Screening for fungi intensively mineralizing 2,4,6-trinitrotoluene

Within a screening program, 91 fungal strains belonging to 32 genera of different ecological and taxonomic groups (wood- and litter-decaying basidiomycetes, saprophytic micromycetes) were tested for their ability to metabolize and mineralize 2,4,6-trinitrotoluene (TNT). All these strains metabolized TNT rapidly by forming monoaminodinitrotoluenes (AmDNT). Micromycetes produced higher amounts of AmDNT than did wood- and litter-decaying basidiomycetes. A significant mineralization of [¹⁴C]TNT was only observed for certain wood- and litter-decaying basidiomycetes. The most active strains, Clitocybula dusenii TMb12 and Stropharia rugosa-annulata DSM11372 mineralized 42 % and 36 % respectively of the initial added [¹⁴C]TNT (100 μM corresponding to 4.75 μCi/l) to ¹⁴CO₂ within 64 days. Micromycetes (deuteromycetes, ascomycetes, zygomycetes) proved to be unable to mineralize [¹⁴C]TNT significantly. Received: 8 August 1996 / Received revision: 16 December 1996 / Accepted: 20 December 1996     

The de novo production of drosophilin A (tetrachloro-4-methoxyphenol) and drosophilin A methyl ether (tetrachloro-1,4-dimethoxybenzene) by ligninolytic basidiomycetes

Ligninolytic basidiomycetes were screened for their ability to produce the tetrachlorinated hydroquinone metabolites drosophilin A (DA, tetrachloro-4-methoxyphenol) and drosophilin A methyl ether (DAME, tetrachloro-1,4-dimethoxybenzene). Five fungal strains produced these metabolites in detectable amounts, including strains from Bjerkandera and Peniophora, which are genera not previously known for DA or DAME production. Phellinus fastuosus ATCC26.125 had the highest and most reliable production of DA and DAME in peptone medium, respectively 15–60 μM and 4–40 μM. This fungus was used to study culture conditions that could increase DAME production. A fourfold increase in DAME production was found after the addition of hydroquinone to growing cultures of P. fastuosus. Therefore, hydroquinone is postulated to be a possible biosynthetic precursor of DAME in the fungus. Antagonising P. fastuosus by adding filter-sterilised culture fluid of a competing fungus, Phlebia radiata, increased DAME production significantly by tenfold. This result suggests that DAME production is elicited by compounds present in the culture fluid of P. radiata, indicating that DAME has an antibiotic function in P. fastuosus. Received: 17 September 1996 / Received revision: 7 February 1997 / Accepted: 15 February 1997     

Saccharomyces cerevisiae mutants selected for increased production of Trichoderma reesei cellulases

 Trichoderma reesei endoglucanase I (EGI) was used as a reporter enzyme for screening mutagenized yeast strains for increased ability to produce protein. Sixteen haploid Saccharomyces cerevisiae strains, transformed with a yeast multicopy vector pALK222, containing the EGI cDNA under the ADH1 promoter, produced EGI activity of 10^-5–10^-4 g/l. On the average 93% of the total activity was secreted into the culture medium. Two strains with opposite mating types were mutagenized, and several mutants were isolated possessing up to 45-fold higher EGI activity. The best mutants were remutagenized and a second-generation mutant, strain 2804, with an additional twofold increase in EGI activity was selected. The mutant strain 2804 grew more slowly and reached a lower final cell density than the parental strain. In the selective minimal medium, the 2804 strain produced 40 mg/l immunoreactive EGI protein, but only 2% was active enzyme. In the rich medium the secreted EGI enzyme stayed active, but without selection pressure the EGI production ceased after 2 days of cultivation, when the strain 2804 had produced 10 mg/l of EGI. A sevenfold difference was found between the parental and the 2804 strain in their total EGI production relative to cell density. The difference in favour of the mutant strain was also detected on the mRNA level. The 2804 mutant was found to be more active than the parental strain also in the production of T. reesei cellulases, cellobiohydrolase I, and cellobiohydrolase II. Received: 22 December 1995/Received revision: 26 February 1996/Accepted: 17 March 1996     

