Process development for degradation of phenol by Pseudomonas putida in hollow-fiber membrane bioreactors

The degradation of phenol (100-2800 mg/L) by cells Pseudomonas putida CCRC14365 in an extractive hollow-fiber membrane bioreactor (HFMBR) was studied, in which the polypropylene fibers were prewetted with ethanol. The effects of flow velocity, the concentrations of phenol, and the added dispersive agent tetrasodium pyrophosphate on phenol degradation and cell growth were examined. It was shown that about 10% of phenol was sorbed on the fibers at the beginning of the degradation process. The cells P. putida fully degraded 2000 mg/L of phenol within 73 h when the cells were immobilized and separated by the fibers. Even at a level of 2800 mg/L, phenol could be degraded more than 90% after 95-h operation. At low phenol levels (< 400 mg/L) where substrate inhibition was not severe, it was more advantageous to treat the solution in a suspended system. At higher phenol levels (> 1000 mg/L), however, such HFMBR-immobilized cells could degrade phenol to a tolerable concentration with weak substrate-inhibition effect, and the degradation that followed could be completed by suspended cultures due to their larger degradation rate. The process development in an HFMBR system was also discussed.     

Anaerobic degradation of phenol in batch and continuous cultures by a denitrifying bacterial consortium

Summary The anaerobic degradation of phenol under denitrifying conditions by a bacterial consortium was studied both in batch and continuous cultures. Anaerobic degradation was dependent on NO_f3 ^p– and concentrations up to 4 mm phenol were degraded within 2–5 days. During continuous growth in a fermenter, steady states could be maintained at eight dilution rates (D) corresponding to residence times between 12.5 and 50 h. Culture wash-out occurred at D=0.084 h^–1. The kinetic parameters obtained for anaerobic degradation of phenol under denitrifying conditions by the consortium were: maximam specific growth rate = 0.091 h^–1; saturation constant = 4.91 mg phenol/l; true growth yield = 0.57 mg dry wt/mg phenol; maintenance coefficient = 0.013 mg phenol/mg dry wt per hour. The Haldane model inhibition constant was estimated from batch culture data giving a value of 101 mg/l. The requirement of CO₂ for the anaerobic degradation of phenol with NO_f3 ^p– indicates that phenol carboxylation to 4-hydroxybenzoate was the first step of phenol degradation by this culture. 4-Hydroxybenzoate, proposed as an intermediate of phenol carboxylation under these conditions, was detected only in continuous cultures at very low growth rates (D=0.02 h^–1), but was never detected as a free intermediary metabolite either in batch or in continuous cultures. Correspondence to: N. Khoury     

Growth kinetics of Pseudomonas putida in cometabolism of phenol and 4-chlorophenol in the presence of a conventional carbon source

Growth kinetics of Pseudomonas putida (ATCC 49451) in cometabolism of phenol and 4-chlorophenol (4-cp) in the presence of sodium glutamate (SG) were studied. In the ternary substrate mixture, phenol and SG are growth substrates while 4-cp is a nongrowth substrate. Cell growth on phenol was found to follow Andrews kinetics and cells displayed substrate inhibition pattern on sodium glutamate in the range of 0-4 g L(-1) as well. A cell growth model for the ternary substrate system was established based on a simplified cell growth mechanism and subsequently modified by experimental results. Model analysis over a wide range of substrate concentrations shows that the inhibition of SG is much larger than phenol at low phenol concentrations (/=600 mg L(-1)). The nongrowth substrate, 4-cp, inhibits cell growth mainly through inactivation of cells (cell decay) and competitive inhibition to cell growth on phenol. In the absence of SG, 4-cp retards cell growth severely and cells cannot grow at 250 mg L(-1) 4-cp. Addition of sodium glutamate, however, greatly attenuates the toxicity of 4-cp and supports cell growth at 4-cp concentration higher than 250 mg L(-1). By using the proposed cell growth model, we were able to optimize the amount of SG needed to enhance cell growth rate and validate model predictions against experimental data.     

