Optimization of Conditions for Feather Waste Biodegradation by Geophilic Trichophyton ajelloi Fungal Strains towards Further Agricultural Use.

The aim of the study was to optimize culture conditions and medium composition to accelerate the biodegradation of chicken feather waste by keratinolytic soil strains of Trichophyton ajelloi, which are poorly known in this respect, as well as to propose hitherto unconsidered culture conditions for these fungi in order to obtain a biopreparation with a high fertilization value. Different pH of the medium, incubation temperatures, amounts of chicken feathers, additional carbon sources, and culture methods were tested. The process of optimizing keratin biodegradation was evaluated in terms of measuring the activity of keratinase, protease, disulfide reductase, concentration of released soluble proteins and peptides, total pool of amino acids, ammonium and sulfate ions, changes in medium pH, and feather weight loss. It was found that the studied fungal strains were capable of decomposing and mineralizing keratin from feather waste. Regarding the fertilizer value of the obtained hydrolysates, it was shown that the release of sulfate and ammonium ions was highest in a stationary culture containing 2% feathers with an initial pH of 4.5 and a temperature of 28 °C. Days 14-21 of the culture were indicated as the optimal culture time for these fungi to obtain biopreparations of high fertilizing value.

Impact of the Cultivation System and Plant Cultivar on Arbuscular Mycorrhizal Fungi of Spelt (Triticum aestivum ssp. Spelta L.) in a Short-Term Monoculture.

Native communities of arbuscular mycorrhizal fungi (AMF) constitute a natural biofertilization, biocontrol, and bioprotection factor for most agricultural crops, including cereals. The present study investigated the native AMF population in cultivated spelt, i.e., a cereal that has not been analyzed in this respect to date. In particular, the aim of the study was to determine the number of spores and the degree of AMF root colonization in two spelt cultivars (Franckenkorn and Badengold) from a 3-year monoculture grown in two different cultivation systems: conventional tillage and no-tillage systems. The study showed considerable accumulation of AMF spores in the soil (on average 1325 in 100 g of air-dry soil), with a wide range of their numbers, and not a very high degree of endomycorrhizal colonization (on average from 3.0% to 31%). The intensity of AMF growth in the subsequent cultivation years gradually increased and depended on the cultivation system as well as the growth stage and cultivar of the spelt. It was found that both analyzed AMF growth indices in the no-tillage system were positively correlated with each other. Moreover, their values were higher in the no-tillage system than in the conventional system, with statistical significance only for the number of spores. This was mainly observed in the variant with the Franckenkorn cultivar. The effect of the growing season was evident in both cultivation systems and spelt cultivars. It was reflected by intensification of sporulation and mycorrhization of spelt roots by AMF in summer (maturation stage) compared with the spring period (flowering stage).

Occurrence and Distribution of Fusarium Communities in the Root Zone in a Post-Bog Permanent Meadow in Relation to Mineral Fertilization and Growing Seasons.

The present study is the first report of a detailed analysis of the frequency of Fusarium and genera related to Fusarium colonizing the root zone of clovers and grasses growing in a permanent meadow established on peat-muck soil in a post-bog habitat. The isolation of fungi was carried out on the Nash and Snyder medium with the plate dilution method. The taxonomic identification of the collection of pure fungal cultures was based on morphological features revealed by macroscopic and microscopic observations. The species dominance coefficients, Marczewski-Steinhaus and Simpson species diversity index were calculated. Eight Fusarium complexes were distinguished. The distribution of the Fusarium population was uneven, which was generally reflected in a higher frequency of the F. oxysporum species complex in the clover root zone and M. nivale, F. avenaceum from the Fusarium tricinctum species complex, and F. culmorum from the F. sambucinum species complex in the grass root zone. The highest similarity of fungi was determined in the rhizoplane and the endorhizosphere. The highest species diversity and the highest population size were determined in the rhizosphere soil. The fertilization treatment reduced the growth rates in the Fusarium sensu lato and in genera related to Fusarium, as evidenced by the decrease in the total abundance and species richness. The root colonization by the Fusarium, especially the F. oxysporum species complex, was not accompanied by plant pathologies, which suggests a saprotrophic and endophytic rather than parasitic character of the relationships with the plant host.

