Degradation of phenol by a mixed culture of Pseudomonas putida and Cryptococcus elinovii adsorbed on activated carbon

Summary Phenol degradation by a defined mixed culture of Pseudomonas putida P8 and Cryptococcus elinovii H 1, which were immobilized by adsorption on activated carbon, was studied.The immobilized mixed culture was able to degrade phenol up to 17 g/l and degraded it faster than the pure cultures, depending on a complementary metabolism of the two microorganisms.Storage experiments revealed an excellent longterm storage capability of the biocatalyst: activated carbon with adsorbed cells of Pseudomonas putida P8 and Cryptococcus elinovii H1 could be stored up to 12 months without decrease on degradation capacity.Scanning electron micrographs showed that Pseudomonas putida P8 had grown through the pore system of the activated carbon into the inside of the carbon particles.     

Development of a microtitre plate method for determination of phenol utilization, biofilm formation and respiratory activity by environmental bacterial isolates

Twenty-five aerobic phenol-degrading bacteria, isolated from different environmental samples on phenol agar after several subcultures in phenol broth, utilized phenol (0.2 g l⁻¹) within 24 h, but removal of phenol was more rapid when other carbon sources were also present. A microtitre plate method was developed to determine growth rate, biofilm formation and respiratory activity of the strains isolated. Pseudomonas putida strains C5 and D6 showed maximum growth (as O.D. at 600 nm), P. putida D6 and unidentified bacterial strain M1 were more stable at high concentrations of phenol (0.8 g l⁻¹), and P. putida C5 formed the greatest amount of biofilm in 0.5 g phenol l⁻¹ medium. Measurement of dehydrogenase activity as reduction of triphenyl tetrazolium chloride supported data on growth rate and biofilm formation. The microtitre plate method provided a selective method for detection of the best phenol degrading and biofilm-forming microorganisms, and was also a rapid, convenient means of studying the effect of phenol concentration on growth rate and biofilm formation.     

Biological treatment of phenolic wastes: Comparison between free and immobilized cell systems

Summary Phenol degradation by free and immobilized cells ofFusarium flocciferum was studied in a chemostat at steady-state conditions. For the free cell system the dilution rates varied from 0.02 to 0.13h^–1, with a total phenol removal up to 0.08h^–1. Wash-out seemed to set in at 0.11h^–1. The immobilized cells showed virtually complete phenol utilization at 1g/l, over a period of four months. At D=0.2h^–1 and above 1g/l phenol, the complete phenol removal is not achieved: a progressive increase in the outlet concentration was observed attaining a value of 284mg/l at 1.5g/l.     

Continuous acetonitrile degradation in a packed-bed bioreactor

A 20-l packed-bed reactor filled with foamed glass beads was tested for the treatment of acetonitrile HPLC wastes. Aeration was provided by recirculating a portion of the reactor liquid phase through an aeration tank, where the dissolved oxygen concentration was kept at 6 mg/l. At a feeding rate of 0.77 g acetonitrile l^–1 reactor day^–1, 99% of the acetonitrile was removed; and 86% of the nitrogen present in acetonitrile was released as NH₃, confirming that acetonitrile volatilization was not significant. Increasing the acetonitrile loading resulted in lower removal efficiencies, but a maximum removal capacity of 1.0 g acetonitrile l^–1 reactor day^–1 was achieved at a feeding rate of 1.6 g acetonitrile l^–1 reactor day^–1. The removal capacity of the system was well correlated with the oxygenation capacity, showing that acetonitrile removal was likely to be limited by oxygen supply. Microbial characterization of the biofilm resulted in the isolation of a Comamonas sp. able to mineralize acetonitrile as sole carbon, nitrogen and energy source. This organism was closely related to C. testosteroni (91.2%) and might represent a new species in the Comamonas genus. This study confirms the potential of packed-bed reactors for the treatment of a concentrated mixture of volatile pollutants.     

