Detoxification of cassava by Aspergillus niger B-1

 The effects of fermentation of cassava by Aspergillus niger B-1 β-glucosidase on its cyanide and protein content, and the optimal conditions for this enzyme’s activity, were examined. This fermentation process reduced the cyanide content of cassava by 95% to 2 mg/kg, and increased its total protein content by 50%, thereby improving its nutritional value. A significant decrease in cyanogenic glycosides was detected after 3 days of fermentation. The optimal pH for A. nigerβ-glucosidase activity on the cyanogenic glycoside linamarin was determined to be 3, the optimal temperature 55 °C, and its K _m 0.3 mM. The findings presented here will facilitate the development of an improved method for detoxification of cassava and for enhancement of its nutritional value. Received: 17 August 1995/Received revision: 27 October 1995/Accepted: 30 October 1995     

Role of Thiobacillus ferrooxidans and sulphur (sulphide)-dependent ferric-ion-reducing activity in the oxidation of sulphide minerals

 Thiobacillus ferrooxidans oxidized the sulphide minerals e.g., pyrite, pyrrhotite and copper concentrate under anaerobic conditions in the presence of ferric ion as sole electron acceptor. Copper and iron were solubilized from sulphide ores by the sulphur (sulphide)-dependent ferric-ion oxidoreductase activity. Treatment of resting cells of T. ferrooxidans with 0.5% phenol for 30 min completely destroyed the iron- and copper-solubilizing activity. The above treatment destroyed the sulphur(sulphide)-dependent ferric-ion-reducing activity completely but did not affect the iron-oxidizing activity. The results suggest that sulphur(sulphide)-dependent ferric-ion-reducing activity actively participates in the oxidation of sulphide minerals under anaerobic conditions. The activity of sulphur(sulphide)-dependent ferric ion reduction in the solubilization of iron and copper from the sulphide ores were also observed under aerobic conditions in presence of sodium azide (0.1 μmol), which completely inhibits the iron-oxidizing activity. Received: 23 May 1995/Received revision: 10 October 1995/Accepted: 16 October 1995     

Construction of an Aspergillus nidulans multicopy transformant for the xlnB gene and its use in purifying the minor X24 xylanase

 Using recombinant DNA techniques, an Aspergillus nidulans multicopy transformant for the gene xlnB coding for the minor X₂₄ xylanase has been constructed. When grown on glucose as sole carbon source this transformant secretes 114 U of xylanase (mg protein)^-1. In this culture condition, X₂₄ is the only xylanase secreted and the predominant protein in the culture filtrate. This strategy has been used to purify the X₂₄ enzyme to homogeneity. The purified xylanase showed a single band on sodium dodecyl sulphate/ polyacrylamide gel electrophoresis with a molecular mass of 24 kDa and had an isoelectric point of approximately 3.5. The enzyme was a non-debranching endo-1,4-β-xylan xylanohydrolase highly specific for xylans and showed optimal activity at pH 5.5 and 52°C. The X₂₄ xylanase had a Michaelis constant, K _m, of 12.43 mg oat spelt xylan ml^-1 and a V _max of 1639 μmol min^-1 (mg protein)^-1. Received: 17 May 1995/Received last revision: 25 September 1995/Accepted: 29 September 1995     

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     

Continuous production of ethanol using yeast cells immobilized in preformed cellulose beads

 Saccharomyces cerevisiae cells were immobilized on preformed cellulose beads by adsorption. The fermentation capacity of the immobilized yeast cells was found to be practically independent of the hydrogen ion concentration between pH 3.1 and 6.25. The fermentation capacity was maximal at 30 °C. The immobilized yeast cells were used for continuous production of ethanol in a fluidized-bead reactor. The average values characteristic for the process were an ethanol concentration of 41.9±0.1 g l^-1, a fermentation efficiency of 82.9±2.1% and a volumetric productivity of 3.94±0.52 g l^-1 h^-1. Received: 9 October 1995/Accepted: 22 April 1996     

Production of a novel copolyester of 3-hydroxybutyric acid and medium-chain-length 3-hydroxyalkanoic acids by Pseudomonas sp. 61-3 from sugars

