Biomass and Biological Activity during the Production of Compost Used as a Substrate in Mushroom Cultivation

The production of a suitable substrate for the cultivation of the common white button mushroom, Agaricus bisporus, is referred to as composting. High microbiological activity causes temperatures of the composting material to rise as high as 80°C. At stacking, an optimal oxygen consumption rate of 140 μmol of O₂ h⁻¹ g (dry weight)⁻¹ was found in the compost at 50°C, whereas the oxygen consumption rate of the end product was lower at all temperatures tested. No significant differences were observed between biomass content and mineralization rate of ¹⁴C-labeled glutamate of the two composts. Biomass content was shown to be a major function of both temperature and the sampling site position in the stack. On the basis of the results reported here, a minimal composting time of 3.3 days for the phase I process was calculated. Further suggestions are made to reduce the time necessary for the production of a substrate for A. bisporus considerably.     

THE RELATIONSHIP BETWEEN BACTERIAL GROWTH AND PHAGE PRODUCTION

1. The effects of temperature and H-ion concentration on the reaction between antistaphylococcus phage and a susceptible staphylococcus have been studied. 2. The temperature optimum for phage production is in the neighborhood of 35°C. and that for bacterial growth is approximately 40°C. 3. With increasing H-ion concentrations there occur: (a) an increase in the lag phase of bacterial growth without any corresponding increase in the lag phase of phage production; (b) a diminution in the total bacterial population accumulating in the medium without any corresponding drop in the total amount of phage formed. 4. With increasing alkalinity there is no pronounced change in the curves of bacterial growth and phage formation. At pH 8.5 the lytic threshold is increased to about 1000 phage units per bacterium instead of 100–140 as is usually the case and the time of lysis is delayed. 5. By adjusting the medium to pH 6 and 28°C. bacterial growth can be completely inhibited while phage production continues at a rapid rate. 6. Apparently, the previously stressed importance of bacterial growth as the prime conditioning factor for phage formation does not hold, for under certain experimental conditions the two mechanisms can be dissociated.     

Compost Grown Agaricus bisporus Lacks the Ability to Degrade and Consume Highly Substituted Xylan Fragments

The fungus Agaricus bisporus is commercially grown for the production of edible mushrooms. This cultivation occurs on compost, but not all of this substrate is consumed by the fungus. To determine why certain fractions remain unused, carbohydrate degrading enzymes, water-extracted from mushroom-grown compost at different stages of mycelium growth and fruiting body formation, were analyzed for their ability to degrade a range of polysaccharides. Mainly endo-xylanase, endo-glucanase, β-xylosidase and β-glucanase activities were determined in the compost extracts obtained during mushroom growth. Interestingly, arabinofuranosidase activity able to remove arabinosyl residues from doubly substituted xylose residues and α-glucuronidase activity were not detected in the compost enzyme extracts. This correlates with the observed accumulation of arabinosyl and glucuronic acid substituents on the xylan backbone in the compost towards the end of the cultivation. Hence, it was concluded that compost grown A. bisporus lacks the ability to degrade and consume highly substituted xylan fragments.     

Lignocellulolytic Enzymes Produced by Volvariella volvacea, the Edible Straw Mushroom

Volvariella volvacea, commonly known as the straw or paddy mushroom, had the following growth characteristics: minimum temperature, 25°C; optimal temperature, 37°C; maximal temperature, 40°C; pH optimum 6.0. Optimal pH for cellulase production was 5.5. The optimal initial pH for cellulase production and mycelial growth was found to be 6.0. The pH and temperature optima for cellulolytic activity were 5.0 and 50°C, respectively. Maximal cellulolytic activity was obtained within 5 days in shake-flask culture. The cellulases were found to be partly cell free and partly cell bound during growth on microcrystalline cellulose. The endoglucanase activity was primarily extracellular, and β-glucosidase activity was found exclusively extracellularly. Weak cellulase activity was detected when cells were grown on cellobiose and lactose. V. volvacea could not digest the lignin portion of newspaper in shake-flask cultivation. Phenol oxidase, an important enzyme in lignin biodegradation, also was lacking in the cell-free filtrate. However, the organism oxidized phenolic compounds when it was cultured on agar plates containing commercial lignin.     

