A new filter for cell separation

Summary A novel filter housing has been designed that permits the use of a coarse sintered stainless steel cylindrical filter element as a cross-flow filtration unit. Cultures of the fungal micro-organism Trichoderma reesei have been used to study the performance characteristics of the unit and filtration rates of 1.0m³/m²/h have been achieved in a sterile manner.     

Indigenous bacteria with antagonistic and plant-growth-promoting activities improve slow-filtration efficiency in soilless cultivation

In tomato soilless culture, slow filtration allows one to control the development of diseases caused by pathogenic microorganisms. During the disinfecting process, microbial elimination is ensured by mechanical and biological factors. In this study, system efficacy was enhanced further to a biological activation of filter by inoculating the pozzolana grains contained in the filtering unit with 5 selected bacteria. Three strains identified as Pseudomonas putida and 2 as Bacillus cereus came from a filter whose high efficiency to eliminate pathogens has been proven over years. These 5 bacteria displayed either a plant growth promoting activity (P. putida strains) or antagonistic properties (B. cereus strains). Over the first months following their introduction in the filter, the bacterial colonisation of pozzolana grains was particularly high as compared to the one observed in the control filter. Conversely to Bacillus spp. populations, Pseudomonas spp. ones remained abundant throughout the whole cultural season. The biological activation of filter unit very significantly enhanced fungal elimination with respect to the one displayed by the control filter. Indeed, the 6-month period needed by the control filter to reach its best efficacy against Fusarium oxysporum was shortened for the bacteria-amended filter; in addition, a high efficacy filtration was got as soon as the first month. Fast colonization of pozzolana grains by selected bacteria and their subsequent interaction with F. oxysporum are likely responsible for filter efficiency. Our results suggest that Pseudomonas spp. act by competition for nutrients, and Bacillus spp. by antibiosis and (or) direct parasitism. Elimination of other fungal pathogens, i.e., Pythium spp., seems to differ from that of Fusarium since both filters demonstrated a high efficacy at the experiment start. Pythium spp. elimination appears to mainly rely on physical factors. It is worth noting that a certain percentage of the 5 pozzolana-inoculated bacteria failed to colonise the filter unit and were, thus, driven to the plants by the nutrient solution. Their contribution to the establishment of a beneficial microbial community in the rhizosphere is discussed.     

Automation of microbial enumeration: development of a disposable hydrophobic grid-membrane filter unit.

A disposable filter unit containing a hydrophobic grid-membrane filter (HGMF) was developed. The unit is liquid tight to serve as a specimen transport container and, by removal of the funnel extender (175- or 300-ml capacity), the unit becomes less than the height of two stacked petri plates to save space during in situ incubation. The polyethylene mesh which supports the HGMF facilitates rinse removal of any substance(s) that would interfere with microbial growth. The correlations between a pour plate, a conventional square HGMF, and a disposable filter unit on microbial enumeration were examined. Characteristics (e.g., clumping, spreading, etc.) of some microorganisms limit the linear counting range to less than 1,000 CFU per filter.     

Automation of microbial enumeration: development of a disposable hydrophobic grid-membrane filter unit

A disposable filter unit containing a hydrophobic grid-membrane filter (HGMF) was developed. The unit is liquid tight to serve as a specimen transport container and, by removal of the funnel extender (175- or 300-ml capacity), the unit becomes less than the height of two stacked petri plates to save space during in situ incubation. The polyethylene mesh which supports the HGMF facilitates rinse removal of any substance(s) that would interfere with microbial growth. The correlations between a pour plate, a conventional square HGMF, and a disposable filter unit on microbial enumeration were examined. Characteristics (e.g., clumping, spreading, etc.) of some microorganisms limit the linear counting range to less than 1,000 CFU per filter.     

Effects of ruminal protozoa on cellulose degradation and the growth of an anaerobic ruminal fungus, Piromyces sp. strain OTS1, in vitro.

