Bioleaching of chalcopyrite by pure and mixed cultures of Acidithiobacillus spp. and Leptospirillum ferriphilum

Bioleaching is an economical method for the recovery of metals that requires low investment and operation costs. Furthermore, it is generally more environmentally friendly than many physicochemical metal extraction processes. The bioleaching of chalcopyrite in shake flasks was investigated with pure and mixed cultures of Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Acidithiobacillus caldus, and Leptospirillum ferriphilum. The mixed cultures containing both iron- and sulfur-oxidizing bacteria were more efficient than the pure culture alone. The presence of sulfur-oxidizing bacteria positively increased the dissolution rate and the percentage recovery of copper from chalcopyrite. Mixed cultures consisting of moderately thermophilic L. ferriphilum and A. caldus leached chalcopyrite more effectively than mesophilic A. ferrooxidans pure and mixed cultures. The decrease of the chalcopyrite dissolution rate in leaching systems containing A. ferrooxidans after 12–16 days coincided with the formation of jarosite precipitation as a passivation layer on the mineral surface during bioleaching. Low pH significantly reduces jarosite formation in pure and mixed cultures of L. ferriphilum and A. caldus.     

Degradation of olestra, a non caloric fat replacer, by microorganisms isolated from activated sludge and other environments

Olestra is a non-caloric fat substitute consisting of fatty acids esterified to sucrose. Previous work has shown that olestra is not metabolized in the gut and is excreted unmodified in human feces. To better understand the fate of olestra in engineered and natural environments, aerobic bacteria and fungi that degrade olestra were enriched from sewage sludges, soils and municipal solid waste compost not previously exposed to olestra. Various mixed and pure cultures were obtained from these sources which were able to utilize olestra as a sole carbon and energy source. The fastest growing enrichment was obtained from activated sludge and later yielded an olestra-degrading pure culture of Pseudomonas aeruginosa. This mixed culture extensively degraded both ¹⁴C-fatty acid labeled olestra and ¹⁴C-sucrose labeled olestra during 8 days of incubation. Longer-term incubation with pure cultures of P. aeruginosa demonstrated that >98% of ¹⁴C-sucrose labeled olestra and >72% of ¹⁴C-fatty acid labeled olestra was mineralized to CO₂ after 69 days. These results indicate that olestra degraders are present in environments not previously exposed to olestra and that olestra can serve as a sole carbon and energy source. Furthermore, a common bacterial species was isolated from activated sludge and shown to have the ability to degrade olestra.     

Response to oxygen of diazotrophic Azospirillum brasilense — Arthrobacter giacomelloi mixed batch culture

Azospirillum brasilense and Arthrobacter giacomelloi were grown together in batch culture under different oxygen pressures. The response to oxygen of growth, nitrogenase activity and respiration rate was determined. The two microorganisms were found to be able to coexist all over the range of partial oxygen pressures examined, that is from 0.004–0.20 bar. Nitrogenase activity by mixed culture of A. brasilense and A. giacomelloi always appeared higher than that of A. brasilense pure culture. Low respiratory activity at partial oxygen pressures higher than 0.02 bar by both pure and mixed cultures seemed not to account for the high nitrogenase activity and improved oxygen tolerance of the mixed culture.Abbreviations pO₂ partial oxygen pressure     

Changes in the antibiotic production by co-culture of Rhizopus peka P8 and Bacillus subtilis IFO3335

The co-culture of Bacillus subtilis IFO 3335 with Rhizopus peka P8 or Rhizopus oligosporus P12 in liquid medium was found to increase production of antibiotic activity and to alter the spectrum of activity relative to the pure cultures. However, a mixed culture of Rhizopus arrhizus P7 and Rhizopus oryzae P17 did not produce antibiotic activity. The concentration, ratio, and time of addition of B. subtilis to the R. peka culture was found to influence antibiotic yields. Solid-state fermentations using mixed cultures of R. peka and B. subtilis were investigated. The growth of Escherichia coli IFO 3792 as a target bacterium was inhibited by the mixed culture. These results suggest the possibility of biopreservation of fermented foods by novel co-culture systems.     

