Interactions between a soil fungus,Trichoderma harzianum,and IIb metals—adsorption to mycelium and production of complexing metabolites

Fungi are capable of accumulating metals and, in soil, such accumulation may influence metal speciation and transport. The interactions between a common soil fungus, Trichoderma harzianum, and IIb elements were studied in the present investigation. The accumulation of the metals zinc, cadmium and mercury by starved and non-starved mycelium at different pH was determined by a batch technique using radioactive tracers; uptake of the metals was found to be large, with respective distribution coefficients of about 10^3.5, 10^2.5 and 10^4.0 for zinc, cadmium and mercury, respectively. Metal accumulation by a starved system was largely independent of pH in the range 3–9, where in a non-starved system an increased accumulation of zinc (at 10^– m) was observed at low pH (3–5). Potentiometric titrations performed on the two systems revealed significant differences in acid capacities, i.e. values close to zero for the starved system and 500–800 meq kg^– for the non-starved system. The maximum metal uptake was at least 50 mmol kg^– at pH 6.5 (calculated from adsorption isotherms). The present findings suggests that in the non-starved system a metabolite is produced and then released when the pH is within a certain range.     

Application of X-ray microanalysis to cell suspensions of oil palm (Elaeis guineensis Jacq.)

X-ray microanalysis has been used to determine the elemental composition of oil-palm (Elaeis guineesis) cell suspensions without the use of cryoprotectants. Results based on individual cells were gathered over a typical growth cycle of 14 d. During the log phase (5–7 d) there is an increase in the number of cells containing high concentrations of both K (400 mmol kg^-1 dry weight) and P (400 mmol kg^-1 dry weight). Morphologically these cells had thin cell walls and were frequently joined to other cells (two to five cells per clump).     

Accumulation of heavy metals in the metal-tolerant fern,Athyrium yokoscense,growing on various environments

The accumulation of copper, zinc and cadmium inA. yokoscense collected from Ashio (copper-contaminated area), Bandai (zinc- and cadmium-contaminated area) and Tama (non-contaminated area), has been investigated. Copper and zinc were accumulated most highly in the root, whilst cadmium was accumulated more in the leaf. The root ofA. yokoscense growing in areas contaminated with metals contained maximum amounts of Cu (5, 989 mg. kg⁻¹ dry weight) and Zn (6,384 mg.kg⁻¹ dry weight), while in the leaf from the Bandai area 164.8 mg Cd.kg⁻¹ dry weight was accumulated. These amounts are far greater than those found inA yokoscense growing on the non-metalliferous habitat (Tama). Twenty five times more zinc and three times more cadmium were found in the dead leaf than in the living leaf. InA. yokoscense growing on soils containing more than 1,000 mg Cu or Zn.kg⁻¹ dry weight, the uptake of copper by the root increased considerably with increasing copper content in the soils, while the uptake of zinc increased only slightly compared with the increase of zinc in the soils.     

Heavy metal tolerance by ectomycorrhizal fungi and metal amelioration by Pisolithus tinctorius

Five ectomycorrhizal fungi, Pisolithus tinctorius, Thelephora terrestris, Cenococcum geophilum, Hymenogaster sp. and Scleroderma sp., which were demonstrated previously to be capable of forming ectomycorrhizas with some pine, eucalypt and fagaceous tree species were grown in vitro in liquid cultures for 3 weeks at six different concentrations of nine heavy metals, aluminium, iron, copper, zinc, nickel, cadmium, chromium, lead and mercury. Measurements of mean mycelial dry weight yields indicated that the local isolates of Hymenogaster sp. and Scleroderma sp., as well as the introduced fungal species P. tinctorius, were able to withstand high concentrations of Al, Fe, Cu and Zn and might, therefore, have potential for revegetation schemes in metal-contaminated soils. The metal amelioration mechanism in the metal-tolerant fungal species P. tinctorius was observed to involve extrahyphal slime and, as demonstrated by energy-dispersive X-ray spectrometry, was achieved by polyphosphate linkage of Cu and Zn.     