An anaerobic rumen fungus, Piromyces sp. strain OTS1, was incubated in the presence or absence of a mixed, A-type, protozoal population obtained from a goat, in a medium containing filter paper cellulose as energy source and antibiotics to suppress bacterial growth. Fermentation end products, cellulose degradation, and chitin as an indicator of fungal biomass were examined. In the presence of protozoa, total volatile fatty acids, notably propionate and butyrate, increased, and lactate decreased. In fungus-protozoan coincubations, formate was not detected at the end of the experiment and the amount of reducing sugars remained low throughout the incubation period. The fungal growth in the coincubations was negatively affected. While protozoal predation on zoospores was one mechanism of inhibition, mature fungal cells were also affected. Total cellulose degradation was greater in fungal monocultures, but the amount of cellulose degraded per unit of fungal biomass was 25% larger in the coincubations. The negative effects that the protozoal predatory activity had on the fungal growth and subsequently on the amount of cellulose degraded by Piromyces sp. strain OTS1 were partially attenuated by the protozoal fibrolytic activity or by an enhanced fungal activity due to a more favorable environment.     

Biomass accumulation and carbon utilization in layered sand filter biofilm systems receiving milk fat and detergent mixtures

This research evaluated utilization of organic compounds and biomass accumulation in three different layered sand filter biofilm systems renovating a mixture of detergent and milk fat. Organic compounds were measured as chemical oxygen demand (COD) and biomass as ATP. A two-layer sand filter with coarse sand on the top and fine sand at the bottom; a three-layer sand filter with pea gravel on the top, coarse sand in the middle, and fine sand at the bottom; and a two-filters in-series sand filter biofilm system each received mixtures of butterfat and detergent. While the three-layer sand filter system had greater COD utilization capacity due to a longer filter run, the two-filters in-series sand filter system had greater COD utilization efficiency. The COD utilization was highly associated with the period of filter run to clogging. The decrease of COD utilization rate could lead to the clogging of sand filters. To maintain long filter runs without clogging, a COD loading rate of 2100 mg O(2)/m(2)/h was recommended for three-layer sand filters and 1800 mg O(2)/m(2)/h for two-layer sand filters for high fat content wastewater. In the two-layer sand filters and the three-layer sand filters, biomass did not seem to accumulate in any particular layer on a unit volume of sand. However, based on unit surface area, the coarse sand layer accumulated more biomass than the fine sand layer and the pea gravel layer.     

Fungal Colonization of Air Filters and Insulation in a Multi-Story Office Building: Production of Volatile Organics

Secondary air filters in the air-handling units on four floors of a multi-story office building with a history of fungal colonization of insulation within the air distribution system were examined for the presence of growing fungi and production of volatile organic compounds. Fungal mycelium and conidia of Cladosporium and Penicillium spp. were observed on insulation from all floors and both sides of the air filters from one floor. Lower concentrations of volatile organics were released from air filter medium colonized with fungi as compared with noncolonized filter medium. However, the volatiles from the colonized filter medium included fungal metabolites such as acetone and a carbonyl sulfide-like compound that were not released from noncolonized filter medium. The growth of fungi in air distribution systems may affect the content of volatile organics in indoor air. Received: 2 June 1997 / Accepted: 13 June 1997     

Chemistry and microbial activity of forest and pasture riparian-zone soils along three Pacific Northwest streams

Throughout the United States, agricultural practices are responsible for large quantities of nutrients entering lakes and streams. Previous studies have shown that forested riparian areas can filter nutrients from surface runoff and groundwater that may potentially contaminate lakes and streams. This study examined seasonal differences in soil chemistry and soil microorganisms in paired mixed-forest riparian and pasture systems, the aim being to gain understanding of the sequestering of N and P. The forest soils retained higher levels of organic C and N, mineralizable N, extractable P, and fungal biomass, and had higher respiration rates than pasture soils. These findings suggest that forested riparian zones have a greater capacity than pasture soils to sequester C and retain nutrients. In past studies, fungal biomass has been shown to be less than bacterial biomass in grassland soils, but in this study, fungal biomass was greater than bacterial biomass throughout the year in both forest and pasture soils.     