Dispersal of Aphanoascus keratinophilus by the rook Corvus frugilegus during breeding in East Poland.

The process of dispersal of the potentially disease-causing, geophilic and keratinolytic fungal strain Aphanoascus keratinophilus (the perfect, sexual stage of Chrysosporium keratinophilum) by the rook Corvus frugilegus was studied. The source of A. keratinophilus strains was pellets of the rook, thus far not considered a carrier of this particular opportunistic pathogen. Pellets collected from breeding colonies of rooks were analysed in terms of the occurrence of keratinolytic fungi with the application of the native keratin bait method. Among the 83 rook pellets analysed, 24 (29%) were infected by keratinophilic fungi. Pure cultures of the fungi were identified to species based on traditional morphological features. Traditional mycological identification was verified by the PCR-RFLP molecular identification method as well as DNA sequencing. The obtained results showed the presence of 90 Aphanoascus keratinophilus strains, 6 Chrysosporium tropicum strains, and 3 Chrysosporium pannicola strains. The PCR melting profile (PCR-MP) method was used to identify intraspecies variations of the 90 analysed A. keratinophilus strains. The dispersal of genotypes and possible pathways of A. keratinophilus dispersal and infection via rook pellets were analysed.

Arthroderma tuberculatum and Arthroderma multifidum Isolated from Soils in Rook (Corvus frugilegus) Colonies as Producers of Keratinolytic Enzymes and Mineral Forms of N and S.

Keratinolytic fungi representing the genus Arthroderma that were isolated from the soils of a rook (Corvus frugilegus) colony were used as biological agents for the disposal of waste feathers. The aim of this study was to assess the abilities of Arthroderma tuberculatum and Arthroderma multifidum fungi with a varied inflow of keratin matter to biodegrade waste feathers. The evaluation was based on the determination of feather mass loss, the activity of keratinolytic enzymes, and the content of mineral N and S forms. It was found that the activity of protease released by the fungi contributed to an increase in the level of soluble proteins and peptides and the concentration of ammonium ions, as well as alkalization of the culture medium. Keratinase activity was significantly correlated with sulfate release, especially in A. tuberculatum cultures. The strains of A. tuberculatum fungi isolated from the soil with the highest supply of organic matter, i.e., strains III, IV, and V, had the lowest enzymatic activity, compared to the A. multifidum strains, but they released mineral nitrogen and sulfur forms that are highly important for fertilization, as well as nutritionally important peptides and amino acids. A. tuberculatum strains can be used for the management of waste feathers that can be applied in agricultural practice.

Modification of post-industrial lignin by fungal strains of the genus Trichoderma isolated from different composting stages.

One of the links in the environmental management chain is the environmentally friendly utilization of the emerging post-industrial waste and improvement of the methods of processing thereof. The aim and novelty of this research was to evaluate the potential of fungi to purify wastewater containing post-industrial lignin, i.e. waste originating from the pulp and paper industry. Trichoderma were dominant in the composts with different qualities and quantities of lignocellulosic compounds. The Trichoderma strains used in the research were isolated from two lignocellulosic composts at three different time points (from 10-, 20- and 30-week-old composting mass). Eighteen strains of the genus Trichoderma were tested for their ability to biodegrade 0.2% post-industrial lignin. It was evaluated by determination of decolorization, activities of ligninolytic enzymes, and concentration of phenolic compounds in the post-culture liquid. The Trichoderma strains isolated from 10-week-old compost I and 30-week-old compost II showed the highest decolorization activity and biotransformation of dark post-industrial lignin. All strains secreted horseradish-like peroxidase (HRP-like), superoxide dismutase-like (SOD-like), xylanase, and phenolic compounds. Strains isolated from 30-week-old compost I and from 10-week-old compost II released the greatest amounts of phenolic compounds into the culture liquid containing post-industrial lignin. The strains isolated from 10- and 20-week-old compost were characterized by high SOD-like and HRP-like activity, respectively. The concentration of phenolic compounds measured with HPLC in Trichoderma fungus culture VII from compost I corresponded with the decolorization degree and high HRP-like activity. The study results indicate that the genus Trichoderma with decolorization activity isolated from the first composting stages can be used in the biotransformation of post-industrial lignin waste.

Changes in mineral forms of nitrogen and sulfur and enzymatic activities during composting of lignocellulosic waste and chicken feathers.

