was observed that the addition of these particles resulted in an increased phenol production of more than a fourfold while the commercial resin (XAD-4) which is widely used in aromatic removal from aqueous phases, only gave a 2.5-fold increase in volumetric production.     

Efficient production of D-glucosaminic acid from D-glucosamine by Pseudomonas putida GNA5

D-glucosaminic acid was produced efficiently from glucosamine by oxidative fermentation using a newly isolated strain, Pseudomonas putida GNA5. After optimization of the fermentation process, 51.5 g L(-1) D-glucosaminic acid was produced from an initial concentration of 60 g L(-1) D-glucosamine-HCl after 72 h of oxidative fermentation, which corresponded to a molar yield of 95.4%. This production process is potentially of considerable economic significance because very few by-products were detected. Furthermore, D-glucosaminic acid was accumulated stably during the oxidative fermentation process without the addition of an inhibitor of D-glucosaminic acid breakdown, even though D-glucosamine was exhausted. These results suggest that the mechanisms of D-glucosaminic acid-related metabolism differ between Pseudomonas putida GNA5 and the strain Pseudomonas genera, which was previously reported to produce D-glucosaminic acid.     

Epoxidation of 1,7‐octadiene by Pseudomonas oleovorans in a membrane bioreactor

A growing cell culture of Pseudomonas oleovorans was used to biotransform 1,7‐octadiene to 1,2‐epoxy‐7,8‐octene in a continuous‐flow bioreactor with an external membrane module. A dense silicone rubber membrane was used to contact an organic phase, containing both the reactant (1,7‐octadiene) and the growth substrate (heptane), with an aqueous biomedium phase containing the biocatalyst. Heptane and octadiene delivery to the aqueous phase, and epoxide extraction into the solvent, occurred by diffusion across the dense membrane under a concentration‐driving force. In addition, a liquid feed of heptane and octadiene was pumped directly into the bioreactor to increase the rate of delivery of these compounds to the aqueous phase. In this system 1,2‐epoxy‐7,8‐octene accumulated in a pure solvent phase, thus, product recovery problems associated with emulsion formation were avoided. Furthermore, no phase breakthrough of either liquid across the membrane was observed. In this system, the highest volumetric productivity obtained was 30 U.L⁻¹, and this was achieved at a dilution rate of 0.07 h⁻¹, 70 m².m⁻³ of membrane area, and a steady‐state biomass concentration of 2.5 g.L⁻¹. The system was stable for over 1250 h. Decreasing the dilution rate led to an increased biomass concentration, however, the specific activity was significantly reduced, and therefore, an optimal dilution rate was determined at 0.055 h⁻¹. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 601–611, 1999.     

A novel pathway for the biodegradation of 3-nitrotoluene in Pseudomonas putida

Abstract: 3-Nitrotoluene was degraded when incubated with the resting cells of Pseudomonas putida OU83. Most of the 3-nitrotoluene (70%) was metabolized via reduction of the nitro group to form 3-aminotoluene (3-AT). A minor portion (30%) was degraded through a novel pathway involving oxidation of 3-NT to form 3-nitrophenol through a series of intermediary metabolites: 3-nitrobenzyl alcohol, 3-nitrobenzaldehyde and 3-nitrobenzoic acid. Degradation of 3-nitrophenol occurred with the formation of a transient intermediary metabolite, hydroxynitroquinone, which was further degraded with the near stoichiometric release of nitrite into the medium. 3-Nitrotoluene-induced cells showed increased oxygen consumption with 3-nitrotoluene, 3-nitrobenzaldehyde, 3-nitrobenzoate, and 3-nitrophenol as substrates in comparison to uninduced cells. Cell extracts prepared from strain OU83 contained benzylalcohol dehydrogenase and benzaldehyde dehydrogenase activities. The experimental evidence suggests a novel pathway for the degradation of 3-NT in which C-1 elimination is catalyzed by a cofactor-independent deformylase, rather than a decarboxylase or dioxygenase.     

