Growth conditions of Aspergillus sp. ATHUM-3482 for polygalacturonase production

A wild type of Aspergillus sp. ATHUM-3482 produced extracellular polygalacturonase when grown in liquid medium containing citrus pectin as sole carbon source. A number of factors affecting enzyme activity were investigated. Polygalacturonase activities as high as␣4.3 U␣ml⁻¹(reducing-group-releasing activity) and 17␣U␣ml⁻¹ (viscosity-diminishing activity) were obtained under optimum growth conditions. With sugar-beet as sole carbon source the respective activities were 6.5 U␣ml⁻¹ and 40 U ml⁻¹, the highest achieved in this work. Under these conditions no pectin lyase or pectinesterase activity was detected. The above yields of polygalacturonase activity compare favourably with those reported for fungi grown under similar growth conditions. Received: 5 March 1996 / Received last revision: 29 October 1996 / Accepted: 2 November 1996     

Enantioselective reduction of 3,4-methylene-dioxyphenylacetone using Candida famata and Zygosaccharomyces rouxii

In an effort to prepare 3,4-methylene-dioxyphenyl-(S)-isopropanol from 3,4-methylene-dioxyphenylacetone, an initial screen of microbes indicated that Candida famata could catalyze this reaction efficiently at low substrate concentration. A dilute, large-scale process was developed to provide experimental material for the chemical synthesis to be explored. However, the productivity number of this process [0.134 g product (g␣wet␣weight cells)⁻¹ day⁻¹ was too low to be practical. C.␣famata was also extremely sensitive to concentrations of both the ketone and the alcohol greater than 2 g/l. A more extensive screen of yeast and fungi revealed that Zygosaccharomyces rouxii was more tolerant to higher substrate concentrations and had a higher productivity number [0.8 g (g wet weight cells)⁻¹ day⁻¹]. These characteristics suggested that Z. rouxii could be used in a large-scale process at high substrate concentrations. Received: 8 July 1996 / Received revision: 9 September 1996 / Accepted: 18 September 1996     

Effect of feed composition and upflow velocity on aggregate characteristics in anaerobic upflow reactors

Two upflow anaerobic hybrid reactors treated lactose and a mixture of ethanol, propionate and butyrate, respectively, at a volumetric loading rate of 3.7 kg chemical oxygen demand (COD) m⁻³day⁻¹, a hydraulic retention time of 5 days and a liquid upflow velocity of 0.01 m/h. Under steady-state conditions, the lactose-fed sludge had much higher (20%–100%) specific methanogenic conversion rates than the volatile-fatty acid␣(VFA)/ethanol-fed sludge for all substrates tested, including VFA. In both reactors, a flocculant sludge developed, although a much higher content of extracellular polysaccharide was measured in the lactose-fed sludge [1900 μg compared to 305 μg uronic acid/g volatile suspended solids (VSS)]. When the liquid upflow velocity of a third, VFA/ethanol-fed reactor was increased to 0.5 m/h, granulation of the sludge occurred, accompanied by a large increase (200%–500%) in the specific methanogenic conversion rates for the syntrophic and methanogenic substrates studied. Granulation reduced the susceptibility of the sludge to flotation. Glucose was degraded at a high rate (100 mg glucose gVSS⁻¹h⁻¹) by the sludge from the third reactor, despite not having been exposed to a sugar-containing influent for 563␣days. Received: 7 June 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996     

Manganese uptake and toxicity in magnesium-supplemented and unsupplemented Saccharomyces cerevisiae

The magnesium content of Saccharomyces cerevisiae was found to vary by up to fivefold at differing␣ stages of batch growth and during growth in the presence of differing magnesium concentrations. Excess Mg was primarily sequestered in vacuoles. Mn²⁺-uptake experiments revealed that Mg-enriched cells had a markedly reduced capacity for Mn²⁺ accumulation. For example, after 6 h incubation in the presence of 50 μM Mn²⁺, Mn levels were approximately twofold higher in cells previously grown in unsupplemented medium than in those from Mg-supplemented medium. These differences were further accentuated at higher Mn²⁺ concentrations and were not attributable to altered cell-surface charge or altered cell-surface Mn²⁺ binding. Cellular Mg status also influenced Mn toxicity towards S. cerevisiae. During exposure to 5 mM Mn²⁺, 50% reductions in the viability of cells with initial Mg contents of approximately 1400 and 2700 nmol (10⁹ cells)⁻¹ occurred after approximately 1.6 h and 3.6 h respectively. In cells containing 3300 nmol Mg (10⁹ cells)⁻¹, more than 75% viability was still maintained after 7 h incubation with 5 mM Mn²⁺. It is concluded that Mn²⁺ uptake and toxicity in S. cerevisiae are strongly influenced by intracellular Mg, possibly through Mg-dependent regulation of divalent-cation transport activity. Received: 15 May 1996 / Received revision: 13 September 1996 / Accepted: 22 September 1996     