 Pseudomonas sp. 61-3 (isolated from soil) produced a polyester consisting of 3-hydroxybutyric acid (3HB) and of medium-chain-length 3-hydroxyalkanoic acids (3HA) of C₆, C₈, C₁₀ and C₁₂, when sugars of glucose, fructose and mannose were fed as the sole carbon source. The polyester produced was a blend of homopolymer and copolymer, which could be fractionated with boiling acetone. The acetone-insoluble fraction of the polyester was a homopolymer of 3-hydroxybutyrate units [poly (3HB)], while the acetone-soluble fraction was a copolymer [poly(3HB-co-3HA)] containing both short- and medium-chain-length 3-hydroxyalkanoate units ranging from C₄ to C₁₂:44 mol% 3-hydroxybutyrate, 5 mol% 3-hydroxyhexanoate, 21 mol% 3-hydroxyoctanoate, 25 mol% 3-hydroxydecanoate, 2 mol% 3-hydroxydodecanoate and 3 mol% 3-hydroxy-5-cis-dodecenoate. The copolyester was shown to be a random copolymer of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoate units by analysis of the ¹³C-NMR spectrum. The poly(3HB) homopolymer and poly (3HB-co-3HA) copolymer were produced simultaneously within cells from glucose in the absence of any nitrogen source, which suggests that Pseudomonas sp. 61-3 has two types of polyhydroxy-alkanoate syntheses with different substrate specificities. Received: 9 June 1995/Received last revision: 30 October 1995/Accepted: 6 November 1995     

Use of an industrial effluent as a carbon source for denitrification of a high-strength wastewater

Denitrification of a high-strength synthetic wastewater (150 g NO^- ₃ l^-1) was carried out using a wine distillery effluent as an example of an industrial carbon source (22.7 g chemical oxygen demand l^-1). Two configurations were tested: one consisted of an acidogenesis reactor followed by a denitrifying reactor and the other was a single reactor directly fed with the raw effluents. In both cases, denitrification was achieved at a nitrate load of 9.54 g NO^- ₃ l^-1 day^-1 (2.19 g N as NO^- ₃ l^-1 day^-1) with good specific reduction rates: 32.6 mg and 35.2 mg N as NO _x  g volatile suspended solids h^-1, calculated on a single day, for the two-step and the one-step process respectively. Dissimilatory nitrate reduction to ammonium did not occur, even in the one-step process. Received: 26 October 1995/Received revision: 15 February 1996/Accepted: 20 February 1996     

Cloning,sequencing and overexpression in Escherichia coli of a xylanase gene,xynA from the thermophilic bacterium Rt8B.4 genus Caldicellulosiruptor

 A genomic library of the extremely thermophilic eubacterial strain Rt8B.4 was constructed in λZapII and screened for the expression of xylanase activity. One recombinant bacteriophage showed xylanase, xylosidase and arabinosidase activity. Sequence analysis and homology comparisons showed that this plasmid derivative, pNZ2011, was composed of 6.7 kb thermophilic DNA and contained what appeared to be an operon-like structure involving genes associated with xylose metabolism. The xylanase gene, xynA was shown to code for a multi-domain protein. Xylanase activity was shown to be associated with the carboxy-terminal domain (domain 2) by deletion analysis and also by selective polymerase chain reaction (PCR) amplification and expression of the individual domains. Denaturing polyacrylamide gel analysis of the protein encoded by the PCR product showed three main overexpressed proteins to be present in cell extracts, presumably caused by proteolytic degradation in the Escherichia coli host. The xylanase activity from domain 2 is associated with a 36-kDa protein, which is stable at 70^°C for at least 12 h at pH 7. The small size of this active enzymatic domain and its temperature stability suggest that it may be of value in the enzyme-enhanced bleaching of kraft pulp. Received: 18 April 1995/Received revision: 4 August 1995/Accepted: 22 August 1995     

A comparative study on the transformation of Aspergillus nidulans by microprojectile bombardment of conidia and a more conventional procedure using protoplasts treated with polyethyleneglycol

 An Aspergillus nidulans strain, auxotrophic for pyrimidine, was transformed to prototrophy by means of microprojectile bombardment. The transformation frequency was somewhat lower than conventional polyethyleneglycol-mediated transformation of protoplasts. However, the percentage of stable transformants was considerably higher with the biolistic approach. Typically, integrations of several copies of the plasmid introduced into chromosomal DNA were observed. The effect of several parameters, like the concentration of conidia, chamber pressure during bombardment and size of microprojectiles, on transformation frequencies were investigated and compared to previously published data on microprojectile bombardment of fungal conidia. Optimum results (6 transformants/μg plasmid DNA) were obtained when 10⁸ conidia were bombarded with a helium pressure of 5.5–8.3 MPa (800–1200 lb/in²). M5, M10 and M17 tungsten particles were equally efficient. Received: 9 August 1995/Received revision: 27 September 1995/Accepted: 4 October 1995     

