Fungal decomposition of oat straw during liquid and solid state fermentation

White rot fungi (Coriolus hirsutus, Coriolus zonatus, and Cerrena maxima from the collection of the Komarov Botanical Institute of the Russian Academy of Sciences) and filamentous fungi (Mycelia sterilia INBI 2-26 and Trichoderma reesei 6/16) were grown on oat straw-based liquid and solid media, as well as in a bench-scale reactor, either individually or as co-cultures. All fungi grew well on solid agar medium supplemented with powdered oat straw as the sole carbon source. Under these conditions, the mould Trichoderma reesei fully suppressed the growth of all basidiomycetes studied; conversely, Mycelia sterilia neither affected the development of any of the cultures, nor did it show any substantial susceptibility to suppression by their presence. Pure solid cultures of basidiomycetes, as well as the co-culture of Coriolus hirsutus and Cerrena maxima caused a notable bleaching of the oat straw during its consumption. When grown on the surface of oat straw-based liquid medium, the basidiomycetes consumed up to 40% polysaccharides without measurable lignin degradation (a concomitant process). Under these conditions, Mycelia sterilia decomposed no more than 25% lignin in 60 days, but this was observed only after polysaccharide exhaustion and biomass accumulation. In contrast, during solid state straw fermentation, white rot fungi consumed up to 75% cellulose and 55% lignin in 83 days (C. zonarus), whereas the corresponding consumption levels for co-cultures of Mycelia sterilia and Trichoderma reesei equaled 70 and 45%, respectively (total loss of dry weight ranged from 55 to 60%). Carbon dioxide-monitored solid-state fermentation of oat straw by the co-culture of filamentous fungi was successfully performed in an aerated bench-scale reactor.     

Estimation of cell volume and biomass of penicillium chrysogenum using image analysis

A methodology for the estimation of biomass for the penicillin fermentation using image analysis is presented. Two regions of hyphae are defined to describe the growth of mycelia during fermentation: (1) the cytoplasmic region, and (2) the degenerated region including large vacuoles. The volume occupied by each of these regions in a fixed volume of sample is estimated from area measurements using image analysis. Areas are converted to volumes by treating the hyphae as solid cylinders with the hyphal diameter as the cylinder diameter. The volumes of the cytoplasmic and degenerated regions are converted into dry weight estimations using hyphal density values available from the literature. The image analysis technique is able to estimate biomass even in the presence of nondissolved solids of a concentration of up to 30 gL(-1). It is shown to estimate successfully concentrations of mycelia from 0.03 to 38 gL(-1). Although the technique has been developed for the penicillin fermentation, it should be applicable to other (nonpellected) fungal fermentations.     

Image analysis of Daphnia populations: non-destructive determination of demography and biomass in cultures

SUMMARY 1. An image analysis technique was developed for the semiautomatic determination of abundance, size distribution and biomass in Daphnia cultures. This allowed detailed observations of growth, demography and biomass accumulation in live populations, avoiding artifacts caused by subsampling and sampling losses.
2. The image analysis method gave fast, non-destructive and reliable individual counts, even in cultures with high density and a large fraction of juveniles.
3. In Daphnia , animal width changes with nutritional status and growth within instar, while length changes only at the moult. Thus, estimation of individual biomass using an ellipsoidal model based on animal width gave improved biomass calculations compared to manual counting, sizing, and length : weight regressions.
4. The power of the image analysis technique for assessing population growth and size structure was demonstrated in two 40-day experiments, with Daphnia magna feeding on the green algae Selenastrum capricornutum in a two-stage chemostat system.     

