Membrane-associated particle aggregates in extracts of plant: infected with some viruses of the potato Y group

Membrane-associated aggregates of filamentous virus particles were found occasionally in sap from plants infected with each of five distinct viruses of the potato Y group. The aggregates measured up to 960 × 700 nm and contained from four to twenty-four virus particles. Their occurrence is apparently specifically associated with potato Y and allied viruses, observations suggesting that they may be either fragments of virus-containing cytoplasmic strands or groups of particles enveloped during cellular disruption by pieces of tonoplast with which they are associated in vivo.     

Classification and measurement of fungal pellets by automated image analysis

An automated image analysis method for classifying and measuring pellets of filamentous fungi growing in submerged fermentations has been developed. The method discriminates between pelleted mycelial growth and loose aggregates of dispersed hyphae. Pellets are classified into smooth and hairy types. In both cases, the core of the pellet is identified and its shape and size characterized. For hairy pellets the annular region is also characterized. The method was tested on pellets of Aspergillus niger ATCC 11414 grown in a defined medium in shake flasks. This rapid method makes practical extensive studies on the morphology of pellets in submerged fermentations and the influence of fermentation conditions on that morphology.     

Yeasts and filamentous fungi carried by the gynes of leaf-cutting ants

Insect-associated microbes exhibit a wide range of interactions with their hosts. One example of such interactions is the insect-driven dispersal of microorganisms, which plays an essential role in the ecology of several microbes. To study dispersal of microorganisms by leaf-cutting ants (Formicidae: Attini), we applied culture-dependent methods to identify the filamentous fungi and yeasts found in two different body parts of leaf-cutting ant gynes: the exoskeleton and the infrabuccal pocket. The gynes use the latter structure to store a pellet of the ants’ symbiotic fungus during nest founding. Many filamentous fungi (n = 142) and yeasts (n = 19) were isolated from the gynes’ exoskeleton. In contrast, only seven filamentous fungi and three yeasts isolates were recovered from the infrabuccal pellets, suggesting an efficient mechanism utilized by the gynes to prevent contamination of the symbiotic fungus inoculum. The genus Cladosporium prevailed (78%) among filamentous fungi whereas Aureobasidium, Candida and Cryptococcus prevailed among yeasts associated with gynes. Interestingly, Escovopsis, a specialized fungal pathogen of the leaf-cutting ant-fungus symbiosis, was not isolated from the body parts or from infrabuccal pellets of any gynes sampled. Our results suggest that gynes of the leaf-cutter ants Atta laevigata and A. capiguara do not vertically transmit any particular species of yeasts or filamentous fungi during the foundation of a new nest. Instead, fungi found in association with gynes have a cosmopolitan distribution, suggesting they are probably acquired from the environment and passively dispersed during nest foundation. The possible role of these fungi for the attine ant–microbial symbiosis is discussed.     

Soil aggregate sequestration of cover crop root and shoot-derived nitrogen

Cover crop roots and shoots release carbon (C) and nitrogen (N) compounds in situ during their decomposition. Depending upon the season, these C and N compounds may be sequestered, the C may be respired or the N may be leached below the root zone. A field study was established to identify the contributions of cover crop root and shoot N to different regions within aggregates in the A_p horizon of a Kalamazoo loam soil. Fall-planted rye plants (Secale cerealeL.) were labeled the next May with foliar applications of solutions containing 99% atom (¹⁵NH₄)₂SO₄. Isotopic enrichment of soil aggregates ranging from 2.0 to 4.0, 4.0–6.3 and 6.3–9.5 mm across was determined following plant residue applications. Concentric layers of aggregates were removed from each aggregate by newly designed meso soil aggregate erosion (SAE) chambers. Non-uniform distributions of total N and recently derived rye N in soil macroaggregates, across time, suggested that the formations and functions of macroaggregates are very dynamics processes and soil aggregates influence where N is deposited. Early in the season, more ¹⁵N migrated to the interior regions of the smallest aggregates, 2–4 mm across, but it was limited to only surfaces and transitional regions of the larger aggregates, 6.3–9.3 mm across. Exterior layers of aggregates between 6.0 and 9.5 mm retained 1.6% of the N_{derived from roots} in July 1999, which was three times more than their interior regions. This was slightly greater than the % N_{derived from shoot.} One month later, as the maize root absorption of N increased rapidly, % N_{derived from roots} and % N_{derived from shoot} were nearly equal in exterior layers and interior regions of soil aggregates. This equilibrium distribution may have been from either greater diffusion of N within the aggregates and/or maize root removal form aggregate exteriors. Results supported that most of roots grew preferentially around surfaces of soil aggregates rather than through aggregates. Cover crop roots contributed as much N as cover crop shoots to the total soil N pool. Subsequent crops use N from the most easily accessible zones of soil structure, which are surfaces of larger soil aggregates. Therefore maintaining active plant roots and aggregated soil structure in the soil enhances N sequestration and maximize soil N availability. These studies suggest that the rapid and perhaps bulk flow of soil N solutions may bypass many of the central regions of soil aggregates, resulting in greater leaching losses.     

