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     

Characterization and optimization of a two-phase partitioning bioreactor for the biodegradation of phenol

An electrochemical reactor employing activated carbon fibers (ACF) was constructed for the disinfection of bacteria in drinking water. The application of an alternating potential of 1.0 V and −0.8 V versus a saturated calomel electrode, for disinfecting and desorbing bacteria, enabled reactor operation for 840 h. Drinking water was passed through the reactor in stop/flow mode: 300 ml/min flow for 12 h and no flow for 12 h, alternately. The bacterial cell density in treated water was always been less than 20 cells/ml. It was also found that the formation of biofilm on the ACF reactor caused an increase in current, enabling the self-detection of microbial fouling. Received: 19 February 1996 / Received last revision: 23 July 1996 / Accepted: 2 September 1996     

Corrosion inhibition by aerobic biofilms on SAE 1018 steel

Carbon steel (SAE 1018) samples were exposed to complex liquid media containing either the aerobic bacterium Pseudomonas fragi or the facultative anaerobe Escherichia coli DH5α. Compared to sterile controls, mass loss was consistently 2- to 10-fold lower in the presence of these bacteria which produce a protective biofilm. Increasing the temperature from 23 °C to 30 °C resulted in a 2- to 5-fold decrease in corrosion inhibition with P. fragi whereas the same shift in temperature resulted in a 2-fold increase in corrosion inhibition with E. coli DH5α. Corrosion observed with non-biofilm-forming Streptomyces lividans TK24 was similar to that observed in sterile media. A dead biofilm, generated in situ by adding kanamycin to an established biofilm, did not protect the metal (corrosion rates were comparable to those in the sterile control), and mass loss in cell-free, spent Luria-Bertani (LB) medium was similar to that in sterile medium. Confocal laser scanning microscopy analysis confirmed the presence of a biofilm consisting of live and dead cells embedded in a sparse glycocalyx matrix. Mass-loss measurements were consistent with microscopic observations of the metal surface after 2 weeks of exposure, indicating that uniform corrosion occurred. The biofilm was also able to withstand mild agitation (60 rpm), provided that sufficient time was given for its development. Received: 3 May 1996 / Received revision: 8 August 1996 / Accepted: 24 August 1996     

Non-toluene-associated respiration in a Pseudomonas putida 54G biofilm grown on toluene in a flat-plate vapor-phase bioreactor

Non-toluene-associated respiration (NTAR) within a Pseudomonas putida 54G biofilm growing on toluene as sole external carbon source was evaluated using oxygen microelectrodes in a flat-plate vapor-phase biological reactor. Two fluorescent probes, 2,4-diamidino-2-phenylindole and 5-cyano-2,3-ditolyltetrazolium chloride, were used to evaluate the number of total and respiring cells respectively within the biofilm. Biofilm samples were also analyzed for viable and toluene-culturable cells by spread-plating on non-selective and selective media respectively. Fractions of viable stressed, respiring and non-respiring cells within the biofilm were evaluated. The NTAR rate was positively correlated with the fraction of viable stressed and non-respiring cells within the biofilm, which suggested the capability of some cells to grow at the expense of leakage and lysis products coming from injured and dead cells. This effect was more pronounced at higher toluene concentration. Results suggest that NTAR should be incorporated into mathematical models of biofilm reactors degrading volatile organic carbon compounds. Received: 4 January 1997 / Received revision: 20 March 1997 / Accepted: 27 March 1997     

Isolation and characterization of Rhodococcus rhodochrous for the degradation of the wastewater component 2-hydroxybenzothiazole

2-Hydroxybenzothiazole (OBT) is present in wastewaters from the industrial production of the rubber vulcanization accelerator 2-mercaptobenzothiazole (MBT). We have achieved the first isolation of axenic bacterial cultures capable of the degradation of OBT and growth on this substrate as the sole source of carbon, nitrogen and energy. All isolates had similar characteristics corresponding to one particular isolate, which was studied in more detail and identified as Rhodococcus rhodochrous. The strains were also capable of degrading benzothiazole (BT) but not MBT or benzothiazole-2-sulphonate (BTSO₃). OBT was degraded at a concentration of up to 600 mg · l⁻¹. BT was toxic above 300 mg · l⁻¹. MBT inhibited OBT degradation. Growth on OBT was not significantly different at pH values of between 6.3 and 7.9 or salt concentrations between 1 % and 3 %. In shake flasks the cells clumped together, which resulted in a lower rate of oxygen transfer and slower degradation as compared to cells grown on OBT in a stirred reactor. Received: 22 August 1996 / Received revision: 29 November 1996 / Accepted: 29 November 1996     

