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.     

Bioregeneration by mixed microorganisms of granular activated carbon loaded with a mixture of phenols

The role of mixed microorganisms on the bioregeneration of granular activated carbon (GAC) loaded with a mixture of phenol and 2,4-dichlorophenol was investigated. In a biological activated-carbon, sequencing batch reactor (BAC-SBR), bioregeneration efficiency for phenol was enhanced from 39 to 48% and for 2,4-dichlorophenol from 38 to 43% by increasing solid retention time from 3 to 8 days. Prolonging the sludge retention time induced both progressive desorption of adsorbates due to biodegradation in the bulk solution and direct assimilation of adsorbates on GAC by attached microorganisms.     

Catechol and phenol degradation by a methanogenic population of bacteria

An anaerobic population of bacteria became acclimated to catechol and phenol in 32 and 18 days, respectively. Evidence from carbon balance measurements indicates that the aromatic ring is cleaved and that the products are stoichiometrically fermentable to methane and carbon dioxide.     

Catechol and phenol degradation by a methanogenic population of bacteria.

An anaerobic population of bacteria became acclimated to catechol and phenol in 32 and 18 days, respectively. Evidence from carbon balance measurements indicates that the aromatic ring is cleaved and that the products are stoichiometrically fermentable to methane and carbon dioxide.     

Anaerobic degradation of long-chain dicarboxylic acids by methanogenic enrichment cultures

Abstract Methanogenic enrichment cultures fermented the long-chain dicarboxylates adipate, pimelate, suberate, azelate, and sebacate (C₆-C₁₀) stoichiometrically to acetate and methane. After several transfers, the cultures contained cells of only a few morphologically distinguishable types. During anaerobic degradation of dicarboxylic acids with even-numbered carbon atoms, propionate accumulated intermediately, and butyrate was the intermediate product of degradation of those with an odd number of carbon atoms. Degradation of the long-chain dicarboxylates depended strictly on the presence of hydrogenotrophic methanogens. The primary attack in these processes was β-oxidation rather than decarboxylation. A general scheme of anaerobic degradation of long-chain dicarboxylic acids has been deduced from these results.     

Acidophilic degradation of methanol by a methanogenic enrichment culture

Abstract An acidophilic methanogenic enrichment culture was obtained in a continuous up-flow anaerobic sludge blanket reactor operated at pH 4.2 with methanol as the sole carbon source. The specific methylotrophic methanogenic activity of the enriched reactor sludge at pH 5 was 3.57 g COD g⁻¹ volatile suspended solids day⁻¹ and the apparent doubling time of the biomass was 15.8 h. Acidic conditions were obligatory, since the enrichment culture was not able to produce methane or to grow at pH 7. Based on morphological characteristics, the dominant methanogenic species in the enrichment culture was a Methanosarcina .     

Anaerobic degradation of isovalerate by a defined methanogenic coculture

Isovalerate-oxidizing strictly aneerobic bacteria were isolated from marine sediment and sewage sludge in coculture with Desulfovibrio sp. Cells stained Gram positive and behaved Gram positive also in Gram classification with KOH. Isovalerate degradation depended on interspecies hydrogen transfer to syntrophic hydrogen-oxidizing sulfate reducers or methanogens. Isovalerate was the only substrate utilized and was fermented to 3 mol acetate and 1 mol hydrogen per mol substrate. The degradation pathway was studied by enzyme assays in crude cell extracts, and included acetyl-CoA dependent activation of isovalerate, oxidation to methylcrotonyl-CoA and carboxylation to methylgluta-conyl-CoA which is hydrated and cleaved to acetoacetate and acetyl-CoA. Studies with inhibitors and ionophores suggest that energy conservation with this organism depends on either acetate efflux-driven proton symport or on an ion-gradient driven carboxylation mechanism.     

Effects of some alkyl phenols on methanogenic degradation of phenol

The effects of six phenolic compounds (o-, m-, and p-cresol and 2-, 3-, and 4-ethylphenol) on the anaerobic biodegradation of phenol was examined in batch methanogenic cultures. Results showed that ethylphenols were more inhibitory of phenol degradation than were cresols. The inhibitory effects of the three isomers of cresol and ethylphenol did not vary with the isomer but rather with the substituted functional group.     