Studies of a pelleted growth form of Rhizopus nigricans as a biocatalyst for progesterone 11α-hydroxylation

The noncoagulative type of pellet formation can be induced in submerged cultivation of the filamentous fungus Rhizopus nigricans. The size and constitution of the hyphal agglomerates obtained varied with changes in inoculum size and agitation speed for given media composition and cultivation conditions. The physiological state of mycelium, used for a further process of biotransformation, was estimated by following the growth kinetics, pH value and substrate utilization during submerged cultivation. Namely, differences in pellet morphology and physiology affect the ability of R. nigricans to hydroxylate progesterone at the 11α position. A repeated batch procedure revealed the best maintenance of biotransformation capacity for pellets, obtained from the growth phase of cultivation at high agitation speed and with low inoculum size.     

Corn Stunt Spiroplasma in the Leafhopper Euscelidius variegatus

Corn stunt spiroplasma (CSS) multiplied in all leafhoppers Euscelidius variegatus injected with a culture of CSS, reaching titres of over 1x10⁶ colony forming units (cfu) per insect and 2x10⁴ cfu per salivary gland of each insect. CSS could be isolated from the haemolymph and the salivary glands at any time after injection. The growth of CSS in culture was inhibited by insect extract at concentrations greater than the equivalent of 0.1 insect/ml. Transmission of CSS to sterile feeding solution and to broad beans were compared using 24 h feeding periods. A porportion of 1.7 % of injected leafhoppers began to transmit to sterile feeding solution through membranes by the 4th day after injection, and reached a maximum of 30 % by day 14. Similar insects started transmitting to broad bean plants on day 12 (2 %), reaching a maximum of 7.5 % by day 14. The number of spiroplasmas transmitted by each insect to sterile feeding solution increased from 3, cfu on day 4 to a maximum of 80 cfu by day 14. Helices were seen in the haemolymph at any time after injection. However, partially deformed cells were not present until the 1st week and clumps of 3–4 cells and small aggregates until the 3rd week after injection. The salivary glands of injected insects contained membrane-bound “pockets” or “colonies” packed with pleomorphic, filamentous (helical and non-helical) cells and aggregates. Intracellular colonies were always at the periphery of the acini and were easily detectable by fluorescence microscopy after staining with a DNA-binding fluorescent stain. Pleomorphic and filamentous cells were also seen intercellularly in the salivary glands.     

Fate of Tobacco Mosaic Virus after Entering the Host Cell

Tobacco leaves were inoculated with tobacco mosaic virus labeled with ³²P or ³⁵S. After various intervals, extracts of the leaves were prepared. In extracts from leaves infected for 5 to 360 min, about 40 to 60% of the virus retained on leaves was recovered in the pellet of the homogenate centrifuged at 12 000 × g. The virus associated with the 12 000 × g pellet was dissociable by treatment with pancreatic RNase, alkali or sodium dodecyl sulfate (SDS). The parental virus extracted by SDS from the pellet at 12 000 × g had a large amount of partially uncoated virus possessing naked RNA. Analysis by density gradient centrifugation suggested that, in addition to partially uncoated virus, some fragmented RNA was also associated with the 12 000 × g pellet. This fragmented RNA seemed to be derived from partially uncoated virus. Density gradient analysis of SDS extracts from the 12 000 × g pellet suggested that some of the virus underwent uncoating at the internal regions of the virus particle.     

