Tetrachloroethene dechlorination kinetics by Dehalospirillum multivorans immobilized in upflow anaerobic sludge blanket reactors

Tetrachloroethene (C₂Cl₄) dechlorination kinetics in upflow anaerobic sludge blanket (UASB) reactors was determined after introducing de novo activities into the granular sludge. These activities were introduced by immobilizing Dehalospirillum multivorans in a test reactor containing unsterile granular sludge, and in a reference reactor, R1, containing sterile granular sludge. A second reference reactor, R2, contained only unsterile granular sludge and served as a control. The kinetic experiments were performed by pulsing the reactors with C₂Cl₄ in a recirculating batch mode. Formate and acetate were added as electron donor and carbon source. Both reactors inoculated with D. multivorans dechlorinated C₂Cl₄ to an equimolar amount of C₂H₂Cl₂ with only traces of C₂HCl₃ in the effluent. In the control reactor, C₂HCl₃ accumulated before C₂H₂Cl₂ was produced. A computer simulation program (AQUASIM) was used to estimate the kinetic parameters. The half-saturation constants (K _s) for C₂Cl₄ and C₂HCl₃ were almost equal in the reactors containing D.␣multivorans (17 μM and 18 μM for C₂Cl₄; 26 μM and 28 μM for C₂HCl₃), indicating no influence of sludge bacteria on the affinity of D. multivorans for C₂Cl₄ and C₂HCl₃. The maximum dechlorination rates (k _m X _B) were about twice as high in the reactor containing D.␣multivorans immobilized in sterile sludge (11 mmol C₂Cl₄ l sludge⁻¹ day⁻¹ and 27 mmol C₂HCl₃ l sludge⁻¹ day⁻¹) than in the test reactor (4.4 mmol C₂Cl₄ l sludge⁻¹ day⁻¹ and 15 mmol C₂HCl₃ l sludge⁻¹ day⁻¹). Compared to other C₂Cl₄-degrading systems, the dechlorination rates of the inoculated reactors and their affinities for C₂Cl₄ and C₂HCl₃ were high. Therefore, introduction of de novo activity is promising for the use of anaerobic reactors to bioremediate C₂Cl₄-polluted water. Received: 5 November 1998 / Received revision: 25 January 1999 / Accepted: 31 January 1999     

Complete degradation of tetrachloroethene in coupled anoxic and oxic chemostats

Anaerobic tetrachloroethene(C₂Cl₄)-dechlorinating bacteria were enriched in slurries from chloroethene-contaminated soil. With methanol as electron donor, C₂Cl₄ and trichloroethene (C₂HCl₃) were reductively dechlorinated to cis-1,2-dichloroethene (cis-C₂H₂Cl₂), whereas, with l-lactate or formate, complete dechlorination of C₂Cl₄ via C₂HCl₃, cis-C₂H₂Cl₂ and chloroethene (C₂H₃Cl) to ethene was obtained. In oxic soil slurries with methane as a substrate, complete co-metabolic degradation of cis-C₂H₂Cl₂ was obtained, whereas C₂HCl₃ was partially degraded. With toluene or phenol both of the above were readily co-metabolized. Complete degradation of C₂Cl₄ was obtained in sequentially coupled anoxic and oxic chemostats, which were inoculated with the slurry enrichments. Apparent steady states were obtained at various dilution rates (0.02–0.4 h⁻¹) and influent C₂Cl₄-concentrations (100–1000 μM). In anoxic chemostats with a mixture␣of␣formate and glucose as the carbon and electron source, C₂Cl₄ was transformed at high rates (above␣140 μmol l⁻¹ h⁻¹, corresponding to 145 nmol Cl⁻ min⁻¹ mg protein⁻¹) into cis-C₂H₂Cl₂ and C₂H₃Cl. Reductive dechlorination was not affected by addition of 5 mM sulphate, but strongly inhibited after addition of 5 mM nitrate. Our results (high specific dechlorination rates and loss of dechlorination capacity in the absence of C₂Cl₄) suggest that C₂Cl₄-dechlorination in the anoxic chemostat was catalysed by specialized dechlorinating bacteria. The partially dechlorinated intermediates, cis-C₂H₂Cl₂ and C₂H₃Cl, were further degraded by aerobic phenol-metabolizing bacteria. The maximum capacity for chloroethene (the sum of tri-, di- and monochloro derivatives removed) degradation in the oxic chemostat was 95 μmol l⁻¹ h⁻¹ (20 nmol min⁻¹ mg protein⁻¹), and that of the combined anoxic → oxic reactor system was 43.4 μmol l⁻¹ h⁻¹. This is significantly higher than reported thus far. Received: 17 April 1997 / Received revision: 6 June 1997 / Accepted: 7 June 1997     