Temperature-Dependent Growth of Geomyces destructans,the Fungus That Causes Bat White-Nose Syndrome

White-nose syndrome (WNS) is an emergent disease estimated to have killed over five million North American bats. Caused by the psychrophilic fungus Geomyces destructans, WNS specifically affects bats during hibernation. We describe temperature-dependent growth performance and morphology for six independent isolates of G. destructans from North America and Europe. Thermal performance curves for all isolates displayed an intermediate peak with rapid decline in performance above the peak. Optimal temperatures for growth were between 12.5 and 15.8°C, and the upper critical temperature for growth was between 19.0 and 19.8°C. Growth rates varied across isolates, irrespective of geographic origin, and above 12°C all isolates displayed atypical morphology that may have implications for proliferation of the fungus. This study demonstrates that small variations in temperature, consistent with those inherent of bat hibernacula, affect growth performance and physiology of G. destructans, which may influence temperature-dependent progression and severity of WNS in wild bats.     

A New Operation for Producing Disease-Suppressive Compost from Grass Clippings

This study evaluated the use of grass clippings discharged from golf courses as the raw material for production of a suppressive compost to control Rhizoctonia large-patch disease in mascarene grass. Bacillus subtilis N4, a mesophilic bacterium with suppressive effects on the pathogenic fungus Rhizoctonia solani AG2-2, was used as an inoculum in a procedure developed with the aim of controlling composting temperatures and inoculation timing. The population density of mesophilic bacteria in the raw material was reduced to around 5 log₁₀ CFU/g (dry weight) of composting material in the self-heating reaction at the initial stage of composting by maintaining a temperature of 80°C for 1 day. The inoculum was applied immediately, and the composting material was maintained at 40°C for 3 days. This served both to highly concentrate the suppressive bacterium and to achieve sporulation. The temperature was then raised to 60°C and maintained, enabling hygienic, high-speed composting while maintaining the population density of the suppressive bacterium as high as 8 log₁₀ CFU/g (dry weight) in the compost. The suppressiveness of compost made in this way was confirmed in a turf grass disease prevention assay.     

Thermal Inactivation of Bacillus anthracis Spores in Cow's Milk

Decimal reduction time (time to inactivate 90% of the population) (D) values of Bacillus anthracis spores in milk ranged from 3.4 to 16.7 h at 72°C and from 1.6 to 3.3 s at 112°C. The calculated increase of temperature needed to reduce the D value by 90% varied from 8.7 to 11.0°C, and the Arrhenius activation energies ranged from 227.4 to 291.3 kJ/mol. Six-log-unit viability reductions were achieved at 120°C for 16 s. These results suggest that a thermal process similar to commercial ultrahigh-temperature pasteurization could inactivate B. anthracis spores in milk.     

Effects of Temperature, pH, and NaCl on Growth and Pectinolytic Activity of Pseudomonas marginalis

The interaction of temperature (4, 10, 18, and 30°C), pH (6, 7, and 8), and NaCl (0, 2.5, and 5%) and their effects on specific growth rate, lag phase, and pectinolytic enzymes of Pseudomonas marginalis were evaluated. Response surface methodology was adapted to describe the response of growth parameters to environmental changes. To obtain good conditions of storage, the combined action of salt and temperature is necessary. At 4°C with an NaCl concentration of 5% and a pH of 7, the lag time was 8 days and no growth was observed at 4°C with 5% NaCl and a pH of 6. In the absence of salt, P. marginalis could grow regardless of temperature and pH. Pectate lyase and pectin lyase were produced by P. marginalis, while pectin methyl esterase activity was not observed in our culture conditions. The enzyme production depended on temperature, pH, and salt concentration but also on the age of the culture. Pectinolytic enzymes were abundantly excreted during the stationary phase, and even at 4°C, after 2 weeks of storage, enzyme activities in supernatant culture were sufficient to damage vegetables. Both bacterial growth and enzymatic production have to be taken into account in order to estimate correctly the shelf life of vegetables.     