Micropropagation of 21 species of Mexican cacti by axillary proliferation

Summary We have developed micropropagation systems for 21 species of Mexican cacti using explants from seedlings germinatedin vitro or shoot segments of juvenile 2–3-yr-old greenhouse plants. The species propagated belong to the generaAstrophytum, Cephalocereus, Coryphantha, Echinocactus, Echinocereus, Echinofossulocactus, Ferocactus, Mammillaria, Nyctocereus, andStenocactus. Multiple shoot formation from areoles was achieved in Murashige and Skoog (MS) medium supplemented with either 1 or 2 mg N⁶-benzyladenine (BA) per 1 (4.44 or 8.87 μM) or BA at 1 or 2 mg/l plus naphthaleneacetic acid at 0.1 or 1 mg/l (0.54 or 5.37 μM). The requirements of growth regulators for optimal shoot proliferation, the velocity of the response, and the number of buds produced by explant were different among the genera and species studied. Rooting of the shoots generatedin vitro was achieved in MS medium supplemented with indoleacetic acid at 0.5–1 mg/l (2.85–5.71 μM) or indolebutyric acid at 0.5–1 mg/l (2.46–4.90 μM). Finally, 70–95% of the rooted plants transferred to potting medium survived.     

Microbial sensors for naphthalene using Sphingomonas sp. B1 or Pseudomonas fluorescens WW4

 Amperometric biosensors for naphthalene were developed using either immobilized Sphingomonas sp. B1 or Pseudomonas fluorescens WW4 cells. The microorganisms were immobilized within a polyurethane-based hydrogel, which was used for a microbial biosensor for the first time. Both strains were shown to be equally suited for the quantification of naphthalene in aqueous solutions. The biosensors were tested in a flow-through system and a stirred cell (batch method). In both systems a linear response down to the detection limit was obtained. Measurements in the flow-through system gave sensitivities of up to 1.2 nA mg⁻¹ l⁻¹ and a linear range from 0.03 mg/l to 2.0 mg/l. The response time (t ₉₅) was 2 min and the sample throughput six per hour; the repeatability was within ±5 %. With the batch method, sensitivities of between 3 nA mg⁻¹ l⁻¹ and 5 nA mg⁻¹l⁻¹ and a linear range of 0.01–3.0 mg/l were obtained; the response time was between 3 min and 5 min. The sensors reached an operational lifetime of up to 20 days. The sensitivity of both sensors for naphthalene was, in most cases, more than four times higher than for various other substrates. Received: 18 October 1995/Received revision: 22 December 1995/Accepted: 22 January 1996     

Direct fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of direct fermentation for lactic acid production by fungal species of Rhizopus arrhizus 3,6017 and Rhizopus oryzae 2,062 was studied with respect to growth pH, temperature and substrate. The direct fermentation was characterized by starch hydrolysis, accumulation of reducing sugar, and production of lactic acid and fungal biomass. Starch hydrolysis, reducing sugar accumulation, biomass formation and lactic acid production were affected with the variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30°C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.87–0.97 g/g starch associated with 1.5–2.0 g/l fungal biomass produced in 36 h fermentation. R. arrhizus 3,6017 had a higher capacity to produce lactic acid, while R. oryzae 2,062 produced more fungal biomass under similar conditions.     

Influence of edaphic and climatic factors on dynamics of root colonization and spore density of vesicular-arbuscular mycorrhizal fungi in Acacia farnesiana Willd. and A. planifrons W.et.A.

 A study was conducted to assess the dynamics of vesicular-arbuscular mycorrhizal (VAM) fungi associated with Acacia farnesiana and A. planifrons in moderately fertile alkaline soils. The intensity of root colonization by VAM fungi and the distribution of VAM fungal structures varied with host species over a period of time. The occurrence of vesicles with varied morphology in the mycorrhizal roots indicates infection by different VAM fungal species. This was further confirmed from the presence of spores belonging to different VAM fungal species in the rhizosphere soils. Root colonization and spore number ranged from 56% – 72% and 5 – 14 g^{–  1}soil in A. farnesiana and from 60% – 73% and 5 – 15 g^{–  1} soil in A. planifrons. Per cent root colonization and VAM spore number in the rhizosphere soil were inversely related to each other in both the Acacia species. However, patterns of the occurrence of VAM fungal structures were erratic. Spores of Acaulospora foveata, Gigaspora albida, Glomus fasciculatum, G. geosporum and Sclerocystis sinuosa were isolated from the rhizosphere of A. farnesiana whereas A. scrobiculata, G. pustulatum, G. fasciculatum, G. geosporum and G. microcarpum were isolated from that of A. planifrons. The response of VAM status to fluctuating edaphic factors varied with host species. In A. farnesiana though soil nitrogen (N) was positively correlated with root colonization, soil moisture, potassium and air temperature were negatively correlated to both root colonization and spore number. Per cent root colonization and spore number in A. planifrons were negatively related to each other. Further, in A. planifrons as the soil phosphorus and N were negatively correlated with the density of VAM fungal spores, the same edaphic factors along with soil moisture negatively influenced root colonization. Received: 16 May 1995 / Accepted: 7 February 1996     

