A polymerase chain reaction-based test for the identification of Trichoderma harzianum biotypes 2 and 4, responsible for the worldwide green mold epidemic in cultivated Agaricus bisporus

We describe a polymerase chain reaction (PCR)-based test that is specific for the pathogenic European biotype 2 (Th2) and North American biotype 4 (Th4) of Trichoderma harzianum, responsible for the green mold epidemic in the cultivated mushroom, Agaricus bisporus. A PCR primer pair was designed that targets a 444-bp arbitrary sequence in the genome of Th4. The primers also amplified the same product with Th2, but showed no reactivity with other biotypes of T. harzianum, several biocontrol Trichoderma, or with 31 other genera and species of fungi. The PCR-based test should have application in disease management programs, and in the evaluation of biocontrol Trichoderma for potential pathogenicity on mushrooms. Received: 23 November 1998 / Received revision: 19 February 1999 / Accepted: 5 March 1999     

Immobilisation of Thiobacillus ferrooxidans cells on nickel alloy fibre for ferrous sulfate oxidation

The immobilisation of the iron-oxidising bacteria Thiobacillus ferrooxidans on nickel alloy fibre as support is described. This matrix showed promise for application in iron oxidation under strongly acidic conditions. The influence on the colonisation process of T. ferrooxidans exerted by the initial pH of the medium and by temperature has also been studied. Results showed that immobilisation of T. ferrooxidans cells was affected by changes of temperature between 30 °C and 40 °C and in pH from 1.4 to 2.0. Received: 25 January 2000 / Received version: 20 April 2000 / Accepted: 1 May 2000     

Evaluation of cell recycling in continuous fermentation of enzymatic hydrolysates of spruce with Saccharomyces cerevisiae and on-line monitoring of glucose and ethanol

The maximum growth rate of Saccharomyces cerevisiae ATCC 96581, adapted to fermentation of spent sulphite liquor (SSL), was 7 times higher in SSL of hardwood than the maximum growth rate of bakers' yeast. ATCC 96581 was studied in the continuous fermentation of spruce hydrolysate without and with cell recycling. Ethanol productivity by ATCC 96581 in continuous fermentation of an enzymatic hydrolysate of spruce was increased 4.6 times by employing cell recycling. On-line analysis of CO₂, glucose and ethanol (using a microdialysis probe) was used to investigate the effect of fermentation pH on cell growth and ethanol production, and to set the dilution rate. Cell growth in the spruce hydrolysates was strongly influenced by fermentation pH. The fermentation was operated in continuous mode for 210 h and a theoretical ethanol yield on fermentable sugars was obtained. Received: 25 May 1998 / Received revision: 11 August 1998 / Accepted: 12 August 1998     

Production of β-Xylosidase Activity by Trichodermaharzianum Strains

Nine Trichoderma harzianum strains were screened for β-xylosidase activity when grown in solid-state cultures on media containing wheat bran as the carbon source. All strains produced β-xylosidase activity, the most active being in extracts of cultures of T. harzianum strain 4. A β-xylosidase was purified by ammonium sulfate precipitation, ultrafiltration, gel filtration, and ion exchange chromatography from solid-state cultures of T. harzianum strain C. Enzyme preparations yielded a single band when stained for protein following eletrophoresis. The molecular weight value, calculated following SDS-PAGE, was determined to be 60 kDa. β-Xylosidase was most active at pH 4.0–4.5 and 70°C. This enzyme had a K _m value of 0.053 mM. The phenol-sulfuric acid method detected the presence of a small amount of carbohydrate in the purified enzyme preparation. β-Xylosidase was active against some p-nitrophenylglycosides. The enzyme was inactive against xylan and PNPG. β-xylosidase activity was inhibited by xylose and SDS. Iodoacetamide, dithiothreitol, gluconolactone, glucose, and mercuric chloride failed to inactivate this enzyme's activity. A synergistic effect was observed when β-xylosidase from T. harzianum strain C and β-xylanase from Aspergillus fumigatus were incubated with pretreated arabinoxylan. Received: 1 December 1995 / Accepted: 11 December 1995     