Heat Shock Proteins of Cyanobacterium Anacystis nidulans

Cells of Anacystis nidulans grown at 30°C were incubatedwith ¹⁴C-Chlorella protein hydrolysate at the elevated temperatures(30–55°C) and the effect of heat shock treatment onprotein synthesis was studied. Incubation temperatures higherthan 45°C caused a significant decrease in the incorporationof amino acids into proteins. Further, the heat shock treatmentinduced significant changes in the fluorographic profile ofthe newly synthesized proteins. (Received October 25, 1985; Accepted December 4, 1985)     

Production of ethanol at 45°C on starch-containing media by mixed cultures of the thermotolerant,ethanol-producing yeast Kluyveromyces marxianus IMB3 and the thermophilic filamentous fungus Talaromyces emersonii CBS 814.70

 The thermotolerant, ethanol-producing yeast strain, Kluyveromyces marxianus IMB3, was shown to produce ethanol at 45°C on starch-containing media supplemented with a crude amylase preparation derived from the thermophilic, filamentous fungus Talaromyces emersonii CBS 813.70. Ethanol production on media containing 4% (w/v) starch increased to a maximum of 15 g/l with 40 h, and this represented 74% of the maximum theoretical yield. Subsequent experimentation involving growth of both organisms in fermentations on starch-containing media (4% w/v) demonstrated that the mixed-culture system was capable of ethanol production at 45°C with maximum yields at 12 g/l obtained with 65 h. The advantages associated with ethanol production by this system are discussed. Received: 16 May 1994/Accepted: 22 October 1994     

Cloning and analysis of a gene from Streptomyces lividans 66 encoding a novel secreted protease exhibiting homology to subtilisin BPN′

 Amino-terminal degradation has been observed for many of the secreted heterologous proteins produced by S. lividans 66. We, therefore, set out to characterize the relevant proteinases and their genes. A tripeptide chromogenic substrate was used to identify a gene that was shown to encode a secreted protein which removed tripeptides from the amino terminus of extracellular proteins (tripeptidyl aminopeptidase, Tap; Butler et al. 1995). This activity was removed by a homologous gene deletion replacement and the ability of the S. lividans strain to remove N-terminal tripeptides was greatly reduced, but still significant. When the tap-deleted strain was used as a host for the rescreening of a S. lividans 66 genomic DNA library, a number of other genes encoding proteases with aminopeptidase activities were discovered. One clone (P5-4) produced a 45-kDa secreted protein (Ssp), which showed activity against Ala-Pro-Ala-β-naphthylamide (APA-βNH-Nap) substrate. Further analysis of the cloned DNA showed an open-reading frame encoding a protein larger than 45 kDa. Direct Edman degradation of the secreted protein confirmed that it was encoded within the cloned DNA and probably processed from a larger precursor. Protein sequence analysis revealed a striking homology to subtilisin BPN′ in three regions around the active-site residues suggesting that the protein is a serine protease. As expected, the protease activity was inhibited by phenylmethylsulphonyl fluoride. Mutant strains with most of the ssp gene deleted exhibited reduced activity against APA-βNH-Nap substrate compared to their non-deleted parental strains. Received: 15 May 1995/Received last revision: 2 October 1995/Accepted: 4 October 1995     

Kinetic coefficients for simultaneous reduction of sulfate and uranium by Desulfovibrio desulfuricans

 Previously it was demonstrated that bacteria are capable of transforming soluble uranyl ion, U(VI), to insoluble uraninite, U(IV); however, the rate for this transformation has not been determined. We report the kinetic coefficients for Desulfovibrio desulfuricans DSM 1924 grown in a continuous-flow chemostat where pyruvate was the electron donor and sulfate was the electron acceptor. The medium was supplemented with 1 mM uranyl nitrate, and the chemostat flow rate ranged from 1.12 ml/h to 4.75 ml/h with incubation at 28°C. The maximum rate of pyruvate utilization (k) was determined to be 4.7 days^-1, while the half-velocity constant (K _s) was 127 mg/l. The yield coefficient (Y) of cells per mole of pyruvate oxidized was calculated to be 0.021 g, while the endogenous decay coefficient (k _d) was determined to be 0.072 days^-1. More than 90% of U(VI) was transformed to U(VI) in the chemostat under the conditions employed. Received: 7 September 1995/Received last revision: 10 January 1996/Accepted: 5 February 1996     