The aim of this study was to show the dynamics of changes in the activity of enzymes responsible for C, N, and S metabolism, i.e., cellulase, protease, urease, and arylsulfatase in two lignocellulosic composts as well as changes in the concentration of mineral forms important in plant nutrition (N-NH4+, N-NO3-, S-SO42-). Most of the enzyme activity was higher during 10 weeks of composting in compost I, containing higher amounts of easily available organic matter than in compost II. Enzymatic activities in compost II remained at a higher level for a longer time, but they increased at a slower rate. Mineral content changes in the compost mass consisted primarily of an increase in N-NO3- concentration and a decrease in N-NH4+ and S-SO42- levels, especially in compost I. The concentration of mineral nitrogen and sulfur forms in compost water extracts was about 10-100 times lower than in the compost mass. At the end of composting, the amount of sulfates in the compost mass was 30 and 150 mg kg-1 dw in compost II and I, respectively. In this context, the composts obtained should be considered valuable for fertilizing soils poor in this component and for cultivating plants with high sulfate S demand.

Synthesis of Indoleacetic Acid, Gibberellic Acid and ACC-Deaminase by Mortierella Strains Promote Winter Wheat Seedlings Growth under Different Conditions.

The endogenous pool of phytoregulators in plant tissues supplied with microbial secondary metabolites may be crucial for the development of winter wheat seedlings during cool springs. The phytohormones may be synthesized by psychrotrophic microorganisms in lower temperatures occurring in a temperate climate. Two fungal isolates from the Spitzbergen soils after the microscopic observations and "the internal transcribed spacer" (ITS) region molecular characterization were identified as Mortierella antarctica (MA DEM7) and Mortierella verticillata (MV DEM32). In order to study the synthesis of indoleacetic acid (IAA) and gibberellic acid (GA), Mortierella strains were grown on media supplemented with precursor of phytohormones tryptophan at 9, 15 °C, and 20 °C for nine days. The highest amount of IAA synthesis was identified in MV DEM32 nine-day-culture at 15 °C with 1.5 mM of tryptophan. At the same temperature (15 °C), the significant promoting effect (about 40% root and shoot fresh weight) of this strain on seedlings was observed. However, only MA DEM-7 had the ACC (1-aminocyclopropane-1-carboxylate) deaminase activity with the highest efficiency at 9 °C and synthesized IAA without tryptophan. Moreover, at the same conditions, the strain was confirmed to possess the strong promoting effect (about 40% root and 24% shoot fresh weight) on seedlings. Both strains synthesized GA in all tested terms and temperatures. The studied Mortierella strains had some important traits that led them to be considered as microbial biofertilizers components, improving plant growth in difficult temperate climates.

Microbial strategies and biochemical activity during lignocellulosic waste composting in relation to the occurring biothermal phases.

Typically, hardly-degradable lignocellulosic waste is a component or a co-component of the composted mass. The aim of the work was to present the dynamics and succession of microbial communities during small temperature changes, conditioned by the availability of lignocellulosic polymer and feather waste (the presence of N) in composts with qualitatively and quantitatively different lignocellulosic waste, but most of all, to draw attention to the previously not considered microbial strategies in the composted mass. Decomposition of lignocellulose during composting was similar to the priming effect in the soil, because it was associated with the successive occurrence of two groups of microorganisms. The first group of microorganisms, using easily accessible fraction of the lignocellulose complex, was named the first-strategist group, i.e., non-nutritionally specialized group of microorganisms. The second group, utilizing the hard-to-degrade fraction of lignocellulose, was named second-strategist microorganisms, i.e., nutritionally-specialized group of microorganisms. Biodegradation of the lignocellulose complex in compost I enriched with grass (42.86% pine bark, 34.28% grass, 20.0% sawdust and 2.86% chicken feathers) was faster than in compost II that did not contain any grass, but included more hardly degradable components (25.54% pine bark, 10.63% wheat straw, 51.07% sawdust, 12.76% chicken feathers). In compost I, a higher temperature in the thermophilic phase was recorded; larger amounts of non-specialized mesophilic and thermophilic bacteria in the first weeks of composting and a higher abundance of ligninolytic, xylanolytic fungi and cellulolytic bacteria were observed already in biothermal phase 3 with limited access to easily available C and energy sources. During this period, phosphatase, dehydrogenase and respiratory activities were higher in compost I than compost II. This work demonstrates that the succession of particular groups of microorganisms may help determine the start of biodegradation of recalcitrant ligninocellulosic components during composting.