Enhanced continuous production of lovastatin using pellets and siran supported growth of Aspergillus terreus in an airlift reactor

Lovastatin, a hypocholesterolemic agent, is a secondary metabolite produced by filamentous microorganism Aspergillus terreus in submerged batch cultivation. Lovastatin production by pellets and immobilized siran cells was investigated in an airlift reactor. The process was carried out by submerged cultivation in continuous mode with the objective of increasing productivity using pellet and siran supported growth of A terreus. The continuous mode of fermentation improves the rate of lovastatin production. The effect of dilution rate and aeration rate were studied in continuous culture. The optimum dilution rate for pellet was 0.02 h⁻¹ and for siran carrier was 0.025 h⁻¹. Lovastatin productivity using immobilized siran carrier (0.0255 g/L/h) was found to be greater than pellets (0.022 g/L/h). The productivity by both modes of fermentation was found higher than that of batch process which suggests that continuous cultivation is a promising strategy for lovastatin production.     

SPATIAL SEGREGATION OF CO2 FIXATION IN TRICHODESMIUM SPP.: LINKAGE TO N2 FIXATION POTENTIAL1

The aggregate-forming, nonheterocystous, filamentous blue-green alga (cyanobacteria) Trichodesmium spp. is a widespread and important planktonic N₂ fixer and primary producer in tropical and subtropical oceans. It is unique among nonheterocystous genera because it conducts N₂ and CO₂ fixation (O₂ evolution) simultaneously; a notable achievement, because O₂ is a potent inhibitor of N₂ fixation. Spatial and temporal CO₂ fixation patterns were examined in trichomes and aggregates from natural and cultured populations, utilizing microautoradiographic detection of ¹⁴CO₂ incorporation. Parallel N₂ fixation (acetylene reduction) measurements were also made. Diel N₂ and CO₂ fixation patterns were similar, with co-optimization of both processes near midday. Microautoradiographs revealed several trichome-level ¹⁴CO₂ incorporation patterns: 1)uniform, heavy labeling, 2)uniform, light labeling, 3) heavier labeling in distal as opposed, to proximal regions, and 4) virtually no labeling throughout. Similar patterns were observed in natural and cultured populations. Given previous immunochemical findings that N₂ fixation potential is widespread in Trichodesmium spp. trichomes and aggregates, current results suggest a high degree of individuality, and possibly a “division of labor” in terms of CO₂ fixation, among trichomes comprising active N₂-fixing aggregates. Segregation of photosynthesis within and among trichomes facilitates simultaneous N₂ and CO₂ fixation in Trichodesmium spp. trichomes and aggregates.     

A rapid method for harvesting and immobilization of oleaginous microalgae using pellet-forming filamentous fungi and the application in phytoremediation of secondary effluent

A rapid method for harvesting and immobilization of oleaginous microalgae using pellet-forming filamentous fungi was developed. The suitable conditions for pellet formation by filamentous fungi were determined. Among the strains tested, Trichoderma reesei QM 9414 showed superior pellet forming ability. Its pellets were used to harvest oleaginous microalga Scenedesmus sp. With increasing volume ratio of fungal pellets to microalgae culture up to 1:2, >94% of microalgal cells were rapidly harvested within 10 min. The ratio of fungal pellets could manipulate both harvesting time and initial concentration of microalgal cells in the pellets. The microalgae–fungal pellets were successfully used as immobilized cells for effective phytoremediation of secondary effluent from seafood processing plants under nonsterile condition. The chemical oxygen demand, total nitrogen, and total phosphorus removal were >74%, >44%, and >93%, respectively. The scanning electron microscopy showed that the microalgal cells were not only entrapped in the pellets but also got attached to the fungal hyphae with sticky exopolysaccharides, possibly secreted by the fungi. The extracted lipids from the pellets were mainly composed of C16–C18 (>83%) with their suitability as biodiesel feedstocks. This study has shown the promising strategy to rapidly harvest and immobilize microalgal cells and the possible application in phytoremediation of industrial effluent.     