Phenol degradation by microorganisms adsorbed on activated carbon

Summary The phenol degradation by Candida sp. and Pseudomonas sp. immobilized on activated carbon was investigated. Thanks to its great adsorptive surface, activated carbon is suited as supporting material for microorganisms and also provides a high adsorption capacity for phenol.The immobilization by adsorption avoids any unphysiological treatment of the microorganisms. One gram activated carbon adsorbed in 10 h about 4×10⁹ Pseudomonas cells and 3×10⁸ Candida cells. While the free cells did not tolerate more than 1.5 g/l phenol, the adsorbed microorganisms survived at temporary high phenol concentrations up to 15 g/l, and they degraded about 90% of the adsorbed phenol.The activated carbon operated like a depot: the adsorbed phenol diffused out of the carbon and could be metabolized by the microorganisms. The results give an explanation of the stimulating effect of activated carbon in the treatment of waste waters observed until now.     

Rapid Degradation of Isolated Lignins by Phanerochaete chrysosporium

Phanerochaete chrysosporium degraded purified Kraft lignin, alkali-extracted and dioxane-extracted straw lignin, and lignosulfonates at a similar rate, producing small-molecular-weight (~1,000) soluble products which comprised 25 to 35% of the original lignins. At concentrations of 1 g of lignin liter⁻¹, 90 to 100% of the acid-insoluble Kraft, alkali straw, and dioxane straw lignins were degraded by 1 g of fungal mycelium liter⁻¹ within an active ligninolytic period of 2 to 3 days. Cultures with biomass concentrations as low as 0.16 g liter⁻¹ could also completely degrade 1 g of lignin liter⁻¹ during an active period of 6 to 8 days. The absorbance at 280 nm of 2 g of lignosulfonate liter⁻¹ increased during the first 3 days of incubation and decreased to 35% of the original value during the next 7 days. The capacity of 1 g of cells to degrade alkali-extracted straw lignin under optimized conditions was estimated to be as high as 1.0 g day⁻¹. This degradation occurred with a simultaneous glucose consumption rate of 1.0 g day⁻¹. When glucose or cellular energy resources were depleted, lignin degradation ceased. The ability of P. chrysosporium to degrade the various lignins in a similar manner and at very low biomass concentrations indicates that the enzymes responsible for lignin degradation are nonspecific.     

Semicontinuous and continuous degradation of phenol by Pseudomonas putida P8 adsorbed on activated carbon

Summary The semicontinuous and continuous degradation of phenol by Pseudomonas putida P8 which was immobilized on activated carbon was investigated. The amount of bacteria immobilized on the activated carbon surface dependend on the cell concentration in the suspension and on the type of activated carbon. In a continuous process running for four weeks the biomass, which accumulated in the activated carbon fixed bed, was removed periodically. The average phenol degradation rate in this process was 360 mg/1 h. The degradation activity of the bacteria for phenol, measured by the activity of the catechol-2,3-dioxygenase, was stimulated by the activated carbon. During the fermentation processes the carbon particles were covered with a biofilm. The bacteria grew, especially in the caverns and the entrances of the macropores, whereby the phenol adsorption by the activated carbon was decreased.     

Substrate-dependent autoaggregation of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> CP1 during the degradation of mono-chlorophenols and phenol

A bacterium, CP1, identified as Pseudomonas putida strain, was investigated for its ability to grow on and degrade mono-chlorophenols and phenols as sole carbon sources in aerobic shaking batch culture. The organism degraded up to 1.56 mM 2- and 3-chlorophenol, 2.34 mM 4-chlorophenol and 8.5 mM phenol using an ortho-cleavage pathway. P. putida CP1, acclimated to degrade 2-chlorophenol, was capable of 3-chlorocatechol degradation, while P. putida, acclimated to 4-chlorophenol degradation, degraded 4-chlorocatechol. Growth of P. putida CP1 on higher concentrations of the mono-chlorophenols, ≥1.56 mM 4-chlorophenol and ≥0.78 mM 2- and 3-chlorophenol, resulted in decreases in cell biomass despite metabolism of the substrates, and the formation of large aggregates of cells in the culture medium. Increases in cell biomass with no clumping of the cells resulted from growth of P. putida CP1 on phenol or on lower concentrations of mono-chlorophenol. Bacterial adherence to hydrocarbons (BATH) assays showed cells grown on the higher concentrations of mono-chlorophenol to be more hydrophobic than those grown on phenol and lower concentrations of mono-chlorophenol. The results suggested that increased hydrophobicity and autoaggregation of P. putida CP1 were a response to toxicity of the added substrates. Journal of Industrial Microbiology & Biotechnology (2002) 28, 316–324 DOI: 10.1038/sj/jim/7000249 Received 27 June 2001/ Accepted in revised form 09 February 2002     