Carbohydrate Level and Growth of Tomato Plants: II. The Effect of Irradiance and Temperature

The growth response of tomato (Lycopersicon esculentum L.) to temperature and irradiance may be related to carbohydrate concentration. Plants in the exponential phase of vegetative growth were grown under temperatures ranging from 9 to 36°C and under low or high irradiances of approximately 110 or 370 microeinsteins per square meter per second photosynthetically active radiation for a 12 hour photoperiod. The relative growth rate, leaf area ratio, net assimilation rate and whole plant carbohydrate levels were measured. At high irradiance, relative growth rate was 43% faster and total nonstructural carbohydrate concentration was 41% greater than at low irradiance. The change in carbohydrate with irradiance could explain the growth response. Plant growth was fastest at 25°C and decreased parabolically at lower and higher temperatures with a half-maximal rate at 13 and 36°C. Total nonstructural carbohydrate decreased between 13 and 23°C and remained constant at higher temperatures. Soluble sugar concentrations varied little with temperature above 13°C except for sucrose, whose level rose above 30°C. The change in carbohydrate with temperature could not explain the growth response. Above 23°C tomato plants appeared to regulate growth rate to maintain a relatively constant nonstructural carbohydrate concentration.     

Use of Gradient Plates To Study Combined Effects of Temperature, pH, and NaCl Concentration on Growth of Monascus ruber van Tieghem, an Ascomycetes Fungus Isolated from Green Table Olives

The effect of temperature, pH, and sodium chloride concentration on the growth of the Ascomycetes fungus Monascus ruber van Tieghem, the main spoilage microorganism during storage of table olives, was studied by using the gradient plate technique. Gradients of NaCl (3 to 9%, wt/vol) at right angles to gradients of pH (2 to 6.8) were prepared for the plates, which were incubated at 25, 30, and 35°C. Visible fungal growth, expressed in optical density units, was recorded by image analysis and graphically presented in the form of three-dimensional grids. Results obtained from the plates indicated that the fungus was salt and acid tolerant, being able to grow at NaCl concentrations of up to 9% (wt/vol) and pH values of as low as 2.2, depending on the incubation temperature. The inhibitory effect of NaCl increased as the pH decreased progressively at 25 and 30°C but not at 35°C. Growth was better at 30 and 25°C as judged by the larger extent of the plates covered by mycelium compared with that at 35°C, where no growth was observed at pHs below 3.7. Differentiation between vegetative (imperfect-stage) and reproductive (perfect-stage) growth was evident on all plates, providing useful information about the effect of environmental conditions on the form of fungal growth. When the growth/no-growth surface model was obtained by applying linear logistic regression, it was found that all factors (pH, NaCl, and temperature) and their interactions were significant. Plots of growth/no-growth interfaces for P values of 0.1, 0.5, and 0.9 described the results satisfactorily at 25 and 35°C, whereas at 35°C the model predicted lower minimum pH values for growth in the range of 7 to 10% NaCl than those observed on the plates. Overall, it is suggested that the fungus cannot be inhibited by any combination of pH and NaCl within the limits of the brine environment, so further processing is required to ensure product stability in the market.     