Trichloroethylene degradation and mineralization by pseudomonads and Methylosinus trichosporium OB3b

 To examine the trichloroethylene (C₂HCl₃)-degrading capability of five microorganisms, the maximum rate, extent, and degree of C₂HCl₃ mineralization were evaluated for Pseudomonas cepacia G4, Pseudomonas cepacia G4 PR1, Pseudomonas mendocina KR1, Pseudomonas putida F1, and Methylosinus trichosporium OB3b using growth conditions commonly reported in the literature for expression of oxygenases responsible for C₂HCl₃ degradation. By varying the C₂HCl₃ concentration from 5 μM to 75 μM, V _max and K _m values for C₂HCl₃ degradation were calculated as 9 nmol/(min mg protein) and 4 μM for P. cepacia G4, 18 nmol/(min mg protein) and 29 μM for P. cepacia G4 PR1, 20 nmol/(min mg protein) and 10 μM for P. mendocina KR1, and 8 nmol/(min mg protein) and 5 μM for P. putida F1. This is the first report of these Michaelis-Menten parameters for P. mendocina KR1, P. putida F1, and P. cepacia G4 PR1. At 75 μM, the extent of C₂HCl₃ that was degraded after 6 h of incubation with resting cells was 61%–98%; the highest degradation being achieved by toluene-induced P. mendocina KR1. The extent of C₂HCl₃ mineralization in 6 h (as indicated by concentration of chloride ion) was also measured and varied from 36% for toluene-induced P. putida F1 to 102% for M. trichosporium OB3b. Since C₂HCl₃ degradation requires new bio-mass, the specific growth rate (μ_max) of each of the C₂HCl₃-degradation microorganisms was determined and varied from 0.080/h (M. trichosporium OB3b) to 0.864/h (P. cepacia G4 PR1). Received: 1 May 1995/Received revision: 11 July 1995/Accepted: 26 July 1995     

Removal of tetrachloroethylene in an anaerobic column bioreactor

Removal of tetrachloroethylene (perchloroethylene; C₂Cl₄) by microbial consortia from two sites with different C₂Cl₄ exposure histories was examined in a bench-scale anaerobic column bioreactor. It was hypothesized that optimal removal would be observed in the reactor packed with sediments having an extensive exposure history. Microbial consortia were enriched from hyporheic-zone (HZ) sediments from the Portneuf aquifer near Pocatello, Idaho, and from industrial-zone (IZ) sediments from a highly contaminated aquifer in Portland, Oregon. Lactate and acetate were the electron donors during experiments conducted over 9 and 7 months for HZ and IZ sediments, respectively. In the HZ bioreactor, the retention time ranged from 31 h to 81 h, and inlet C₂Cl₄ concentrations ranged from 0.1 ppm to 1.0 ppm. Dechlorination of C₂Cl₄ averaged 60% and reached a maximum of 78%. An increase in C:N from 27:1 to 500:1 corresponded to an 18% increase in removal efficiency. Trichloroethylene production corresponded to decreased effluent C₂Cl₄; further intermediates were not detected. In the IZ bioreactor, the retention time varied from 34 h to 115 h; the inlet C₂Cl₄ concentration was 1.0 ppm. C₂Cl₄ removal averaged 70% with a maximum of 98%. Trichloroethylene and cis-dichloroethylene were detected in the effluent. Increases in C:N from 50:1 to 250:1 enhanced dechlorination activity. Received: 3 February 1997 / Received revision: 15 May 1997 / Accepted: 1 June 1997     