Secretion of the sweet-tasting protein thaumatin by recombinant strains of Aspergillus niger var. awamori

A recombinant form of the sweet-tasting protein thaumatin has been produced in the filamentous fungus Aspergillus niger var. awamori. Expression cassettes containing a synthetic gene encoding thaumatin II were prepared and used to transform Aspergillus niger var. awamori strain NRRL312. Several fungal strains capable of synthesizing and secreting thaumatin into the culture medium were generated, and their production capabilities were determined, first in shake flasks and later in a laboratory fermentor. We report the expression and secretion of thaumatin in concentrations of 5–7 mg/l. This recombinant thaumatin is sweet. Received: 7 October 1997 / Received revision: 21 November 1997 / Accepted: 21 November 1997     

高磷土壤中丛枝菌根真菌的分离鉴定

【背景】丛枝菌根(arbuscular mycorrhiza,AM)真菌具有广泛的寄主范围、环境适应性和优良的植物促生能力。然而,土壤的高磷水平严重抑制了AM真菌生长及AM形成。【目的】分离鉴定出耐较高有效磷含量的华南土著AM真菌菌株,为菌根学研究工作提供新颖材料。【方法】采用经典形态学和分子系统学方法鉴定高磷土壤中AM真菌。【结果】从有效磷含量为53-131 (平均值±标准差为88.2±17.6) mg/kg的根区土壤中鉴定出7属25种AM真菌,包括无梗囊霉属(Acaulospora) 12种、球囊霉属(Glomus) 7种、隔球囊霉属(Septoglomus) 2种、近明球囊霉属(Claroideoglomus) 1种、根孢囊霉属(Rhizophagus) 1种、硬囊霉属(Sclerocystis) 1种和类球囊霉属(Paraglomus) 1种,其中幼套近明球囊霉(Claroideoglomus etunicatum)和蜜色无梗囊霉(Acaulospora mellea)是优势种。在(87.7±8.0) mg/kg的高磷水平下,AM真菌仍能形成丛枝和泡囊。但当有效磷含量达到(99.7±1.2) mg/kg时,菌根侵染率和丛枝丰度显著下降,但仍能够形成泡囊。【结论】从广州市南沙区有效磷含量为(88.2±17.6) mg/kg的耕地植物根区土壤中,鉴定出具有耐高磷潜力的7属25种AM真菌,幼套近明球囊霉和蜜色无梗囊霉等分离株可作为后续高磷抑制机制解析及耐高磷AM真菌菌剂研发工作的试验菌株。     

Production of L-arabitol from L-arabinose by Candida entomaea and Pichia guilliermondii

