Evolution of novel metabolic pathways for the degradation of chloroaromatic compounds

Chlorobenzenes are substrates not easily metabolized by existing bacteria in the environment. Specific strains, however, have been isolated from polluted environments or in laboratory selection procedures that use chlorobenzenes as their sole carbon and energy source. Genetic analysis indicated that these bacteria have acquired a novel combination of previously existing genes. One of these gene clusters contains the genes for an aromatic ring dioxy-genase and a dihydrodiol dehydrogenase. The other contains the genes for a chlorocatechol oxidative pathway. Comparison of such gene clusters with those from other aromatics degrading bacteria reveals that this process of recombining or assembly of existing genetic material must have occurred in many of them. Similarities of gene functions between pathways suggest that incorporation of existing genetic material has been the most important mechanism of expanding a metabolic pathway. Only in a few cases a horizontal expansion, that is acqui sition of gene functions to accomodate a wider range of substrates which are then all transformed in one central pathway, is observed on the genetic level. Evidence is presented indicating that the assembly process may trigger a faster divergence of nearby gene sequences. Further fine-tuning, for example by developing a proper regulation, is then the next step in the adaptation.     

Kinetics of carbendazim degradation in a horizontal tubular biofilm reactor

Bioprocess and Biosystems Engineering - The fungicide carbendazim is an ecotoxic agent affecting aquatic biota. Due to its suspected hormone-disrupting effects, it is considered a “priority...     

Phenol degradation in a three-phase biofilm fluidized sand bed reactor

A previous three phase fluidized sand bed reactor design was improved by adding a draft tube to improve fluidization and submerged effluent tubes for sand separation. The changes had little influence on the oxygen transfer coefficients(K _L a), but greatly reduced the aeration rate required for sand suspension. The resulting 12.5 dm³ reactor was operated with 1 h liquid residence time, 10.2dm³/min aeration rate, and 1.7–2.3 kg sand (0.25–0.35 mm diameter) for the degradation of phenol as sole carbon source. The K _La of 0.015 s^–1 gave more than adequate oxygen transfer to support rates of 180g phenol/h · m³ and 216 g oxygen/h · m³. The biomass-sand ratios of 20–35 mg volatiles/g gave estimated biomass concentrations of 3–6 g volatiles/dm³. Offline kinetic measurements showed weak inhibition kinetics with constants ofK _s=0.2 mg phenol/dm³, K _o2=0.5 mg oxygen/dm³ and KinI= 122.5 mg phenol/dm³. Very small biofilm diffusion effects were observed. Dynamic experiments demonstrated rapid response of dissolved oxygen to phenol changes below the inhibition level. Experimentally simulated continuous stagewise operation required three stages, each with 1 h residence time, for complete degradation of 300 mg phenol/dm³ · h.     

Selected factors limiting the microbial degradation of recalcitrant compounds

Summary The focus of this review is to examine some of the reasons biodegradation may not take place in the environment even though its occurrence in the laboratory has been demonstrated. Some approaches for dealing with chemical persistence will be discussed. In addition, the potential of bioremediation as an in situ clean-up technology will be considered.     

Activity of synchronized cells of a steady-state biofilm recirculated reactor during xenobiotic biodegradation.

The maintenance of a steady-state biofilm in a continuous-flow fixed-bed reactor, as a consequence of the reproduction-detachment of cells (an interfacial cell physiology phenomenon of steady-state biofilm) during the biodegradation of 2,4,6-trichlorophenol by Pseudomonas cells, was determined. After cell adhesion on an open-pore glass support, the biofilm was formed in a packed-bed recirculated reactor. After the steady-state biofilm was reached, the mechanisms of the interfacial cell detachment (at the biofilm-liquid interface) were determined. It was established that (i) the hydrophobicity of immobilized sessile cells (parent cells) increased (from 50 to 80%) as the dilution rate increased, while the hydrophobicity of detached suspended cells (daughter cells) remained constant (about 45%); and (ii) the immediately detached suspended cells showed a synchronized growth in about three generations. These results indicate that (i) the immobilized sessile and suspended detached cells grew synchronically at the end and at the beginning of the cell cycle, respectively; and (ii) the hydrophobicity difference of immobilized sessile and suspended detached cells permitted the cells detachment. Therefore, it is probable that independent of shear stress (due to recirculated flow), the synchronized growth and hydrophobicity of cells (which vary during the cell cycle) are the main factors permitting the maintenance of a steady-state xenobiotic-degrading biofilm reactor (in which the overall accumulation of biofilm is determined by the average growth rate of the biofilm cells minus the rate of detachment of cells from the biofilm).     