Response on scallop culture to enhanced nutrient supply by experimental fertilisation of a landlocked bay

The effect of an enhanced nutrient supply to coastal waters of a landlocked bay, Hopavågen in Central Norway, on the phytoplankton production and biomass, and on growth of scallops (Pecten maximus) was studied in 1997–1999. Nitrogen, silicon and phosphorous (N:Si:P = 16:8:1, atomic) were added daily between May and October in 1998 at a level of 0.4 mg P m^–3 day^–1. The concentration of nutrient addition was doubled in 1999 during the same period. High addition of nutrients (1999) resulted in a significantly higher phytoplankton biomass in the summer period, expressed as chlorophyll a content, than without nutrient (1997) and low nutrient (1998). The respective mean chlorophyll a levels were 2.4 in 1999, 1.6 in 1998 and 1.2 g l^–1 in 1997. The mean primary production during the summers generally increased with the addition of nutrients from an average level of 320 mg carbon m^–2 day^–1 in 1997 to 1200 mg carbon m^–2 day^–1 in 1999. Scallops placed at 10 m depth in Hopavågen showed an increase in growth rate of the outer scallop shell in the period July–September from 0.16% day^–1 in 1997 to 0.53% day^–1 in 1998. Scallops grown in an unfertilised control station in the fjord outside Hopavågen had a significantly lower growth rate than those grown in the fertilised water of Hopavågen. The results showed decreased growth rate with increasing shell sizes. However, for all size groups studied a higher growth rate of the scallops was observed when nutrients were added to the bay. The tissue dry weight content of scallops grown in Hopavågen was 2–4 times higher than in the control scallops.     

Feasibility study of exponential feeding strategy in fed-batch cultures for phenol degradation using Cupriavidus taiwanensis

An indigenous phenol-degrading bacterial isolate Cupriavidus taiwanensis R186 was used to degrade phenol from an aqueous solution under fed-batch operation. An exponential feeding strategy combined with dissolved oxygen control was applied based on kinetic characteristics of cell growth and phenol degradation to meet sufficient metabolic needs for cellular growth and achieve the best phenol removal efficiency. Without the stress of phenol inhibition, the optimal set point of specific growth rate of exponential feeding for fed-batch phenol degradation was found to be 0.50–0.55μ_max (μ_max denotes the maximum specific growth rate from Monod model). Meanwhile, the sufficient set point of dissolved oxygen for maximal phenol degradation efficiency was approximately at 10–55% air saturation. With the optimal operation conditions, the best phenol degradation rate was 0.213 g phenol h⁻¹, while a shortest treatment time of 15 h was achieved for complete degradation of 11.35 mM (ca. 3.20 g) of phenol.     

Thiocyanate degradation by Acremonium strictum and inhibition by secondary toxicants

Acremonium strictum, capable of degrading 7.4 g thiocyanate l^–1, was isolated from wastewater condensate from coke-oven gas. Ammonia and sulfate were the final products from thiocyanate degradation with a stoichiometric ratio of near 1:1. The highest degradation activity was at pH 6. Although the degradation rate started to be inhibited above 4 g thiocyanate l^–1, thiocyanate was completely degraded up to 7.4 g l^–1 within 85 h in shake-flask cultures. The degradation of thiocyanate was inhibited by phenol above 625 mg l^–1, by cyanide above 16 mg l^–1, and by nitrite above 100 mg l^–1. However, ammonia and nitrate had negligible inhibition on thiocyanate degradation up to 3 g l^–1 and 1.5 g l^–1, respectively.     

Simultaneous biodegradation ofp-cresol and phenol by the basidiomycetePhanerochaete chrysosporium

Summary The fungusPhanerochaete chrysoporium BKM-F-1767 was able to degrade high concentrations ofp-cresol (up to 150 mg L^–1) provided that glucose was added as a carbon and energy source and conditions favourable to ligninolytic enzyme activities were used, i.e. a nitrogen-limited medium. The fungus also simultaneously degradedp-cresol (50 mg L^–1) and phenol (50 mg L^–1) in a mixture at similar rates. Kinetics ofp-cresol biodegradation were almost identical whether the compound was tested individually or in a mixture with phenol.     