Transposon insertion mutation of Antarctic psychrotrophic fungus for red pigment production adaptive to normal temperature

Polar regions are rich in microbial and product resources. Geomyces sp. WNF-15A is an Antarctic psy chrotrophic filamentous fungus producing high quality red pigment with potential for industrial use. However, efficient biosynthesis of red pigment can only realize at low temperature, which brings difficult control and high cost for the large-scale fermentation. This study aims to develop transposon insertion mutation method to improve cell growth and red pigment production adaptive to normal temperature. Genetic manipulation system of this fungus was firstly developed by antibiotic marker screening, protoplast preparation and transformation optimization, by which transformation efficiency of ∼50% was finally achieved. Then transposable insertion systems were established using Helitron, Fot1, and Impala transposons. The transposition efficiency reached 11.9%, 9.4%, and 4.6%, respectively. Mutant MP1 achieved the highest red pigment production (OD₅₂₀ of 39) at 14°C, which was 40% higher than the wild-type strain. Mutant MP14 reached a maximum red pigment production (OD₅₂₀ of 14.8) at 20°C, which was about twofold of the wild-type strain. Mutants MP2 and MP10 broke the repression mechanism of red pigment biosynthesis in the wild-type and allowed production at 25°C. For cell growth, eight mutants grew remarkably better (12%∼30% biomass higher) than the wild-type at 25°C. This study established an efficient genetic manipulation and transposon insertion mutation platform for polar filamentous fungus. It provides reference for genetic breeding of psychrotrophic fungi from polar and other regions.     

Mutant of Methylomonas methanolica and Its Characterization with Respect to Biomass Production from Methanol

A stable variant of Methylomonas methanolica, with a higher temperature optimum for growth, was obtained after mutagenic treatment and selection. The mutant strain M13V has an optimum growth temperature of 35 to 37°C and a maximum at 43°C, as compared with 30 and 40°C for the wild strain. Strain M13V and M. methanolica have similar basic characteristics and cell composition. An extracellular polysaccharide is produced by both strains, but this property is more pronounced in strain M13V. In strain M13V the production is favored by high temperature, low growth rate, and oxygen limitation. In continuous culture of strain M13V, the polysaccharide production was partly growth associated and partly independent of the growth rate. The extracellular polysaccharide acted as a flocculating agent. A relationship between polysaccharide concentration and sedimentation rate was found. Biomass production from strain M13V is most effective at 35°C with respect to both growth rate and substrate utilization. It was found that the yield coefficient for methanol was independent of the dilution rate, whereas the yield coefficient for oxygen increased and the production coefficient for carbon dioxide decreased at increasing dilution rates. These results are discussed in connection with the polysaccharide production.     

Temperature Adaptations in the Terminal Processes of Anaerobic Decomposition of Yellowstone National Park and Icelandic Hot Spring Microbial Mats

The optimum temperatures for methanogenesis in microbial mats of four neutral to alkaline, low-sulfate hot springs in Yellowstone National Park were between 50 and 60°C, which was 13 to 23°C lower than the upper temperature for mat development. Significant methanogenesis at 65°C was only observed in one of the springs. Methane production in samples collected at a 51 or 62°C site in Octopus Spring was increased by incubation at higher temperatures and was maximal at 70°C. Strains of Methanobacterium thermoautotrophicum were isolated from 50, 55, 60, and 65°C sites in Octopus Spring at the temperatures of the collection sites. The optimum temperature for growth and methanogenesis of each isolate was 65°C. Similar results were found for the potential rate of sulfate reduction in an Icelandic hot spring microbial mat in which sulfate reduction dominated methane production as a terminal process in anaerobic decomposition. The potential rate of sulfate reduction along the thermal gradient of the mat was greatest at 50°C, but incubation at 60°C of the samples obtained at 50°C increased the rate. Adaptation to different mat temperatures, common among various microorganisms and processes in the mats, did not appear to occur in the processes and microorganisms which terminate the anaerobic food chain. Other factors must explain why the maximal rates of these processes are restricted to moderate temperatures of the mat ecosystem.     