A resting-cell assay for cholesterol reductase activity inEubacterium coprostanoligenes ATCC 51222

 A resting-cell assay was established to evaluate the cholesterol reductase activity of Eubacterium coprostanoligenes ATCC 51222. Cell suspensions from cholesterol-free media rapidly reduced cholesterol to coprostanol. Optimal assay conditions in a 1-ml reaction mixture were determined to be up to 1 h of incubation and up to 0.25 mg bacterial protein/assay with at least 1 mM cholesterol as substrate. The cholesterol reductase activity in cells decreased as a function of storage time at 22°C, 4°C and −20°C. Filling the headspace of the reaction mixture with H₂ increased the activity about 20%. Optimal cholesterol reductase activity occurred at pH 7.5 in sodium phosphate buffer. Pyruvate and reducing agents in the buffer increased the activity. This study has validated assay conditions for determination of cholesterol reductase activity in resting cells of E. coprostanoligenes. Received: 2 August 1994/Received revision: 15 November 1994/Accepted: 8 December 1994     

Kelp degradation by Paractora trichosterna (Thomson) (Diptera: Helcomyzidae) at sub-Antarctic South Georgia

 The Diptera are one of the dominant insect consumer groups on sub-Antarctic islands and are thought to contribute significantly to terrestrial ecological processes at many of these islands. The life-cycle of Paractora trichosterna and its contribution to kelp degradation at Husvik Harbour, South Georgia were therefore investigated in the laboratory and in two artificial wrack beds in the field. Duration of the larval stage was approximately 2 months at 10°C, during which time larvae attained a maximum individual mass of ca. 90 mg. Larvae had a relative consumption rate of 0.734 mg dry mass kelp mg dry mass larva^-1 day^-1. Based on this rate and information on larval densities of P. trichosterna, and a smaller species, Antrops truncipennis, kelp consumption was estimated to be 714–870 g dry mass kelp m^-2 over the 7-week study period. During this time, kelp dry biomass declined to 30% of its original value, both in a bed protected from trampling by vertebrates and in an exposed one. P. trichosterna was directly responsible for 12% of this loss in the protected bed and 20% in the exposed one. A. truncipennis was responsible for an additional 3% loss in the exposed bed and 8% in the protected one. These fly species therefore contributed significantly to kelp degradation. Differences in biomass of the larvae and adults of the two species between the beds suggested that P. trichosterna prefers more exposed wrack than does A. truncipennis. Received: 27 March 1995/Accepted: 4 July 1995     

Seasonal photosynthetic performance of the endemic antarctic red algaPalmaria decipiens (Reinsch) Ricker

 A male gametophyte of the endemic Antarctic red macroalga Palmaria decipiens (Reinsch) Ricker was cultivated under fluctuating daylengths, simulating the seasonal changes at the site of collection (King George Island, Antarctica). The plant was maintained at 0±1°C, an irradiance of 25 μmol m^-2 s^-1 and under growth-saturating nutrient conditions. Samples were taken at intervals of 3–6 weeks to measure growth, photosynthesis, dark respiration and pigment content. The growth optimum in spring coincided with a higher photosynthetic activity. Whereas dark respiration was constantly low over the year, there was a rapid increase in maximum photosynthetic rate (P_max) in conditions corresponding to September and October. This was correlated with a change in the initial slope (α) of the photosynthesis versus irradiance (P vs I) curve. Higher activity in photosynthesis mainly resulted from higher Chl a and phycobilin concentrations during Antarctic spring, an indication of an increase in absorption cross-section areas of photosynthetic reaction centres. These changes in physiology are discussed in relation to the seasonal growth “strategy” of the species, which is controlled by seasonal variation in daylength. Received: 27 February 1995/Accepted 3 October 1995     

Degradation of pyrene by Mycobacterium flavescens

 A strain of Mycobacterium flavescens was isolated from polluted sediments. It was capable of utilizing pyrene as a sole source of carbon and energy. When pyrene was supplied as a suspension at 50 μg/ml, the generation time was 9.6 h and the rate of pyrene utilization was 0.56 μg ml^-1 day^-1. In addition to pyrene, the strain could mineralize phenanthrene (17.7%) and fluoranthene (17.9%), but failed to mineralize naphthalene, chrysene, anthracene, fluorene, acenaphthene and benzo[a]pyrene, as determined by recovery of radiolabeled CO₂ in incubations conducted for 2 weeks under growth conditions. Metabolites produced during growth on pyrene were detected and characterized by HPLC and GC-MS. The product of initial ring oxidation, 4,5-dihydroxy-4,5-dihydropyrene was identified, as well as ring-fission products including 4-phenanthroic acid, phthalic acid, and 4,5-phenanthrenedioic acid. Received: 3 October 1995/Received last revision: 1 April 1996/Accepted: 15 April 1996     