Degradation of acenaphthene, phenanthrene and pyrene in a packed-bed biofilm reactor

Biofilm reactors are particularly suitable for the treatment of large amounts of diluted effluent, such as groundwater contaminated with scarcely soluble pollutants. A packed-bed column reactor was tested for the degradation of acenaphthene, phenanthrene and pyrene provided at their aqueous solubility concentrations. Acenapthene and phenanthrene were removed to more than 99% efficiency from this reactor whilst pyrene was removed to 90%. Pollutant disappearance was also recorded in the control reactor and was probably caused by the adsorption of pollutants into the reactor. The measurement of oxygen consumption in both reactors confirmed that microbial degradation of the pollutants was indeed occurring in the inoculated reactor. Physical adsorption is not however unwanted, as it could help with the formation of a biofilm at an early stage of the treatment. Received: 29 February 2000 / Received revision: 30 May 2000 / Accepted: 3 June 2000     

Immobilisation of whole bacterial cells for anaerobic biotransformations

Anaerobically grown cells of Escherichia coli were immobilised within a range of entrapment matrices and packed into a column under standard conditions, and the ability of the immobilised cells to reduce nitrite (0.5 mM) was measured at a range of flow rates using sodium formate (20 mM) as the electron donor for nitrite reduction. A flow-rate/activity plot was constructed for each flow-through reactor and RA_1/2 values (residence time corresponding to 50 % nitrite removal) calculated for each reactor type. Cells immobilised in flat and hollow-fibre membranes were the most effective (RA_1/2 = 0.35 h and 0.47 h respectively), with cells entrapped by dialysis membrane (1.53 h), alginate beads (1.93 h), Hypol foam (2.31 h) and polyacrylamide gel (50 % nitrite not removed at maximum residence time tested: 4.9 h) performing progressively less effectively. Cells grown as a biofilm on a range of support materials were also tested in comparable packed-bed reactors. Cell loss from these supports was extensive and contributed to poor performance of the reactors despite high initial biomass loadings (RA_1/2 values using raschig rings, coke and activated-carbon supports: 1.6 h, 2.3 h and 1.0 h respectively). Biofilms grown on Pharmacia microcarrier supports and used in packed and also fluidised beds were more stable and the performance of these reactors was superior to that of biofilm reactors using other supports, and comparable to that of the membrane reactors (RA_1/2 values for Cytoline 2, Cytopore 2 and Cytodex 3: 0.76 h, 0.56 h, 0.68 h respectively). Received: 12 August 1996 / Received revision: 14 November 1996 / Accepted: 15 November 1996     

Nitrification, denitrification, and dechlorination in bleached kraft pulp mill wastewater

This study deals with combining the biologi cal removal of organic halogens with the removal of nitrogen from bleached kraft pulp mill wastewater in fluidized-bed reactors under nitrifying and denitrifying conditions. Untreated and biotreated bleached kraft pulp mill wastewaters had no detrimental effect on nitrification or denitrification. The nitrifying biofilm reactor, pregrown on synthetic inorganic feed with ammonia, removed without a lag phase adsorbable organic halogens [7.2 mg Cl (g biomass volatile solids)⁻¹day⁻¹] from bleached kraft pulp mill wastewater and selected chlorophenols from synthetic wastewater. Electron microscopical examination of the biofilm showed that bacteria, morphologically similar to the nitrifying species Nitrosomonas or Nitrobacter, and Nitrosospira were dominant. The denitrifying fluidized-bed reactor, pregrown on nitrate and methanol, denitrified without a lag phase bleached kraft pulp mill wastewater. Under denitrifying conditions, 35% of the total organic carbon content of untreated bleached kraft pulp mill waste water was removed. The reducing power delivered by untreated bleached kraft pulp mill wastewater for denitrification was 2 mmol electrons/mmol carbon mineralized. Dechlorination under denitrifying conditions was negligible. Received: 21 November 1996 / Received revision: 27 January 1997 / Accepted: 1 February 1997     

