Genome and physiology of the ascomycete filamentous fungus Xeromyces bisporus, the most xerophilic organism isolated to date.

Xeromyces bisporus can grow on sugary substrates down to 0.61, an extremely low water activity. Its genome size is approximately 22 Mb. Gene clusters encoding for secondary metabolites were conspicuously absent; secondary metabolites were not detected experimentally. Thus, in its 'dry' but nutrient-rich environment, X. bisporus appears to have relinquished abilities for combative interactions. Elements to sense/signal osmotic stress, e.g. HogA pathway, were present in X. bisporus. However, transcriptomes at optimal (∼ 0.89) versus low aw (0.68) revealed differential expression of only a few stress-related genes; among these, certain (not all) steps for glycerol synthesis were upregulated. Xeromyces bisporus increased glycerol production during hypo- and hyper-osmotic stress, and much of its wet weight comprised water and rinsable solutes; leaked solutes may form a protective slime. Xeromyces bisporus and other food-borne moulds increased membrane fatty acid saturation as water activity decreased. Such modifications did not appear to be transcriptionally regulated in X. bisporus; however, genes modulating sterols, phospholipids and the cell wall were differentially expressed. Xeromyces bisporus was previously proposed to be a 'chaophile', preferring solutes that disorder biomolecular structures. Both X. bisporus and the closely related xerophile, Xerochrysium xerophilum, with low membrane unsaturation indices, could represent a phylogenetic cluster of 'chaophiles'.

Growth inhibition of various Enterobacteriaceae species by the yeast Hansenula anomala during storage of moist cereal grain.

Eleven of 13 Enterobacteriaceae species tested grew in moist stored wheat, highlighting a potential risk of this energy-saving airtight storage method. When Hansenula anomala was coinoculated, all Enterobacteriaceae species were significantly inhibited after 2 months of storage, six of them to below the detection limit.

Description of Holtermanniella gen. nov., including Holtermanniella takashimae sp. nov. and four new combinations, and proposal of the order Holtermanniales to accommodate tremellomycetous yeasts of the Holtermannia clade.

The novel genus Holtermanniella is proposed here to accommodate four Cryptococcus species closely related to Holtermannia corniformis that are included in the Holtermannia clade (Basidiomycota, Agaricomycotina). Thus, four novel combinations are proposed: Holtermanniella nyarrowii comb. nov., Holtermanniella festucosa comb. nov., Holtermanniella mycelialis comb. nov. and Holtermanniella wattica comb. nov. In addition, a novel anamorphic yeast species was studied with 15 isolates obtained from different habitats around the world. Analysis of the sequences of the D1/D2 region of their large subunit rDNA showed that the novel species is placed phylogenetically within the Holtermannia clade of the Tremellomycetes (Agaricomycotina, Basidiomycota). PCR fingerprinting and sequencing of ITS1-5.8S-ITS2 showed genetic intraspecific variability among the strains: three groups were formed, which did not correlate with geographical origin or substrate. This novel species, designated the type species of Holtermanniella gen. nov., is described as Holtermanniella takashimae sp. nov.; the type strain is CBS 11174(T) (=HB 982(T) =DBVPG 8012(T)). The order Holtermanniales ord. nov. is proposed here to include Holtermannia (the type genus) and Holtermanniella.

Pichia anomala J121: a 30-year overnight near success biopreservation story.