Production of flavour compounds by yogurt starter cultures

The present work studied the production of carbonyl compounds and saturated volatile free fatty acids by pure cultures of Streptococcus thermophilus and Lactobacillus bulgaricus, and by starter cultures for Bulgarian yogurt during cultivation and cooling. The mixed cultures formed volatile aromatic compounds more actively than the pure cultures. A guiding factor in the preparation of the starter cultures was the biochemical activity of Lactobacillus bulgaricus in synthesizing the major carbonyl compounds, acetaldehyde, diacetyl and the volatile fatty acids C₂–C₁₀. The activity of the yogurt cultures in synthesizing carbonyl compounds was at its highest during milk coagulation and cooling, up to 7 h. However, maximum concentration was reached by 22–31 h. In the cooled 22–h starter cultures, acetaldehyde predominated (1415.0–1734.2 μg per 100 g) followed by diacetyl (165.0–202.0 μg per 100 g), acetoin (170.0–221.0 μg per 100 g), acetone (66.0–75.5 μg per 100 g), ethanol (58.0 μg per 100 g), and butanone-2 (3.6–3.8 μg per 100 g). The thermophilic streptococcus and lactobacillus cultures, and the starter cultures contained predominantly acetic, butyric and caproic acids. Received 19 June 1997/ Accepted in revised form 10 January 1998     

Complete transformation of 1,1,1-trichloroethane to chloroethane by a methanogenic mixed population

A methanogenic mixed population in a packed-bed reactor completely transformed 1,1,1-trichloroethane (10 μM) to chloroethane by a cometabolic process. Chloroethane was not further transformed. Acetate and methanol served as electron donors. Complete transformation of 1,1,1-trichloroethane to chloroethane only occurred when sufficient electron donor was fed into the reactor. Otherwise, besides chloroethane, 1,1-dichloroethane was also found as a product. The products of 1,1,1-trichloroethane transformation also depended on the type of electron donor present. With acetate, the degree of dechlorination was higher, i.e. more 1,1,1-trichloroethane was transformed to chloroethane than with methanol. In an enrichment culture obtained from the reactor contents, 1,1,1-trichloroethane was only transformed to 1,1-dichloroethane and was not further metabolized. Methanol, acetate, formate, ethanol, 2-propanol, trimethylamine and H₂, but not dimethylamine and methylamine, served as electron donors for 1,1,1-trichloroethane transformation by this enrichment culture. Both nitrate and nitrite inhibited 1,1,1-trichloroethane transformation; while nitrate completely inhibited 1,1,1-trichloroethane dechlorination, some conversion did occur in the presence of nitrite. The product(s) of this conversion remain unknown, since no chlorinated hydrocarbons were detected. Received: 19 June 1998 / Received revision: 14 September 1998 / Accepted: 17 September 1998     

Anaerobic dechlorination of perchloroethene in an extractive membrane bioreactor

An extractive membrane bioreactor (EMB) is described that used an undefined anaerobic culture to dechlorinate tetrachloroethene (C₂Cl₄) reductively in a synthetic wastewater. Comparable reactors described in the literature use set-ups where the bacteria are in direct contact with the wastewater, and thus would require the addition of significant quantities of nutrients to the wastewater stream in practical application. In the EMB, a silicone rubber membrane separates the microbial culture from the wastewater stream, so that addition of nutrients can be minimised. The EMB was operated continuously for 48 days and dechlorinated 359 mol C₂Cl₄/(l biomedium⁻¹ day⁻¹) on average. Lactate was fed as an electron donor and C₂Cl₄ dechlorination was verified by chloride measurements. Particular attention was paid to the reduction of transmembrane C₂Cl₄ flux caused by a membrane-attached biofilm. Following a start-up period, the reactor operation was stable and remained largely unaffected by biofilm thickness and oxygen contamination from the wastewater. Received: 19 January 1998 / Received revision: 8 May 1998 / Accepted: 8 May 1998     