A third xylanase from Trichoderma reesei PC-3-7

A third xylanase (Xyn III) from Trichoderma reesei PC-3–7 was purified to electrophoretic homogeneity by gel filtration and ion-exchange chromatographies. The enzyme had a molecular mass of 32 kDa, and its isoelectric point was 9.1. The pH optimum of Xyn III was 6.0, similar to that of Xyn II, another basic xylanase of  T. reesei. The purified Xyn III showed high activity with birchwood xylan but no activity with cellulose and aryl glycoside. The hydrolysis of birchwood xylan by Xyn III produced mainly xylobiose, xylotriose and other xylooligosaccharides. The amino acid sequences of the N-terminus and internal peptides of Xyn III exhibited high homology with the family F xylanases, showing that they were distinct from those of Xyn I and Xyn II of  T. reesei, which belong to family G. These results reveal that Xyn III is a new specific endoxylanase, differing from Xyn I and Xyn II in  T. reesei. It is noteworthy that this novel xylanase was induced only by cellulosic substrates and l-sorbose but not by xylan and its derivarives. Furthermore,  T. reesei PC-3-7 produced Xyn III in quantity when grown on Avicel or lactose as a carbon source, while  T. reesei QM9414 produced little or no Xyn III. Received: 7 November 1997 / Received last revision: 2 February 1988 / Accepted: 23 February 1998     

pH-mediated release of betalains from transformed root cultures of Beta vulgaris L.

Brief exposure of Beta vulgaris root cultures to acidic medium resulted in release of betalain pigments while the capability for regrowth and continued pigment accumulation was retained. A 10-min exposure to pH 2 followed by return to standard growth medium (pH 5.5, 1.1 mM PO₄) resulted in release of 0.59 mg pigment/g dry weight over the subsequent 24-h period. The released pigment corresponds to 36.8% of the total pigments. Further improvement in culture productivity was achieved through phosphate limitation. Specific pigment productivity increased fivefold for cultures grown in phosphate-free medium as compared to cultures grown in control medium (1.1 mM PO₄). A maximum total pigment production of 25.2 mg/l was observed at an initial medium phosphate level 0.3 mM. When combined with phosphate limitation, low pH facilitated the release of 3.03 mg pigment/g dry weight, which corresponds to 50% of the total pigment. The permeabilized roots were capable of regrowth and continued pigment accumulation. A cytochemical assay for respiratory activity revealed that the basis of regrowth was lateral root initials that were unaffected during the acidic pH treatment. Received: 16 December 1997 / Received revision: 7 May 1998 / Accepted: 16 May 1998     

Optimization of agitation and aeration conditions for maximum virginiamycin production

To maximize the productivity of virginiamycin, which is a commercially important antibiotic as an animal feed additive, an empirical approach was employed in the batch culture of Streptomyces virginiae. Here, the effects of dissolved oxygen (DO) concentration and agitation speed on the maximum cell concentration at the production phase, as well as on the productivity of virginiamycin, were investigated. To maintain the DO concentration in the fermentor at a certain level, either the agitation speed or the inlet oxygen concentration of the supply gas was manipulated. It was found that increasing the agitation speed had a positive effect on the antibiotic productivity independent of the DO concentration. The optimum DO concentration, agitation speed and addition of an autoregulator, virginiae butanolide C (VB-C), were determined to maximize virginiamycin productivity. The optimal strategy was to start the cultivation at 450 rpm and to continue until the DO concentration reached 80%. After reaching 80%, the DO concentration was maintained at this level by changing the agitation speed, up to a maximum of 800 rpm. The addition of an optimal amount of the autoregulator VB-C in an experiment resulted in the maximal production of virginiamycin M (399 mg/l), which was about 1.8-fold those obtained previously. Received: 13 July 1998 / Received revision: 19 August 1998 / Accepted: 13 September 1998     