 Lignocellulosic biomass, particularly corn fiber, represents a renewable resource that is available in sufficient quantities from the corn wet milling industry to serve as a low cost feedstock for production of fuel alcohol and valuable coproducts. Several enzymatic and chemical processes have potential for the conversion of cellulose and hemicellulose to fermentable sugars. The hydrolyzates are generally rich in pentoses (D-xylose and L-arabinose) and D-glucose. Yeasts produce a variety of polyalcohols from pentose and hexose sugars. Many of these sugar alcohols have food applications as low-calorie bulking agents. During the screening of 49 yeast strains capable of growing on L-arabinose, we observed that two strains were superior secretors of L-arabitol as a major extracellular product of L-arabinose. Candida entomaea NRRL Y-7785 and Pichia guilliermondii NRRL Y-2075 produced L-arabitol (0.70 g/g) from L-arabinose (50 g/l) at 34°C and pH 5.0 and 4.0, respectively. Both yeasts produced ethanol (0.32–0.33 g/g) from D-glucose (50 g/l) and only xylitol (0.43–0.51 g/g) from D-xylose (50 g/l). Both strains preferentially utilized D-glucose>D-xylose>L-arabinose from mixed substrate (D-glucose, D-xylose and L-arabinose, 1:1:1, 50 g/l, total) and produced ethanol (0.36–0.38 g/g D-glucose), xylitol (0.02–0.08 g/g D-xylose) and L-arabitol (0.70–0.81 g/g L-arabinose). The yeasts co-utilized D-xylose (6.2–6.5 g/l) and L-arabinose (4.9–5.0 g/l) from corn fiber acid hydrolyzate simultaneously and produced xylitol (0.10 g/g D-xylose) and L-arabitol (0.53–0.54 g/g L-arabinose). Received: 24 April 1995/Received revision: 9 August 1995/Accepted: 7 September 1995     

Distribution of carotenoids and sterols in relation to the taxonomy of Taphrina and Protomyces

Species of the genera Taphrina Fr. and Protomyces Unger were screened for the presence of carotenoid pigments and the sterols ergosterol and brassicasterol. All strains produced carotenoids in variable amounts: Taphrina: 0.3–39 g/g dry weight; Protomyces: 65–99 g/g dry weight. It was concluded that the tow genera cannot be separated on the basis of presence or absence of carotenoids. Thirty strains (24 species) of Taphrina produced brassicasterol as the principal sterol; twenty-one strains (17 species) did not form ergosterol. Only four isolates (4 species) produced ergosterol without formation of brassicasterol. Brassicasterol was the major sterol in 3 species of Protomyces, whereas ergosterol was absent. Brassicasterol is a rather unique sterol within the fungal kingdom and has hitherto not been found in the red yeasts. Therefore, this sterol is of taxonomic significance in contrast with ergosterol, which is widespread among fungi.     

Isolation of mutants of Penicillium purpurogenum resistant to catabolite repression

 The strain Penicillium purpurogenum P-26 was subjected to UV irradiation and N-methyl-N′-nitro-N-nitrosoguanidine treatment and mutants were isolated capable of synthesizing cellulase under the conditions of a high concentration of glucose. Initially mutants resistant to catabolite repression by 2-deoxy-D-glucose were isolated on Walseth’s cellulose/agar plates containing 15–45 mM 2-deoxy-D-glucose. These mutants were again screened for resistance to catabolite repression by glycerol or glucose on Walseth’s cellulose/agar plates containing 50 g/l glycerol or 50 g/l glucose respectively. Four mutants with different sizes of clearing zone on Walseth’s cellulose/agar plates containing 50 g/l glucose were selected for flask culture. Among them, the mutant NTUV-45-4 showed better carboxymethylcellulase activity in flask culture containing 1% Avicel plus 3% glucose than did the parental strain. Received: 9 October 1995/Received revision: 27 November 1995/Accepted: 8 January 1996     

Production of prodigiosin and chitinases by tropical Serratia marcescens strains with potential to control plant pathogens

The potential of three Serratia marcescens strains (CFFSUR-B2, CFFSUR-B3 and CFFSUR-B4) isolated from tropical regions in Mexico to inhibit the mycelial growth and conidial germination of Colletotrichum gloeosporioides, causal agent of fruit anthracnose, was evaluated. The ability of these strains to produce prodigiosin and chitinases when cultivated in oil seed-based media (peanut, sesame, soybean and castor bean) and in Luria–Bertani medium was determined. All of the strains exhibited similar fungal antagonistic activities and inhibited myceliar growth by more than 40% while inhibiting conidial germination by 81–89% (P = 0.01). The highest level of prodigiosin (40 μg/ml) was produced in the peanut-based medium while growth in soybean-based medium allowed the highest production of chitinases (56 units/ml), independent of the strain used. Strain CFFSUR-B2 grown in peanut medium was used to evaluate the effect of inoculum density and initial pH on metabolite production. The amount of prodigiosin produced increased with greater inoculum densities, with an initial density of 1 × 10¹² resulting in the highest production (60 μg/ml). Prodigiosin production was not affected by pH. The strains studied have the advantage of being adapted to tropical climates and are able to produce chitinases in the absence of chitin induction in vitro. These characteristics suggest their potential as biocontrol agents for fungal pathogens in tropical regions of the world.     