A GAC biofilm reactor for the continuous degradation of 4-chlorophenol: treatment efficiency and microbial analysis

Using a continuous enrichment technique, a bacterial consortium capable of degrading 4-chlorophenol (4-CP) was obtained from the rhizosphere of Phragmites australis. A granular activated carbon (GAC) biofilm reactor was established using this consortium, and the degradation of 4-CP was investigated under continuous flow operation using a feed of 20-50 mg l(-1) with a hydraulic residence time of 17 min over a 6-month period. Chloride liberation occurred throughout the operation, and the reactor had 4-CP removal efficiencies of 69-100%. Periods of lower performance were attributed to clogging of the column with biomass and the formation of channels. Subsequently, the immobilized biofilm was subjected to a starvation period of 5 months, after which its degradative capacity was still maintained. The microbial consortium was characterized during the continuous flow experiment and dynamic population changes were observed throughout. One isolate recovered from the biofilm was shown to be capable of degrading 4-CP as a sole carbon and energy source.     

Complete degradation of polychlorinated hydrocarbons by a two-stage biofilm reactor.

A two-stage anaerobic-aerobic biofilm reactor successfully degraded a mixture of chlorinated organic compounds to water-soluble metabolic intermediates and carbon dioxide. Reductive dechlorination of hexachlorobenzene (HCB), tetrachloroethylene (PCE), and chloroform (CF) occurred on all tested primary carbon sources such as glucose, methanol, and acetate. However, the extent of dechlorination was maximum when the anaerobic biofilm column was fed acetate as a primary carbon source. HCB, PCE, and CF were dechlorinated to the levels of tri- and dichlorinated products (99, 80, and 32%, respectively) with acetate in the feed. This is important, since these less-chlorinated compounds can be metabolized by the aerobic biofilm. The effluent from the anaerobic biofilm column was fed directly into the aerobic column. After both columns, the total amount transformed into nonvolatile intermediates and carbon dioxide was 94, 96, and 83% for [14C]HCB, [14C]trichloroethylene, and [14C]CF, respectively. This research shows the potential application of this novel two-stage bioreactor system for treating groundwaters and industrial effluents composed of highly chlorinated aliphatic and aromatic hydrocarbons.     

Complete degradation of polychlorinated hydrocarbons by a two-stage biofilm reactor.

A two-stage anaerobic-aerobic biofilm reactor successfully degraded a mixture of chlorinated organic compounds to water-soluble metabolic intermediates and carbon dioxide. Reductive dechlorination of hexachlorobenzene (HCB), tetrachloroethylene (PCE), and chloroform (CF) occurred on all tested primary carbon sources such as glucose, methanol, and acetate. However, the extent of dechlorination was maximum when the anaerobic biofilm column was fed acetate as a primary carbon source. HCB, PCE, and CF were dechlorinated to the levels of tri- and dichlorinated products (99, 80, and 32%, respectively) with acetate in the feed. This is important, since these less-chlorinated compounds can be metabolized by the aerobic biofilm. The effluent from the anaerobic biofilm column was fed directly into the aerobic column. After both columns, the total amount transformed into nonvolatile intermediates and carbon dioxide was 94, 96, and 83% for [14C]HCB, [14C]trichloroethylene, and [14C]CF, respectively. This research shows the potential application of this novel two-stage bioreactor system for treating groundwaters and industrial effluents composed of highly chlorinated aliphatic and aromatic hydrocarbons.     

Dynamic modeling of an enzymatic membrane reactor for the treatment of xenobiotic compounds

A membrane enzymatic reactor, consisting of a stirred tank coupled to an ultrafiltration membrane was set up for the enzymatic oxidation of xenobiotic compounds. The azo dye Orange II was selected for the model compound and manganese peroxidase for the oxidative enzyme. The ligninolytic cycle was initiated and maintained by the controlled addition of all factors (reactants, mediators, and stabilizers) at suitable rates. Considering the distinctiveness of this process, in which the substrate to be oxidized is not the primary substrate for the enzyme, a kinetic model was developed. The azo dye concentration and hydrogen peroxide addition rate were found to be the main factors affecting the process. The reaction kinetics was defined using a Michaelis-Menten model with respect to the Orange II concentration and a first-order linear dependence relative to the H(2)O(2) addition rate. The dynamic model, which takes into account both the kinetics and the hydraulics of the system, was validated by comparing the experimental results in continuous operation under steady and non-steady state to model predictions. In particular, the model predicted the behavior of the system when unexpected alterations in steady-state operation occurred. Furthermore, the model allowed us to obtain the most appropriate H(2)O(2)/Orange II ratio in the feed to maximize the process efficiency.     