Antagonism against Rhizoctonia solani and fungitoxic metabolite production by some Penicillium isolates

A number of Penicillium isolates were recovered in association to Rhizoctonia solani strains pathogenic on tobacco and from soil on plates pre-colonized by the pathogen itself. Their antagonism toward R. solaniAG-2-1 was evaluated in dual cultures in vitro. Inhibition of growth was evident to some extent in most pairings, while hyphal interactions referable to mycoparasitic relationships were not observed. However, the occurrence of plasmolysis and/or vacuolisation and the induction of monilioid cells were indicative of the release of bioactive compounds. Therefore, production of fungitoxic metabolites was tested by adding concentrated culture filtrates of each Penicillium isolate to the growth medium of R. solani. Complete and lasting inhibition was incited by culture filtrates of some isolates belonging to P. brevicompactum, P. expansum, and P. pinophilum. Three purified compounds, respectively mycophenolic acid, patulin and 3-O-methylfunicone, which were extracted from culture filtrates, were able to inhibit R. solani in vitro. Their production was also detected in dual cultures of the same Penicilliumstrains with R. solani prepared in sterilized soil and when the Penicilliumstrains were cultured directly on R. solani mycelium harvested from liquid cultures. The possible role of such metabolites in antagonism of the above-mentioned Penicilliumspecies against R. solani is discussed.     

Synergism of cellulase enzymes in mixed culture solid substrate fermentation

Sugar cane bagasse was subjected to a mixed culture, solid substrate fermentation with Trichoderma reesei QM9414 and Aspergillus terreus SUK-1 to produce cellulase and reducing sugars. The highest cellulase activity and reducing sugar amount were obtained in mixed culture. The percentage of substrate degradation achieved employing mixed culture was 26% compared to 50% using separate cultures of the two molds. This suggests that the synergism of enzymes in mixed culture solid substrate fermentation have lower synergism than in pure culture.     

Antagonistic interactions between fungal pathogens and phylloplane fungi of guava

Dominant phylloplane fungi of guava (Psidium guajava L.) were screened for their antagonistic activities against the two pathogens,Colletotrichum gloeosporioides andPestalotia psidii, bothin vitro andin vivo. Culture filtrates ofAspergillus niger, Fusarium oxysporum andPenicillium citrinum caused more than 50% growth inhibition ofC. gloeosporioides. Filtrates ofCephalosporium roseo-griseum andF. oxysporum were most effective in reducing the growth ofP. psidii. Volatiles produced from the cultures ofA. niger, F. oxysporum, P. citrinum andP. oxalicum inhibited the growth ofC. gloeosporioides, whereas volatiles fromC. roseo-griseum, F. oxysporum andTrichoderma harzianum inhibited the growth ofP. psidii. The inhibitory effect of volatiles decreased with increase in incubation time. In general, the maximum effect of volatiles was noticed after 48 h incubation. Different grades of colony interactions in dual cultures were recognised between the two pathogens and the phylloplane fungi examined. Maximum inhibition ofC. gloeosporioides was caused byAureobasidium pullulans, Cladosporium cladosporioides, epicoccum purpurascens, F. oxysporum andMyrothecium roridum, whereasAspergillus terreus, C. roseo-griseum andP. oxalicum significantly reduced the growth ofP. psidii. Application of a spore suspension of each test fungus inhibited lesion development of guava leaves caused by the test pathogensin vitro. Inhibition was more pronounced when the spore concentration was increased.A. pullulans, C. cladosporioides, E. purpurascens, F. oxysporum, andT. harzianum were found to be strongly antagonistic toC. gloeosporioides. A. niger, A. terreus, C. roseo-grisem andT. harzianum were strongly antagonistic toP. psidii.     

Influence of wine-related physicochemical factors on the growth and metabolism of non-<Emphasis Type="Italic">Saccharomyces</Emphasis> and <Emphasis Type="Italic">Saccharomyces</Emphasis> yeasts in mixed culture

The influence of two physicochemical factors involved in winemaking, temperature and SO₂, on the kinetics and metabolic behavior of Kloeckera apiculata and Saccharomyces cerevisiae was examined. Highest biomass was reached at 15 and 25°C for K. apiculata and S. cerevisiae, respectively. Pure cultures of K. apiculata died off early with increasing temperature, but in co-culture with S. cerevisiae it showed higher viability and a change in the death curve from exponential to linear. Statistical analysis revealed that metabolite production was significantly different for the three cultures and also at the different fermentation temperatures. Besides, the interaction between culture type and temperature was significant. At temperatures from 15 to 30°C the mixed culture showed similar ethanol and lower acetic acid production compared with a pure culture of K. apiculata. SO₂ addition slightly increased survival of the non-Saccharomyces species in pure and mixed cultures. Statistical evaluation indicated that culture type and SO₂ addition significantly affected metabolite production, but the interaction between culture and SO₂ was not significant. These results contribute to current knowledge of enological factors and their effect on prevalence and fermentative activities of the composite yeast flora and the statistical significance emphasizes the importance of the combined influence of the culture type and physicochemical factors on the production of fermentation metabolites.     