Heat Shock Proteins in Tobacco Cell Suspension during Growth Cycle

Tobacco (Nicotiana tabacum L. cv Wisconsin 38) cells grown in suspension culture at 26°C produce heat shock proteins (HSPs) when exposed to elevated temperature of 34 to 42°C. At 34 and 38°C, synthesis of normal proteins is maintained while HSPs are expressed within 30 minutes after initiation of the shock. At 42°C, HSPs are still expressed but normal proteins are made at a reduced rate or not at all. Exposure of cells to 38°C allows for a full expression of HSPs without inhibition of the synthesis of normal proteins. Induced synthesis of HSPs at 38°C is maximal 1 to 2 hours after elevation of temperature and diminishes thereafter through at least 6 hours. Cells growing asynchronously in the logarithmic phase of growth produce HSPs at a much higher rate than those in the stationary phase. The ability to synthesize HSPs disappears about one generation time before the cells reach a growth plateau.     

Studies on the origin of bacterial viruses. V. The effect of temperature on the terramycin-resistant and phage-producing cells of Bacillus megatherium cultures

The growth rates, the mutation frequency rate constants of the terramycin-resistant cells, the burst size of the phage-producing cells, and the ratio of phage to cells all have a temperature coefficient of about 2 from 20 to 35° (µ = 9 x 10³ calories), with a maximum at 40°. The mutation frequency rate constant (or time rate constant) of the phage-producing cells increases from 20 to 45° with a temperature coefficient of about 3 (µ = 2 to 3 x 10⁴ cal.). The change in the values for the growth rate, mutation rate, and cell volume occurs in less than 1 hour, after the temperature is changed. The value for the burst size of phage-producing cells changes for 3 to 4 hours. Prolonged growth of megatherium 899 at 48 to 50° results in the production of C + S phage, in place of T. Returning the culture to 25° results in the production of small T phage.     

Synergistic Effects of High Hydrostatic Pressure,Mild Heating,and Amino Acids on Germination and Inactivation of Clostridium sporogenes Spores

The synergistic effects of high hydrostatic pressure (HHP), mild heating, and amino acids on the germination of Clostridium sporogenes spores were examined by determining the number of surviving spores that returned to vegetative growth after pasteurization following these treatments. Pressurization at 200 MPa at a temperature higher than 40°C and treatment with some of the 19 l-amino acids at 10 mM or higher synergistically facilitated germination. When one of these factors was omitted, the level of germination was insignificant. Pressures of 100 and 400 MPa were less effective than 200 MPa. The spores were effectively inactivated by between 1.8 and 4.8 logs by pasteurization at 80°C after pressurization at 200 MPa at 45°C for 120 min with one of the amino acids with moderate hydrophobicity, such as Leu, Phe, Cys Met, Ala, Gly, or Ser. However, other amino acids showed poor inactivation effects of less than 0.9 logs. Spores in solutions containing 80 mM of either Leu, Phe, Cys, Met, Ala, Gly, or Ser were successfully inactivated by pasteurization by more than 5.4 logs after pressurization at 200 MPa at 70°C for 15 to 120 min. Ala and Met reduced the spore viability by 2.8 and 1.8 logs, respectively, by pasteurization at a concentration of 1 mM under 200 MPa at 70°C. These results indicate that germination of the spores is facilitated by a combination of high hydrostatic pressure, mild heating, and amino acids.     

Nitrate Transport and Not Photoinhibition Limits Growth of the Freshwater Cyanobacterium Synechococcus Species PCC 6301 at Low Temperature

The effect of low temperature on cell growth, photosynthesis, photoinhibition, and nitrate assimilation was examined in the cyanobacterium Synechococcus sp. PCC 6301 to determine the factor that limits growth. Synechococcus sp. PCC 6301 grew exponentially between 20°C and 38°C, the growth rate decreased with decreasing temperature, and growth ceased at 15°C. The rate of photosynthetic oxygen evolution decreased more slowly with temperature than the growth rate, and more than 20% of the activity at 38°C remained at 15°C. Oxygen evolution was rapidly inactivated at high light intensity (3 mE m⁻² s⁻¹) at 15°C. Little or no loss of oxygen evolution was observed under the normal light intensity (250 μE m⁻² s⁻¹) for growth at 15°C. The decrease in the rate of nitrate consumption by cells as a function of temperature was similar to the decrease in the growth rate. Cells could not actively take up nitrate or nitrite at 15°C, although nitrate reductase and nitrite reductase were still active. These data demonstrate that growth at low temperature is not limited by a decrease in the rate of photosynthetic electron transport or by photoinhibition, but that inactivation of the nitrate/nitrite transporter limits growth at low temperature.     