Alternative electron acceptors in microbial coal-conversion wastewater treatment

Biotreatment experiments with solutions of autoxidized phenolic compounds as well as coal-conversion wastewater stored for 30 years and rich in humic matter were performed under nitrate-reducing, sulphate-reducing and methanogenic conditions. The removal of total organic carbon in fractions of different molecular mass and of monomeric phenolic compounds in the wastewater was determined. A comparison of biotransformation potentials and rates indicated a relationship between these aspects and the availability of electron acceptors in the system. The capacities of the microbial consortia increased significantly with the energy microorganisms could gain from their respective respiration process and can be expressed by the order: aerobic process – nitrate reduction – sulphate reduction – methanogenesis. Received: 25 April 1996 / Received revision: 23 July 1996 / Accepted: 5 August 1996.     

Microbial removal of chlorinated phenols during aerobic treatment of effluents from radiata pine kraft pulps bleached with chlorine-based chemicals,with or without hemicellulases

  The removal of chlorophenolic compounds from kraft mill effluents bleached with chlorine (cBKME) or chlorine plus hemicellulases (bBKME) was studied in reactors of aerobic treatment lagoons. In these laboratory models, a stable microbial population removed biochemical oxygen demand at similar rates of the mill lagoon. Complete removal of nine chlorophenols and chloroguaiacols during microbial treatment of these effluents was detected by gas chromatography. Abiotic removal was only observed with 2,4-dichlorophenol and 2,4,5-trichlorophenol. There were no significant differences in degradative ability between microorganisms acclimated to grow in reactors fed with cBKME or bBKME. The latter had a lower content of adsorbable organic halogen and chlorophenols than cBKME. Microorganisms acclimated to cBKME or bBKME were only able to grow on phenol or guaiacol as sole carbon source. However, these microorganisms removed (0.1–0.5 mM) 4-chlorophenol, 2,4-dichlorophenol and 2,4-dichlorophenoxyacetate with BKME as primary carbon source. Under these conditions, 2,4,6- and 2,4,5-trichlorophenol, 4,5-dichloroguaiacol, 4,5,6-trichloroguaiacol and tetrachloroguaiacol were not removed. These results suggest that the microbial removal of bleaching chlorophenols and chloroguaiacols during aerobic treatment, probably takes place only because of their very low concentration (1–200 ppb) in BKME. Received: 12 February 1996 / Received revision: 10 June 1996 / Accepted: 22 June 1996     

Pyrazine production by Bacillus subtilis in solid-state fermentation on soybeans

2,5-Dimethylpyrazine (2,5-DMP) and tetramethylpyrazine (TTMP) were produced using Bacillus subtilis IFO 3013 grown on soybeans. Solid-state cultivations were carried out either in 100-ml bottles or in a fixed-bed column reactor, both systems being at 27 °C. Optimization studies showed that the best way to produce the two above aroma compounds involved two separate processes. 2,5-DMP was obtained using soybeans enriched with 75 g threonine/kg initial dry weight (i.d.w.), giving 0.85 g metabolite/kg i.d.w. after 6 days. TTMP production involved addition of 90 g/kg i.d.w. acetoin to soybeans, and 2.5 g/kg i.d.w. was recovered after 14 days. These results demonstrated the suitability of solid-state cultivation for production of high-added-value compounds. Received: 30 September 1996 / Received revision: 23 December 1996 / Accepted: 30 December 1996     

Catabolite-repressor-like protein regulates the expression of a gene under the control of the Escherichia coli lac promoter in the plant pathogen Xanthomonas campestris pv. campestris

  Xanthomonas campestris pv. campestris, the causal agent of black-rot disease of cruciferous plants, and an important industrial microbe, was able to express the Escherichia coliβ-glucuronidase reporter gene (uidA) when fused to the E. coli lactose operon promoter on a wide-host-range plasmid vector. The gene fusion is expressed constitutively at high levels in both complex and defined media using a wide range of carbon sources, and is not repressible by glucose or inducible by the gratuitous lac inducer isopropyl β-d-thiogalactoside. An X. campestris campestris strain with a lesion in the clp (catabolite-repressor-like protein) locus, and containing the plac/uidA fusion, was tested for β-glucuronidase activity. We found that the expression of the plac/uidA fusion gene is dependent on the presence of catabolite-repressor-like protein, with an approximately 75% reduction of expression in the clp -deficient mutant. Received: 1 April 1996 / Received revision: 21 June 1996 / Accepted: 15 July 1996     