Dechlorination of polychlorinated biphenyl congeners by an anaerobic microbial consortium

  An anaerobic methanogenic microbial consortium, developed in a granular form, exhibited extensive dechlorination of defined polychlorinated biphenyl (PCB) congeners. A 2,3,4,5,6-pentachlorobiphenyl was dechlorinated to biphenyl via 2,3,4,6-tetrachlorobiphenyl, 2,4,6-trichlorobiphenyl, 2,4-dichlorobi-phenyl and 2-chlorobiphenyl (CB). Removal of chlorine atoms from all three positions of the biphenyl ring, i.e., ortho, meta and para, was observed during this reductive dechlorination process. Biphenyl was identified as one of the end-products of the reductive dechlorination by GC-MS. After 20 weeks, the concentrations of the dechlorination products 2,4,6-CB, 2,4-CB, 2-CB and biphenyl were 8.1, 41.2, 3.0 and 47.8 μM respectively, from an initial 105 μM 2,3,4,5,6-CB. The extent and pattern of the dechlorination were further confirmed by the dechlorination of lightly chlorinated congeners including 2-CB, 3-CB, 4-CB, 2,4-CB and 2,6-CB individually. This study indicates that the dechlorination of 2,3,4,5,6-CB to biphenyl is due to ortho, meta and para dechlorination by this anaerobic microbial consortium. Received: 30 April 1996 / Received revision: 26 July 1996 / Accepted: 5 August 1996     

Biodegradation of atrazine under denitrifying conditions

Anaerobic biodegradation of atrazine by the bacterial isolate M91-3 was characterized with respect to mineralization, metabolite formation, and denitrification. The ability of the isolate to enhance atrazine biodegradation in anaerobic sediment slurries was also investigated. The organism utilized atrazine as its sole source of carbon and nitrogen under anoxic conditions in fixed-film (glass beads) batch column systems. Results of HPLC and TLC radiochromatography suggested that anaerobic biotransformation of atrazine by microbial isolate M91-3 involved hydroxyatrazine formation. Ring cleavage was demonstrated by ¹⁴CO₂ evolution. Denitrification was confirmed by detection of ¹⁵N₂ in headspace samples of K¹⁵NO₃-amended anaerobic liquid cultures. In aquatic sediments, mineralization of uniformly ring-labeled [¹⁴C]atrazine occurred in both M91-3-inoculated and uninoculated sediment. Inoculation of sediments with M91-3 did not significantly enhance anaerobic mineralization of atrazine as compared to uninoculated sediment, which suggests the presence of indigenous organisms capable of anaerobic atrazine biodegradation. Results of this study suggest that the use of M91-3 in a fixed-film bioreactor may have applications in the anaerobic removal of atrazine and nitrate from aqueous media. Received: 3 September 1997 / Received revision: 4 December 1997 / Accepted: 2 January 1998     

Biodegradation of the pesticide 4,6-dinitro-ortho-cresol by microorganisms in batch cultures and in fixed-bed column reactors