The action of antibiotics on the anaerobic digestion process

Antibiotics can disturb the production of biogas during anaerobic digestion. This study shows a systematic approach to understanding how the different bacterial populations involved in the final conversion of organic matter into methane are inhibited by 15 antimicrobial agents with different specificities and modes of action. The results obtained show the following trends: (i) some inhibitors, such as the macrolide erythromycin, lack any inhibitory effect on biogas production; (ii) some antibiotics, with different specificities, have partial inhibitory effects on anaerobic digestion and decrease methane production by interfering with the activity of propionic-acid- and butyric-acid-degrading bacteria,␣(e.g. antibiotics that interfere with cell wall synthesis, RNA polymerase activity and protein synthesis, especially the aminoglycosides); (iii) the protein synthesis inhibitors chlortetracycline (IC₅₀ 40 mg l⁻¹) and chloramphenicol (IC₅₀ 15–20 mg l⁻¹) are very powerful inhibitors of anaerobic digestion. The majority of the antibiotics tested lacked activity against acetoclastic methanogens, being active only on the acetogenic bacteria. However, chloramphenicol and chlortetracycline could cause the complete inhibition of the acetoclastic methanogenic archaea. Received: 6 February 1996 / Received revision: 24 July 1996 / Accepted: 5 August 1996     

Efficient mediator-independent hydrogenation of carbon-carbon double bonds,using the obligate anaerobe,Acetobacterium woodii,with fructose as an electron donor

Fructose and H₂ were compared as electron donors for hydrogenation of carbon-carbon double bonds using Acetobacterium woodii. Caffeate was used as a model substrate. An electron donor was required and both fructose and H₂ were suitable. With fructose as the donor, the K _s for caffeate was 0.5 mM and the V _max was 678 mmol kg_dry weight ⁻¹ h⁻¹.␣Fructose oxidation was coupled very efficiently to caffeate reduction by an alteration in the fructose fermentation so that acetate was no longer produced. Received: 24 June 1996 / Accepted: 1 July 1996     

Purification and properties of a thermoactive and thermostable pullulanase from Thermococcushydrothermalis, a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent

The extremely thermophilic archaeon Thermococcus hydrothermalis, isolated from a deep-sea hydrothermal vent in the East Pacific Rise at 21°N, produced an extracellular pullulanase. This enzyme was purified 97-fold to homogeneity from cell-free culture supernatant. The purified pullulanase was composed of a single polypeptide chain having an estimated molecular mass of 110 kDa (gel filtration) or 128 kDa (sodium dodecyl sulfate/polyacryl amide gel electrophoresis). The enzyme showed optimum activity at pH 5.5 and 95 °C. The thermostability and the thermoactivity were considerably increased in the presence of Ca²⁺. The enzyme was activated by 2-mercaptoethanol and dithiothreitol, whereas N-bromosuccinimide and α-cyclodextrin were inhibitors. This enzyme was able to hydrolyze, in addition to the α-1,6-glucosidic linkages in pullulan, α-1,4-glucosidic linkages in amylose and soluble starch, and can therefore be classified as a type II pullulanase or an amylopullulanase. The purified enzyme displayed Michaelis constant (K _m) values of 0.95 mg/ml for pullulan and 3.55 mg/ml for soluble starch without calcium and, in the presence of Ca²⁺, 0.25 mg/ml for pullulan and 1.45 mg/ml for soluble starch. Received: 19 November 1997 / Received revision: 9 March 1998 / Accepted: 14 March 1998     

Biodehalogenation of low concentrations of 1,3-dichloropropanol by mono- and mixed cultures of bacteria