Microbiological analysis of bioaerosols collected from Hospital Emergency Departments and ambulances.

INTRODUCTION AND OBJECTIVE: The goal of the study was a microbiological, qualitative and quantitative analysis of bioaerosol at the workplace of medical personnel (Health Emergency Departments (HEDs), ambulances), and comparative administration offices with an expected neutral occupational exposure to biological agents measured with individual Button Sampler. MATERIAL AND METHODS: Personal sampling was performed with Button Sampler instrument loaded with gelatine filters in 10 HEDs, in 9 ambulances and in 9 offices to assess the occupational biological agents' exposure in air. Sampling was conducted from March until April 2016. Samples were quantitatively assessed for viable and total number of bacteria and fungi. Routine procedures for microbiological diagnostics were implemented. Data were analysed using Kruskal-Wallis and Mann-Whitney statistical tests with α=0.05. P value less than 0.05 were considered significant. RESULTS: At the workplaces assessed, the concentrations of viable microorganisms in HEDs were 1.3×102 - 4.2×103 CFU/m3 for bacteria, 3.4×100 - 8.1×101 CFU/m3 for fungi; in ambulances 1.3×102 - 1.4×103 CFU/m3 (bacteria), 6.7×100 - 6.5×102 CFU/m3 (fungi) and in offices 4.2×101 - 5.0×103 CFU/m3 (bacteria), 0 - 7.9×102 CFU/m3(fungi). In outdoor air, the number of microorganisms reached the level: 1.0×102 - 5.9×102 CFU/m3 for bacteria and 1.5×102 - 8.2×102 CFU/m3 for fungi. The predominant isolated bacteria were Gram-positive cocci. The prevalent fungi species belonged to the genus Aspergillus and Penicillium. CONCLUSIONS: The quantitative assessment of examined indoor air was similar to control outdoor air, and were relatively low. The level of microbiological contamination did not exceed 5×103 CFU/m3 which is recommended as an admissible level in public spaces in Poland.

Identification and characteristics of biological agents in work environment of medical emergency services in selected ambulances.

OBJECTIVES: Assessment of microbial air quality and surface contamination in ambulances and administration offices as a control place without occupational exposure to biological agents; based on quantitative and qualitative analysis of bacteria, yeasts and filamentous fungi found in collected samples. MATERIAL AND METHODS: The sampling was done by wet cyclone technology using the Coriolis recon apparatus, imprint and swab methods, respectively. In total, 280 samples from 28 ambulances and 10 offices in Warszawa were tested. Data was analyzed using Shapiro-Wilk normality test, Kruskal-Wallis test with α = 0.05. P value ≤ 0.05 was considered as significant. RESULTS: The levels of air contamination were from 0 to 2.3×101 colony-forming unit (CFU)/m3 for bacteria and for yeast and filamentous fungi were from 0 to 1.8×101 CFU/m3. The assessment of office space air samples has shown the following numbers of microorganisms: bacteria from 3.0×101 to 4.2×101 CFU/m3 and yeast and filamentous fungi from 0 to 1.9×101 CFU/m3. For surface contamination the mean bacterial count in ambulances has been between 1.0×101 and 1.3×102 CFU/25 cm2 and in offices - between 1.1×101 and 8.5×101 CFU/25 cm2. Mean fungal count has reached the level from 2.8×100 to 4.2×101 CFU/25 cm2 in ambulances and 1.3×101 to 5.8×101 CFU/25 cm2 in offices. The qualitative analysis has revealed the presence of Acinetobacter spp. (surfaces), coagulase - negative Staphylococci (air and surfaces), Aspergillus and Penicillium genera (air and surfaces). CONCLUSIONS: The study has revealed a satisfactory microbiological quantity of analyzed air and surface samples in both study and control environments. However, the presence of potentially pathogenic microorganisms in the air and on surfaces in ambulances may endanger the medical emergency staff and patients with infection. Disinfection and cleaning techniques therefore should be constantly developed and implemented. Int J Occup Med Environ Health 2017;30(4):617-627.