Biodegradation of Phenol by Actinobacillus Sp.: Mathematical Interpretation and Effect of Some Growth Conditions

Models of the cultivation process of Actinobacillus sp. cells in two media, rich (NB) and minimal (M9) that includes phenol as a sole carbon source, have not been described in the available literature. For these reasons, several single-substrate inhibition models (Monod, Andrew, and Tesseir) were investigated in order to determine the mathematical expression of Actinobacillus sp. growth rate. The experimental data for both nutrient broth and M9 media were fitted to the above models mentioning that Andrews' model best fits these data adequately for both media with regression coefficient of 0.973 and 0.962, respectively. The maximum predicted growth rate by this model is 0.37 h^{- 1} for both media obtained when the initial concentration of phenol is 100 mg/L. The half-saturation concentration constant, K_P, is 1.00 mg/L, which represents the phenol concentration when μ is equal to half μ_max. On the other hand, the inhibition constant, K_p is 13,000.00 mg/L for broth medium and 12,000 mg/L for M9 medium, which is a measure of sensitivity to inhibition by inhibitory substances. When cells are grown in nutrient broth and minimal media, the rate of cell production with time can be expressed by the Reccati and Voltera models. Voltera model better fits in the case of M9 minimal medium plus phenol as sole carbon source. The pH of 7, the incubation temperature of 35°C to 37°C, and the agitation rate of 150 rpm are the optimal conditions for achieving the higher percentage of phenol degradation by Actinobacillus sp. Succinic acid and glycine as carbon and nitrogen source, respectively, were the most efficient of the cosubstrates (out of 10 substrates tested) for removal of phenol on an mg/L basis.     

Phenol degradation by Ralstonia eutropha: colorimetric determination of 2-hydroxymuconate semialdehyde accumulation to control feed strategy in fed-batch fermentations.

Phenol biodegradation by Ralstonia eutropha was modeled in different culture modes to assess phenol feeding in biotechnological depollution processes. The substrate-inhibited growth of R. eutropha was described by the Haldane equation with a Ks of 2 mg/L, a Ki of 350 mg/L and a mumax of 0.41 h(-1). Furthermore, growth in several culture modes was characterized by the appearance of a yellow color, due to production of a metabolic intermediate of the phenol catabolic pathway, 2-hydroxymuconic semialdehyde (2-hms) which was directly correlated to the growth rate and/or the phenol-degradation rate, because these two parameters are coupled (as seen by the constant growth yield of 0.68 g biomass/g phenol whatever the phenol concentration). This correlation between color appearance and metabolic activity was used to develop a control procedure for optimal phenol degradation. A mass-balance equation modeling approach combined with a filtering step using an extended Kalman filter enabled state variables of the biological system to be simulated. A PI controller, using the estimation of the phenol concentration provided by the modeling step, was then built to maintain the phenol concentration at a constant set-point of 0.1 g/L which corresponded to a constant specific growth rate of 0.3 h(-1), close to the maximal specific growth value of the strain. This monitoring strategy, validated for two fed-batch cultures, could lead, in self-cycling fermentation systems, to a productivity of more than 19 kg of phenol consumed/m(3)/d which is the highest value reported to date in the literature. This system of monitoring metabolic activity also protected the bacterial culture against toxicity problems due to the transient accumulation of phenol.     

Degradation of phenol by aerobic granules and isolated yeast Candida tropicalis

Aerobic granules effectively degrade phenol at high concentrations. This work cultivated aerobic granules that can degrade phenol at a constant rate of 49 mg-phenol/g x VSS/h up to 1,000 mg/L of phenol. Fluorescent staining and confocal laser scanning microscopy (CLSM) tests demonstrated that an active biomass was accumulated at the granule outer layer. A strain with maximum ability to degrade phenol and a high tolerance to phenol toxicity isolated from the granules was identified as Candida tropicalis via 18S rRNA sequencing. This strain degrades phenol at a maximum rate of 390 mg-phenol/g x VSS/h at pH 6 and 30 degrees C, whereas inhibitory effects existed at concentrations >1,000 mg/L. The Haldane kinetic model elucidates the growth and phenol biodegradation kinetics of the C. tropicalis. The fluorescence in situ hybridization (FISH) and CLSM test suggested that the Candida strain was primarily distributed throughout the surface layer of granule; hence, achieving a near constant reaction rate over a wide range of phenol concentration. The mass transfer barrier provided by granule matrix did not determine the reaction rates for the present phenol-degrading granule.     