Adding talc microparticles to Aspergillus terreus ATCC 20542 preculture decreases fungal pellet size and improves lovastatin production

Changing fungal morphology with the use of morphological engineering techniques leads to improving the production of metabolites by filamentous fungi in the submerged culture. Adding mineral microparticles is one such simple method to change fungal pellet size. Here, it was studied for a lovastatin producer, Aspergillus terreus ATCC 20542. The experiments were conducted in shake flasks and 10 μm talc microparticles were added to the preculture. Intrapellet oxygen concentration profiles were determined by an oxygen microprobe. Talc microparticles caused a decrease of A. terreus pellets diameter from about 2000 to 900 μm, dependent on their concentration in the preculture. Smaller pellets produced more lovastatin, whose titre exceeded then 120 mg L^?1, utilising more lactose. The decrease in pellet size resulted in changes of oxygen concentration profiles in the pellets. The estimated critical pellet diameter, at which the non‐oxygenated zone was observed in the centre of the pellets, was 1700 μm. Smaller pellets were fully penetrated by oxygen. To conclude, facilitated diffusion of oxygen into the pellets of smaller diameter and their less dense structure made lactose utilisation by A. terreus more efficient, which ultimately increased lovastatin production in the runs with talc microparticles added, compared to the control runs.     

Ultrastructural changes associated with the accumulation and secretion of sanguinarine in Papaver bracteatum suspension cultures treated with fungal elicitor

Suspension cultures of Papaver bracteatum Arya II Lindl., grown without hormone in the presence of conidial extracts of Verticillium dahliae Kleb., accumulate millimolar quantities of the benzophenanthridine alkaloid, sanguinarine. Under the fluorescence microscope, the elicitor-treated cells display an orange-yellow fluorescence characteristic of sanguinarine, primarily near the periphery of the cells. Electron-microscopic inspection showed the presence of slightly dilated endoplasmic reticulum and of electron-dense protuberances on the tonoplast of large central vacuoles. These osmiophilic aggregates lining the tonoplast bud into spherical bodies, appear to become detached from the membrane and are released into the vacuole. Upon subcellular fractionation of elicited cells on Renografin step gradients, sanguinarine was found to be distributed in all bands but with 86% concentrated in the gradient pellet. Analysis of the pellet by electron microscopy showed that it contained electron-dense fragments similar to the osmiophilic bodies observed on the tonoplast of intact elicited cells. In elicited cell cultures, most of the sanguinarine was recovered from medium in a 100·g sedimenting, cell-free, particulate fraction accounting for as much as 85% of the media sanguinarine and 62% of the total sanguinarine. The sanguinarine-rich 100·g media pellet was determined to be two-thirds protein, one-third RNA and was essentially devoid of phenolics, phospholipid and DNA. The pellet consisted of electrondense material and cytoplasmic remnants resembling those found in the Renografin pellet and tonoplast aggregates of intact cells. When placed under hypotonic conditions or extracted with aqueous buffer, pH 3–11, the pellet did not release sanguinarine. These observations provide evidence for storage of sanguinarine at electron-dense deposits which occur on the tonoplast and as freely floating bodies in vacuoles.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - EM electron microscopy - ER endoplasmic reticulum - HPLC high-pressure liquid chromatography - MRST Murashige and Skoog's revised tobacco medium     

A simple staining procedure for the characterisation of Basidiomycetes pellets by image analysis

Summary A simple staining procedure, associated with image analysis, was used to discriminate two zones (the filamentous zones and the compacted cores) in aggregates of fungal biomass according to their accessibility for an agent of the medium. The application of the method to batch fermentations of Schizophyllum commune showed a higher proportion of loosely entangled particles (clumps) and thinner filamentous outer zones obtained at a high agitation speed (500 rpm) compared to low agitation speeds (120–200 rpm).     

Comparative ultrastructure of a filamentous mutant and the wild type ofAgmenellum quadruplicatum

Summary A filamentous variant of the marine coccoid cyanophycean alga,Agmenellum quadruplicatum has been produced after treatment of cells with the chemical mutagen, N-methyl-N-nitro-N-nitrosoguanidine (NTG). The mutation to the filamentous condition is stable and has been compared ultrastructurally with the unicellular wild type. The structural basis for the maintenance of the filamentous state is due to a failure of the homogeneously-structured transverse wall to differentiate into the stratified longitudinal wall. A manifestation of this condition is the continued synthesis of the investment membrane resulting in proliferations at each constriction. The wild type cells are nearly spherical while the filamentous mutant cells are smaller and elongate. An analysis of the structure and morphology of the nucleoplasmic regions is presented for both strains and a correlation between the morphology of these regions and the unicellular and filamentous conditions is made.     