1,2,3-Trichlorobenzene transformation by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase

 Transformation of 1,2,3- and 1,2,4-trichlorobenzene in the presence of 20 mM sodium formate, by the methanotrophic bacterium Methylosinus trichosporium OB3b, was studied using cells grown in batch and continuous culture. Only 1,2,3-trichlorobenzene was transformed and transformation was strictly co-metabolic, only catalysed in the presence of the soluble form of methane monooxygenase. The kinetics of transformation could be described by simple first-order kinetics (0.00193 l min^-1 g^-1). Also the kinetics of transformation were found to be linearly proportional to cell density. No chloride ion release was observed during the reaction and the products of transformation (2,3,4- and 3,4,5-trichlorophenol) were identified by gas chromatography/mass spectroscopy and ¹H-NMR and a 1.84:1 ratio of products in favour of para hydroxylation was observed. It was also observed that the relationship between mass of substrate transformed and cell density was linear giving a transformation capacity of 88.8±11.8 μmol g^-1, after which the transformation of 1,2,3-trichlorobenzene was inhibited. This inhibition was not due to O₂ limitation, co-substrate (CHOONa) limitation or product inhibition. Recovery and washing of the cells did not reverse this inhibition, indicating that inhibition was irreversible. During transformation a substantial decrease in the endogenous and formate-dependent O₂ consumption rates was observed, although the methanol-dependent O₂ consumption rate varied little between fresh cell samples and samples that had been used to transform 1,2,3-trichlorobenzene. Received: 22 June 1995 / Received last revision: 26 October 1995 / Accepted: 30 October 1995     

Thermophilic microbial leaching of heavy metals from municipal sludge using indigenous sulphur-oxidizing microbiota

 It was demonstrated in shake-flask experiments that the sulphur-oxidizing microbiota of municipal sludges can be used at 53°C for heavy-metal leaching. Five sludges were tested and the average final pH, oxidation/reduction potential and SO^2- ₄ concentration after 30 days were 2.8, 237 mV and 5668 mg/l respectively. Ferric chloride was added to enhance the redox potential and to lower pH, which resulted in average values of 409 mV and 1.86 respectively. The average solubilisation of Cd, Cu, Cr, Mn, Ni, Pb, Zn, K and P after ferric chloride addition was 35.6±20.6%, 65.9±15.1%, 28.5±13.5%, 74.0±10.0%, 60.3± 13.1%, 33.7±27.6%, 83.9±6.2%, 39.0±16.6 and 18.2±15.8% respectively. The present process enhanced the sludge dewaterability compared to the conventional thermophilic digestion. During the leaching batch process, the volatile and volatile suspended solids were degraded to the same level as observed when the conventional thermophilic digestion was used as control. Received: 7 June 1995/Received revision: 8 September 1995/Accepted: 29 September 1995     

Shake-flask culture of Laccaria laccata,an ectomycorrhizal basidiomycete

 Large-scale exploitation of the potential benefits of ectomycorrhizal fungi in improving plantation yields means that fermentation techniques for these fungi will be required. Starting with a base performance on a rich, complex medium, the effect of variations in some physicochemical culture parameters on biomass yield was studied. It was possible to reduce the amount of phosphate salts (to 1/9th) and other ingredients (to 1/3rd) in the medium. A shaking speed of either 100 rpm or 200 rpm in an orbital incubator was satisfactory and biomass yield responded to an increase in carbon substrate (glucose, from 10 g l^-1 and 20 g l^-1) though Y _x/s declined. An increase in inoculum size shortened culture time but decreased biomass yield. The upper limit of the incubation temperature was between 25°C and 30°C. Biomass yields were about 12 g l^-1 dry weight (Y _x/s=0.63) when 20 g l^-1 glucose was supplied, and about 7 g l^-1 (Y _x/s=0.74) when 10 g l^-1 glucose was supplied. Received: 9 October 1995/Accepted: 4 December 1995