Survey of bovine mycotic mastitis in dairy herds in the State of São Paulo,Brazil

The purpose of this study was to isolate fungi from the quarter milk of cow udders from several dairy herds and to identify the different genera and species involved in mastitis. A total of 2078 milk samples from normal, clinical and subclinical mastitis quarters from 22 dairy herds of 16 districts in the State of São Paulo, Brazil was utilized in this survey. Two hundred and fifty one (12.07%) fungi were isolated from the samples. Two hundred and eight of these (82.86%) were yeasts and 30 (11.95%) were moulds. The fungi were isolated in pure culture (24.77%) or in cultures mixed with bacteria (72.22%). The yeasts isolated were:Cryptococcus spp. (71 strains),Rhodotorula spp. (40),Candida spp. (68),Trichosporon cutaneum (21),Aureobasidium pullulans (7), andPichia ohmeri (1). Moulds classified in following genera were also isolated:Aspergillus (3),Penicillium (3),Alternaria (3),Phoma (3),Epicoccum (2), andGeotrichum (16).     

Diazotrophic mixed culture ofAzotobacter vinelandii andRhodobacter capsulatus

The question, whetherAzotobacter vinelandii can provide fixed N for the growth of other organisms, was studied with mixed cultures ofA. vinelandii andRhodobacter capsulatus, grown with aeration in the light. N₂-fixation byR. capsulatus was prevented by growing the cultures on either mannitol, glycerol or ethanol, which cannot be used by this organism. In the course of growth with mannitol, cell numbers of both organisms increased largely in parallel and attained a maximal ratio of about oneA. vinelandii per tenR. capsulatus. Prolonged growth of mixed cultures with mannitol did not lead to an adaptation ofR. capsulatus to this compound. After growth on either one of the three alcohols, mixed cultures exhibited almost twice as high protein levels as pure cultures ofA. vinelandii. Up to 80% of the protein of mixed cultures was incorporated intoR. capsulatus. The results suggest thatA. vinelandii provided an organic N-source for the growth ofR. capsulatus.     

Fusarium udum parasitic onAspergillus luchuensis andSyncephalastrum racemosum

Summary Mycoparasitic behaviour ofF. udum on two soil-inhabiting microfungi,Aspergillus luchuensis andSyncephalastrum racemosum, was studied in dual cultures.Fusarium udum coiled and penetrated the host fungi and formed chlamydospores inside their hyphae and reproductive structures. The vegetative hyphae ofA. luchuensis showed swellings due to diffusible toxic substances ofF. udum in the medium and formed vesicle-like structures. This is the first record of these fungi being hosts of the mycoparasiteF. udum.     

Division of the generative nucleus in cultured pollen tubes of the Bromeliaceae

Pollen germination, division of the generative nucleus and position of the generative nucleus in the pollen tube during in vitro germination were examined for six bromeliad cultivars. The influence of mixed amino acids (casein hydrolysate) and individual amino acids (Arg, Asn, Asp, Glu, Gly, Met, Phe, Orn, Tyr) were tested. Aechmea fasciata and A. chantinii pollen tubes showed more generative nuclear division in cultured pollen tubes than the other four cultivars tested. Casein hydrolysate did not stimulate generative nuclear division. In general arginine (1 mM) improved division of the Aechmea generative nucleus and to a lesser extent this of Vriesea `Christiane', Guzmania lingulata and Tillandsia cyanea. A concentration of 2 mM arginine reduced pollen tube growth of Aechmea. The vegetative nucleus was ahead of the generative nucleus in approximately 50% of the pollen tubes of all cultivars studied. In about 25% of the pollen tubes, the generative nucleus was ahead and in ±25% pollen tubes the vegetative and generative nuclei were joined together. The distance between the two generative nuclei and the distance from the generative nuclei to the pollen tube tip differed significantly for Aechmea fasciata and A. chantinii. The influence of different amino acids for Aechmea fasciata and A. chantinii varied with respect to pollen germination and generative nuclear division. Arg and Met improved nuclear division of both Aechmea cultivars. Pollen germination and sperm cell production were not linked. This information is important to ameliorate in vitro pollination methods used to overcome fertilization barriers in Bromeliaceae and other higher plants.     