Production of (R )-3-pentyn-2-ol through stereospecific hydrolysis of racemic 3-pentyn-2-ol esters with microbial enzymes

Microorganisms and commercial enzymes were screened for their ability to produce (R)-3-pentyn-2-ol from racemic 3-pentyn-2-ol esters through stereospecific hydrolysis. Among the esters formed with acetic acid, propionic acid, hexanoic acid and benzoic acid, the acetate was most effectively hydrolyzed by microbial cells and commercial lipases with high stereospecificity. Rhodococcus rubropertinctus AKU NOC082 was a good catalyst for (R)-3-pentyn-2-ol production through the hydrolytic resolution of racemic 3-pentyn-2-yl acetate. With 15%, 25% and 50% (v/v) racemic 3-pentyn-2-yl acetate as the substrate, 42.6%, 40.8% and 40.0% was hydrolyzed in 5 h, 10 h and 98 h respectively, under the optimized conditions (pH 7.0, 30 °C, 7.5% wet cell concentration), the (R) enantiomer of 3-pentyn-2-ol being formed with an optical purity of 97.8%, 98.0% and 94.2% respectively. Received: 2 June 1998 / Received revision: 3 August 1998 / Accepted: 3 September 1998     

Influence of soil impoverishment on the interaction between Glomus mosseae and saprobe fungi

 The effect of the saprobe fungi Wardomyces inflatus (Marchal) Hennebert, Paecilomyces farinosus (Holm & Gray) A. H. S. Brown & G. Sm., Gliocladium roseum Bain., Trichoderma pseudokoningii Rifai and T. harzianum Rifai, isolated from sporocarps of Glomus mosseae, on arbuscular mycorrhizal (AM) colonisation and plant dry matter of soybean was studied in 2/3 and 1/5 diluted soils in a greenhouse trial. Soil dilution to 1/5 had no effect on shoot dry matter of soybean but decreased AM colonisation and root dry weight of plants. CFU of saprobe fungi, except T. harzianum, were higher in 1/5 than in 2/3 diluted soils. W. inflatus and Gliocladium roseum decreased the shoot dry weight of soybean plant when inoculated together with Glomus mosseae. The saprobe fungi P. farinosus and T. pseudokoningii increased the shoot dry weights of plants grown in 1/5 diluted soil. The shoot dry weight and AM colonisation in 1/5 diluted soil were also increased when T. harzianum was inoculated together with Glomus mosseae. Thus, saprobe fungi increased AM colonisation of soybean plants by indigenous endophytes. The AM colonisation of plants at both soil dilutions was increased by Glomus mosseae. The highest level of AM colonisation was observed when P. farinosus and T. pseudokoningii were inoculated together Glomus mosseae. The dilution of soils influenced the interaction between inoculated microorganisms and their effect on plant growth. Accepted: 7 June 1999     

Intermediary sulfur compounds in pyrite oxidation: implications for bioleaching and biodepyritization of coal

Accumulation of elemental sulfur during pyrite oxidation lowers the efficiency of coal desulfurization and bioleaching. In the case of pyrite bioleaching by Leptospirillum ferrooxidans, an iron(II)-ion-oxidizing organism without sulfur-oxidizing capacity, from the pyritic sulfur moiety about 10% elemental sulfur, 2% pentathionate, and 1% tetrathionate accumulated by a recently described cyclic pyrite oxidation mechanism. In the case of pure cultures of Thiobacillus ferrooxidans and mixed cultures of L. ferrooxidans and T. thiooxidans, pyrite was nearly completely oxidized to sulfate because of the capacity of these cultures to oxidize both iron(II) ions and sulfur compounds. Pyrite oxidation in acidic solutions, mediated chemically by iron(III) ion, resulted in an accumulation of similar amounts of sulfur compounds as obtained with L. ferrooxidans. Changes of pH to values below 2 or in the iron ion concentration are not decisive for diverting the flux of sulfur compounds. The literature on pyrite bioleaching is in agreement with the findings indicating that the chemistry of direct and indirect pyrite leaching is identical. Received: 20 April 1998 / Received revision: 27 August 1998 / Accepted: 3 September 1998     