Thirty years ago, the ascomycetous yeast Pichia anomala strain J121 was isolated from moist wheat grain stored under conditions of restricted air access. Early observations indicated that an inverse relationship existed between mould and P. anomala colony forming units in grain. This yeast strain was later found to have strong antifungal properties in laboratory, pilot and farm studies with high-moisture wheat under malfunctioning airtight storage. P. anomala had the highest inhibitory activity of 60 yeast species evaluated against the mould Penicillium roqueforti. It also demonstrated strong inhibitory effects against certain Gram-negative bacteria. P. anomala J121 possesses a number of physiological characteristics, i.e. capacity to grow under low pH, low water activity and low oxygen tension and ability to use a wide range of carbon and nitrogen sources, enabling it to act as an efficient biopreservative agent. The biocontrol effect in grain was enhanced by addition of glucose, mainly through formation of the volatile antimicrobial ethyl acetate. Animal feeding trials with P. anomala J121 inoculated grains, fed to chickens and beef cattle, demonstrated that mould control observed in vitro in small scale laboratory experiments could be extended to large scale farm trials. In addition, no adverse effects on animal weight gain, feed conversion, health or behaviour were observed. We have now studied P. anomala J121 biology, ecology and grain preservation ability for 30 years. Over this period, more than 40 scientific publications and five PhD theses have been written on different aspects of this yeast strain, extending from fundamental research on metabolism, genetics and molecular biology, all the way to practical farm-scale level. In spite of the well documented biopreservative ability of the yeast, it has to date been very difficult to create the right constellation of technical, agricultural and biotechnical industries necessary to reach a commercial launch of a P. anomala J121 based biopreservation system. Additionally, the complications caused by a complex EU regulatory system remain a significant barrier to practical applications.

Formulation and stabilisation of the biocontrol yeast Pichia anomala.

The yeast Pichia anomala has antifungal activities and its potential in biocontrol and biopreservation has previously been demonstrated. To practically use an organism in such applications on a larger scale the microbe has to be formulated and stabilised. In this review we give an overview of our experience of formulating and stabilising P. anomala strain J121 in a wider perspective. The stabilisation techniques we have evaluated were liquid formulations, fluidised bed drying, lyophilisation (freeze-drying) and vacuum drying. With all methods tested it was possible to obtain yeast cells with shelf lives of at least a few months and in all cases the biocontrol activity was retained. Fluidised bed drying was dependent on the addition of cottonseed flour as a carrier during the drying process. In liquid formulations a sugar, preferentially trehalose, was a required additive. These two kinds of microbial stabilisation are easily performed and relatively inexpensive but in order to keep the cells viable the biomaterial has to be stored at cool temperatures. However, there is room for optimization, such as improving the growth conditions, or include preconditioning steps to enable the cells to produce more compatible solutes necessary to survive formulation, desiccation and storage. In contrast, lyophilisation and vacuum drying require a lot of energy and are thus expensive. On the other hand, the dried cells were mostly intact after one year of storage at 30°C. Inevitably, the choice of formulation and stabilisation techniques will be dependent also on the intended use.

Past, present and future research directions with Pichia anomala.

The first International Pichia anomala Symposium provided a survey of past, recent and ongoing research on this yeast. The research community working with this yeast has focussed on several areas. Based on molecular data, a revision of the taxonomy is required: the name P. anomala is no longer applicable, as the genus Pichia is polyphyletic. The current debate centres on whether the yeast should be designated as Wickerhamomyces anomalus or if the previous name, Hansenula anomala, should be re-instated. The anti-microbial activities of this yeast received considerable attention during the symposium. H. anomala has been extensively studied as a biopreservation agent in many different post-harvest systems. Several mechanisms account for its anti-microbial activities, including the production of killer proteins and toxic volatile metabolites. Anti-idiotypic antibodies generating an "internal image" of a killer protein have been found to possess therapeutic activity against a broad range of microorganisms. A great diversity of H. anomala strains was reported at the symposium. Strains have been isolated from several food and feed systems and even from the intestine and reproductive organs of a malaria vector (Anopheles stephensi). Feed and food supplemented with certain H. anomala strains show an improved quality due, for example, to the addition of advantageous proteins and phytase activity. However, a number of apparent opportunistic pathogenic strains have also been isolated. Strain differentiation, especially the recognition of potentially pathogenic isolates, is an important challenge for the future commercialisation of this yeast. Future industrial and agricultural application of this yeast also raises questions of the economics of large-scale production, its survival during storage (formulation) and of safety regulations, all of which require further investigation.

Fermentation characteristics of Dekkera bruxellensis strains.