Environmental filtering structures fungal endophyte communities in tree bark

The factors that control the assembly and composition of endophyte communities across plant hosts remains poorly understood. This is especially true for endophyte communities inhabiting inner tree bark, one of the least studied components of the plant microbiome. Here, we test the hypothesis that bark of different tree species acts as an environmental filter structuring endophyte communities, as well as the alternative hypothesis, that bark acts as a passive reservoir that accumulates a diverse assemblage of spores and latent fungal life stages. We develop a means of extracting high‐quality DNA from surface sterilized tree bark to compile the first culture‐independent study of inner bark fungal communities. We sampled a total of 120 trees, spanning five dominant overstorey species across multiple sites in a mixed temperate hardwood forest. We find that each of the five tree species harbour unique assemblages of inner bark fungi and that angiosperm and gymnosperm hosts harbour significantly different fungal communities. Chemical components of tree bark (pH, total phenolic content) structure some of the differences detected among fungal communities residing in particular tree species. Inner bark fungal communities were highly diverse (mean of 117–171 operational taxonomic units per tree) and dominated by a range of Ascomycete fungi living asymptomatically as putative endophytes. Together, our evidence supports the hypothesis that tree bark acts as an environmental filter structuring inner bark fungal communities. The role of these potentially ubiquitous and plant‐specific fungal communities remains uncertain and merits further study.     

Biodiversity and concentration of airborne fungi in a hospital environment

The biodiversity and concentration of airborne fungi were monitored over a period of 6 months in a special-care unit of a hospital. Air sampling was performed in a corridor that was also accessible to visitors and in an adjacent bone-marrow transplantation (BMT) unit using an air sampler and two isolation media. Altogether, 98 fungal species could be identified, among them Aspergillus fumigatus and A. terreus as well as 48 other species reported as potential pathogens. The average contamination values of the corridor air ranged from 124 to 485 cfu m⁻³. Neither the degree of fungal air contamination nor the species composition inside the special care unit differed from those found in the corridor. By means of data obtained with a light-activated sensor, a possible influence of human activities on diurnal changes of fungal propagule concentration was shown. This revised version was published online in July 2006 with corrections to the Cover Date.     

棉秸秆降解高温菌株的筛选及产酶分析

从新疆地区分离具有降解棉秸秆纤维素功能的菌株,得到4株耐高温真菌(50°C)。纤维素酶学性质分析表明,该4株菌的纤维素酶具有良好的耐酸性(最适pH为4.5)和耐高温性(最高达60°C)。以羧甲基纤维素钠(CMC-Na)、微结晶纤维素、棉花、滤纸、淀粉、果胶为底物测定酶活力,滤纸酶活力(FPA)最高达2.63 U/mL、淀粉酶活力最高达6.17 U/mL、果胶酶活力最高达5.86 U/mL。4株真菌酶学特性分析表明,该系列菌株在秸秆生物质利用方面有很大的应用潜力。     

Development of a novel disposable filter bag for separation of biomolecules with functionalized magnetic particles

The integration of disposable magnetic filters in combination with functionalized magnetic particles represents a fast and cost‐effective alternative for enzyme purification in comparison to solid/liquid separation by means of centrifugation followed by chromatographic purification. The main advantage of the particle‐based process is the solid/solid/liquid separation in one step combined with disposable equipment. Furthermore this combination provides the possibility to also process biocatalytic reactions in cell‐containing media into disposable equipment with preimmobilized enzymes onto the magnetic particles. The focus of the presented study is on the design and performance of a disposable filtration unit consisting of a plastic bag with an inlet and outlet and a stainless steel filter matrix. During magnetic separation, the magnetic particles selectively retard at the filter matrix due to the magnetic force, which counteracts the drag force. It was found that the length of a lengthwise aligned filter matrix should be longer than the magnetic pole surfaces in fluid flow direction. Hereby, a filtration capacity of 5.6 g magnetic particles was measured with a loss of below 0.5%. Introducing a two‐phase flow optimizes the cleaning of the bag after a magnetic filtration. The procedure offered a high cleaning efficiency. Herewith, the cleaned filter unit could be discarded with minimum losses of product and magnet particles.     

Topography-specific emergence of fungal fruiting bodies in warm temperate evergreen broad-leaved forests on Yakushima Island,Japan

We conducted line route censuses of fungal fruiting bodies from August to September in 2005 and 2006 along ridges and valleys and compared the differences in the encounter rates of fungal fruiting bodies (= fruiting bodies seen per census kilometer) between types of topography and between fungal functional groups (i.e., ectomycorrhizal and saprobic fungi) in warm temperate evergreen broad-leaved forests on Yakushima Island, Japan. We found 251 fungal fruiting bodies (26 families, 50 genera, and 65 species) in total, including 51 bodies from Tricholomataceae, 41 from Russulaceae, 25 from Boletaceae, and 19 from Amanitaceae. The encounter rate of ectomycorrhizal fungi was greater at the ridge route (26.7 unit/km) than at the valley route (8.7 unit/km) and that of saprobic fungi was greater at the valley route (25.0 unit/km) than at the ridge route (12.5 unit/km). In addition, we conducted 7-year intermittent sampling and identified 40 families, 96 genera, and 142 species. The topography-specific emergence pattern of the intermittent sampling method was similar to that of the line census method. The fungal species composition in this study was possibly affected by a topographic gradient for both fungal functional groups through soil moisture, nutrient availability, and host tree distribution.     