BACTERIAL ASSOCIATIONS WITH MARINE OSCILLATORIA SP. (TRICHODESMIUM SP.) POPULATIONS: ECOPHYSIOLOGICAL IMPLICATIONS1

On three separate occasions we investigated morphological and physiological aspects of bacterial associations with planktonic aggregates of the ubiquitous marine N₂ fixing cyanobacterium Trichodesmium sp. Close associations generally characterized Trichodesmium blooms; associations were present during day- and night-time. Colonization by both rod-shaped and filamentous heterotrophic bacteria occurred on Trichodesmiun aggregates actively fixing N₂ (acetylene reduction). Scanning electron and optical microscopy showed bacteria located both around and within aggregates. Microautoradiography demonstrated that associated bacteria largely mediated utilization of trace additions of ³H-labeled carbohydrates (fructose, glucose, mannitol) and amino acids, whereas Trichodesmium utilized amino acids only. Oxygen measurements using microelectrodes revealed high localized oxygen consumption among aggregates, with rapid (within a minute) changes from supersaturated to subsaturated oxygen following the transition from photosynthetic illuminated to dark periods. Stab culturing techniques confirmed the presence of heterotrophic N₂ fixers among aggregate-associated bacteria. Parallel deployment of oxygen microelectrodes, the tetrazolium salt 2,3,5 triphenyl tetrazolium chloride (TTC) and acetylene reduction assays demonstrated microaerophilic requirements for expression of nitrogenase activity among cultured bacteria. Trichodesmium aggregates are characterized by dynamic nutrient and oxygen regimes, which promote and maintain simultaneous and contiguous oxygenic photosynthesis and N₂ fixation. In part, the above-mentioned consortial interactions with a variety of heterotrophic bacteria facilitate Trichodesmium biomass production and bloom formation in nitrogen depleted, oligotrophic tropical/subtropical waters.     

An X-ray microtomography-based method for detailed analysis of the three-dimensional morphology of fungal pellets

Filamentous fungi are widely used in the production of biotechnological compounds. Since their morphology is strongly linked to productivity, it is a key parameter in industrial biotechnology. However, identifying the morphological properties of filamentous fungi is challenging. Owing to a lack of appropriate methods, the detailed three-dimensional morphology of filamentous pellets remains unexplored. In the present study, we used state-of-the-art X-ray microtomography (µCT) to develop a new method for detailed characterization of fungal pellets. µCT measurements were performed using freeze-dried pellets obtained from submerged cultivations. Three-dimensional images were generated and analyzed to locate and quantify hyphal material, tips, and branches. As a result, morphological properties including hyphal length, tip number, branch number, hyphal growth unit, porosity, and hyphal average diameter were ascertained. To validate the potential of the new method, two fungal pellets were studied—one from Aspergillus niger and the other from Penicillium chrysogenum. We show here that µCT analysis is a promising tool to study the three-dimensional structure of pellet-forming filamentous microorganisms in utmost detail. The knowledge gained can be used to understand and thus optimize pellet structures by means of appropriate process or genetic control in biotechnological applications.     

Organic aggregates formed by benthopleustophyte brown alga Acinetospora crinita (Acinetosporaceae,Ectocarpales)

This work presents the elemental, polysaccharide, and fatty acid compositions of benthic aggregates formed by the filamentous brown alga Acinetospora crinita, which are widely spread on the rocky bottoms of the Mediterranean Sea. The aggregates can be characterized as mineralized centers in which regeneration of nutrients and recycling of dissolved organic matter actively occur and favor the development of an abundant phytoplankton community. Analyses of the stable isotopes of C and N display their marine origin and could provide evidence of the processes that occur inside/outside of the aggregates. The monosaccharide compositions of Adriatic and Tyrrhenian mucilages produced by brown alga A. crinita were quite similar. In particular, the Adriatic sample compositions resembled the average composition of the Tyrrhenian high molecular weight exopolymers, and the observed differences could be ascribed to different degradation stages. The fatty acid patterns found for the aggregates were similar to those observed in the isolated A. crinita algae with variable contributions from embedded diatom species. The bacterial contribution to the fatty acid pool was quite low, most likely due to the known poor conditions for their heterotrophic growth.     