The influence of pH, temperature and carbon source (glucose and maltose) on growth rate and ethanol yield of Dekkera bruxellensis was investigated using a full-factorial design. Growth rate and ethanol yield were lower on maltose than on glucose. In controlled oxygen-limited batch cultivations, the ethanol yield of the different combinations varied from 0.42 to 0.45 g (g glucose)(-1) and growth rates varied from 0.037 to 0.050 h(-1). The effect of temperature on growth rate and ethanol yield was negligible. It was not possible to model neither growth rate nor ethanol yield from the full-factorial design, as only marginal differences were observed in the conditions tested. When comparing three D. bruxellensis strains and two industrial isolates of Saccharomyces cerevisiae, S. cerevisiae grew five times faster, but the ethanol yields were 0-13% lower. The glycerol yields of S. cerevisiae strains were up to six-fold higher compared to D. bruxellensis, and the biomass yields reached only 72-84% of D. bruxellensis. Our results demonstrate that D. bruxellensis is robust to large changes in pH and temperature and may have a more energy-efficient metabolism under oxygen limitation than S. cerevisiae.

Screening of yeast strains for phytase activity.

A screening method was developed to elucidate the ability of different yeast strains to utilize phytic acid as sole phosphorus source. The growth test in liquid culture in a microtiter plate with phytic acid as sole phosphorus source was shown to be a reliable, fast and easy-to-use screening method. We tested 122 strains from 61 species with our method and observed growth differences among species and strains that were not detectable on solid medium. Specific phytase activities were measured for 10 yeasts strains, selected due to their strong growth in the liquid medium. Strains of Arxula adeninivorans and Pichia anomala reached the highest volumetric phytase activities. Arxula adeninivorans also displayed the highest intra- and extracellular specific activities. There were large differences in both extra- and intracellular phytase activities among species. Strain-specific extracellular phytase activities were detected in P. anomala. The presence of free phosphate in the media completely suppressed the extracellular phytase activity and also reduced intracellular phytase activity for all tested yeast strains.

Cryptococcus cerealis sp. nov. a psychrophilic yeast species isolated from fermented cereals.

Two yeast strains isolated in 2007 from fermented pig feed were studied, including the analysis of sequences of the D1/D2 and ITS-regions of the rDNA-repeats, their morphology and nutritional physiology. Sequence comparison of the D1/D2 and ITS regions demonstrated that the strains do not belong to any known species. Therefore, a new species, Cryptococcus cerealis with the type strain CBS 10505, is proposed. The species belongs to Filobasidiales (Agaricomycetes, Basidiomycota), and has Cryptococcus saitoi as the closest related species. The new species is psychrophilic, showing significant growth at 4 and 10 degrees C.

Population diversity of yeasts and lactic acid bacteria in pig feed fermented with whey, wet wheat distillers' grains, or water at different temperatures.

The diversity of populations of yeast and lactic acid bacteria (LAB) in pig feeds fermented at 10, 15, or 20 degrees C was characterized by rRNA gene sequencing of isolates. The feeds consisted of a cereal grain mix blended with wet wheat distillers' grains (WWDG feed), whey (W feed), or tap water (WAT feed). Fermentation proceeded for 5 days without disturbance, followed by 14 days of daily simulated feed outtakes, in which 80% of the contents were replaced with fresh feed mixtures. In WWDG feed, Pichia galeiformis became the dominant yeast species, independent of the fermentation temperature and feed change. The LAB population was dominated by Pediococcus pentosaceus at the start of the fermentation period. After 3 days, the Lactobacillus plantarum population started to increase in feeds at all temperatures. The diversity of LAB increased after the addition of fresh feed components. In W feed, Kluyveromyces marxianus dominated, but after the feed change, the population diversity increased. With increasing fermentation temperatures, there was a shift toward Pichia membranifaciens as the dominant species. L. plantarum was the most prevalent LAB in W feed. The WAT feed had a diverse microbial flora, and the yeast population changed throughout the whole fermentation period. Pichia anomala was the most prevalent yeast species, with increasing occurrence at higher fermentation temperatures. Pediococcus pentosaceus was the most prevalent LAB, but after the feed change, L. plantarum started to proliferate. The present study demonstrates that the species composition in fermented pig feed may vary considerably, even if viable cell counts indicate stable microbial populations.

Verrucine F, a quinazoline from Penicillium verrucosum.