The Degradation of Dimethoate and the Mineral Immobilizing Function for Cd2+ by Pseudomonas putida

Organophosphorus pollution and heavy metal pollution are prominent in China and have caused increasingly severe environmental pollution. This research used Pseudomonas putida to degrade dimethoate so as to induce the formation of calcium carbonate (CaCO₃) and calcium phosphate (Ca₃(PO₄)₂) in beef extract peptone medium. In addition, the mineral immobilizing function of the generated Ca₃(PO₄)₂ and CaCO₃ for Cd²⁺ was studied by adding different concentrations of Cd²⁺ to the culture solution. Meanwhile, transmission electron microscopy (TEM), scanning electronic microscopy (SEM), X-ray diffraction, gas chromatography and atomic absorption spectrophotometry were used to investigate the biodegradation of dimethoate, the concentration variation of Ca²⁺ and Cd²⁺, the mineral and chemical compositions of the precipitates. The results showed that the growth of P. Putida could increase the pH value of the culture solution and effectively degrade the organophosphorus pesticide dimethoate. Besides, the concentration of Ca²⁺ in the culture solution decreased significantly in the first four days and then tended to be stable. Moreover, the TEM and SEM results presented that there were large amounts of biogenic sedimentary CaCO₃ and a little Ca₃(PO₄)₂ in the precipitates. Furthermore, in the employed culture system, the removal rates of Cd²⁺, when added at two different concentrations (6 ppm and 15 ppm), reached 100%. Therefore, this study provided a new idea for treating wastewater polluted with organophosphorus pesticide and heavy metals by using microorganisms.     

Improved performance of Pseudomonas putida in a bioelectrochemical system through overexpression of periplasmic glucose dehydrogenase

It was recently demonstrated that a bioelectrochemical system (BES) with a redox mediator allowed Pseudomonas putida to perform anoxic metabolism, converting sugar to sugar acids with high yield. However, the low productivity currently limits the application of this technology. To improve productivity, the strain was optimized through improved expression of glucose dehydrogenase (GCD) and gluconate dehydrogenase (GAD). In addition, quantitative real‐time RT‐PCR analysis revealed the intrinsic self‐regulation of GCD and GAD. Utilizing this self‐regulation system, the single overexpression strain (GCD) gave an outstanding performance in the electron transfer rate and 2‐ketogluconic acid (2KGA) productivity. The peak anodic current density, specific glucose uptake rate and 2KGA producing rate were 0.12 mA/cm², 0.27 ± 0.02 mmol/g_CDW/hr and 0.25 ± 0.02 mmol/g_CDW/hr, which were 327%, 477%, and 644% of the values of wild‐type P. putida KT2440, respectively. This work demonstrates that expression of periplasmic dehydrogenases involved in electron transfer can significantly improve productivity in the BES.     

Miniaturized systems for evaluating enzyme activity in polymeric membrane bioreactors

Enzyme‐coated polymeric membranes are versatile catalysts for biofuel production and other chemical production from feedstock, like plant biomass. Such bioreactors are more energy efficient than high temperature methods because enzymes catalyze chemical reactions near room temperature. A major challenge in processing plant biomass is the presence of lignin, a complex aromatic polymer that resists chemical breakdown. Therefore, membranes coated with enzymes such as laccase that can degrade lignin are sought for energy extraction systems. We present an experimental study on optimizing an enzyme‐based membrane bioreactor and investigate the tradeoff between high flow rate and short dwell time in the active region. In this work, zero flow rate voltammetry experiments confirm the electrochemical activity of Trametes versicolor laccase on conductive polymer electrodes, and a flow‐through spectroscopy device with laccase‐coated porous nylon membranes is used with a colorimetric laccase activity indicator to measure the catalysis rate and percent conversion as a function of reactant flow rate. Membrane porosity before and after laccase coating is verified with electron microscopy.     