Changes in production of lignin degrading enzymes during interactions between mycelia of the tropical decomposer basidiomycetes Marasmiellus troyanus and Marasmius pallescens

During the interaction of two tropical agaric fungi, Marasmius pallescens and Marasmiellus troyanus, on agar media, initial deadlock between the two mycelia was ultimately followed by take-over by M. troyanus. When shaken liquid cultures of these two fungi were mixed, a rapid increase in laccase and manganese peroxidase activity, but no lignin peroxidase, was detected in the culture supernatant. Even more rapid and elevated induction of laccase occurred when filter-sterilized supernatant of Marasmius pallescens was added to Marasmiellus troyanus cultures, but the reciprocal experiment (addition of M. troyanus supernatant to M. pallescens cultures), did not lead to any increase in laccase activity. Addition of autoclaved supernatant of M. pallescens also induced laccase activity from M. troyanus cultures, but over a period of days rather than hours. Although both M. troyanus, and to a lesser extent M. pallescens, are able to produce laccases in shaken liquid culture following addition of the inducer 2,5-dimethylalanine, these experiments suggest that the presence of heat-stable and heat-labile laccase inducers secreted by M. pallescens mycelia lead to induction of laccases by M. troyanus.     

Detection and identification of ferricrocin produced by ectendomycorrhizal fungi in the genusWilcoxina

The ectendomycorrhizal fungiWilcoxina mikolae isolates CSY-14 and RMD-947 andW. rehmii isolate CSY-85 were grown in pure culture under iron-limiting conditions. All three isolates tested positive for siderophore formation using both the ferric perchlorate assay and a sensitive HPLC iron-binding assay. A peptide siderophore was isolated from the culture medium by HPLC and shown to contain the amino acids serine, glycine and ornithine in a 1:2:3 ratio. This siderophore was identified as ferricrocin on the basis of electrospray mass spectroscopy and its co-chromatography in two different HPLC systems with ferricrocin isolated fromAspergillus fumigatus. Ferricrocin was the only siderophore isolated from theseWilcoxina cultures. This is the first report of siderophore formation by ectendomycorrhizal fungi.     

Effect of oxygen on batch yogurt cultures

A yogurt culture (Streptococcus thermophilus 15HA + Lactobacillus delbrueckii subsp. bulgaricus 2-11) was studied in conditions of aerobic batch fermentation (10–40% dissolved oxygen in milk). The growth and acidification of S. thermophilus 15HA were stimulated at 20% oxygen concentration and the lactic acid process in a mixed culture was shortened by 1 h (2.5 h for the aerobic culture and 3.5 h for the anaerobic mixed culture). Streptococcus thermophilus 15HA oxygen tolerance was significantly impaired at oxygen concentrations in the milk above 30%. Though S. thermophilus 15HA was able to overcome to some extent the impact of high oxygen concentration (40%), the lactic acid produced was insufficient to coagulate the milk casein (4.0 g lactic acid l⁻¹ in the mixed culture and 3.8 g lactic acid l⁻¹ in the pure culture). A dramatic decrease in the viable cell count of L. delbrueckii subsp. bulgaricus 2-11 in the pure and mixed cultures was recorded at 30% dissolved oxygen. This revised version was published online in July 2006 with corrections to the Cover Date.     

Controlled formation of volatile components in cider making using a combination of Saccharomyces cerevisiae and Hanseniaspora valbyensis yeast species

The effect of pure and mixed fermentation by Saccharomyces cerevisiae and Hanseniaspora valbyensis on the formation of major volatile components in cider was investigated. When the interaction between yeast strains of S. cerevisiae and H. valbyensis was studied, it was found that the two strains each affected the cell growth of the other upon inoculation of S. cerevisiae during growth of H. valbyensis. The effects of pure and mixed cultures of S. cerevisiae and H. valbyensis on alcohol fermentation and major volatile compound formation in cider were assessed. S. cerevisiae showed a conversion of sugar to alcohol of 11.5%, while H. valbyensis produced alcohol with a conversion not exceeding 6%. Higher concentrations of ethyl acetate and phenethyl acetate were obtained with H. valbyensis, and higher concentrations of isoamyl alcohol and isobutyl were formed by S. cerevisiae. Consequently, a combination of these two yeast species in sequential fermentation was used to increase the concentration of ethyl esters by 7.41–20.96%, and to decrease the alcohol concentration by 25.06–51.38%. Efficient control of the formation of volatile compounds was achieved by adjusting the inoculation time of the two yeasts.     