Verrucine F (3), a quinazoline similar to verrucines A (1) and B (2), contains one anthranilic acid residue, one L-glutamine residue, and one alpha,beta-unsaturated phenylalanine residue, as determined by NMR, MS, and chemical methods. Compounds 1 and 3, but not 2, were produced by Penicillium verrucosum J255 and eight additional P. verrucosum strains. Verrucines were typically more concentrated in the inner (older) parts of the colony, and 3 peaked 4-8 days after 1, but at 10-fold lower concentrations (approximately 200 microg/g). Verrucine F (3) formed spontaneously from 1 in buffered water solutions, probably by oxidation at C-1 followed by water elimination.

Antifungal compounds from cultures of dairy propionibacteria type strains.

Antifungal compounds from cultures of five type strains of dairy propionibacteria, as well as from the cultivation medium, were studied. Cell-free supernatants and medium were fractionated by C(18) solid phase extraction. The aqueous 95% acetonitrile fractions were analyzed by GC-MS or subjected to reversed-phase HPLC, to identify, quantify or isolate antifungal substances. The resulting HPLC fractions were screened for antifungal activity against the mold Aspergillus fumigatus and the yeast Rhodotorula mucilaginosa. Active fractions were further separated by HPLC and the structures of the compounds were determined by spectroscopic and chromatographic methods. All five strains produced 3-phenyllactic acid, at concentrations ranging from 1.0 microg mL(-1) (Propionibacterium freudenreichii ssp. shermanii) to 15.1 microg mL(-1) (Propionibacterium thoenii), and at L/D -ratios ranging from 2 : 3 (Propionibacterium acidipropionici) to 9 : 1 (Propionibacterium freudenreichii). A number of active compounds found in cultures of propionibacteria were also present in noninoculated growth medium: two antifungal diketopiperazines, cyclo(L-Phe-L-Pro) and cyclo(L-Ile-L-Pro), and seven antifungal linear peptides. Three of the linear peptides corresponded to sequences found in the medium component casein, suggesting their origin from this component, whereas the diketopiperazines were suggested to be formed from medium peptides by heat treatment.

Production of volatile compounds by Rhizopus oligosporus during soybean and barley tempeh fermentation.

Rhizopus oligosporus Saito can ferment soybeans or cereal grains to tempeh, a sliceable cake with improved nutritional properties. Volatiles produced by different R. oligosporus strains grown on malt extract agar (MEA), barley and soybean were investigated. The effect of co-cultivation with Lactobacillus plantarum on the production of volatiles was also studied. Volatile compounds were collected in situ by headspace diffusion and identified by GC-MS. The ten R. oligosporus strains that had different colony morphologies on MEA produced very similar volatile profiles, except for slight variations among the minor volatile compounds (e.g. sesquiterpenes). Likewise, practically no differences in volatile profiles were observed between three of the strains grown on soybeans. In contrast, the R. oligosporus volatile profile on soybean was different from that on barley from the same strain. Co-cultivation with L. plantarum did not influence volatile production by R. oligosporus. The dominant compounds produced on all three substrates were ethanol, acetone, ethyl acetate, 2-butanone, 2-methyl-1-propanol, 3-methyl-1-butanol and 2-methyl-1-butanol. Acetaldehyde and 2-methyl-propanal were also produced on MEA and barley, while 2-pentanone, methyl acetate, 2-butanol and 3-methyl-3-buten-1-ol were observed on soybeans. Ethanol, 2-methyl-1-butanol and 3-methyl-1-butanol were the most abundant volatile compounds produced on MEA and barley, while 2-butanone was the dominant volatile metabolite on soybean. The mushroom odour compounds, 3-octanone and 1-octen-3-ol, were only detected from soybean and soybean tempeh.

Fungal survival during anaerobic digestion of organic household waste.