A mixed culture of microorganisms able to utilize 4,6-dinitro-ortho-cresol (DNOC) as the sole source of carbon, nitrogen and energy was isolated from soil contaminated with pesticides and from activated sludge. DNOC was decomposed aerobically in batch cultures as well as in fixed-bed column reactors. Between 65% and 84% of the substrate nitrogen was released as nitrate into the medium, and 61% of the carbon from uniformly ¹⁴C-labelled DNOC was recovered as ¹⁴CO₂. The mixed microbial culture also decomposed 4-nitrophenol and 2,4-dinitrophenol but not 2,3-dinitrophenol, 2,6-dinitrophenol, 2,4-dinitrotoluene, 2,4-dinitrobenzoic acid or 2-sec-butyl-4,6-dinitrophenol (Dinoseb). Maximal degradation rates for DNOC by the bacterial biofilm immobilized on glass beads in fixed-bed column reactors were 30 mmol day⁻¹ (l reactor volume)⁻¹, leaving an effluent concentration of less than 5 μg l⁻¹ DNOC in the outflowing medium. The apparent K _s value of the immobilized mixed culture for DNOC was 17 μM. Degradation was inhibited at DNOC concentrations above 30 μM and it ceased at 340 μM, possibly because of the uncoupling action of the nitroaromatic compound on the cellular energy-transducing mechanism. Received: 27 March 1997 / Received revision: 5 June 1997 / Accepted: 7 June 1997     

The effect of growth conditions on the biodegradation of tributyl phosphate and potential for the remediation of acid mine drainage waters by a naturally-occurring mixed microbial culture

The biodegradation of tributyl phosphate (Bu₃-P, TBP), releasing phosphate at a high enough concentration locally to precipitate uranium from solution, was demonstrated by a mixed culture consisting primarily of pseudomonads. The effect of various parameters on Bu₃-P biodegradation by growing cells is described. Growth at the expense of Bu₃-P as the carbon and phosphorus source occurred over a pH range from 6.5 to 8, and optimally at pH 7. Bu₃-P biodegradation was optimal at 30 °C, reduced at 20 °C and negligible at 4 °C and 37 °C. Incorporation of Cu or Cd inhibited, and Ni, Co and Mn reduced its degradation. Inorganic phosphate (above 10 mM) and kerosene (up to 1 g/l) reduced Bu₃-P biodegradation significantly, but nitrate had no effect. Sulphate (10–100 mM) was inhibitory. When pregrown biomass was used the fastest rates of tributyl and dibutyl phosphate biodegradation were 25 μmol h⁻¹ mg protein⁻¹ and 37 μmol h⁻¹ mg protein⁻¹ respectively. Microcarrier-immobilised biomass decontaminated uranium-bearing acid mine waste water by uranium phosphate precipitation at the expense of Bu₃-P hydrolysis in the presence of 35 mM SO₄ ²⁻. At pH 4.5, 79% of the UO₂ ²⁺ was removed at a flow rate of 1.4 ml/h on a 7-ml test column. Received: 2 June 1997 / Received revision: 15 September 1997 / Accepted: 19 September 1997     

Aerobic degradation of a hydrocarbon mixture in natural uncontaminated potting soil by indigenous microorganisms at 20 °C and 6 °C

A hydrocarbon mixture containing p-xylene, naphthalene, Br-naphthalene and straight aliphatic hydrocarbons (C₁₄ to C₁₇) was aerobically degraded without lag phase by a natural uncontaminated potting soil at 20 °C and 6 °C. Starting concentrations were approximately 46 ppm for the aromatic and 13 ppm for the aliphatic compounds. All aliphatic hydrocarbons were degraded within 5 days at 20 °C, to levels below detection (ppb levels) but only down to 10% of initial concentration at 6 °C. Naphthalene was degraded within 12 days at 20 °C and unaffected at 6 °C. At 20 °C p-xylene was degraded within 20 days, but no degradation occurred at 6 °C. Br-naphthalene was only removed down to 30% of initial concentration at 20 °C, with no significant effect at 6 °C. The biodegradation was monitored with head space solid-phase microextraction and gas chromatography–mass spectrometry. Received: 5 October 1998 / Received revision: 4 December 1998 / Accepted: 5 December 1998     