Heterologous production of the Piromyces equi cinnamoyl esterase in Trichoderma reesei for biotechnological applications

Aims:  The objective of the study was to produce and characterize the cinnamoyl esterase EstA from the anaerobic fungus Piromyces equi for potential industrial applications.
Methods and Results:  The catalytic domain EstA was produced in Trichoderma reesei. Because the two fungi displayed different genome features, including different codon usage and GC content, a synthetic gene was designed and expressed, leading to the production of the corresponding protein at around 33 mg per litre in the T. reesei culture medium. After the recombinant protein was purified, biochemical characterization showed that EstA presents peak activity at pH 6·5 and at 50–60°C. Furthermore, EstA remained stable at pH 6–8 and below 50°C. EstA was compared to cinnamoyl esterases FaeA and FaeB from Aspergillus niger in terms of ferulic acid (FA) release from wheat bran (WB), maize bran (MB) and sugar beet pulp (SBP).
Conclusion:  The synthetic gene was successfully cloned and overexpressed in T. reesei. EstA from P.   equi was demonstrated to efficiently release FA from various natural substrates.
Significance and Impact of the Study:  Recombinant EstA produced in an industrial enzyme producer, T. reesei , was biochemically characterized, and its capacity to release an aromatic compound (FA) for biotechnological applications was demonstrated.     

Spectrophotometric determination of mycelial biomass

The optical density (450 nm) of samples of homogenized fungal biomass correlated linearly with the dry weight of the biomass in the samples. As shown for broths of the filamentous microfungus Neurospora sitophila, the sensitivity of the technique depended on the extent of fragmentation of fungal hyphae during homogenization: increased fragmentation increased sensitivity. The method applied during all phases of growth, was as accurate as the conventional dry weight technique and permitted rapid and simple measurement of biomass concentration.     

Measurement of biofilm formation by clinical isolates of Escherichia coli is method-dependent

Aims:  In this study, we have evaluated the impact of methodological approaches in the determination of biofilm formation by four clinical isolates of Escherichia coli in static assays.
Methods and Results:  The assays were performed in microtitre plates with two minimal and two enriched broths, with one- or two-steps protocol, and using three different mathematical formulas to quantify adherent bacteria. Different biofilm formation patterns were found depending on the E. coli strain, culture medium and reading optical density on one- and two-steps protocol. Strong or moderate biofilm formation occurred mostly in minimal media. The mathematical formulas used to quantify biofilm formation also gave different results and bacterial growth rate should be taken into account to quantify biofilm.
Conclusions:  Escherichia coli forms biofilms on static assays in a method-dependent fashion, depending on strain, and it is strongly modulated by culture conditions.
Significance and Impact of the Study:  As verified in the studied E. coli strains, biofilm formation by any organism should be cautiously interpreted, considering all variables in the experimental settings.     

Morphological characterization and viability assessment of Trichoderma reesei by image analysis

The production of cellulase from the filamentous fungus Trichoderma reesei is a critical step in the industrial process leading to cellulose ethanol. As a result of the lack of quantitative analysis tools, the intimate relationship that exists between the morphological and physiological states of the microorganism, the shear field in the bioreactor, and the process performance is not yet fully understood. A semiautomatic image analysis protocol was developed to characterize the mycelium morphology and to estimate its percentage viability during the fermentation process based on four morphological types (unbranched, branched, entangled, and clumped microorganisms). Pictures taken under bright field microscopy combined with images of fluorescein diacetate stained fungi were used to assess the morphological parameters and the percentage viability of microorganisms simultaneously. The method was tested during the course of fed-batch fermentation in a reciprocating plate bioreactor. The use of the image analysis protocol was found to be successful in quantifying the variations in the morphology and the viability of T. reesei throughout the fermentation.     

The effect of particle morphology and concentration on the directly measured yield stress in filamentous suspensions

The influence of concentration (mass and volume fraction) and particle morphology on the yielding properties of filamentous broths ofAspergillus niger and Strepto-myces levoris was investigated using the rotating vane technique and compared with those of pulp suspensions. Two methods were applied to determine the volume fraction of the cells growing in filamentous form: the measurement of interstitial volume using a high molecular weight dye or dextran, and the measurement of dewatered broth filter cake volume by displacement using a pycnometer. The latter method provided the most reliable results. Cell concentrations ranging from 3 to 20 g dw/L, with corresponding volume fractions between 0.005 and 0.05, were obtained with broths generated in stirred tank and shake flask fermentations. The yield stress values obtained using the vane technique (0.1 相似文献   