Anaerobic digestion of organic waste yields energy rich biogas and retains nutrients (N, P, K, S, etc.) in a stabilised residue. For the residue to be used as a soil fertiliser, it must be free from pollutants and harmful microorganisms. Fungal survival during sanitation and anaerobic treatment of source-separated organic household waste and during aerobic storage of the residue obtained was investigated. Decimal reduction times were determined for inoculated fungi (Aspergillus flavus and Aspergillus fumigatus, Penicillium roqueforti, Rhizomucor pusillus, Thermoascus crustaceus and Thermomyces lanuginosus). Several different fungal species were found after waste sanitation treatment (70 degrees C, 1 h), with Aspergillus species dominating in non-inoculated waste. Anaerobic waste degradation decreased the diversity of fungal species for processes run at both 37 and 55 degrees C, but not total fungal colony forming units. Fungi surviving the mesophilic anaerobic digestion were mainly thermotolerant Talaromyces and Paecilomyces species. T. crustaceus and T. lanuginosus were the only inoculated fungi to survive the thermophilic anaerobic degradation process. Aerobic storage of both types of anaerobic residues for one month significantly decreased fungal counts.

Co-cultivation of antifungal Lactobacillus plantarum MiLAB 393 and Aspergillus nidulans, evaluation of effects on fungal growth and protein expression.

The fungal inhibitory effects of strain Lactobacillus plantarum MiLAB 393, producing broad-spectrum antifungal compounds, were evaluated. A co-cultivation method was set up to monitor effects on fungal growth and protein expression of growing Aspergillus nidulans with L. plantarum MiLAB 393. The effects of inhibitory metabolites produced by L. plantarum MiLAB 393, cyclo(l-Phe-l-Pro), lactic acid and 3-phenyllactic acid, were also investigated by addition of pure compounds to the growth medium of A. nidulans. The co-cultivation strongly affected the morphology of the fungal mycelium and decreased the biomass to 36% of control. Co-cultivation with Lactobacillus coryniformis MiLAB 123 gave only marginal morphological changes and minor biomass reduction, suggesting specific effects of L. plantarum MiLAB 393. The amount of several A. nidulans-proteins was increased during co-cultivation and by all of the inhibiting substances. This study shows that the growth of A. nidulans is inhibited during co-cultivation with L. plantarum MiLAB 393 and that the expression of fungal proteins is altered.

Antifungal effect of dairy propionibacteria--contribution of organic acids.

Large amounts of food and feed are lost every year due to spoilage by moulds and yeasts. Biopreservation, i.e. the use of microorganisms as preservatives instead of chemicals, has gained increased interest. Lactic acid bacteria and propionibacteria might be particularly useful due to their important role in many food fermentations. Knowledge of the antifungal effects of the organic acids produced by these bacteria is necessary to understand their inhibitory activity. We evaluated the antifungal activity of the type strains of five dairy propionibacteria, Propionibacterium acidipropionici, P. jensenii, P. thoenii, P. freudenreichii subsp. freudenreichii and P. freudenreichii subsp. shermanii against eight food- and feedborne moulds and yeasts. A dual culture system assayed the inhibitory activity on three different agar media, sodium lactate (SL), de Man Rogosa Sharp (MRS) and MRS without acetate (MRS-ac). The amounts of organic acids produced during growth of propionibacteria in liquid SL, MRS and MRS-ac were also determined. The minimal inhibitory concentration (MIC) values of propionic, acetic and lactic acid were established for all fungi at pH 3, 5 and 7. Propionic acid, followed by acetic acid, was the most potent antifungal acid. Inhibition at pH 7 generally required concentrations above 500 mM for all three acids, at pH 5 the MIC values for propionic and acetic acids were 20-120 mM and above 500 mM for lactic acid. At pH 3, the MIC values were, with one exception, below 10 mM for both propionic and acetic acid and above 160 mM for lactic acid. The yeast Pichia anomala was the fungus most resistant to organic acids. The propionibacteria exhibited a pronounced species variation in antifungal activity on MRS (+/-acetate) agar, with P. thoenii being the most potent. Four of the five propionibacteria species produced more propionic and acetic acid in liquid SL medium than in MRS (+/-acetate) broth. However, when SL agar was used as the growth medium, none of the propionibacteria inhibited fungal growth.

Growth of lactic acid bacteria and Rhizopus oligosporus during barley tempeh fermentation.