Enrichment and granulation of Anammox biomass started up with methanogenic granular sludge

A sequencing batch reactor (SBR) seeded with methanogenic granular sludge was started up to enrich Anammox (Anaerobic Ammonium Oxidation) bacteria and to investigate the feasibility of granulation of Anammox biomass. Research results showed that hydraulic retention time (HRT) was an important factor to enrich Anammox bacteria. When the HRT was controlled at 30 days during the initial cultivation, the SBR reactor presented Anammox activity at t = 58 days. Simultaneously, the methanogenic granular sludge changed gradually from dust black to brown colour and its diameter became smaller. At t = 90 days, the Anammox activity was further improved. NH₄⁺-N and NO₂⁻N were removed simultaneously with higher speed and the maximum removal rates reached 14.6 g NH₄⁺-N /(m³ _reactor·day) and 6.67 g NO₂⁻-N /(m³ _reactor·day), respectively. Between t = 110 days and t = 161 days, the nitrogen load was increased to a HRT of 5 days (70 mg/l NH₄⁺ and 70 mg/l NO₂), the removal rates of ammonium and nitrite were 60.6% and 62.5% respectively. The sludge changed to red and formed Anammox granulation with high nitrogen removal activity.     

Effects of some alkyl phenols on methanogenic degradation of phenol.

The effects of six phenolic compounds (o-, m-, and p-cresol and 2-, 3-, and 4-ethylphenol) on the anaerobic biodegradation of phenol was examined in batch methanogenic cultures. Results showed that ethylphenols were more inhibitory of phenol degradation than were cresols. The inhibitory effects of the three isomers of cresol and ethylphenol did not vary with the isomer but rather with the substituted functional group.     

Effect of temperature and additional carbon sources on phenol degradation by an indigenous soil <Emphasis Type="Italic">Pseudomonad</Emphasis>

A new indigenous soil bacterium Pseudomonas sp. growing on phenol and on a mixture of phenol, toluene, o-cresol, naphthalene and 1,2,3-trimethylbenzene (1,2,3-TMB) was isolated and characterized. Phylogenetic analysis suggested its classification to Pseudomonadaceae family and showed 99.8% DNA sequence identity to Pseudomonas pseudoalcaligenes species. The isolate was psychrotroph, with growth temperatures ranging from ca. 0 to 40 °C. The GC–MS structural analysis of metabolic products of phenol degradation by this microorganism indicated a possible ortho cleavage pathway for high concentrations (over 200 mg L^–1) of phenol. Biodegradation rates by this species were found to be three times more effective than those previously reported by other Pseudomonas strains. The effect of temperature on phenol degradation was studied in batch cultures at temperatures ranging from 10 to 40°C and different initial phenol concentrations (up to 500mgL^–1). Above 300mgL^–1 of initial phenol concentration no considerable depletion was recorded at both 10 and 40°C. Maximum degradation rates for phenol were recorded at 30°C. The biodegradation rate of phenol was studied also in the presence of additional carbon sources (o-cresol, toluene, naphthalene, 1,2,3-TMB) at the optimum growth temperature and was found significantly lower by a factor of eight in respect to the strong competitive inhibition between the substrates and the more available sources of carbon and energy. The Haldane equation =_m S/(K_S+S+S²/K_I) was found to best fit the experimental data at the optimum temperature of 30°C than the Monod equation with kinetic constants _m=0.27h^–1, K_S=56.70mgL^–1, K_I=249.08mgL^–1.     

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     

Anaerobic degradation of isobutyrate by methanogenic enrichment cultures and by a Desulfococcus multivorans strain

Methanogenic enrichment cultures with isobutyrate as sole source of carbon and energy were inoculated with sediment and sludge samples from freshwater and marine origin. Over more than 20 transfers, these cultures fermented 2 mol isobutyrate with 1 mol CO₂ via an intermediate formation of n-butyrate to 4 mol acetate and 1 mol CH₄. The primary isobutyrate-fermenting bacteria could not be purified. From one of the marine enrichment cultures, a sulfate-reducing bacterium was isolated which oxidized isobutyrate with sulfate completely to CO₂. Based on its physiological and morphological properties, this strain was assigned to the known species Desulfococcus multivorans. It also oxidized many other fatty acids without significant release of short-chain intermedeates. The enzymes involved in isobutyrate degradation by this bacterium were assayed in cell-free extracts. The results indicate that isobutyrate is activated to its CoA derivative and oxidized via methylmalonate semialdehyde to propionyl-CoA. Propionyl-CoA is further converted via the methylmalonyl-CoA pathway to acetyl-CoA which is finally cleaved by the CO-dehydrogenase system. It is evident that this is not the pathway used by the fermenting bacteria prevailing in the methanogenic enrichment cultures. There results are discussed on the basis of energetical considerations.     