Improvement of Trichoderma strains for biocontrol

The use of the fungal genus Trichoderma to control fungal plant diseases is a promising alternative to the use of chemical compounds. The aim of this work has been to obtain Trichoderma strains with improved capacity as biological control agents. To do so, the hydrolytic capacity on fungal cell walls of strains of the fungus Trichoderma harzianum has been increased. On one hand, transformation experiments with genes which coded for chitinases and glucanases have been carried out in T. harzianumstra ins. On the other hand, the medium composition has also been modified in order to eliminate proteolytic degradation of some of the overproduced enzymes. Finally, hybrid chitinolytic enzymes with substrate-binding domains have been produced as an alternative to obtain improved biocontrol strains. The transformant strains, when compared with the wild type, showed improved antifungal capacity against the phytopathogenic fungus Rhizoctonia solani, in in vitro experiments.     

Ectomycorrhizal fungal response to warming is linked to poor host performance at the boreal‐temperate ecotone

Rising temperatures associated with climate change have been shown to negatively affect the photosynthetic rates of boreal forest tree saplings at their southern range limits. To quantify the responses of ectomycorrhizal (EM) fungal communities associated with poorly performing hosts, we sampled the roots of Betula papyrifera and Abies balsamea saplings growing in the B4Warmed (Boreal Forest Warming at an Ecotone in Danger) experiment. EM fungi on the root systems of both hosts were compared from ambient and +3.4 °C air and soil warmed plots at two sites in northern Minnesota. EM fungal communities were assessed with high‐throughput sequencing along with measures of plant photosynthesis, soil temperature, moisture, and nitrogen. Warming selectively altered EM fungal community composition at both the phylum and genus levels, but had no significant effect on EM fungal operational taxonomic unit (OTU) diversity. Notably, warming strongly favored EM Ascomycetes and EM fungi with short‐contact hyphal exploration types. Declining host photosynthetic rates were also significantly inversely correlated with EM Ascomycete and EM short‐contact exploration type abundance, which may reflect a shift to less carbon demanding fungi due to lower photosynthetic capacity. Given the variation in EM host responses to warming, both within and between ecosystems, better understanding the link between host performance and EM fungal community structure will to clarify how climate change effects cascade belowground.     

EFFECTS OF AM FUNGI AND WATER STRESS ON DROUGHT RESISTANCE OF ARTEMISIA ORDOSICA IN DIFFERENT SOILS

 利用两种不同土壤研究了水分胁迫和接种AM真菌(摩西球囊霉(Glomus mosseae)和油蒿(Artemisia ordosica)根际土著AM真菌)对毛乌素沙地重要演替物种油蒿生长和抗旱性的影响。结果表明, 两种土壤中水分胁迫没有显著影响油蒿的植株形态和含水量, 但严重抑制了菌根侵染率。水分胁迫促使油蒿提高叶片保水能力, 抑制N、P在地上部的分配。在胁迫前期SOD活性较高, 而POD活性在后期较高。同一水分条件下接种AM真菌显著提高了AM真菌侵染率, 土壤中孢子数显著增多, 提高了植株分枝数并促进侧根发育, 显著提高根冠比和植株保水能力, 加强了根系对全磷、全氮的吸收。接种AM真菌的植株可溶性糖和丙二醛含量较低, 可溶性蛋白含量无显著变化, SOD和POD活性提高, 油蒿抗旱性加强。水分胁迫下在不同土壤中接种不同AM真菌对油蒿的促进效应差异较大, 接种土著AM真菌的效果优于摩西球囊霉单一接种。干旱导致菌根侵染率下降是宿主植物吸水能力下降的原因之一, 在植物生长前期接种AM真菌可以增强植物抵抗生长中后期环境干旱的能力。     

Effectiveness of host cell protein removal using depth filtration with a filter containing diatomaceous earth