Proteome reference map for the plant growth‐promoting bacterium Pseudomonas putida UW4

A proteome reference map containing 326 2‐D gel spots representing 275 different proteins was constructed for the plant growth‐promoting bacterium Pseudomonas putida UW4. Protein identifications were obtained using Q‐TOF MS/MS spectra matching to homologous proteins from other Pseudomonas strains and confirmed by PMF analysis. This data set is accessible at http://world‐2dpage.expasy.org/repository/ and will aid in further characterization of Pseudomonas strains and interactions of plant growth‐promoting bacterium with the plant rhizosphere environment.     

Putting cells under pressure: a simple and efficient way to enhance the productivity of medium-chain-length polyhydroxyalkanoate in processes with Pseudomonas putida KT2440

The success of bioprocess implementation relies on the ability to achieve high volumetric productivities and requires working with high‐cell‐density cultivations. Elevated atmospheric pressure might constitute a promising tool for enhancing the oxygen transfer rate (OTR), the major growth‐limiting factor for such cultivations. However, elevated pressure and its effects on the cellular environment also represent a potential source of stress for bacteria and may have negative effects on product formation. In order to determine whether elevated pressure can be applied for enhancing productivity in the case of medium‐chain‐length polyhydroxyalkanoate (mcl‐PHA) production by Pseudomonas putida KT2440, the impact of a pressure of 7 bar on the cell physiology was assessed. It was established that cell growth was not inhibited by this pressure if dissolved oxygen tension (DOT) and dissolved carbon dioxide tension (DCT) were kept below ～30 and ～90 mg L^?1, respectively. Remarkably, a little increase of mcl‐PHA volumetric productivity was observed under elevated pressure. Furthermore, the effect of DCT, which can reach substantial levels during high‐cell‐density processes run under elevated pressure, was investigated on cell physiology. A negative effect on product formation could be dismissed since no significant reduction of mcl‐PHA content occurred up to a DCT of ～540 mg L^?1. However, specific growth rate exhibited a significant decrease, indicating that successful high‐cell‐density processes under elevated pressure would be restricted to chemostats with low dilution rates and fed‐batches with a small growth rate imposed during the final part. This study revealed that elevated pressure is an adequate and efficient way to enhance OTR and mcl‐PHA productivity. We estimate that the oxygen provided to the culture broth under elevated pressure would be sufficient to triple mcl‐PHA productivity in our chemostat system from 3.4 (at 1 bar) to 11 g L^?1 h^?1 (at 3.2 bar). Biotechnol. Bioeng. 2012; 109:451–461. © 2011 Wiley Periodicals, Inc.     

The role of fatty acid biosynthesis and degradation in the supply of substrates for poly(3-hydroxyalkanoate) formation in Pseudomonas putida

Abstract The relationship between fatty acid metabolism and PHA biosynthesis in P. putida is described. Detailed ¹H and ¹³C NMR studies were performed to investigate the structures of poly(3-hydroxyalkanoates) (PHAs) formed from carbohydrates and fatty acids. On the basis of these results, it is proposed that during growth on glucose the 3-hydroxyacyl-acyl carrier protein intermediates of the de novo fatty acid biosynthetic pathway are diverted to PHA biosynthesis. Similarly, further evidence is presented that during cultivation on fatty acids, intermediates of the β-oxidation cycle serve as precursors of PHA biosynthesis.     

Co-metabolic degradation of trichloroethylene by Pseudomonas putida in a fibrous bed bioreactor

Co-metabolic degradation of trichloroethylene (TCE) by Pseudomonas putida F1 was investigated in a novel bioreactor with a fibrous bed. A pseudo-first-order rate constant for TCE degradation was 1.4 h^–1 for 2.4 to 100 mg TCE l^–1. Competitive inhibition of toluene on TCE removal could be prevented in this bioreactor. 90% TCE was removed over 4 h when 95 mg toluene l^–1 was presented simultaneously.