Lipid accumulation in Trichoderma species

Abstract Two filamentous fungi, Trichoderma harzianum and Trichoderma viride , were compared for their ability to synthesize lipids on different carbon and nitrogen sources. Three culture media were selected for each strain after preliminary screening. All the test media were nitrogen-deficient (C/N = 60) so as to stimulate lipid accumulation. For both microorganisms the glucose-ammonium sulphate medium was the most conducive to lipid production: a lipid accumulation of 17% (w/w) of biomass dry weight was obtained for T. harzianum and of 32% (w/w) of biomass dry weight for T. viride . In sucrose-sodium nitrate medium T. harzianum was able to accumulate almost 25% (w/w) of its biomass in lipid form. However the small quantity of biomass produced (2 g dry weight/l) limited the quantity of lipid obtained. Neutral lipids, free fatty acids and phospholipids were monitored during 8 days of cultivation of the two fungi.     

Enhanced in vitro and in vivo antioxidant activity and mobilization of free phenolic compounds of soybean flour fermented with different β-glucosidase-producing fungi

Aims:  To evaluate the soybean polyphenol glucosides bioconversion to aglycone forms by different β-glucosidases-producing filamentous fungi to enhance their antioxidant activity.
Methods and Results:  Soybean defatted flour was submitted to solid-state fermentation with Aspergillus niger , Aspergillus niveus and Aspergillus awamori . The fungi studied produced approximately the same β-glucosidase activity units amount when p- nitrophenyl-β- d -glucopyranoside was used as substrate for the assay. However, electrophoretic analysis, using 4-methylumbellipheryl-β- d -glucopyranoside as substrate, showed that β-glucosidase produced by A.   niveus was more active. Fermented methanolic extracts showed an increase in polyphenol and genistein contents and antioxidant activities. The highest genistein content was found in soybean fermented by A. niveus . Methanolic extracts of the soybean fermented by the different fungi showed a similar capacity of scavenging H₂O₂ generated in vivo by the tumour promoter 12- O- tetradecanoyl phorbol-13-acetate.
Conclusions:  A.   niveus synthesized a β-glucosidase with higher specificity to hydrolyse genistin β-glycosidic bond than those produced by A .  awamori and A. niger .
Significance and Impact of the Study:  The utilization of these β-glucosidases-producing fungi in soybean fermentation processes resulted in the obtaining of methanolic extracts with different antioxidant potentials that could be used either therapeutically or as an antioxidant in nonphysiological oxidative stress conditions, as the one induced in skin by UV radiation.     

Steric hindrance of growth of filamentous fungi in solid substrate fermentation of wheat straw

Cellulolytic fungi, such as Trichoderma reesei, T. lignorum, and Chaetomium cellulolyticum reach a low packing density of mycelia when grown on straw under conditions of solid substrate fermentation. The low packing density is shown to be caused partially by the geometric limitation of the growth of mycelia in the substrate and particularly in its pores, but the exact contribution of this limitation and other limitations such as mass transfer and substrate availability cannot be easily distinguished. The combined effect of such limitations is called steric hindrance. This steric hindrance is associated with and may be the principal cause of low biomass concentration in SSF.     

Continuous growth of the giant grass Zizaniopsis bonariensis in subtropical wetlands

1.  Zizaniopsis bonariensis (giant grass) is an emergent macrophyte species endemic to subtropical wetlands in Brazil, Argentina, Paraguay and Uruguay. In this study, we show the effects of its continuous clonal reproduction and its 'phalanx' growth strategy in the Taim Wetland (southern Brazil).
2. The continuous clonal growth of this 3-m high grass gave rise to the emergence of 11.7 new shoots m⁻² per month and a high total above ground production (2870 g dry weight m⁻² year⁻¹). The biomass of the new shoots emerging every month formed a wave of growth, moderated by only weak seasonal variation.
3. We show its phalanx growth strategy by analysing the variations in population density and shoot height within a transect through the stand canopy. The inverse relation between density and height from the border to the interior indicates self-regulation of biomass.
4. The plants modified their environment, enhancing resistance to drought within the stand and thus facilitating their dominance. This positive feedback suggests that the dominance of the plant might constitute an alternative state in subtropical wetlands.     