The degradation of low concentrations of 1,3-dichloro-2-propanol (1,3-DCP) and related halohydrins by whole cells and cell-free extracts of soil bacteria has been investigated. Three bacteria (strains A1, A2, A4), isolated from the same soil sample, were distinguished on the basis of cell morphology, growth kinetics and haloalcohol dehalogenase profiles. Strain A1, probably an Agrobacterium sp., dehalogenated 1,3-DCP with the highest specific activity (0.33 U mg protein⁻¹) and also had the highest affinity for 1,3-DCP (K _m, 0.1 mM). Non-growing cells of this bacterium dehalogenated low concentrations of 1,3-DCP with a first-order rate constant (k ₁) of 1.13 h⁻¹ . The presence of a non-dehalogenating bacterium, strain G1 (tentatively identified as Pseudomonas mesophilius), did not enhance the dehalogenation rate of low 1,3-DCP concentrations. However, the mixed-species consortium of strains A1 and G1 had greater stability than the mono-species culture at DCP concentrations above 1.0 gl⁻¹. Received: 30 April 1996 / Received revision: 30 July 1996 / Accepted: 5 August 1996     

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     

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     

Transformation of indole and quinoline by Desulfobacterium indolicum (DSM 3383)

Degradation of indole and quinoline by Desulfobacterium␣indolicum was studied in batch cultures. The first step in the degradation pathway of indole and quinoline was a hydroxylation at the 2 position to oxindole and 2-hydroxyquinoline respectively. These hydroxylation reactions followed saturation kinetics. The kinetic parameters for indole were an apparent maximum specific transformation rate (V _Amax) of 263 μmol mg total protein⁻¹ day⁻¹ and an apparent half-saturation constant (K _Am) of 139 μM. The V _Amax for quinoline was 170 μmol mg total protein⁻¹ day⁻¹ and K _Am was 92 μM. Oxindole inhibited indole hydroxylation whereas 2-hydroxyquinoline stimulated quinoline hydroxylation. An adaptation period of approximately 20 days was required before transformation of 2-hydroxyquinoline in cultures previously grown on quinoline. Indole and quinoline were hydroxylated with a lag phase shorter than 4 h in a culture adapted to ethanol. Chloramphenicol inhibited the hydroxylation of indole and quinoline in ethanol-adapted cells, indicating an inducible enzyme system. Chloramphenicol had no effect on the hydroxylation of indole in quinoline-adapted cells or on the hydroxylation of quinoline in indole-adapted cells. This indicated that it was the same inducible enzyme system that hydroxylated indole and quinoline. Received: 16 July 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996     

Influence of cell immobilization on the production of benzaldehyde and benzyl alcohol by the white-rot fungi Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens

Three white-rot basidiomycetes, Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens, were cultivated on a liquid medium supplemented with l-phenylalanine, a precursor for benzaldehyde (bitter almond aroma) and benzyl alcohol. Remarkable amounts of benzaldehyde (587 mg l⁻¹) were found in cultures of B. adusta. Immobilization of this fungus on polyurethane foam cubes allowed an 8.3-fold increase of the production of benzaldehyde and a 15-fold increase of the productivity as compared with non-immobilized cells. Aryl-alcohol oxidase activity was only detected in B. adusta. This activity was also significantly enhanced in immobilized cells, suggesting that it plays an important role in benzaldehyde biosynthesis. Conversely, consistent amounts of benzyl alcohol (340 mg l⁻¹ for B. adusta and I. benzoinum and 100 mg l⁻¹ for D. squalens) were produced by the three fungi when immobilized. Laccase activity was found only in the strains I. benzoinum and D. squalens. This activity was markedly enhanced in free cells cultures. Immobilization of the fungi did not promote benzyl alcohol production by comparison with free cell cultures (500 mg l⁻¹). Received: 10 December 1996 / Received revision: 17 February 1997 / Accepted: 22 February 1997     

High rates of microbial sulphate reduction in a mesophilic ethanol-fed expanded-granular-sludge-blanket reactor