低温高效苯酚降解菌的分离与降解特性

从哈尔滨太平污水厂活性污泥中筛选到7株高效苯酚降解菌,可利用苯酚作为唯一碳源和能源。通过对这7株菌在不同温度、pH值、以及不同苯酚浓度下生长和苯酚降解情况的考察,确定了这7株菌的最适生长温度为10°C,最适pH值为7.5,最大可降解苯酚浓度为3000mg/L。通过对这7株苯酚降解菌降解性能的研究表明:其具有较强的苯酚降解能力,在10°C、pH值为7.5、装液量为50mL、接种量15%、摇床振荡速度160r/min的条件下,反应48h后可使500mg/L的苯酚降解率达90%以上。葡萄糖对菌体的生长及苯酚降解能力均有一定的影响,当葡萄糖浓度是500mg/L时,该菌对苯酚的降解率仍在80%以上。该研究对处理含有其它碳源的含酚废水具有一定的意义。通过DGGE图谱条带的分析表明,其亮度可以说明这些菌在各个系统中均表现为优势菌,且在污水环境中表现出较强的活性,其优势地位能够稳定地存在。其中2、4、24、28条带丰富,表现出它们在污水环境系统中的多样性。     

Growth and phenol activity of Pseudomonas aeruginosa strain 101/1 in batch cultures

Strain 101/1, isolated from petroleum wastewater sediment was classified as Pseudomonas aeruginosa. In wild type condition the strain tolerated phenol in concentration 1,000 mg/L under aerobic conditions and 800 mg/L under denitrifying conditions. As a result of adaptation to phenol the resistance of the strain to the compound increased to 1,600 and 1,400 mg/L, respectively. Maximum phenol activity under aerobic and denitrifying conditions was 350 and 65 mg/L x day-1, respectively. Under denitrifying conditions a reduction in incubation temperature from 30 degrees C to 20 degrees C resulted in two-fold drop in phenol activity of the adapted strain and reduction in tolerance to phenol by 400 mg/L.     

假单胞菌诱导筛选菌株PhA苯酚降解动力学及SDS对其影响

为提高苯酚降解速率,由假单胞菌(Pseudonomonas．sp)诱导筛选得到了一株能以苯酚为唯一碳源生长的新菌株Pha,并使其苯酚选择压力从400mg／L逐步提高到了700mg／L。且Pha菌株降解苯酚过程符合一级反应动力学方程。使用十二烷基磺酸钠(SDS)作为增溶剂来促进降解时,发现在SDS浓度为50～150mg／L时,降解苯酚的速率随SDS浓度增加而提高。SDS在低浓度时对其生长影响很小,但浓度达到300mg／L时,对其生长开始有了明显的抑制作用。结果标明PhA菌株有着较高的苯酚耐受浓度,SDS可以显著的提高苯酚的降解速率。SDS的理论最佳投放量为150mg／L。     

Kinetic Modeling of cometabolic degradation of ethanethiol and phenol by Ralstonia eutropha

Cometabolism, as a complex phenomenon in microbial world, is a special mechanism for transformation of many compounds of environmental and toxicological significance. Several models have been proposed to describe the cometabolic transformations of non-growth substrates in the absence or presence of growth substrates. In this study, a model was proposed to simulate the degradation kinetics of phenol and ethanethiol (ET) by a pure culture of Ralstonia eutropha, including the effects of cell growth, endogenous cell decay, loss of transformation activity, competitive inhibition between growth and non-growth substrates, and self-inhibition of non-growth substrate. The model parameters were determined independently and were then used for evaluating the applicability of the model by comparing experimental data with model predictions. The model successfully predicted ET transformation and phenol utilization for a wide range of concentrations of ET (0 ～ 40 mg/L) and phenol (0 ～ 100 mg/L).     

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.     

Optimal microbial adaptation routes for the rapid degradation of high concentration of phenol

Pseudomonas fluorescence KNU417 was able to degrade up to 700 mg/L of phenol in 65 h but could not degrade 1,000 mg/L of phenol. Phenol degradation rate was noticeably enhanced by pre-adaptation. In addition, the cell was able to degrade up to 1,300 mg/L of phenol by pre-adapting to 700 mg/L of phenol. Repeated adaptations to the same concentration of phenol showed negligible increase in degradation rate. Also, relatively low concentration of phenol (100–700 mg/L) required only one pre-adaptation while high concentration (1,000 mg/L) did two consecutive stepwise pre-adaptations for rapid degradation. Optimal adaptation routes were suggested for the fast phenol degradation. For example, 1,000 mg/L of phenol was degraded as fast as in 48 h when the cell was pre-adapted to 100 and 300 mg/L of phenol sequentially. The mechanism of adaptation was explained in terms of catechol 1,2-dioxygenase induction, related to aromatic ring cleavage.     