Effects of oxygen on invertase expression in continuous culture of recombinant Saccharomyces cerevisiae containing the SUC2 gene

The yeast SUC2 gene, cloned on a multicopy plasmid pRB58, was used to study the effect of oxygen on the invertase expression of the recombinant Saccharomyces cerevisiae. Glucose repression was not the only factor affecting the invertase expression. The results obtained from the single-stage continuous cultures under microaerobic conditions showed that invertase expression was also strongly dependent on oxygen availability, and moving from anaerobic to aerobic conditions led to a five-fold increase in specific invertase activity. However, the cell yields under anaerobic conditions were quite low compared to those under aerobic conditions. These opposite effects of oxygen on cell growth and gene expression offer a strategy for maximizing invertase productivity by a two-stage continuous culture. The first stage was operated at a low level of glucose, around 100 mg/l, under aerobic conditions in order to obtain a high yield of yeast biomass, and the second stage maintained anaerobic conditions with residual glucose levels of 50 mg/l to derepress and fully induce invertase expression. The two-stage continuous culture resulted in a 2.5-fold increase in invertase productivity over that of a single-stage continuous culture. Received: 28 July 1998 / Received revision: 22 September 1998 / Accepted: 7 November 1998     

Physiological aspects of continuous lactic acid fermentations at high dilution rates

The effects of the substrate conditions on the volumetric productivity of Lactobacillus helveticus at different cell densities up to 60 g l⁻¹ in a continuous stirred-tank reactor with microfiltration to retain the biomass were investigated. At low dilution rates, D, the steady-state volumetric productivity, r _p, gradually increased to a maximum at D = 1.2–1.5 h⁻¹, because of reduced product inhibition. At higher D values, r _p unexpectedly decreased, although the substrate conditions further improved. The maxima of r _p at different cell densities coincided with a critical specific substrate utilization rate beyond which the cell metabolism seems to be controlled through a catabolic modulator factor, and r _p decreases. Received: 8 September 1997 / Received last revision: 31 December 1997 / Accepted: 2 January 1998     

Agrobacterium-mediated transformation of cotyledonary explants of Chinese cabbage (Brassica campestris L. ssp. pekinensis)

 A procedure for producing transgenic Chinese cabbage plants by inoculating cotyledonary explants with Agrobacterium tumefaciens strain EHA101 carrying a binary vector pIG121Hm, which contains kanamycin-resistance and hygromycin-resistance genes and the GUS reporter gene, is described. Infection was most effective (highest infection frequency) when explants were infected with Agrobacterium for 15 min and co-cultivated for 3 days in co-cultivation medium at pH 5.2 supplemented with 10 mg/l acetosyringone. Transgenic plants of all three cultivars used were obtained with frequencies of 1.6–2.7% when the explants were regenerated in shoot regeneration medium solidified with 1.6% agar. A histochemical GUS assay and PCR and Southern blot analyses confirmed that transformation had occurred. Genetic analysis of T1 progeny showed that the transgenes were inherited in a Mendelian fashion. Received: 15 December 1998 / Revision received: 2 July 1999 · Accepted: 8 July 1999     

Modelling the influence of pH, temperature, glucose and lactic acid concentrations on the kinetics of lactic acid production by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour

A kinetic model of the fermentative production of lactic acid from glucose by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour has been developed. The model consists of terms for substrate and product inhibition as well as for the influence of pH and temperature. Experimental data from fermentation experiments under different physical conditions were used to fit and verify the model. Temperatures above 30 °C and pH levels below 6 enhanced the formation of by-products and d-lactic acid. By-products were formed in the presence of maltose only, whereas d-lactic acid was formed independently of the presence of maltose although the amount formed was greater when maltose was present. The lactic acid productivity was highest between 33 °C and 35 °C and at pH 6. In the concentration interval studied (up to 180 g l⁻¹ glucose and 89  g l⁻¹ lactic acid) simulations showed that both substances were inhibiting. Glucose inhibition was small compared with the inhibition due to lactic acid. Received: 28 October 1997 / Received revision: 3 February 1998 / Accepted: 6 February 1998     