Phenol biodegradation by free and immobilized Acinetobacter

Acinetobacter sp. strain W-17, immobilized on porous sintered glass completely degraded 500 mg phenol l^–1 in 40 h, but free cells required 120 h for this to be achieved. Immobilized cells can be used 7 times without losing their activity.     

Differential Rhizosphere Establishment and Cyanide Production by Alginate-Formulated Weed-Deleterious Rhizobacteria

The effects of Pseudomonas putida ATH2-1RI/9 and Acidovorax delafieldii ATH2-2RS/1 on rhizosphere colonization, cyanide production, and growth of velvetleaf and corn was examined. When formulated in alginate beads and inoculated onto velvetleaf and corn plants (10⁹ CFU/plant), only P. putida ATH2-1RI/9 consistently reduced velvetleaf growth. Neither isolate inhibited corn growth. Interestingly the levels of P. putida ATH2-1RI/9 in the velvetleaf rhizosphere were 1000-fold higher (7 × 10⁷ CFU/g root) than the A. delafieldii ATH2-2RS/1 populations. Cyanide (53–68 mM/g root) was recovered from the P. putida ATH2-1RI/9-inoculated velvetleaf plants. In contrast both A. delafieldii ATH2-2RS/1 and P. putida ATH2-1RI/9 colonized the corn rhizosphere to the same extent (1–5 × 10⁷ CFU/g root), producing 1 mM and 14 mM/g root respectively. These results suggest that bacterial formulation methods can influence the effectiveness of deleterious rhizobacteria in reducing weed growth.     

Kinetics of BTEX biodegradation by a coculture of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> and<Emphasis Type="Italic"> Pseudomonas fluorescens</Emphasis> under hypoxic conditions

Pseudomonas putida and Pseudomonas fluorescens present as a coculture were studied for their abilities to degrade benzene, toluene, ethylbenzene, and xylenes (collectively known as BTEX) under various growth conditions. The coculture effectively degraded various concentrations of BTEX as sole carbon sources. However, all BTEX compounds showed substrate inhibition to the bacteria, in terms of specific growth, degradation rate, and cell net yield. Cell growth was completely inhibited at 500mgl^–1 of benzene, 600mgl^–1 of o-xylene, and 1000mgl^–1 of toluene. Without aeration, aerobic biodegradation of BTEX required additional oxygen provided as hydrogen peroxide in the medium. Under hypoxic conditions, however, nitrate could be used as an alternative electron acceptor for BTEX biodegradation when oxygen was limited and denitrification took place in the culture. The carbon mass balance study confirmed that benzene and toluene were completely mineralized to CO₂ and H₂O without producing any identifiable intermediate metabolites.     

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     

Increased removal capacity for 1,2-dichloroethane by biological modification of the granular activated carbon process

The removal of 5 mg 1^–1 1,2-dichloroethane [(CH₂Cl)₂] was studied in two granular activated carbon (GAC) reactors run with hydraulic retention times of below 1 h. One reactor was operated abiotically. The other one was inoculated with microorganisms able to degrade (CH₂Cl)₂. While the (CH₂Cl)₂-adsorption capacity of the non-inoculated GAC reactor was exhausted after 20 days, it apparently did not exhaust for at least 170 experimental days in the biologically activated system because (CH₂Cl)₂ was removed to over 95% as a result of the microbial degradation. The biodegradation was quantified: during the passage through the biologically activated GAC reactor, (CH₂Cl)₂ (5± mg l^–1) disappeared, chloride ions (3.3±0.2 mg l^–1) were produced, and oxygen (4 to 6 mg l^–1) was consumed. Removal of 30% of GAC at the entrance of the reactor, which visibly carried most of the biomass, and its replacement by virgin GAC at the end of the column did not change the apparent (CH₂Cl)₂removal capacity of the GAC column, indicating that still enough biomass was available to degrade most of the chemical fed. After the addition of the virgin carbon, the effluent concentration fell for a short period of time from about 200 g l^–1 to below 100 g l^–1, indicating partial adsorption of the non-degraded (CH₂Cl)₂ at the end of the reactor by the virgin carbon. Thus, the modification of the adsorption process by inoculation and maintenance of bacteria with special degradation capabilities resulted in a lower consumption of GAC and thus led to an extended service life of the GAC columns.     