The effect of concentration of phenols on their batch methanogenesis

The biodegradability of phenol and six other phenolic compounds (o-, m-, and p-cresol, 2-, 3-, and 4-ethylphenol) was examined in batch methanogenic cultures. The effect of concentration of these alkyl phenols on the anaerobic biodegradation of phenol was also evaluated. The inoculum used in this study was cultivated in a continuous flow laboratory fermenter with phenol as the primary substrate. Phenol, at initial concentrations as high to 1400 mg/L was completely degraded to methane and carbondioxide after 350 hours incubation. Complete degradation of m- and p-cresol was also observed while the ethylphenols and o-cresol were not significantly degraded.At initial concentrations exceeding 600 mg/L, phenol inhibited the phenol-degrading microorganisms but not the methanogens. At about 600 mg/L, cresols reduced the rate of phenol degradation to 50% of that observed in a control culture containing only 200 mg/L phenol. Ethylphenols were more inhibitory than cresols. Phenol degrading microorganisms were more susceptible to inhibition by cresols and ethylphenols than were the methanogens. The inhibitory effects of the three isomers of cresol and ethylphenol did not vary with the isomer but rather with the substituted functional group.     

一株苯酚降解菌的鉴定及其降解特性

【目的】鉴定从某化工厂附近土样中分离到的一株耐高浓度苯酚的菌株T10,通过优化菌株的培养条件提高菌株对苯酚的降解率。【方法】根据菌株的形态、生理生化鉴定及16S rDNA测序分析确定其种属,以液体摇瓶培养菌株T10对苯酚的降解率为指标,对菌株的生长条件进行优化。【结果】菌株T10属恶臭假单胞菌(Pseudomonas putida)。添加葡萄糖、蛋白胨能有效缩短T10菌的生长周期,并使苯酚的降解率提高1.7倍。在菌体初始接种浓度为10%、温度为30°C、转速为180 r/min条件下,对初始苯酚浓度、pH和装液量的响应面优化结果如下:初始苯酚浓度3 000 mg/L、pH 7.5和装液量80 mL/250 mL,苯酚去除率最高可达到87.56%。【结论】T10菌能够耐受较高浓度的含酚废水,并且对苯酚有较强的降解能力,为下一步利用生物法处理含酚废水提供科学依据。     

Application of dried sewage sludge as phenol biosorbent

The aim of this work was to determine the potential application of dried sewage sludge as a biosorbent for removing phenol from aqueous solution. Results showed that biosorption capacity was strongly influenced by the pH of the aqueous solution with an observed maximum phenol removal at pH around 6-8. Biosorption capacity increased when initial phenol concentration was increased to 110 mg/L but beyond this concentration, biosorption capacity decreased suggesting an inhibitory effect of phenol on biomass activity. Biosorption capacity decreased from 94 to 5 mg/g when biosorbent concentration was increased from 0.5 to 10 g/L suggesting a possible competitive effect of leachable heavy metals from the sludge. The effect of Cu2+ on biosorption capacity was also observed and the results confirmed that the phenol biosorption capacity decreased when concentration of Cu2+ in the sorption medium was increased up to 15 mg/L. Desorption of phenol using distilled deionized water was less than 2% suggesting a strong biosorption by the biomass.     

SEC-HPLC法测定重组人生长激素注射液中苯酚含量

采用SEC-HPLC法测定重组人生长激素注射液中苯酚的含量。色谱柱为TSK G2000SWxL（7.8×300mm,5μ）,流动相为PB（pH值7.0）-异丙醇（97∶3）,流速0.6 mL/min,检测波长214nm,柱温25℃。苯酚在0.10～1.00 mg/mL浓度范围内线性关系良好,r=0.99915;最低检测限为0.5 ng/mL;平均回收率101.48%,RSD=0.58%（n=9）;3批重组人生长激素注射液中苯酚的含量分别为2.98、3.02和2.96 mg/mL,分别为标示量的99.33%、100.67%和98.67%。SEC-HPLC法环保、准确、快速、可靠,可用于重组人生长激素注射液中苯酚含量的测定。     

Biodegradation Kinetic Studies on Phenol in Internal Draft Tube (Inverse Fluidized Bed) Biofilm Reactor Using Pseudomonas fluorescens: Performance Evaluation of Biofilm and Biomass Characteristics

The bioremediation potential of Pseudomonas fluorescens was studied in an internal draft tube (inverse fluidized bed) biofilm reactor (IDTBR) under batch recirculation conditions using synthetic phenol of various concentrations (400, 600, 800, 1000, and 1200 mg/L). The performance of IDTBR was investigated and the characteristics of biomass and biofilm were determined by evaluating biofilm dry density and thickness, bioparticle density, suspended and attached biomass concentration, chemical oxygen demand, and phenol removal efficiency. Biodegradation kinetics had been studied for the suspended biomass culture and biofilm systems. Suspended biomass followed substrate inhibition kinetics, and the experimental data fitted well with the Haldane model. The correlation coefficient, R ², and root-mean-square error (RMSE) obtained for the Haldane model with respect to specific growth rate were .9389 and .00729, respectively, and with respect to specific phenol consumption rate were .9259 and .00972, respectively. It was also observed experimentally that biofilm overcame substrate inhibition effect and fitted the same to the Monod model (R ² = .9831, RMSE = .00884 for specific growth rate and R ² = .9686, RMSE = .00912 for specific phenol consumption rate).     