Enhanced degradation of phenol in floating treatment wetlands by plant-bacterial synergism

Phenol is a commonly found organic pollutant in industrial wastewaters. Its ecotoxicological significance is well known and, therefore, the compound is often required to be removed prior to discharge. In this study, plant-bacterial synergism was established in floating treatment wetlands (FTWs) in an attempt to maximize the removal of phenol from contaminated water. A common wetland plant, Typha domingensis, was vegetated on a floating mat and augmented with three phenol-degrading bacterial strains, Acinetobacter lwofii ACRH76, Bacillus cereus LORH97, and Pseudomonas sp. LCRH90, to develop FTWs for the remediation of water contaminated with phenol. All of the strains are known to have phenol-reducing properties, and grow well in FTWs. Results showed that T. domingensis was able to remove a small amount of phenol from the contaminated water; however, bacterial augmentation enhanced the removal potential significantly, i.e., 0.146 g/m²/day vs. 0.166 g/m²/day, respectively. Plant biomass also increased in the presence of bacterial consortia; and inoculated bacteria displayed successful colonization/survival in the rhizosphere, root interior and shoot interior of the plant. Similarly, highest reduction in chemical oxygen demand (COD), biochemical oxygen demand (BOD₅), and total organic carbon (TOC) was achieved by the combined application of plants and bacteria. The study demonstrates that the plant-bacterial synergism in a FTW may be a more effective approach for the remediation of phenol-contaminated water.     

Continuous anaerobic phenol degradation using an adapted mixed culture

A mixed culture derived from cow dung and sewage sludge and adapted to phenol was used for anaerobic phenol degradation. The phenol degradation rate depended on the period of adaptation of the mixed culture to phenol. In the continuous process, a higher degradation rate (2500 mg.1^-1 d^-1) and better reactor stability was achieved with a granular activated-carbon-packed bed reactor than with a stirred tank reactor.The authors are with the Department of Biochemical Engineering & Biotechnology, Indian Institute of Technology, Delhi Hauz Khas, New Delhi, India.     

Economic comparison of enzymatic reactors and advanced oxidation processes applied to the degradation of phenol as a model compound

Abstract

Some micropollutants present in wastewaters are barely removed in sewage treatment plants. In many cases a post-treatment process based on separation and/or oxidation has to be applied. The aim of this study was the technical and economic comparison of enzymatic technologies with other advanced oxidation processes (AOPs) for the degradation of phenol. Batch and continuous enzymatic reactors, using free and immobilized manganese peroxidase (MnP, EC 1.11.1.13), were considered. Continuous degradation of phenol in an enzymatic membrane reactor was shown to be the fastest process and degradation in a continuous reactor with immobilized enzyme involved the lowest consumption of enzyme. However, the immobilization process increased the enzyme cost 100-fold. A continuous enzymatic membrane reactor gave high degradation efficiency and may be a viable technology for phenol removal when compared with other AOPs from both technical and economic points of view.     

Studies on composition and stability of a large membered bacterial consortium degrading phenol

A ten member microbial consortium (AS) consisting of eight phenol-degrading and two non-phenol-degrading strains of bacteria was developed and maintained in a fed-batch reactor by feeding 500 mg l⁻¹ phenol for four years at 28 ± 3 °C. The consortium could degrade 99% of 500 mg l⁻¹ phenol after 24 hours incubation with a biomass increase of 2.6 × 10⁷ to 4 × 10¹² CFU ml⁻¹. Characterization of the members revealed that it consisted of 4 principal genera, Bacillus, Pseudomonas, Rhodococcus, Streptomyces and an unidentified bacterium. Phenol degradation by the mixed culture and Bacillus subtilis, an isolate from the consortium was compared using a range of phenol concentrations (400 to 700 mg l⁻¹) and by mixing with either 160 mg l⁻¹ glucose or 50 mg l⁻¹ of 2,4-dichlorophenol in the medium. Simultaneous utilization of unrelated mixed substrates (glucose/2,4-dichlorophenol) by the consortium and Bacillus subtilis, indicated the diauxic growth pattern of the organisms. A unique characteristic of the members of the consortia was their ability to oxidize chloro aromatic compounds via meta pathway and methyl aromatic compounds via ortho cleavage pathway. The ability of a large membered microbial consortia to maintain its stability with respect to its composition and effectiveness in phenol degradation indicated its suitability for bioremediation applications.     

Kinetics of phenol degradation using Pseudomonas putida MTCC 1194

Pseudomonas putida (MTCC 1194) has been used to degrade phenol in water in the concentration range 100–1000?ppm. The inhibition effects of phenol as substrate have become predominant above the concentration of 500?ppm (5.31?mmoles/dm³). The optimum temperature and initial pH required for maximum phenol biodegradation were 30?°C and 7.00 respectively. From the degradation data the activation energy (E _a ) was found to be equal to 13.8?kcal/g mole substrate reacted. The most suitable inoculum age and volume for highest phenol degradation were 12?hrs and 7% v/v respectively. Surfactants had negligible effect on phenol biodegradation process for this microorganism. Monod model has been used to interpret the free cell data on phenol biodegradation. The kinetic parameters have been estimated upto initial concentration of 5.31?mmoles/dm³. μ _max and K _S gradually increased with higher concentration of phenol. However, beyond the phenol concentration of 5.31?mmoles/dm³, the inhibition became prominant. The μ _max has been to be a strong function of initial phenol concentration. The simulated and the experimental phenol degradation profiles have good correspondence with each other.