In a mesophilic (30–35 °C), sulphidogenic, ethanol-fed expanded-granular-sludge-blanket reactor, sulphate, at loading rates of up to 10.0–12.0 g Sl⁻¹␣day⁻¹, was removed with an average efficiency of more than 80%. The pH was between 7.7 and 8.3 and the maximal total dissolved sulphide concentration was up to 20 mM S (650 mg S/l). The alkaline pH was maintained by either a pH-control unit with sodium hydroxide or by stripping part of the sulphide and CO₂ from the recycle with nitrogen gas. The superficial upstream liquid velocity (v _up) was 3.0–4.5 m/h. The ratio of ethanol to sulphur was near stoichiometry. At alkaline pH, the activity of the acetotrophic sulphate-reducing bacteria, growing on acetate, was strongly enhanced, whereas at pH below 7.7 the acetotrophic sulphate-reducing bacteria were inhibited by aqueous H₂S. With regard to the removal efficiency and operational stability, external stripping with N₂ and pH control were equally successful. Received: 2 December 1996 / Received revision: 13 March 1997 / Accepted: 15 March 1997     

Nitrate and phosphate ion removal from water by Phormidium laminosum immobilized on hollow fibres in a photobioreactor

Removal of nitrate and phosphate ions from water, by using the thermophilic cyanobacterium Phormidium laminosum, immobilized on cellulose hollow fibres in the tubular photobioreactor at 43 °C, was studied by continuously supplying dilute growth medium for 7 days and then secondarily treated sewage (STS) for 12 days. The concentrations of NO⁻ ₃ and PO³⁻ ₄ in the effluent from the dilute growth medium decreased from 5.0 mg N/l to 3.1 mg N/l, and from 0.75 mg P/l to 0.05 mg P/l respectively, after a residence time of 12 h. The concentrations of NO⁻ ₃ and PO³⁻ ₄ in the effluent from STS decreased from 11.7 mg N/l to 2.0 mg N/l, and from 6.62 mg P/l to 0.02 mg P/l respectively, after a residence time of 48 h. The removal rates of nitrogenous␣and phosphate ions from STS were 0.24 and 0.11 mmol day⁻¹ l reactor⁻¹ respectively, under the same conditions. Although, among nitrogenous ions, nitrate and ammonium ions were efficiently removed by P.␣laminosum, the nitrite ion was released into the effluent when STS was used as influent. Treatment of water with thermophilic P. laminosum immobilized on hollow fibres thus appears to be an appropriate means for the removal of inorganic nitrogen and phosphorus from treated wastewater. Received: 15 August 1997 / Received last revision: 18 November 1997 / Accepted: 29 November 1997     

Pentachlorophenol biodegradation kinetics of an oligotrophic fluidized-bed enrichment culture

A fluidized-bed reactor (FBR) was used to enrich an aerobic chlorophenol-degrading microbial culture. Long-term continuous-flow operation with low effluent concentrations selected oligotrophic microorganisms producing good-quality effluent for pentachlorophenol(PCP)-contaminated water. PCP biodegradation kinetics was studied using this FBR enrichment culture. The results from FBR batch experiments were modeled using a modified Haldane equation, which resulted in the following kinetic constants: q _max = 0.41 mg PCP mg protein⁻¹ day⁻¹, K _S = 16 μg l⁻¹, K _i = 5.3 mg l⁻¹, and n = 3.5. These results show that the culture has a high affinity for PCP but is also inhibited by relatively low PCP concentrations (above 1.1 mg PCP l⁻¹). This enrichment culture was maintained over 1 year of continuous-flow operation with PCP as the sole source of carbon and energy. During continuous-flow operation, effluent concentrations below 2 μg l⁻¹ were achieved at 268 min hydraulic retention time (t _HR) and 2.5 mg PCP l⁻¹ feed concentration. An increase in loading rate by decreasing t _HR did not significantly deteriorate the effluent quality until a t _HR decrease from 30 min to 21 min resulted in process failure. Recovery from process failure was slow. Decreasing the feed PCP concentration and increasing t _HR resulted in an improved process recovery. Received: 10 October 1996 / Received revision: 21 January 1997 / Accepted: 24 January 1997     

Cloning and expression of the Bacillus sphaericus 2362 mosquitocidal genes in a non-toxic unicellular cyanobacterium, Synechococcus PCC6301