Formation of Filamentous Mn Oxide Particles by the Alphaproteobacterium Bosea sp. Strain BIWAKO-01

Mn-rich filamentous particles present in stratified water environments are considered bacteriogenic; however, little is known about their causative agents. This study investigated the production of these particles by an alphaproteobacterium, Bosea sp. strain BIWAKO-01. Particle formation was promoted in static cultures with slightly viscous medium at pH 6.0?6.3 under low-O₂ conditions. The Mn(II) oxidation in cultures was slower in higher O₂ concentration. These results suggested that pH and O₂ concentration are important factors affecting filamentous Mn particle formation in the Mn(II) oxidizer. Lectin staining followed by fluorescence microscopy revealed the presence of specific carbohydrates in the filamentous structures. In addition, transmission electron microscopy, high angle annular dark field scanning transmission electron microscopy, and electron energy loss spectroscopy revealed the structural and spatial associations of Mn with O and C on a nanometer scale in filaments. The results suggested the occurrence of sheet-type Mn oxide likely due to the catalytic activity in exopolymeric substances including acidic polysaccharides.     

Bacterial aggregates in the tentacles of the sea anemone <Emphasis Type="Italic">Metridium senile</Emphasis>

This paper provides first information on organ-like bacterial aggregates in the tentacles of the sea anemone Metridium senile. The specimens were collected from waters near Helgoland (German Bight, North Sea) and the Orkney Islands. Tentacles were prepared for morphological inspection by light and scanning electron microscopy as well as for the phylogenetic analysis of endocytic bacteria. Bacterial aggregates are located in caverns of the tentacles’ epidermis. The aggregates are enwrapped in thin envelopes, which contain coccoid and/or rod-shaped tightly packed bacteria of different division states. Most of the bacterial cells are connected by fine filamentous structures. The phylogenetic determination is based on the sequence data of the 16S rDNA derived from tentacle material. Sequence analysis revealed three different subgroups of intratentacular proteobacteria. The dominant band, detected in all of the samples tested, showed a close relationship (98%) to a gram-negative Endozoicimonas elysicola. Two bands, only detected in tentacles of M. senile from Helgoland were assigned to Pseudomonas saccherophilia (99%), a knallgas bacterium, and to Ralstonia pickettii (100%). The bacteria represent a specific bacterial community. Their DGGE profiles do not correspond to the profiles of the planktonic bacteria generated from seawater close to the habitats of the anemones. The allocation of DNA sequences to the different morphotypes, their isolation, culturing and the elucidation of the physiological functions of intratentacular bacteria are in progress.     

Morphological engineering of Streptomyces hygroscopicus var. geldanus: regulation of pellet morphology through manipulation of broth viscosity

Actinomycetes, especially members of the genus Streptomyces, are responsible for producing the majority of known antibiotics. The production of antibiotics by filamentous organisms is often dependent on the morphology and size distribution of the pellet population within the culture. Particle interaction and subsequent pellet formation are primarily dependent on the rate of collision of particles in culture, which is in turn, a function of fluid turbulence. The microbial polysaccharide xanthan gum was used to artificially regulate the apparent viscosity (_a) of S. hygroscopicus fermentation broths with the aim of controlling particle interaction, aggregation and hence pellet formation. An increase in both pellet count and biomass concentration from approximately 2,000 to 8,000 pellets ml^–1 and 0.9–2.1 g l^–1 dry weight of biomass, as well a decrease in the mean pellet volume from 0.014 to 0.004 mm³ was observed in cultures supplemented with 3 g l^–1 xanthan gum. The addition of xanthan gum significantly alters fluid rheology by increasing the _a. Counter-intuitively, an increase in the _a within the experimental range examined resulted in an increase in the rate of gas–liquid mass transfer. This was attributed to the predominantly diffusive nature of oxygen transfer in shake flask cultures.