An aerobic fixed-phase biofilm reactor system for the degradation of the low-molecular weight aromatic compounds occurring in the effluents of anaerobic digestors treating olive mill wastewaters

An aerobic co-culture, prepared by combining Ralstonia sp. LD35 and Pseudomonas putida DSM1868, was recently found to be capable of extensively degrading many of the hydroxylated and/or methoxylated benzoic, phenylacetic and 3-phenyl-2-propenoic acids occurring in the olive mill wastewaters (OMWs). In the perspective of developing a biotechnological process for the degradation of low-molecular weight (MW) aromatic compounds occurring in the effluents of anaerobic digestors treating OMWs, the capability of this bacterial co-culture of biodegrading a synthetic mix of the above mentioned compounds and the aromatic compounds of an anaerobic OMW-treatment plant effluent in the physiological state of immobilised cells was investigated. Two aerobic fixed-bed biofilm reactors were developed by immobilising the co-culture cells on Manville silica beads and on polyurethane foam cubes. Both supports were found to give rise to a microbiologically stable and biologically active biofilm. The two biofilm reactors were found to be similarly capable of rapidly and completely biodegrading the components of a synthetic mix of nine monocyclic aromatic acids typically present in OMWs and the low-MW aromatic compounds occurring in the anaerobic effluent in batch conditions. However, in the same conditions, the silica bead-packed reactor was found to be more effective in the removal of high-MW phenolic compounds from the anaerobic effluent with respect to the polyurethane cube-packed reactor. These results are encouraging in the perspective of using the co-culture as immobilized cells for developing a continuous biotechnological process for the post-treatment of effluents with low-MW aromatic compounds produced by anaerobic digestors treating OMWs.     

Kinetics of anaerobic methane oxidation coupled to denitrification in the membrane biofilm reactor

Anaerobic oxidation of methane coupled to denitrification (AOM-D) in a membrane biofilm reactor (MBfR), a platform used for efficiently coupling gas delivery and biofilm development, has attracted attention in recent years due to the low cost and high availability of methane. However, experimental studies have shown that the nitrate-removal flux in the CH₄-based MBfR (<1.0 g N/m²-day) is about one order of magnitude smaller than that in the H₂-based MBfR (1.1–6.7 g N/m²-day). A one-dimensional multispecies biofilm model predicts that the nitrate-removal flux in the CH₄-based MBfR is limited to <1.7 g N/m²-day, consistent with the experimental studies reported in the literature. The model also determines the two major limiting factors for the nitrate-removal flux: The methane half-maximum-rate concentration (K₂) and the specific maximum methane utilization rate of the AOM-D syntrophic consortium (k_max2), with k_max2 being more important. Model simulations show that increasing k_max2 to >3 g chemical oxygen demand (COD)/g cell-day (from its current 1.8 g COD/g cell-day) and developing a new membrane with doubled methane-delivery capacity (D_m) could bring the nitrate-removal flux to ≥4.0 g N/m²-day, which is close to the nitrate-removal flux for the H₂-based MBfR. Further increase of the maximum nitrate-removal flux can be achieved when D_m and k_max2 increase together.     

Maleylacetate reductase of Pseudomonas sp. strain B13: dechlorination of chloromaleylacetates,metabolites in the degradation of chloroaromatic compounds

The maleylacetate reductase of 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 has been purified 50-fold. The enzyme converted 2-chloromaleylacetate to 3-oxoadipate with temporary occurrence of maleylacetate; 1 mol of chloride was eliminated during the conversion of 1 mol of 2-chloro- and 2,3-dichloromaleylacetate; 2 mol of NADH were consumed per mol of 2-chloro- and 2,3-dichloromaleylacetate while only 1 mol was necessary to catalyze the conversion of maleylacetate or 2-methylmaleylacetate. The maleylacetate reductase failed to use fumarylacetate as a substrate. The role of the enzyme in the chloroaromatics degradation is discussed.     