Isolation and Characterization of a Pseudomonas putida Strain able to Grow with Trimethyl-1,2-dihydroxy-propyl-ammonium as Sole Source of Carbon, Energy and Nitrogen

Trimethyl-1,2-dihydroxypropyl-ammonium (TM) originates from the hydrolysis of the parent esterquat surfactant, which is widely used as softener in fabric care. Based on test procedures mimicking complex biological systems, TM is supposed to degrade completely when reaching the environment. However, no organisms able to degrade TM were isolated nor has the degradation pathway been elucidated so far. We isolated a Gram-negative rod able to grow with TM as sole source of carbon, energy and nitrogen. The strain reached a maximum specific growth rate of 0.4 h^–1 when growing with TM as the sole source of carbon, energy and nitrogen. TM was degraded to completion and surplus nitrogen was excreted as ammonium into the growth medium. A high percentage of the carbon in TM (68% in continuous culture and 60% in batch culture) was combusted to CO₂ resulting in a low yield of 0.54 mg cell dry weight per mg carbon during continuous cultivation and 0.73 mg cell dry weight per mg carbon in batch cultures. Choline, a natural structurally related compound, served as a growth substrate, whereas a couple of similar other quaternary aminoalcohols also used in softeners did not. The isolated bacterium was identified by 16S-rDNA sequencing as a strain of Pseudomonas putida with a difference of only one base pair to P. putida DSM 291^T. Despite their high identity, the reference strain P. putida DSM 291^T was not able to grow with TM and the two strains differed even in shape when growing on the same medium. This is the first microbial isolate able to degrade a quaternary ammonium softener head group to completion. Previously described strains growing on quaternary ammonium surfactants (decyltrimethylammonium, hexadecyltrimethylammonium and didecyldimethylammonium) either excreted metabolites or a consortium of bacteria was required for complete degradation.     

Biofilm build-up of Pseudomonas putida in a chemostat at different dilution rates

Summary A test system was set up where the build-up of a biofilm on a defined surface could be studied in a carbon source limited chemostat.The attachment of P. putida ATCC 11172 to glass when growing on L-asparagine was studied at different dilution rates (specific growth rates) from 0.1 to 1.5 h^–1 The number of attached colony forming units (cfu) increased with dilution rate from 1×10⁶ cfu/cm² at 0.1 h^–1 to 4×10⁷ cfu/cm² at 1.0 h^–1 and then the attachment decreased to about 6×10⁶ cfu/cm² at higher dilution rates (1.1–1.5 h^–1). The number of attached cfu was measured after 24 h exposure. The value of the maximum specific growth rate in batch culture was 0.6 h^–1.The total amount of attached cell-mass followed roughly the same pattern as the viable count.The viable count of the cells suspended in the growth medium showed its lowest value at the same dilution rate as resulted in maximum adhesion.It was shown that the effect of growth rate on the biofilm build-up of P. putida is significant, and ought to be borne in mind when continuous culture systems are set up and results evaluated.     

High efficiency degradation of tetrahydrofuran (THF) using a membrane bioreactor: identification of THF-degrading cultures of<Emphasis Type="Italic"> Pseudonocardia</Emphasis> sp. strain M1 and<Emphasis Type="Italic"> Rhodococcus ruber</Emphasis> isolate M2

A mixed microbial culture capable of growing aerobically on tetrahydrofuran (THF) as a sole carbon and energy source was used as the inoculum in a 10 l working volume membrane bioreactor. Following start-up, the reactor was operated in batch mode for 24 h and then switched to continuous feed with 100% biomass recycle. On average, greater than 96% of THF fed to the reactor was removed during the 8-month study. THF loading rates ranged from 0.62 to 9.07 g l^–1 day^–1 with a hydraulic retention time of 24 h. THF concentrations as high as 800 mg/l were tolerated by the culture. Biomass production averaged 0.28 kg total suspended solids/kg chemical oxygen demand removed, i.e., comparable to a conventional wastewater treatment process. Periodic batch wasting resulted in a solids retention time of 7–14 days. Reactor biomass typically ranged from 4 to 10 g/l volatile suspended solids and the effluent contained no solids. Pure THF-degrading cultures were isolated from the mixed culture based on morphological characteristics, Gram-staining and THF degradation. Based on 16S rDNA analysis the isolates were identified as Pseudonocardia sp. M1 and Rhodococcus ruber M2.