Comparative anatomy of resin ducts of the Pinaceae

 Resin ducts are common in the Pinaceae. The comparative anatomy of stems and leaves of 50 species and two varieties from ten genera has been investigated. The structure and distribution of resin ducts differ among genera. Resin ducts occur in foliage leaves of ten genera of Pinaceae. Cortical resin ducts are absent in the stems of Pseudolarix and Larix. Resin ducts only occur in the secondary xylem of stems of Pinus, Picea, Cathaya, Larix, Pseudotsuga and some Keteleeria species. All of the epithelial and sheath cells are alive and thin-walled in the resin ducts of stem cortex and mesophyll. Except for Pinus the epithelial cells of resin ducts in the secondary xylem of stems have thick, lignified walls. Comparative study shows there are obvious differences in the resin ducts of different genera; apparent differences do not exist, however, in the resin ducts of different species of the same genus. According to the structure and distribution of the resin ducts in ten genera of Pinaceae, a synoptical arrangement of the genera is given and generic relationships of the Pinaceae are discussed. Received: 12 September 1995 / Accepted: 14 March 1996     

Exopolysaccharide production of <Emphasis Type="Italic">Lactobacillus salivarius</Emphasis> BCRC 14759 and <Emphasis Type="Italic">Bifidobacterium bifidum</Emphasis> BCRC 14615

In the present study, the production of exopolysaccharides (EPS) by 13 strains of Lactobacillus and 6 strains of Bifidobacterium in a chemical defined medium (CDM) supplemented with 30 g lactose/l was first compared. The highest EPS production of the Lactobacillus strains was found in L. salivarius BCRC 14759 while among the Bifidobacterium strains examined, B. bifidum BCRC 14615 showed the highest EPS production. Analyzes of the effect of lactose concentration and cultivation temperature on EPS production revealed that L. salivarius produced the highest amount of EPS (45.3 mg/l) in CDM supplemented with 5 g lactose/l at 40°C while B. bifidum produced the highest EPS (17.0 mg/l) in CDM supplemented with 40 g lactose/l at 35°C. α-Phosphoglucomutase, UDP-glucose pyrophosphorylase and UDP-galactose-4-epimerase exhibited a markedly notable activity compared with other enzymes examined in the cell extract of both test organisms. This indicates their possible involvement in the biosynthesis of EPS.     

Strains of<Emphasis Type="Italic">aspergillus versicolor</Emphasis> tiraboschi synthesizing sterigmatocystin and the differentiation of mycotoxic risk dependent on their productivity in housing buildings

In biomasses from 22 moulds isolated in housing buildings a Chromatographie HPLC UV/VIS analysis was carried out to assess the production of sterigmatocystin by fungal isolates. The results are discussed with respect to mycotoxic risk for people exposed to the presence ofAspergillus versicolor. This fungus is often present on building materials, but does not always produce the carcinogenic mycotoxin sterigmatocystin (ST). In this study, 19 (86%) of the 22 strains synthesized ST at detectable levels (>0.03 mg kg₋₁ biomass). Three of the strains (14%) were found to synthesize ST at levels exceeding 100 mg kg₋₁ of the air-dry mould biomass with laboratory medium. One strain proved to be highly productive and synthesized more than 500 mg kg₋₁ ST. Thus, it presented the highest mycotoxic danger for the dwellers of the buildings whereA. versicolor infested the walls. However, most strains are low producers of ST, with 12 of 22 isolates synthesizing less than 10 mg kg₋₁ biomass. Mycotoxigenic and highly productive strains that produced significant amounts of ST (>500 mg kg₋₁ biomass) were less frequently found, with approximately 5% of all isolates from buildings studied for ST production. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Taxonomy of Pinus species based on the seed oil fatty acid compositions