The increased cell density and product titer in biomanufacturing have led to greater use of depth filtration as part of the initial clarification of cell culture fluid, either as a stand-alone unit operation or after centrifugation. Several recent studies have shown that depth filters can also reduce the concentration of smaller impurities like host cell proteins (HCP) and DNA, decreasing the burden on subsequent chromatographic operations. The objective of this study was to evaluate the HCP removal properties of the Pall PDH4 depth filter media, a model depth filter containing diatomaceous earth, cellulose fibers, and a binder. Experiments were performed with both cell culture fluid (CCF) and a series of model proteins with defined pI, molecular weight, and hydrophobicity chosen to match the range of typical HCP. The location of adsorbed (fluorescently labeled) proteins within the depth filters was determined using confocal scanning laser microscopy. Protein binding was greater for proteins that were positively charged and more hydrophobic, consistent with adsorption to the negatively charged diatomaceous earth. The lowest degree of binding was seen with proteins near their pI, which were poorly removed by this filter. These results provide new mechanistic insights into the factors governing the filter capacity and performance characteristics of depth filters containing diatomaceous earth that are widely used in the clarification of CCF.     

Metal loading and enzymatic degradation of fungal cell walls and chitin

The capacity of chitin (from crab shells) and of fungal cell walls from Trichoderma harzianum to accumulate zinc, cadmium and mercury was studied as well as the effects of adsorbed metals on the enzymatic hydrolysis by Novozym 234 of the two substrates. The total adsorbing capacity with respect to these metals was estimated to be at least 10 mmol kg^–1 chitin (dry weight) and 50 mmol kg^–1 fungal cell walls (dry weight), respectively, at pH 6.1. Enzymatic digestion of fungal cell walls preloaded with mercury and cadmium was significantly reduced, while zinc did not cause any significant inhibition. The effect of metal complexation by chitin on the enzymatic digestion was not as pronounced as for fungal cell walls. This could reflect the fact that chitin sorbed a lower total amount of metals. The inhibitory effect of metals on the enzymatic hydrolysis was caused by the association of the metals with the two substrates and not by the presence of free metals in solution.     

Genotypic coadaptation in plant growth promotion of forage species byBacillus polymyxa

A culture system is described to grow mycorrhizal plants which allows experimental measurements to be made on mycorrhizae, and a portion of intact ectomycorrhizal fungi while in symbiosis, but growing apart from the rooting medium and host roots. A portion of the extramatrical hyphae is kept apart from the rooting medium by a restrictive passageway between the root and fungal chambers. The passageway is small enough to restrict coniferous fine roots to the root chamber, but large enough to allow fungal hyphae to grow out of the root chamber onto pre-weighed glass fiber filter paper situated in the fungal chambers. The pre-weighed filter paper allows for gravimetric estimation of the hyphal mass in the fungal chamber. The pieces of the modular Root-Mycocosm can be assembled in various configurations with one or two seedling chambers and one, two or three fungal chambers. Ponderosa pine (Pinus ponderosa Laws.) seedlings were inoculated withHebeloma crustuliniforme (Bull.: St. Amans) Qúel in either commercial-vermiculite inoculum or in plastic growth-pouches and grown in the Root-Mycocosm. Hyphae were allowed to grow into the fungal chambers and after four weeks, amounted to 5.5±0.81 SE and 6.4±1.5 SE mg for pouch and commercial inoculum techniques, respectively.Copies of a detailed design drawing can be obtained by writing to the authors.     

Dynamic bioreactor operation: effects of packing material and mite predation on toluene removal from off-gas

Recent studies have focused on using vapor-phase bioreactors for the treatment of volatile organic compounds from contaminated air streams. Although high removal capacities have been achieved in many studies, long-term operation is often unstable at high pollutant loadings due to biomass accumulation and drying of the packing medium. In this study, three bench-scale bioreactors were operated to determine the effect of packing material and fungal predation on toluene removal efficiency and pressure drop. Toluene elimination capacities (mass toluene removed per unit packing per unit time) above 100 g m(-3) h(-1) were obtained in the fungal bioreactors packed with light-weight, artificial medium, and submersion of the packing in mineral medium once per week was found to provide sufficient moisture and nutrients to the biofilm. The use of mites as fungal predators improved performance by increasing the overall mineralization of toluene to CO(2), and by dislodging biomass along the bioreactor.