Lactic acid production by Lactobacillus delbrueckii in a dual reactor system using packed bed biofilm reactor

Aims:  An integrated dual reactor system for continuous production of lactic acid by Lactobacillus delbrueckii using biofilms developed on reticulated polyurethane foam (PUF) is demonstrated.
Methods and Results:  Lactobacillus delbrueckii was immobilized on PUF, packed in a bioreactor and used in lactic acid fermentation. The rate of lactic acid production was significantly high with a volumetric productivity of 5 g l⁻¹ h⁻¹ over extended period of time. When coupled to a bioreactor, the system could be operated as dual reactor for over 1000 h continuously without augmentation of inoculum and no compromise on productivity.
Conclusions:  Polyurethane foams offer an excellent support for biofilm formation.
Significance and Impact of the Study:  The system was very robust and could be operated for prolonged period at a volumetric productivity of 4–6 g l⁻¹ h⁻¹.     

Biodegradation of acetonitrile by adapted biofilm in a membrane-aerated biofilm reactor

A membrane-aerated biofilm reactor (MABR) was developed to degrade acetonitrile (ACN) in aqueous solutions. The reactor was seeded with an adapted activated sludge consortium as the inoculum and operated under step increases in ACN loading rate through increasing ACN concentrations in the influent. Initially, the MABR started at a moderate selection pressure, with a hydraulic retention time of 16 h, a recirculation rate of 8 cm/s and a starting ACN concentration of 250 mg/l to boost the growth of the biofilm mass on the membrane and to avoid its loss by hydraulic washout. The step increase in the influent ACN concentration was implemented once ACN concentration in the effluent showed almost complete removal in each stage. The specific ACN degradation rate achieved the highest at the loading rate of 101.1 mg ACN/g-VSS h (VSS, volatile suspended solids) and then declined with the further increases in the influent ACN concentration, attributed to the substrate inhibition effect. The adapted membrane-aerated biofilm was capable of completely removing ACN at the removal capacity of up to 21.1 g ACN/m² day, and generated negligible amount of suspended sludge in the effluent. Batch incubation experiments also demonstrated that the ACN-degrading biofilm can degrade other organonitriles, such as acrylonitrile and benzonitrile as well. Denaturing gradient gel electrophoresis studies showed that the ACN-degrading biofilms contained a stable microbial population with a low diversity of sequence of community 16S rRNA gene fragments. Specific oxygen utilization rates were found to increase with the increases in the biofilm thickness, suggesting that the biofilm formation process can enhance the metabolic degradation efficiency towards ACN in the MABR. The study contributes to a better understanding in microbial adaptation in a MABR for biodegradation of ACN. It also highlights the potential benefits in using MABRs for biodegradation of organonitrile contaminants in industrial wastewater.     

Enzymology of Phanerochaete chrysosporium with respect to the degradation of recalcitrant compounds and xenobiotics

The archetypal white-rot fungus Phanerochaete chrysosporium has been shown to degrade a variety of persistent environmental pollutants. Many of the enzymes responsible for pollutant degradation, which are normally involved in the degradation of wood, are extracellular. Thus, P. chrysosporium is able to degrade toxic or insoluble chemicals more efficiently than other microorganisms. P. chrysosporium has a range of oxidative and reductive mechanisms and uses highly reactive, nonspecific redox mediators which increase the number of chemicals that can be effectively degraded. This review gives an overview of the enzymes that are believed to be important for bioremediation and briefly discusses the degradation of some individual chemicals. Received: 25 April 2000 / Received revision: 05 June 2000 / Accepted: 04 July 2000     

Studies on the kinetics of immobilized enzyme using a recycling enzyme reactor system

A method of measuring kinetic parameters of immobilized enzyme with a recycling enzyme reactor system is described. By analyzing the plot of the dimensionless variable Ln(1-X)/X versus the time needed for a unit conversion, t/X, the mechanism of enzymatic reaction can be recognized and then its basic parameters can be evaluated. On the basis of the experimental data measured by P.R. Coulet et al., it has been proposed that the successive degradation of maltodextrins by the collagen membrane-bound amyloglucosidase was a product glucose inhibition reaction and their corresponding constants have been found with this method.     