Isolation ofTricholoma matsutake andT. bakamatsutake cultures from field-collected ectomycorrhizas

Tricholoma matsutake was isolated into pure cultures from field samples of ectomycorrhizas onPinus densiflora. The mycorrhizal tips were collected at different times of the year from a colony ofT. matsutake in aP. densiflora stand. The mycorrhizal tips were continuously washed with sterilized distilled water and diluted Tween 80 solution, surface-sterilized with calcium hypochlorite solution, and inoculated on several kinds of nutrient agar media. Most of the mycorrhizal tips collected in winter and spring produced colonies that were morphologically similar to cultures ofT. matsutake isolated from basidiocarps. The identity of isolates obtained from mycorrhizas was further confirmed to beT. matsutake based on fungal morphology and RFLP patterns of PCR amplified rDNA. The feasibility ofT. bakamatsutake isolation into pure culture from ectomycorrhizas onQuercus serrata was also confirmed. These results indicated that mycelium of matsutake mushrooms can be isolated into pure culture from ectomycorrhizas at different times of the year. Mycorrhizas of bothT. matsutake andT. bakamatsutake were not observed to have any specific association with soil fungi such asMortierella spp.     

Growth and survival ofAzospirillum brasilense andArthrobacter giacomelloi in binary continuous culture

Summary Azospirillum brasilense andArthrobacter giacomelloi were grown in single and mixed succinate-limited continuous cultures at a partial oxygen pressure of 0.01atm. Growth, viability and survival during nutrient starvation were examined at various dilution rates. At D=0.05 h^–1, K_s values for succinate consumed were calculated.Arthrobacter giacomelloi viability was inversely related to dilution rate whereasAzo. brasilense was directly related. Slightly lower values of viability were obtained in mixed culture, but the ratio between the microorganisms was constant. The survival ofArth. giacomelloi in single culture decreased with increasing growth rate while survival ofAzo. brasilense was directly related to dilution rate. Acetylene reduction activity was generally very low in both single and mixed cultures. Respiration rate was also determined and the mixed culture showed an oxygen uptake rate higher than that of single cultures.Research work supported by CNR, Italy. Special grant I.P.R.A. Sub-project 1. Paper N. 317.     

Frontiers in mycoplasma pathogenicity

Four different strains ofLactobacillus delbrueckii subsp.bulgaricus (Ss₁ and Yop₁₂) andStreptococcus salivarius subsp.thermophilus (Ss₂ and Yop₉) were isolated from two different yogurt sources in Argentina. In medium containing different carbon sources: lactose, fructose, sucrose or glucose plus fructose, the growth of a mixed culture (Yop₁₂+Ss₂) shows stimulation ofS. thermophilus and inhibition ofL. bulgaricus with respect to pure cultures. Both microorganisms in mixed culture grew less well on glucose plus galactose. However, in medium with glucose or galactose, both microorganisms were stimulated.     

Phylogenetic relationships among Puccinia hemerocallidis, P. funkiae, and P. patriniae (Uredinales) inferred from ITS sequence data

Puccinia hemerocallidis and P. funkiae resemble each other morphologically; however, they are biologically and taxonomically distinct, with telia of the former being restricted to species of Hemerocallis and the latter to Hosta species. However, both fungi share a macrocyclic and heteroecious life cycle with Patrinia villosa as the spermogonial and aecial host. An additional microcyclic rust fungus, P. patriniae, is also known on P. villosa. This microcyclic fungus is similar to the two macrocyclic fungi in its telial structure and teliospore morphology. These similarities in morphology and host relationships suggest the three fungi may also share a close evolutionary relationship. To determine the phylogenetic relationships of the three species, a portion of the nuclear ribosomal DNA repeat encoding the ITS and 5.8S subunit regions was amplified by PCR, sequenced, and analyzed. The resulting phylogenetic trees showed that P. hemerocallidis and P. funkiae share a recent common ancestor and that P. patriniae is closely allied with P. hemerocallidis. The results suggest a possible evolutionary derivation of microcyclic P. patriniae from macrocyclic heteroecious P. hemerocallidis, which fits the evolutionary interpretation of correlated species known as Tranzschel's law.