Phenol Biodegradation and Metal Removal by a Mixed Bacterial Consortium

This paper reports the tolerance and biodegradation of phenol by a heavy metal–adapted environmental bacterial consortium, known as consortium culture (CC). At the highest tolerable phenol concentration of 1200 mg/L, CC displayed specific growth rate of 0.04 h^?1, phenol degradation rate of 6.11 mg L^?1 h^?1 and biomass of 8.45 ± 0.35 (log₁₀ colony-forming units [CFU]/ml) at the end of incubation. Phenol was degraded via the ortho-cleavage pathway catalyzed by cathechol-1,2-dioxygenase with specific activity of 0.083 (µmol min^?1 mg^?1 protein). The different constituent bacterial isolates of CC preferentially grow on benzene, toluene, xylene, ethylbenzene, cresol, and catechol, suggesting a synergistic mechanism involved in the degradation process. Microtox assay showed that phenol degradation was achieved without producing toxic dead-end metabolites. Moreover, lead (Pb) and cadmium (Cd) at the highest tested concentration of 1.0 and 0.1 mg/L, respectively, did not inhibit phenol degradation by CC. Simultaneous metal removal during phenol degradation was achieved using CC. These findings confirmed the dual function of CC to degrade phenol and to remove heavy metals from a mixed-pollutant medium.     

Removal of phenolic compounds from a petrochemical effluent with a methanogenic consortium.

A methanogenic consortium was used to degrade phenol and ortho- (o-) cresol from a specific effluent of a petrochemical refinery. This effluent did not meet the local environmental regulations for phenolic compounds (178 mg/L), oils and greases (61 mg/L), ammoniacal nitrogen (75 mg/L) or sulfides (3.2 mg/L). The consortium, which degrades phenol via its carboxylation to benzoic acid, was progressively adapted to the effluent. Despite the very high effluent toxicity (EC50 of 2% with Microtox), the adapted consortium degraded 97% of 156 mg/L phenol in the supplemented effluent after 13 days in batch cultures (serum bottle). The addition of proteose peptone to the effluent is essential for phenol degradation. o-cresol was also transformed but not meta- or para-cresols. A continuous flow fixed-film anaerobic bioreactor was developed with the consortium. Treating the effluent with the bioreactor reduced phenol and phenolic compounds concentrations by 97 and 83%, respectively, for a hydraulic residence time of 6 h. This treatment also reduced by about half the effluent toxicity. Oils and greases and ammoniacal nitrogen were not affected. Similar microbiological forms were observed in serum bottles and in the bioreactors with or without the petrochemical effluent. These results indicate that this methanogenic consortium can treat efficiently the phenolic compounds in this specific petrochemical effluent.     

Effect of nitrates on biotransformation of phosphogypsum and phenol uptake in cultures of autochthonous sludge microflora from petroleum refining wastewaters

The effect of nitrates on the biotransformation of phosphogypsum at 30 degrees C in stationary cultures of anaerobic, heterogeneous microflora growing in medium with phenol (250-1,000 mg/L) as sole carbon source was studied. The microorganisms used in this study were isolated from sludge in biological petroleum-refining wastewater treatment plant. Phosphogypsum (a waste product in the chemical industry that contains approximately 95% CaSO4) was added in amount of 5 g/L, the source of nitrates was KNO3 in concentration equivalent to that of phenol (250-1,000 mg N-NO3/L). The presence of nitrates in heterogeneous cultures has an inhibitory effect on the process of phosphogypsum biotransformation and stimulates the uptake of phenol. We have found that in cultures in medium containing phenol, phosphogypsum and nitrates at least three physiological groups of microorganisms were present. These were phenol-biodegrading microorganisms not requiring an external electron acceptor, sulfate-reducing bacteria biodegrading phenol or intermediate products of its breakdown and denitrifying bacteria not utilising phenol as a carbon source. On solid medium these bacteria together formed heterogeneous single colonies. In spite of repeated attempts we were unable to isolate pure strains and the only result of these measures was loss of denitrification ability in medium with phenol.