The zygomycete Rhizopus oligosporus is traditionally used to ferment soybean tempeh, but it is also possible to ferment other legumes and cereals to tempeh. The traditional soybean tempeh harbours a multitude of microorganisms with potentially beneficial or detrimental effects on quality. Lactic acid bacteria (LAB) have positive effects on the safety of soybean tempeh, but the effects of LAB on R. oligosporus growth have not been investigated. We have developed a cereal grain tempeh by fermenting pearled barley with R. oligosporus ATCC 64063. Four LAB species, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri and Lactococcus lactis were assessed for their growth abilities and their effects on R. oligosporus growth during barley tempeh fermentation. Growth of LAB was assayed as colony forming units (cfu), while growth of R. oligosporus was measured as ergosterol content and hyphal length. The two fungal measurements highly correlated (r=0.83, P<0.001, n=90). The ergosterol content of fungal mycelia ranged from 11.7 to 30.1 mg/g fungal dry matter. L. plantarum multiplied from 4.8 to 7.4 log cfu/g dry tempeh and L. fermentum increased from 4.4 to 6.8 log cfu/g during 24 h incubation at 35 degrees C. L. reuteri and L. lactis had significantly slower growth, with increases from 4.8 to 5.6 log cfu/g and 5.0 to 5.4 log cfu/g, respectively. The growth of R. oligosporus and the final pH (4.9) in barley tempeh were not significantly influenced by any of the LAB investigated.

Secondary metabolite and mycotoxin production by the Rhizopus microsporus group.

Fast-growing Zygomycetes, most notably Rhizopus oligosporus, are traditionally used in many food fermentations, for example, for soybean tempeh production. R. oligosporus is considered to belong to the Rhizopus microsporus group. Certain R. microsporus strains have been reported to produce either the pharmaceutically active rhizoxins or the highly toxic rhizonins A and B. In this study was investigated the formation of secondary metabolites by R. microsporus, R. oligosporus, and Rhizopus chinensis grown on a wide range of different semisynthetic and natural substrates. Liquid chromatography, combined with photodiode array detection and high-resolution mass spectrometric techniques, was used to identify secondary metabolites. Growth on maize, brown rice, and Pharma agar gave both the highest amounts and the maximum diversity of rhizoxins and rhizonins. Rhizoxins were produced by all four R. microsporus strains, whereas only one strain produced rhizonins. The six R. oligosporus and four R. chinensis strains investigated did not produce any of these two classes of metabolites.

Broad and complex antifungal activity among environmental isolates of lactic acid bacteria.

More than 1200 isolates of lactic acid bacteria isolated from different environments were screened for antifungal activity in a dual-culture agar plate assay. Approximately 10% of the isolates showed inhibitory activity and 4% showed strong activity against the indicator mould Aspergillus fumigatus. The antifungal spectra for 37 isolates with strong activity and five isolates with low or no activity were determined. Several of the strains showed strong inhibitory activity against the moulds A. fumigatus, Aspergillus nidulans, Penicillium commune and Fusarium sporotrichioides, and also against the yeast Rhodotorula mucilaginosa. Penicillium roqueforti and the yeasts Pichia anomala and Kluyveromyces marxianus were not inhibited. Several isolates showed reduced antifungal activity after storage and handling. The majority of the fungal inhibitory isolates were identified by 16S rDNA sequencing as Lactobacillus coryniformis. Lactobacillus plantarum and Pediococcus pentosaceus were also frequently identified among the active isolates. The degree of fungal inhibition was not only related to production of lactic or acetic acid. In addition, antifungal cyclic dipeptides were identified after HPLC separation and several other active fractions were found suggesting a highly complex nature of the antifungal activity.

Influence of ethyl acetate production and ploidy on the anti-mould activity of Pichia anomala.

A diploid and a haploid strain of Pichia anomala were tested for their biocontrol ability against the spoilage mould Penicillium roqueforti in glass tubes filled with grain at two water activities (aw). At aw 0.98, the two yeast strains grew and inhibited mould growth equally well and showed similar patterns of ethyl acetate production, reaching maximum values of 10-14 microg ml(-1) headspace. At aw 0.95, both growth and biocontrol performance of the haploid strain were reduced. Ethyl acetate formation was also substantially reduced, with maximum headspace concentrations of 4 microg ml(-1). We conclude that ethyl acetate is a major component of the anti-mould activity. The inhibitory effect of ethyl acetate was confirmed in a bioassay where the pure compound reduced biomass production of P. roqueforti.