Expression of the hepatitis B virus surface antigen in mammalian cells using an Epstein-Barr-virus-derived vector

 The hepatitis B virus surface antigen (HBsAg) gene, under control of the inducible mouse metallothionein I gene promoter, was inserted in an expression vector based on the Epstein-Barr virus (EBV). This vector was introduced into human cells by DNA transfection and clones were selected for their resistance to hygromycin B. The recombinant EBV vector replicates efficiently as an episome in human cells and approximately six copies per cell were found in one clone of hygromycin-B-resistant cells. These cells produce high levels of HBsAg in the presence of metals. The protein is mainly found in the cell medium, suggesting that the HBsAg is secreted from the cells. Received: 25 February 1996 / Received revision: 21 June 1996 / Accepted: 15 July 1996     

Fermentability of hemicelluloses extracted from municipal waste and commercial xylans by Clostridium sp.

The fermentability of commercial xylans and municipal waste hemicelluloses in the presence of Clostridium sp. (C.SAIV; ATCC 700188) has been evaluated. Teak, deal wood, banana stalk and bagasse of the municipal waste contained significant amounts (approx. 12 %–23 %) of hemicellulose. Under optimized growth conditions, the growth rate of C.SAIV was improved as indicated by an increase in the concentration of ethanol in the culture broth. Commercial xylans were utilized fairly efficiently and ethanol formed from larch wood xylan and bagasse hemicellulose was at least 64 mM. The amount of ethanol formed from the bagasse hemicellulose was at least three times higher than any other reported value. The current study also indicated that the source and composition of hemicellulose played an important role in determining the fermentability of the substrate for some microorganisms. Received: 19 June 1996 / Received revision: 22 October 1996 / Accepted: 25 October 1996     

Growth performance and pathway flux determine substrate preference of Alcaligenes eutrophus during growth on acetate plus aromatic compound mixtures

During batch growth of Alcaligenes eutrophus on various aromatic compounds in the presence of acetate, several distinct behaviour patterns were observed. The utilization of substrates of the meta pathway (phenol or p-cresol) was inhibited by acetate. When the aromatic was a substrate of the p-hydroxybenzoate branch of the ortho pathway, growth was mixotrophic, i.e. both substrates were consumed simultaneously. For the substrates of the gentisate pathway or the benzoate branch of the ortho pathway, substrate preference was governed by growth performance. Aromatic compounds enabling growth rate and yields higher than those obtained on acetate alone (i.e. benzoate, benzaldehyde, m-hydroxybenzoate and gentisate) inhibited acetate utilization, while acetate was the substrate consumed preferentially in mixtures containing aromatic compounds supporting only slow growth (i.e. benzoyl formate and 4-fluorobenzoate). Received: 18 April 1996 / Received revision: 9 July 1996 / Accepted: 15 July 1996     

Elucidation of anaplerotic pathways in Corynebacterium glutamicum via 13C-NMR spectroscopy and GC-MS

We have obtained direct evidence indicating the presence of pyruvate-carboxylating activity in Corynebacterium glutamicum, a lysine-overproducing bacterium. This evidence was obtained through the use of ¹³C nuclear magnetic resonance (NMR) spectroscopy and gas chromatography/mass spectrometry (GC-MS) of secreted metabolites in a lysine fermentation. The distribution of ¹³C label after multiple turns in the tricarboxylic acid cycle was accounted for properly to obtain predictions for [¹³C] metabolite enrichments that were employed in the interpretation of ¹³C-NMR and GC-MS data. Of critical importance in arriving at the conclusions was the use of C. glutamicum mutants with deletions of the pyruvate kinase and/or phosphoenolpyruvate carboxylase enzymes. Our results demonstrate the presence of pyruvate-carboxylating pathway(s) in C.␣glutamicum operating simultaneously with phosphoenolpyruvate carboxylase, with the latter enzyme contributing approximately 10 % of the total oxaloacetate synthesis during the lysine-production phase with pyruvate and gluconate as carbon sources. These findings are important for developing strategies to increase the total carbon flux for synthesis of amino acids of the aspartate family through metabolic engineering. Received: 11 June 1996 / Received revision: 30 October 1996 / Accepted: 15 November 1996     

Axenic aerobic biofilms inhibit corrosion of SAE 1018 steel through oxygen depletion