Quantifying metabolic activity of filamentous fungi using a colorimetric XTT assay

The filamentous nature and robust cell walls of many fungi render traditional measurements of active biomass (e.g., turbidity, dry cell weight) ineffective for most fungal bioprocesses. To overcome this challenge, an assay for quantification of overall metabolic activity is developed using 2,3-bis(2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT), which in the presence of active mitochondria is converted to a water-soluble formazan derivative that absorbs light in the visible spectrum (430-490 nm). Tests on the model fungus Aspergillus nidulans show that in actively growing cultures XTT absorbance is linearly related to dry cell weight below 0.2 g/kg broth. Validation through growth rate testing shows the developed XTT assay is able to accurately quantify reductions in culture metabolism during damaging physical treatment (heat, high shear, microwaving). Experiments in batch culture demonstrate that the developed XTT assay is capable of reporting on metabolic activity where dry cell weight is not. The developed assay is inexpensive, relatively rapid, and easy to conduct, making it ideally suited for assessment of fungal processes in the biotechnology industry.     

Multiple approaches to assess filamentous bacterial growth in activated sludge under different carbon source conditions

Aims:  To compare molecular and microscopic approaches in determining which filamentous bacteria grow in activated sludge reactors when different carbon sources and different activated sludge mixed liquor inocula are used.
Methods and Results:  Microscopic and molecular (Denaturing Gradient Gel Electrophoresis and Fluorescent In Situ Hybridization) techniques were used to determine which filamentous bacteria became dominant in lab scale reactors treating wastewater composed of different carbon sources. Molecular analysis indicated the presence of Sphaerotilus natans and Thiothrix -related organisms . Microscopy indicated the presence of Nostocoida limicola in some reactors. Sludge volume index increased as filament abundance increased. The detection level of DGGE analysis increased when the abundance levels of the filaments were high.
Conclusions:  Simultaneous application of traditional and molecular methods was effective, and highlighted the advantages and limitations of each method. Readily biodegradable substances favoured the growth of specific filaments in a mixed liquor environment. The origin of inoculum influenced which specific filamentous bacteria grew.
Significance and Impact of the Study:  The study shows the potential problems when using particular techniques, and highlights the need for multiple approaches when studying filaments. The study also provides more information on which filaments will grow under different carbon source conditions for a given inoculum.     

Variation in arbuscular mycorrhizal fungi colonization modifies the expression of tolerance to above-ground defoliation

1.  Plant association with arbuscular mycorrhizal fungi (AMF) has been considered a factor increasing plant tolerance to herbivory. However, this positive effect could decrease with colonization density if the benefit : cost ratio of the AMF–plant association changes. We measured plant performance and tolerance to defoliation across a gradient of commercial AMF ( Glomus sp.) inoculum concentration.
2.  Six genetic families of Datura stramonium were grown under greenhouse conditions and subjected to five increasing levels of AMF inoculum concentration and to defoliation treatments, i.e. the presence/absence of 50% artificial damage, following a full-factorial design.
3.  AMF colonization increased linearly with inoculum concentration while foliar area, root mass, flowering phenology and seed production expressed nonlinear functions. Plant genetic variation in the benefit function of AMF colonization was also detected. We show a negative interaction between AMF concentration and plant tolerance to defoliation.
4.   Synthesis . The negative correlation between plant tolerance and AMF concentration suggests that defoliation can reduce AMF benefits and that natural variations in AMF can limit the evolution of optimum levels of tolerance. Moreover, genetic variation in the shape of the reaction norms to AMF in the presence/absence of defoliation suggests that plants may evolve in response to variation in densities of AMF and herbivores.     