 The fatty acid compositions of the seed oils from ten pine species have been established by capillary gas-liquid chromatography of the methyl esters. With regard to either normal fatty acids or Δ5-olefinic acids, the general pattern of fatty acids did not differ from that of other pine seed oils reported previously. The main fatty acid was linoleic (9,12–18:2) acid (44.4–57.1%), followed by either oleic (9–18:1) acid (13.4–24.5%) or pinolenic (5,9,12–18:3) acid (1.5–25.2%). When applying multivariate analyses to the chemometric data (13 variables) of 49 pine species (ca. 40% of the living pine species), it was possible to distinguish between several sections: Pinea, Longifolia, Halepensis, Ponderosa-Banksiana, Sylvestris, and Cembra. The latter section was clearly divided into two sub-groups. A few species that presented a low overall content of Δ5-olefinic acids, and that grow in warm-temperate regions, were isolated from the bulk of other pine species. It is hypothesized that Δ5-olefinic acids might be related to cold-acclimation. Received: 5 June 1997 / Accepted: 17 August 1997     

Expression and secretion of functional miniantibodies McPC603scFvDhlx in cell-wall-less L-form strains of Proteus mirabilis and Escherichia coli : A comparison of the synthesis capacities of L-form strains with an E. coli producer strain

The paper describes the synthesis of the phosphorylcholine-binding miniantibody McPC603scFvDhl x in cell-wall-less L-form strains of Escherichia coli and Proteus mirabilis. Cells of these strains were transformed with the plasmid pACK02scKan, carrying the miniantibody (miniAb) coding sequence under the control of the lac promoter. L-form transformants of both species were able to synthesize the functional miniAb as an extracellular soluble product. The highest quantities were obtained by P. mirabilis L-form strains after induction with 5 mM isopropyl β-d-thiogalactopyranoside (IPTG). Yields of 45–75 mg/l total antibody protein and of 10–18 mg/l functional miniAb were estimated in the growth medium of shaking cultures 40–80 h after induction with IPTG. About 10% of the active miniAb remained cell-bound. The yields of functional miniAb could be optimized by lowering the growth temperature from 37 °C to 26–32 °C and by supplementation of the medium with 80 mM sodium fumarate. A comparison of the specific activities revealed that the P. mirabilis L-form strains have a similar synthesis capacity (2–4 mg functional miniAb/g cell dry weight) to that of the producer strain E. coli RV308. The results show that the processes of correct folding and assembling of the miniAb molecules are possible without the periplasmic compartment. Received: 14 April 1997 / Received revision: 17 July 1997 / Accepted: 25 August 1997     

The production of eicosapentaenoic acid by representatives of the genus <Emphasis Type="Italic">Mortierella</Emphasis> grown on brewers’ spent grain

Brewers’ spent grain (BSG) was evaluated as substrate for the production of eicosapentaenoic acid (EPA) by solid-state fermentation with 29 fungal strains representing different Mortierella species. The effect of a 10% (w/w) linseed oil (LSO) supplement on EPA production was also determined. All the strains produced EPA on the substrate, while addition of the LSO improved the EPA yield of most strains. The strains producing the most EPA in the absence of additional LSO generally also produced the most EPA when LSO was added to the BSG. The strains, which produced the highest levels of EPA on BSG supplemented with LSO were Mortierella antarctica Mo 67 and Mortierella epicladia Mo 101, which respectively produced 2.8 mg and 2.5 mg EPA per g of BSG.     

Effects of gibberellic acid on hairy root cultures of Artemisia annua: Growth and artemisinin production

Summary Artemisinin (AN), a potent antimalarial drug that has been used for centuries as a folk remedy in China, is an effective treatment against quinine-resistant strains of Plasmodium. It can be produced through the in vitro culture of genetically transformed (hairy) roots. The effect of gibberellic acid (GA₃) on the growth and secondary metabolite production of hairy roots of Artemisia annua was investigated. Six different concentrations of GA₃ were tested in shaker flasks to determine the optimum concentration. GA₃ levels of 0.01–0.001 mg/l (28.9–2.89 μM) provided the most significant increase in biomass, and 0.01 mg/l (28.9 μM) produced the highest amount of AN. Investigation of growth kinetics showed that the use of GA₃ at 0.01 mg/l (28.9 μM) increased the growth rate of hairy roots of A. annua by 24.9%. Thus, the cultures treated with GA₃ reached stationary phase faster than control cultures.