Biodegradation of feather waste keratin by a keratinolytic soil fungus of the genus Chrysosporium and statistical optimization of feather mass loss.

This paper assesses the ability of strains of Aphanoascus fulvescens and Chrysosporium articulatum isolated from soil (phaesol) to degrade native feather keratin. Strains were identified based on phenotypic traits and nucleotide sequencing. Response Surface Methodology was used to optimize cultivation conditions exhibiting the highest keratinolytic activity. The experiments were based on Box-Behnken designs for the loss of substrate mass (chicken feathers). While substrate mass loss is an "economic coefficient" that reliably indicates feather keratin degradation, it has not been studied before. Stationary liquid cultures of five selected strains were conducted in laboratory conditions at 28 °C using poultry feathers (1 g) as the sole source of carbon, nitrogen and energy. Enzymatic activities, keratin mineralization products and substrate mass loss were determined periodically. The mineralization of keratin proteins by strains yielded a high number of ammonium ions alkalinizing the medium. Increased ammonium ions inhibited the activity of caseinian protease and keratinase. A decrease in the concentration of these ions induced proteolytic enzymes, chiefly the activity of keratinase, at the end of fungal cultivation. Keratinase activity was related to protein- and peptide release and that of caseinian protease to sulfate ions. The highest loss of substrate mass in comparison to the reference strain CBS104.62 (35.4%) was recorded for Aphanoascus fulvescens B21/4-5 (65.9%). Based on a Box-Behnken design, the maximum loss of substrate mass for the Aphanoascus fulvescens strain (71.08%) can be achieved at pH 7.58 and temperature 28.7 °C.

Lignocellulose-degrading enzymes, free-radical transformations during composting of lignocellulosic waste and biothermal phases in small-scale reactors.

Environmentally friendly strategies of waste management are both part of legal solutions currently in place and a focus of interest worldwide. Large-scale composting plants are set up across various regions while home composting is becoming increasingly popular. A variety of microbial groups are successively at work during composting and enzymatic activities detected in the composting mass fluctuate accordingly. Changes in the activities of oxidoreductases and hydrolases, i.e. glucose oxidase, horseradish peroxidase, lignin peroxidase, laccase, xylanase, superoxide dismutase and keratinase, low-molecular weight compounds, i.e. methoxyphenolic and hydroxyphenolic compounds, and the relative level of superoxide radicals and glucose were determined periodically in water extracts of composts to investigate the process of biochemical transformations of ligninocellulose in relation to biothermal phases and to identify a potential priming effect in two composts containing different ratios of lignocellulosic waste and chicken feathers. Composting was conducted for 30weeks. An important aim of the study was to demonstrate that a positive priming effect was induced during composting of a variety of lignocellulosic waste types using native keratin (chicken feathers) as a source of N. The effect was more evident in compost containing grass, which was related to a more rapid depletion of easily available sources of C and energy (glucose) during composting. Ligninolytic enzymes known to biodegrade recalcitrant organic matter were induced in subsequent biothermal phases of composting. Compost I enriched with grass (pine bark, grass, sawdust and chicken feathers) exhibited a higher enzymatic activity than compost II which did not contain any grass but which had a greater number of hardly-degradable components (pine bark, wheat straw, sawdust, chicken feathers). Similar observations were made for the concentrations of low-molecular weight compounds. The enzymes activities and concentration of low-molecular weight compounds listed above can be used to estimate the biodegradation of lignocellulose during composting.

Negative correlation between mycological surfaces pollution in hospital emergency departments and blood monocytes phagocytosis of healthcare workers.

The aim of the present study was to find a possible relationship between the presence of yeast and filamentous fungi in hospital emergency departments and the activity levels of blood granulocytes and monocytes in emergency personnel. The study of mycological pollution was conducted in winter; the samples were collected from 10 Warsaw hospitals emergency departments (HE D) and in 10 control locations (office spaces) and included air samples and swabbing of floor and walls. The blood for immunological investigation was taken in spring, from 40 men, 26 to 53 years old, healthcare workers of these departments, who have been working for at least 5 years in their current positions, and from 36 corresponding controls, working in control offices. Evaluation of blood leukocyte subpopulations was done by hematological analyzer and cytometry analysis and monocyte and granulocyte phagocytosis by Phagotest. There were no significant differences in the level of mycological contamination between the test and control places. The qualitative analysis of the surfaces and air samples revealed a prevalence of strains belonging to Aspergillus spp. and Penicillium spp. genus. Statistical analysis revealed the existence of negative correlation between the number of phagocytizing blood monocytes and fungi spores content on floor and wall surfaces in hospital emergency departments (r = -0.3282, p < 0.05 and positive correlation between the number of phagocytizing monocytes in the blood of office workers and fungi pollution of control offices (r = 0.4421, p < 0.01).