Microbial degradation of tetraethyl lead in soil monitored by microcalorimetry

Sieved agricultural soil samples were treated with the anti-knock agent tetraethyl lead (Et₄Pb), and the resulting effects were analyzed by microcalorimetry. Et₄Pb additions resulted in an increase of the heat production rate, provided that oxygen was present and that the soil was not autoclaved. The increased heat production rate was accompanied by degradation of Et₄Pb, as verified by speciation analysis (GC-MS) of the remaining Et₄Pb and its ionic degradation products (triethyl lead and diethyl lead cations). Conclusive evidence was obtained that these transformations were mediated mainly by microbes. At an initial Et₄Pb concentration of 2 g Pb/kg dry weight the biodegradation rate was about 780 μmol day⁻¹ kg dry weight⁻¹, whilst the chemical decomposition was only 50 μmol day⁻¹ kg dry weight⁻¹. A fivefold rise of the initial Et₄Pb concentration resulted in a decrease of the biodegradation rate to 600 μmol day⁻¹ kg dry weight⁻¹ and an increase of the chemical decomposition to 200 μmol day⁻¹ kg dry weight⁻¹. The biodegradation rate was not influenced by the addition of glucose, which means that no indication for a cometabolic attack of Et₄Pb was found. Received: 25 February 1997 / Received revision: 22 April 1997 / Accepted: 27 April 1997     

Enhanced mineralization of pentachlorophenol by κ-carrageenan-encapsulated Pseudomonas sp. UG30

A pentachlorophenol(PCP)-degrading Pseudomonas sp. strain UG30 was encapsulated in κ-carrageenan for use in PCP degradation. Free and encapsulated cells were compared for their ability to dechlorinate and mineralize 100–800 μg/ml sodium pentachlorophenate in broth. Dechlorination was measured with a chloride ion electrode, and mineralization was measured by ¹⁴CO₂ evolution from radiolabelled [U-¹⁴C]PCP. Free and encapsulated Pseudomonas sp. UG30 cells mineralized up to 200 μg/ml and 600 μg/ml PCP, respectively, after 21 days. Encapsulation of UG30 cells provided a protective effect, allowing dechlorination and mineralization of high levels of PCP to occur. Received: 3 May 1996 / Received revision: 4 September 1996 / Accepted: 13 September 1996     

Domoic acid neurotoxicity in hippocampal slice cultures

Summary.  The neurotoxicity of domoic acid was studied in 2–3 week old rat hippocampal slice cultures, derived from 7 day old rat pups. Domoic acid 0.1–100 μM was added to the culture medium for 48 hrs, alone or together with the glutamate receptor antagonists NS-102 (5-Nitro-6,7,8,9-tetrahydrobenzo[G]indole-2,3-dione-3-oxime), NBQX (2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline) or MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate), followed by transfer of the cultures to normal medium for additional 48 hrs. Neuronal degeneration in the fascia dentata (FD), CA3 and CA1 hippocampal subfields was monitored and EC₅₀ values estimated by densitometric measurements of the cellular uptake of propidium iodide (PI). The CA1 region was most sensitive to domoic acid, with an EC₅₀ value of 6 μM domoic acid, estimated from the PI-uptake at 72 hrs. Protective effects of 10 μM NBQX against 3 and 10 μM domoic acid were observed for both dentate granule cells and CA1 and CA3c pyramidal cells. NS102 and MK 801 only displayed protective effects when combined with NBQX. MK801 significantly increased the combined neuroprotective effect of NBQX and NS102 against 10 μM domoic acid in both CA1 and FD, but not in CA3. We conclude, that domoic acid neurotoxicity in CA3 and in hippocampal slice cultures in general primarily involves AMPA/kainate receptors. At high concentrations (10 μM domic acid) NMDA receptors are, however, also involved in the toxicity in CA1 and FD. Received June 29, 2001 Accepted August 6, 2001 Published online June 3, 2002     

Role of Na+/ H+ exchange and HCO3 − transport in pHi recovery from intracellular acid load in cultured epithelial cells of sheep rumen