Breeding of starch-utilizing and itaconic-acid-producing koji molds by interspecific protoplast fusion between Aspergillus terreus and Aspergillus usamii

Interspecific protoplast fusion between␣Aspergillus terreus, an itaconic acid producer, and A.␣usamii, a glucoamylase producer, was done to breed new koji molds producing itaconic acid from starch. Protoplast fusion between auxotrophic mutant strains by poly(ethylene glycol) treatment produced prototrophic fusants with a fusion frequency of 10⁻⁵−10⁻⁴. The stabilities of some fusants obtained were confirmed by successive subcultures. Conidial analyses of DNA contents and the number of nuclei indicated that the fusants obtained were haploids like the parental strains. One of the stable fusants, F-112, morphologically resembled A. terreus, and produced maximally 35.9 mg/ml itaconic acid from soluble starch (120 mg/ml) at day 6 of cultivation. This productivity from soluble starch was five times as high as that of A. terreus and 70 % of that of A. terreus from glucose (120 mg/ml). Received: 28 June 1996 / Received revision: 3 September 1996 / Accepted: 29 September 1996     

Detoxification of wood hydrolysates with laccase and peroxidase from the white-rot fungus Trametes versicolor

Fermentation of wood hydrolysates to desirable products, such as fuel ethanol, is made difficult by the presence of inhibitory compounds in the hydrolysates. Here we present a novel method to increase the fermentability of lignocellulosic hydrolysates: enzymatic detoxification. Besides the detoxification effect, treatment with purified enzymes provides a new way to identify inhibitors by assaying the effect of enzymatic attack on specific compounds in the hydrolysate. Laccase, a phenol oxidase, and lignin peroxidase purified from the ligninolytic basidiomycete fungus Trametes versicolor were studied using a lignocellulosic hydrolysate from willow pretreated with steam and SO₂. Saccharomyces cerevisiae was employed for ethanolic fermentation of the hydrolysates. The results show more rapid consumption of glucose and increased ethanol productivity for samples treated with laccase. Treatment of the hydrolysate with lignin peroxidase also resulted in improved fermentability. Analyses by GC-MS indicated that the mechanism of laccase detoxification involves removal of monoaromatic phenolic compounds present in the hydrolysate. The results support the suggestion that phenolic compounds are important inhibitors of the fermentation process. Received: 3 November 1997 / Received revision: 4 February 1998 / Accepted: 6 February 1998     

Transformation of macromolecules from a brown coal by lignin peroxidase

Indirect evidence has suggested that lignin peroxidase (LiP) of the white-rot fungus Phanerochaete chrysosporium catalyses oxidative decolourisation and depolymerisation of macromolecules from brown coal in vivo. In this study we show that LiP catalyses these transformations in vitro. Unmethylated (USC45 coal) and methylated (MWSC6 coal) fractions of solubilised macromolecules (M _r > 30 000) from a brown coal were treated with a semi-purified preparation of LiP isozymes from P. chrysosporium. Both coal fractions were decolourised, losing between 26% and 39% of their absorbance at both 280 nm and 400 nm, in reactions that had an absolute requirement for H₂O₂ and veratryl alcohol. Neither coal fraction was transformed when the enzyme was heat-inactivated or in the presence of the LiP inhibitor metavanadate. Gel-permeation chromatography showed that MWSC6 coal but not USC45 was depolymerised and yielded low-molecular-mass (M _r < 30 000) fragments. Nine monomeric products were identified by GC-MS. Received: 20 March 1998 / Received revision: 3 September 1998 / Accepted: 3 September 1998     