Corrosion inhibition of SAE 1018 steel by pure-culture biofilms of Pseudomonas fragi and Escheri-chia coli DH5α has been evaluated in complex Luria-Bertani medium, seawater-mimicking medium, and modified Baar's medium at 30 °C. In batch cultures, both bacteria inhibited corrosion three to six fold compared to sterile controls, and the corrosion was comparable to that observed in anaerobic sterile media. To corroborate this result, a continuous reactor and electrochemical impedance spectroscopy were used to show that both P. fragi K and E. coli DH5α decreased the corrosion rate by 4- to 40-fold as compared to sterile controls; this matched the decrease in corrosion found with sterile medium in the absence of oxygen and with E. coli DH5α grown anaerobically. In addition, the requirement for live respiring cells was demonstrated by the increase in the corrosion rate that was observed upon killing the P. fragi K biofilm in continuous cultures, and it was shown that fermentation products do not cause an increase in corrosion. Hence, pure-culture biofilms inhibit corrosion of SAE 1018 steel by depleting oxygen at the metal surface. Received: 16 December 1996 / Received revision: 18 March 1997 / Accepted: 27 March 1997     

An attempt to channel the transformation of vanillic acid into vanillin by controlling methoxyhydroquinone formation in Pycnoporus cinnabarinus with cellobiose

The effects of adding cellobiose on the transformation of vanillic acid to vanillin by two strains of Pycnoporus cinnabarinus MUCL39532 and MUCL38467 were studied. When maltose was used as the carbon source in the culture medium, very high levels of methoxyhydroquinone were formed from vanillic acid. When cellobiose was used as the carbon source and/or added to the culture medium of P. cinnabarinus strains on day 3 just before vanillic acid was added, it channelled the vanillic acid metabolism via the reductive route leading to vanillin. Adding 3.5 g l⁻¹ cellobiose to 3-day-old maltose cultures of P. cinnabarinus MUCL39532 and 2.5 g l⁻¹ cellobiose to 3-day-old cellobiose cultures of P. cinnabarinus MUCL38467, yielded 510 mg l⁻¹ and 560 mg l⁻¹ vanillin with a molar yield of 50.2 % and 51.7 % respectively. Cellobiose may either have acted as an easily metabolizable carbon source, required for the reductive pathway to occur, or as an inducer of cellobiose:quinone oxidoreductase, which is known to inhibit vanillic acid decarboxylation. Received: 24 July 1996 / Received revision: 29 November 1996 / Accepted: 29 November 1996     

Immobilization of pancreatic islet cells with preserved secretory potential

Dispersed pancreatic islet cells from rats were cultured overnight in the presence of macroporous gelatin microcarriers. The cells attached to the microcarriers were then incubated for 90 min in the absence or presence of 15.0 mM d-glucose and/or 1.25 mM theophylline. The release of insulin during incubation was about three times higher in the simultaneous presence of these two secretagogues than in their absence. This procedure could thus be used for the immobilization of pancreatic islet cells with preserved secretory potential. Received: 9 April 1999 / Received revision: 12 July 1999 / Accepted: 13 July 1999     

Localization and characterization of inclusion bodies in recombinant Escherichia coli cells overproducing penicillin G acylase

Various concentrations of isopropyl β-d-thiogalactopyranoside (IPTG) were used to induce production of the enzyme penicillin G acylase by recom binant Escherichia coli harboring plasmid pQEA11. The plasmid pQEA11 carries a wild-type pga gene, which is under the control of the tac promoter and lacI ^q. At low IPTG concentrations (0.025 – 0.1 mM), enzyme activity increased with increasing IPTG concentrations. At higher IPTG concentrations (0.2 and 0.5 mM), enzyme activity declined progressively. Examination of induced recombinant E. coli cells by transmission electron microscopy showed the presence of only periplasmic inclusion bodies at low IPTG concentrations (up to 0.1 mM) and both periplasmic and cytoplasmic inclusion bodies at high IPTG concentrations (0.2 mM and 0.5 mM). Results from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblots of whole-cell proteins, membrane proteins and inclusion body proteins in these cells indicated that cytoplasmic inclusion bodies constituted an accumulation of preproenzyme (i.e., precursor polypeptide containing a signal peptide) and that periplasmic inclusion bodies constituted an accumulation of proenzyme (i.e., precursor polypeptide lacking a signal peptide). Received: 27 March 1996 / Received revision: 2 July 1996 / Accepted: 10 November 1996