This study sought to investigate effects of short-chain fatty acids and CO₂ on intracellular pH (pH_i) and mechanisms that mediate pH_i recovery from intracellular acidification in cultured ruminal epithelial cells of sheep. pH_i was studied by spectrofluorometry using the pH-sensitive fluorescent indicator 2′,7′-bis (carboxyethyl)-5(6′)-carboxyfluorescein acetoxymethyl ester (BCECF/AM). The resting pH_i in N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-buffered solution was 7.37 ± 0.03. In HEPES-buffered solution, a NH₄ ⁺/NH₃-prepulse (20 mM) or addition of butyrate (20 mM) led to a rapid intracellular acidification (P < 0.05). Addition of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 10 μM) or HOE-694 (200 μM) inhibited pH_i recovery from an NH₄ ⁺/NH₃-induced acid load by 58% and 70%, respectively. pH_i recovery from acidification by butyrate was reduced by 62% and 69% in the presence of EIPA (10 μM) and HOE-694 (200 μM), respectively. Changing from HEPES- (20 mM) to CO₂/HCO₃ ⁻-buffered (5%/20 mM) solution caused a rapid decrease of pH_i (P < 0.01), followed by an effective counter-regulation. 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS; 100 μM) blocked the pH_i recovery by 88%. The results indicate that intracellular acidification by butyrate and CO₂ is effectively counter-regulated by an Na⁺/H⁺ exchanger and by DIDS-sensitive, HCO₃ ⁻-dependent mechanism(s). Considering the large amount of intraruminal weak acids in vivo, both mechanisms are of major importance for maintaining the pH_i homeostasis of ruminal epithelial cells. Accepted: 8 March 2000     

The microbial production of poly(hydroxyalkanoates) from tallow

The bacteria Pseudomonas oleovorans, P. resinovorans, P. putida, and P. citronellolis were evaluated for their ability to grow and produce poly(hydroxyalkanoates) (PHA) using tallow free fatty acids and tallow triglyceride as carbon substrates. Tallow free fatty acids supported cell growth and PHA production for all four organisms, yielding PHA contents of 18%, 15%, 19% and 3% of their cell dry weights for P. oleovorans, P.␣resinovorans, P. putida, and P. citronellolis respectively. Only P. resinovorans, however, was able to grow and produce PHA polymer, with cells attaining a PHA content of 15% of their cell dry weight, using unhydrolyzed tallow as the substrate. Extracts from 46-h cultures of P. resinovorans were found to have a higher esterase activity (12.80 units μl⁻¹min⁻¹) compared to the activities found for cultures of P. oleovorans, P. citronellolis, and P. putida ( < 0.03 units μl⁻¹min⁻¹). Polymer repeat-unit compositions were determined by GC analysis of the β-hydroxymethyl esters of hydrolyzed PHA, and ranged in carbon-chain lengths from C₄ to C₁₄, with some mono-unsaturation in the C₁₂ and C₁₄ side-chains. PHA compositions were similar for the polymers obtained from all four organisms, with repeat units of chain lengths C₈ and C₁₀ predominating. Received: 16 February 1996 / Received revision: 23 May 1996 / Accepted: 10 June 1996     

Organic halogen removal from chlorinated humic ground water and lake water by nitrifying fluidized-bed biomass characterised by electron microscopy and molecular methods