Mineralization of low-chlorinated biphenyls by Burkholderia sp. strain LB400 and by a two-membered consortium upon directed interspecies transfer of chlorocatechol pathway genes

The biphenyl-mineralizing bacterium Burkholderia sp. strain LB400 also utilized 3-chloro-, 4-chloro-, 2,3′-dichloro- and 2,4′-dichlorobiphenyl for growth. By the attack of the initial enzyme a chlorine was eliminated dioxygenolytically from position 2 of one of the aromatic rings when hydrogens of both were substituted by chlorine. The strain mineralized 3-chloro- and 2,3′-dichlorobiphenyl via the central intermediate 3-chlorobenzoate through its chlorocatechol pathway enzymes, but excreted stoichiometric amounts of 4-chlorobenzoate from 4-chloro- and 2,4^′-dichlorobiphenyl. These two compounds were mineralized by a co-culture of strain LB400 and a derivative of the (methyl-) benzoate-degrading strain Pseudomonas putida mt-2 (TOL). The complete degradation was achieved upon transfer of a cluster of at least five genes, encoding the regulated chlorocatechol pathway operon, from strain LB400 to strain mt-2. This transfer was demonstrated by the polymerase chain reaction. Received: 15 April 1998 / Received revision: 12 June 1998 / Accepted: 19 June 1998     

Characterization of a gene encoding Trametes versicolor laccase A and improved heterologous expression in Saccharomyces cerevisiae by decreased cultivation temperature

Laccase can be used for enzymatic detoxification of lignocellulosic hydrolysates. A Saccharomyces cerevisiae strain with enhanced resistance to phenolic inhibitors and thereby improved ability to ferment lignocellulosic hydrolysates would presumably be obtained by heterologous expression of laccase. Sequencing of the cDNA for the novel laccase gene lcc2 from the lignin-degrading basidiomycete Trametes versicolor showed that it encodes an isoenzyme of 499 amino-acid residues preceded by a 21-residue signal peptide. By comparison with Edman degradation data, it was concluded that lcc2 encodes an isoenzyme corresponding to laccase A. The gene product of lcc2 displays 71% identity with the previously characterized T. versicolor lcc1 gene product. An alignment of laccase sequences revealed that the T. versicolor isoenzymes in general are more closely related to corresponding isoenzymes from other white-rot fungi than to the other T. versicolor isoenzymes. The multiplicity of laccase is thus a conserved feature of T. versicolor and related species of white-rot fungi. When the T. versicolor lcc2 cDNA was expressed in S. cerevisiae, the production of active enzyme was strongly dependent on the temperature. After 3 days of incubation, a 16-fold higher laccase activity was found when a positive transformant was kept at 19 °C instead of 28 °C. Similar experiments with Pichia pastoris expressing the T. versicolor laccase gene lcc1 also showed that the expression level was favoured considerably by lower cultivation temperature, indicating that the observation made for the S. cerevisiae expression system is of general significance. Received: 8 December 1998 / Received revision: 9 April 1999 / Accepted: 16 April 1999     

Biosynthesis of polyhydroxyalkanoates from low-rank coal liquefaction products by Pseudomonas oleovorans and Rhodococcus ruber

A screening identified several bacteria that were able to use chemically heterogeneous low-rank coal liquefaction products as complex carbon sources for growth. Pseudomonas oleovorans and Rhodococcus ruber accumulated polyhydroxyalkanoic acids (PHA) amounting to 2%–8% of the cell dry weight when the cells were cultivated on these liquefaction products in the absence of any other carbon source. R. ruber accumulated, in addition to PHA, small amounts of triacylglycerols. The accumulated PHA consisted of 3-hydroxyhexanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate (P. oleovorans) or 3-hydroxybutyric acid and 3-hydroxyvaleric acid (R. ruber). Low-rank coal liquefaction products obtained from Trichoderma atroviride were better substrates for P. oleovorans than chemically produced fulvic acids. Received: 13 May 1998 / Received revision: 11 August 1998 / Accepted: 12 August 1998