4-Chlorophenol degradation by a bacterial consortium: development of a granular activated carbon biofilm reactor

A bacterial consortium that can degrade chloro- and nitrophenols has been isolated from the rhizosphere of Phragmitis communis. Degradation of 4-chlorophenol (4-CP) by a consortium attached to granular activated carbon (GAC) in a biofilm reactor was evaluated during both open and closed modes of operation. During the operation of the biofilm reactor, 4-CP was not detected in the column effluent, being either adsorbed to the GAC or biodegraded by the consortium. When 4-CP at 100 mg l⁻¹ was fed to the column in open mode operation (20 mg g⁻¹ GAC total supply), up to 27% was immediately available for biodegradation, the rest being adsorbed to the GAC. Biodegradation continued after the system was returned to closed mode operation, indicating that GAC bound 4-CP became available to the consortium. Biofilm batch cultures supplied with 10–216 mg 4-CP g⁻¹ GAC suggested that a residual fraction of GAC-bound 4-CP was biologically unavailable. The consortium was able to metabolise 4-CP after perturbations by the addition of chromium (Cr VI) at 1–5 mg l⁻¹ and nitrate at concentrations up to 400 mg l⁻¹. The development of the biofilm structure was analysed by scanning electron microscopy and confocal laser scanning microscopy (CLSM) techniques. CLSM revealed a heterogeneous structure with a network of channels throughout the biofilm, partially occupied by microbial exopolymer structures. Received: 17 March 1999 / Received revision: 27 May 1999 / Accepted: 28 May 1999     

Highly efficient phenol degradation in a batch moving bed biofilm reactor: benefiting from biofilm-enhancing bacteria

In this study, the efficiency improvement of three moving bed biofilm reactors (MBBRs) was investigated by inoculation of activated sludge cells (R1), mixed culture of eight strong phenol-degrading bacteria consisted of Pseudomonas spp. and Acinetobacter spp. (R2) and the combination of both (R3). Biofilm formation ability of eight bacteria was assessed initially using different methods and media. Maximum degradation of phenol, COD, biomass growth and also changes in organic loading shock were used as parameters to measure the performance of reactors. According to the results, all eight strains were determined as enhanced biofilm forming bacteria (EBFB). Under optimum operating conditions, more than 90% of initial COD load of 2795 mg L^?1 was reduced at 24 HRT in R3 while this reduction efficiency was observed in concentrations of 1290 mg L^?1 and 1935 mg L^?1, in R1 and R2, respectively. When encountering phenol loading shock—twice greater than optimum amount-R1, R2 and R3 managed to return to the steady-state condition within 32, 24 and 18 days, respectively. SEM microscopy and biomass growth measurements confirmed the contribution of more cells to biofilm formation in R3 followed by R2. Additionally, established biofilm in R3 was more resistant to phenol loading shock which can be attributed to the enhancer role of EBFB strains in this reactor. It has been demonstrated that the bacteria with both biofilm-forming and contaminant-degrading abilities are not only able to promote the immobilization of other favorable activated sludge cells in biofilm structure, but also cooperate in contaminant degradation which all consequently lead to improvement of treatment efficiency.     

An extractive membrane biofilm reactor for degradation of 1,3-dichloropropene in industrial waste water

A bacterial biofilm, capable of mineralising a technical mixture of cis- and trans-1,3-dichloropropene (DCPE), was enriched on the biomedium side of an extractive membrane biofilm reactor (EMBR). The membrane separates the biomedium from the industrial waste water, in terms of pH, ionic strength and the concentration of toxic chemicals. The biofilm, attached to a silicone membrane, is able to mineralise DCPE after its diffusion through the membrane. Five bacterial strains with degradation capabilities were isolated from the metabolically active biofilm and further investigated in batch experiments. Two of them, Rhodococcus erythropolis strains EK2 and EK5, can grow with DCPE as the sole carbon source. Pseudomonas sp. EK1 utilises cis-3-chloroallylalcohol and cis-3-chloroacrylic acid, whereas the metabolite trans-3-chloroacrylic acid represents a dead-end product of the pathway of this strain. The other two strains, Delftia sp. EK3 and EK4, although unable to grow with DCPE as the carbon source, can transform DCPE and its upper-pathway intermediates at reasonable conversion rates. They may represent helper functions of the biofilm consortium, which mineralised up to 12.5 mmol DCPE per hour per gram of biomass protein. Higher feed rates in the EMBR (up to 15 mmol per hour per 100-l bioreactor volume) and shock loads corresponding to concentrations up to 1.8 mmol l⁻¹ led to a significant increase in the freely floating bacterial biomass in the reactor medium (OD₅₄₆= 0.2). At the standard operating feed rate of 1.8 mmol h⁻¹, the free biomass concentration was very low (OD₅₄₆= 0.04). Received: 23 April 1999 / Received revision: 1 July 1999 / Accepted: 5 July 1999