The dechlorinating and genotoxicity-removing activities of nitrifying fluidized-bed reactor biomass towards chlorinated organic compounds in water were shown at level below 1 ppm. The removal rates of adsorbable organic halogens were 200 μg Cl (g VS day)⁻¹ for chlorinated humic ground water and 50 μyg Cl (g VS day)⁻¹ for chlorinated lake water when studied in batch mode. In a sequenced batch mode the removal rates μg Cl (g VS day)⁻¹] were 2000 from chlorohumus, 1400–1800 from chlorophenols in chlorinated ground water, and 430–720 from chlorohumus in chlorinated lake water. Genotoxicity was removed to a large extent (60%–80%) from the chlorinated waters upon incubation with nitrifying reactor biomass. 2,6-Di-, 2,4,6-tri and 2,3,4,6-tetrachlorophenols competed with chlorinated water organohalogens for dechlorination. The dechlorination of chlorophenols and chlorohumus required no ammonia and was not prevented by inhibitors of ammonia oxidation, nitrapyrin, parathion, sodium diethyldithiocarbamate, or allylthiourea. Electron microscopical inspection of the biomass showed the dominance of clusters of bacteria resembling known nitrifying species, Nitrosomonas, Nitrobacter, and Nitrosospira. This was supported by polymerase chain reaction amplification of the biomass DNA with four different primers, revealing the presence of 16S rDNA sequences assignable to the same species. The most intensive band obtained with the Nitroso4E primer was shown to be closely related to Nitrosomonas europaea by restriction analysis. Received: 27 March 1998 / Received revision: 30 July 1998 / Accepted: 31 July 1998     

Biodegradation kinetics of monoterpenes in liquid and soil-slurry systems

Batch experiments were conducted to evaluate the biodegradation rates of limonene, α-pinene, γ-terpinene, terpinolene and α-terpineol at 23 °C under aerobic conditions. Biodegradation was demonstrated by the depletion of monoterpene mass, CO₂ production and a corresponding increase in biomass. Monoterpene degradation in liquid cultures devoid of soil followed Monod kinetics. The maximum specific growth rate (μ_max) was 0.02 h⁻¹ and 0.06 h⁻¹ and the half-velocity constant (K _s ) varied from 32 mg/l to 3 mg/l for the limonene and α-terpineol respectively. The recovery of monoterpenes by solvent extraction from autoclaved and azide-amended soil-slurry samples decreased over time and ranged from 69% to 73% for 120 h of incubation period. Although a significant fraction of monoterpene hydrocarbon could not be extracted, mineralization of these compounds in the soil-slurry systems took place, as shown by CO₂ production. The soil-normalized degradation rates for the hydrocarbon monoterpenes ranged from 0.6 μg g⁻¹ h⁻¹ to 2.1 μg g⁻¹ h⁻¹. A kinetic model – which combined monoterpene biodegradation in the liquid phase and net desorption – was developed and applied to data obtained from soil-slurry assays. Received: 10 September 1996 / Received revision: 16 December 1996 / Accepted: 10 January 1997     

Effects of Mn2+ and NH+ 4 concentrations on laccase and manganese peroxidase production and Amaranth decoloration by Trametes versicolor

Extracellular lignin peroxidase (LiP) was not detected during decoloration of the azo dye, Amaranth, by Trametes versicolor. Approximately twice as much laccase and manganese peroxidase (MnP) was produced by decolorizing cultures compared to when no dye was added. At a low Mn²⁺ concentration (3 M), N-limited (1.2 mM NH₄ ⁺) cultures decolorized eight successive additions of Amaranth with no visible sorption to the mycelial biomass. At higher Mn²⁺ concentrations (200 M), production of MnP increased and that of laccase decreased, but the rate or number of successive Amaranth decolorations was unaffected. There was always a 6-h to 8-h lag prior to decoloration of the first aliquot of Amaranth, regardless of MnP and laccase concentrations. Although nitrogen-rich (12 mM NH₄ ⁺) cultures at an initial concentration of 200 M Mn²⁺ produced high laccase and MnP levels, only three additions of Amaranth were decolorized, and substantial mycelial sorption of the dye occurred. While the results did not preclude roles for MnP and laccase, extracellular MnP and laccase alone were insufficient for decoloration. The cell-free supernatant did not decolorize Amaranth, but the mycelial biomass separated from the whole broth and resuspended in fresh medium did. This indicates the involvement of a mycelial-bound, lignolytic enzyme or a H₂O₂-generating mechanism in the cell wall. Nitrogen limitation was required for the expression of this activity. Received: 19 May 1998 / Received revision: 22 October 1998 / Accepted: 7 November 1998