Fungal Communities Associated with the Biodegradation of Polyester Polyurethane Buried under Compost at Different Temperatures

Plastics play an essential role in the modern world due to their low cost and durability. However, accumulation of plastic waste in the environment causes wide-scale pollution with long-lasting effects, making plastic waste management expensive and problematic. Polyurethanes (PUs) are heteropolymers that made up ca. 7% of the total plastic production in Europe in 2011. Polyester PUs in particular have been extensively reported as susceptible to microbial biodegradation in the environment, particularly by fungi. In this study, we investigated the impact of composting on PUs, as composting is a microbially rich process that is increasingly being used for the processing of green waste and food waste as an economically viable alternative to landfill disposal. PU coupons were incubated for 12 weeks in fresh compost at 25°C, 45°C, and 50°C to emulate the thermophilic and maturation stages of the composting process. Incubation at all temperatures caused significant physical deterioration of the polyester PU coupons and was associated with extensive fungal colonization. Terminal restriction fragment length polymorphism (TRFLP) analysis and pyrosequencing of the fungal communities on the PU surface and in the surrounding compost revealed that the population on the surface of PU was different from the surrounding compost community, suggesting enrichment and selection. The most dominant fungi identified from the surfaces of PU coupons by pyrosequencing was Fusarium solani at 25°C, while at both 45°C and 50°C, Candida ethanolica was the dominant species. The results of this preliminary study suggest that the composting process has the potential to biodegrade PU waste if optimized further in the future.     

Asexual reproduction and vegetative growth of Bionectria ochroleuca in response to temperature and photoperiod

Growth and reproduction are two essential life‐history traits for fungi. Understanding life‐history strategies provides insight into the environmental adaption of species. Here, we investigated the colonial morphology, vegetative growth, and asexual reproduction of the ascomycete fungus Bionectria ochroleuca in response to a variety of environmental conditions. We demonstrated that the increased temperature from 15 to 25°C induced mycelial growth and conidiation in B. ochroleuca. We also found that the optimal temperatures for mycelial growth and conidial formation in this fungus species were 25 and 30°C, respectively. However, as the temperature increased from 25 to 30°C, mycelial growth was suppressed, but the total number of conidia was significantly increased. The shift in light–dark cycles dramatically changed the morphological features of the colonies and affected both vegetative growth and asexual reproduction. Under incubation environments of alternating light and dark (16:8 and 8:16 light:dark cycles), conidiophores and conidia in the colonies formed dense‐sparse rings and displayed synchronous wave structures. When the light duration was prolonged in the sequence of 0, 8, 16, and 24 hr per day, mycelial growth was suppressed, but conidiation was promoted. Together, our results indicate that temperature and light period may trigger a trade‐off between vegetative growth and asexual reproduction in B. ochroleuca.     

Control of Lignin Peroxidase Production by Phanerochaete chrysosporium INA-12 by Temperature Shifting

There are two temperature optima connected with lignin peroxidase synthesis by Phanerochaete chrysosporium INA-12. One, at 37°C, is for the mycelium-growing phase; the other, at 30°C, is for the lignin peroxidase-producing phase. One of six extracellular proteins with ligninase activity increased when cultures were grown at 30°C for the entire fermentation period or when cultures were grown at 37°C for the first 2 days of incubation and then shifted to 30°C, compared with the activity of control cultures grown at 37°C for the entire fermentation period. The unsaturation of fatty acid (Δ/mole) of P. chrysosporium INA-12 mycelium decreased from 1.25 to 1.03 when the growth temperature was shifted from 20 to 40°C.