Genes encoding the mosquitocidal binary toxin of Bacillus sphaericus 2362 were introduced into Synechococcus PCC6301, a cyanobacterium that can tolerate a number of potential variations in the mosquito breeding environment, and can serve as a food source for mosquito larvae. The toxin genes, preceded by a Synechococcus rbcL promoter, were located on a mobilizable Escherichia coli Synechococcus shuttle vector, which was introduced into Synechococcus PCC6301 at frequencies of 10⁻⁵–10⁻⁷ exconjugants/recipient, depending on the selective conditions used. Recombinant Synechococcus exhibited significant toxicity against 2-day-old and 6-day-old Culex quinquefasciatus larvae, the concentration required to kill 50 % of larvae (LC₅₀) being 2.1 × 10⁵ and 1.3 × 10⁵ cells/ml respectively. Mosquitocidal activity decreased tenfold after 20 generations of non-selective growth. Received: 23 July 1996 / Received revision: 11 November 1996 / Accepted: 15 November 1996     

Bioremediation of diesel-oil-contaminated alpine soils at low temperatures

Bioremediation of two diesel-oil-contaminated alpine subsoils, differing in soil type and bedrock, was investigated in laboratory experiments at 10 °C after supplementation with an inorganic fertilizer. Initial diesel oil contamination of 4000 mg kg⁻¹ soil dry matter (dm) was reduced to 380–400 mg kg⁻¹ dm after 155 days of incubation. In both soils, about 30 % of the diesel oil contamination (1200 mg kg⁻¹ dm) was eliminated by abiotic processes. The residual decontamination (60 %–65 %) could be attributed to microbial degradation activities. In both soils, the addition of a cold-adapted diesel-oil-degrading inoculum enhanced biodegradation rates only slightly and temporarily. From C/N and N/P ratios (determined by measuring the contents of total hydrocarbons, NH₄ ⁺ N, NO₃ ⁻ N and PO₄ ³⁻ P) of soils␣it could be deduced that there was no nutrient deficiency during the whole incubation period. Soil biological activities (basal respiration and dehydrogenase activity) corresponded to the course of biodegradation activities in the soils. Received: 9 September 1996 / Accepted: 7 December 1996     

Insulin and glucagon immunoreactivity during high-intensity exercise under opiate blockade

Eight fit men [maximum oxygen consumption (O_2max) 64.6 (1.9) ml · kg⁻¹ · min⁻¹, aged 28.3 (1.7) years (SE in parentheses) were studied during two treadmill exercise trials to determine the effect of endogenous opioids on insulin and glucagon immunoreactivity during intense exercise (80% O_2max). A double-blind experimental design was used with subjects undertaking the two exercise trials in counterbalanced order. Exercise trials were 20 min in duration and were conducted 7 days apart. One exercise trial was undertaken following administration of naloxone (N; 1.2 mg; 3 ml) and the other after receiving a placebo (P; 0.9% NaCl saline; 3 ml). Prior to each experimental trial a flexible catheter was placed into an antecubital vein and baseline blood samples were collected. Immediately after, each subject received either a N or P bolus injection. Blood samples were also collected after 20 min of continuous exercise (running). Glucagon was higher (P < 0.05), while insulin was lower (P < 0.05), during exercise compared with pre-exercise values in both trials. However, glucagon was higher (P < 0.05) in the P than in the N exercise trial [141.4 (8.3) ng · l⁻¹ vs 127.2 (7.6) ng · l⁻¹]. There were no differences in insulin during exercise between the P and N trials [50.2 (4.3) pmol · l⁻¹ vs 43.8 (5) pmol · l⁻¹]. These data suggest that endogenous opioids may augment the glucagon response during intense exercise. Accepted: 15 June 1996     

Extracellular pH affects regulation of the pcbAB gene in Penicillium chrysogenum

The effect of extracellular pH and dissolved oxygen on regulation of the pcbAB gene in P.␣chrysogenum was examined, using Northern analysis and a reporter gene fusion. It was found that ambient pH markedly affected levels of pcbAB mRNA whereas maintenance of dissolved oxygen concentration above 10 % had no detectable effect. The presence of a DNA-binding protein, which binds upstream of the pcbAB translational start codon, was also related to ambient pH. In all fermentations, pcbAB mRNA was most abundant at around the late exponential/early stationary phase of a culture. Received: 10 May 1996 / Received revision: 14 October 1996 / Accepted: 25 October 1996     

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