Nutrient dynamics and successional changes in a lentic freshwater biofilm

SUMMARY 1. Colonisation, species composition, succession of microalgae and nutrient dynamics in biofilms grown under light and dark conditions were examined during the initial phases of biofilm development in a lentic freshwater environment.
2. Biofilms were developed on inert (perspex) panels under natural illuminated and experimental dark conditions and the panels were retrieved for analysis after different incubation periods. Analysed parameters included biofilm thickness, algal density, biomass, chlorophyll a , species composition, total bacterial density and nutrients such as nitrite, nitrate, phosphate and silicate.
3. Biofilm thickness, algal density, biomass, chlorophyll a and species richness were significantly higher in light-grown biofilms, compared with dark-grown biofilms. The light-grown biofilms showed a three-phased succession pattern, with an initial domination of Chlorophyceae followed by diatoms (Bacillariophyceae) and finally by cyanobacteria. Dark-grown biofilms were mostly dominated by diatoms.
4. Nutrients were invariably more concentrated in biofilms than in ambient water. Nutrient concentrations were generally higher in dark-grown biofilms except in the case of phosphate, which was more concentrated in light-grown biofilms. Significant correlations between nutrients and biofilm parameters were observed only in light-grown biofilms.
5. The N : P ratio in the biofilm matrix decreased sharply in the initial 4 days of biofilm growth; ensuing N-limitation status seemed to influence biofilm community structure. The N : P ratios showed significant positive correlations with the chlorophycean fraction in both light and dark-grown biofilms, and low N : P ratio in the older biofilms favoured cyanobacteria. Our data indicate that nutrient chemistry of biofilm matrix shapes community structure in microalgal biofilms.     

Oviposition habitat selection for a predator refuge and food source in a mosquito

Abstract.  1. The influence of filamentous algae on oviposition habitat selection by the mosquito Anopheles pseudopunctipennis and the consequences of oviposition decisions on the diet, development, body size, and survival of offspring were examined.
2. A natural population of An. pseudopunctipennis in Chiapas, Mexico, oviposited almost exclusively in containers with filamentous algae. Algae represented 47% of the gut contents of mosquito larvae sampled from the natural population. Mosquito larvae fed on an exclusive diet of algae developed as quickly and achieved the same adult body size (wing length) as their conspecifics fed on a standard laboratory diet.
3. Multiple regression of survival of mosquito larvae on percentage surface area cover of algae (0–99%) and the density of predatory fish (zero to four fish per container) was best described by a second-order polynomial model. Increasing fish densities resulted in a reduction in mosquito survival in all algal treatments. The highest incidence of survival was observed at intermediate (66%) algal cover in all treatments.
4. The presence of fish significantly extended larval development times whereas algal cover had no significant effect. The presence of fish resulted in emergence of smaller adults due to reduced feeding opportunities and predator avoidance behaviour. Algal cover also affected mosquito wing length but differently at each fish density.
5. Oviposition habitat selection improves survival in the presence of predators and feeding opportunities for An. pseudopunctipennis larvae.     

Real-time PCR detection and quantification of fish probiotic Phaeobacter strain 27-4 and fish pathogenic Vibrio in microalgae, rotifer, Artemia and first feeding turbot (Psetta maxima) larvae

Aims:  To develop a SYBR Green quantitative real-time PCR protocol enabling detection and quantification of a fish probiotic and two turbot pathogenic Vibrio spp. in microcosms.
Methods and Results:  Phaeobacter 27-4, Vibrio anguillarum 90-11-287 and Vibrio splendidus DMC-1 were quantified as pure and mixed cultures and in presence of microalgae ( Isochrysis galbana ), rotifers ( Brachionus plicatilis ), Artemia nauplii or turbot ( Psetta maxima ) larvae by real-time PCR based on primers directed at genetic loci coding for antagonistic and virulence-related functions respectively. The optimized protocol was used to study bioencapsulation and maintenance of the probiont and pathogens in rotifers and for the detection and quantification of Phaeobacter and V. anguillarum in turbot larvae fed rotifers loaded with the different bacteria in a challenge trial.
Conclusions:  Our real-time PCR protocol is reproducible and specific. The method requires separate standard curve for each host organism and can be used to detect and quantify probiotic Phaeobacter and pathogenic Vibrio bioencapsulated in rotifers and in turbot larvae.
Significance and Impact of the Study:  Our method allows monitoring and quantification of a turbot larvae probiotic bacteria and turbot pathogenic vibrios in in vivo trials and will be useful tools for detecting the bacteria in industrial rearing units.