Microsatellite-primed PCR for intra-species genetic relatedness in Trichophyton ajelloi strains isolated in poland from various soil samples.

Trichophyton ajelloi is a geophilic dermatophyte that specializes in the decomposition of native keratin. It exists in soil with a permanent influx of keratin matter. In the present study, two PCR-based methods were used for the identification and intra-species differentiation of T. ajelloi strains isolated from 3 types of soils with different physicochemical properties. The first method, employed for molecular identification, was PCR amplification of the 5.8S rRNA gene and its flanking regions encoding internal transcribed spacers (ITSs), followed by restriction enzyme digestion using endonuclease HinfI. The second method, employed for molecular differentiation, was microsatellite-primed PCR (MSP-PCR) using the repetitive oligonucleotide (GACA)4. All the T. ajelloi strains were also identified using a traditional culture method. Our results showed that molecular identification using the PCR-restriction fragment length polymorphism (PCR-RFLP) method agreed with the identification made using the traditional approach. On the other hand, PCR-RFLP results showed no strain differentiation, while MSP-PCR using the (GACA)4 primer identified different varieties among the T. ajelloi strains. The reasons for the intra-species differentiation of T. ajelloi have been discussed.

Biodegradation of keratin waste: Theory and practical aspects.

Keratin-rich by-products, i.e. bristles, horns and hooves, chicken feathers and similar, are a source of nutrients for animals (amino acids) and plants (N, S). Contemporary developments in the management of keratin waste in feeds and fertilizers comply with human and animal health protection regulations and respect the principles of ecological development. Biotechnological methods employing keratinolytic bacteria and microscopic fungi play a key role in processing keratin waste. This study reviews the current knowledge on the ecology and physiology of keratinolytic microorganisms and presents the biodegradation mechanism of native keratin. The structure and chemical composition of keratin proteins are described, and methods of keratin waste biotransformation into products of practical industrial and natural value, especially composts, are discussed.

Dynamics of growth and succession of bacterial and fungal communities during composting of feather waste.

Succession of communities of different bacteria and fungi, mainly proteolytic and keratinolytic ones, was observed during composting of chicken feathers with pine bark (FB) and with pine bark/rye straw (FBS). The succession was dominated by fungal than bacterial communities. Bacteria, including Actinomycetes, grew intensively during the first 2-4 weeks of composting and included mainly proteolytic, rarely cellulolytic, populations; afterwards, bacteria were gradually replaced by fungi. Meso- and thermophilic ubiquitous fungi, including cellulolytic ones, appeared among fungal representatives as the first, while keratinolytic strains were detected in the compost biomass at the 6th week of the process. The development of strains within the second fungal group was significantly more intensive than that of cellulolytic populations. The intensity of growth of particular ecological-physiological communities was found to be dependent on chemical content and C/N ratio of biomass and was the strongest in C/N=25 composts.

Changes in enzymatic activity in composts containing chicken feathers.

Enzymatic activity, i.e. respiratory activity, dehydrogenase activity, phosphatase activity, caseinian protease activity, BAA protease activity and urease activity, was determined to investigate the process of biochemical transformations and to select enzymatic indices of maturity of composts prepared from feathers and lignocellulose wastes (bark, straw). Composting was conducted for 7 months, with periodic determinations of activity of the enzymes. The study revealed significant differences in the enzymatic activity, related with the duration of composting and with the substrate composition of the composts. Generally, composts enriched with straw were characterised by higher enzymatic activity than composts without any addition of straw. It was found that the activity of such enzymes as cellulase and protease, towards the end of the period of composting decreased and stabilised. The enzymes enumerated can be taken into consideration in estimation of the maturity of composts prepared from feathers and lignocellulose wastes.