Attempted localization of a substrate for chitinases in plant cells reveals abundant N-acetyl-d-glucosamine residues in secondary walls

Ultrathin sections of healthy and fungus-infected plant tissue were treated with either wheat-germ agglutinin (WGA) ovomucoid-gold complex or microbial chitinase-gold complexes for localizing putative chitin-like macromolecules. Fungal cell walls, known to contain chitin, were labeled with both probes and were considered as positive controls. Plant secondary cell walls of both healthy and infected tissues were also intensely labeled whereas compound middle lamella-primary walls and cell cytoplasm were free of labeling. Enzymatic digestion of plant tissues with chitinase from Streptomyces griseus abolished the fungal cell wall labeling but did not interfere with that of plant secondary cell walls. This suggests that polymers analogous to fungal chitin are absent in plant cell walls. Tissue digestions with either proteinase K or lipase led to surprising results as far as the possible nature of N-acetylglucosamine-containing molecules is concerned. The loss of labeling over plant secondary walls following lipase digestion suggests that N-acetylglucosamine residues may be linked to lipids to form glycolipids. However, these results have to be viewed with caution since the possibility that peptides may be present but inacessible to proteinase K should be considered. The role of the detected N-acetylglucosamine containing molecules as possible substrates for plant chitinases is discussed.     

The concentration of cadmium,lead, copper and zinc inChironomus gr.thummi larvae (Diptera,Chironomidae) with deformedversus normal menta

The concentrations of Cd, Pb, Cu and Zn inChironomus gr.thummi were determined for 4th instar larvae from the polluted Dyle River, tributary of the Scheldt River (Belgium). Comparison was made between larvae with deformed and normal menta. Deformed larvae showed higher overall metal concentrations than normal larvae. Especially Pb and Cu had higher concentrations in deformed larvae (16.22 mg kg^–1 dry weight and 39.66 respectively) than in normal larvae (12.80 mg kg^–1 dry weight and 35.70 respectively). No significant differences were found in the concentrations of Cd and Zn (mean [Cd] = 0.81 mg kg^–1 dry weight and mean [Zn] = 313.12 mg kg^–1 dry weight). There was no difference between the two larval groups as far as total length, dry weight and developmental stage of the imaginal discs are concerned.     

Mercury in the marine food chain in the Southern Ocean at Macquarie Island: an analysis of a top predator,Patagonian toothfish (<Emphasis Type="Italic">Dissostichus eleginoides</Emphasis>) and a mid-trophic species,the warty squid (<Emphasis Type="Italic">Moroteuthis ingens</Emphasis>)

The characteristics and habitat of the Patagonian toothfish (Dissostichus eleginoides) are typical of fish that accumulate high concentrations of mercury. In this study, mercury determinations were made on samples of muscle tissue from Macquarie Island toothfish and the Southern Ocean deepwater warty squid (Moroteuthis ingens). The analysis of mercury in the biological tissues was made by cold vapour-atomic absorption spectrometry following acid digestion. Performance of the analytical procedure was assessed by analysis of certified reference material (DORM-2, dogfish muscle). Mercury concentrations of 16 Macquarie Island toothfish ranged from 0.12 mg kg^–1 (550 g, 381 mm TL) to 0.59 mg kg^–1 (6,100 g, 823 mm TL), with a mean concentration of 0.33±0.12 mg kg^–1. A significant correlation was found between mercury and either toothfish weight or total length. The fish analysed were juveniles, which suggests that larger individuals would have higher mercury concentrations well exceeding food standard code limits for mercury in fish (typically 0.5 mg kg^–1). Warty squid, also from around Macquarie Island, had a low mean mercury concentration of 0.086 mg kg^–1 in mantle tissue; no significant correlation existed between mercury concentration and either squid mantle length or total weight. It is postulated that the squid have a mechanism, possibly involving the digestive gland, that prevents bioaccumulation of mercury in the mantle, and presumably other body tissues.     

Effect of 6-benzyladenine and Casein Hydrolysate on Micropropagation of Amorpha fruticosa

Pisum sativum L. cv. Phenomen plants were grown in pots in greenhouse and their growth, and ATP and chlorophyll (Chl) a and b contents were assessed after 9-d exposure to sodium arsenate [0.04 and 0.07 mmol kg^–1(soil)], or to lead acetate [2.0 and 4.0 mmol kg^–1(soil)], or zinc acetate [5.3 and 9.3 mmol kg^–1(soil)]. The luciferin-luciferase method was used for ATP analyses. Soil pollution reduced significantly the growth, but the low toxicant concentrations elevated the cotyledon and shoot ATP concentrations per fresh matter content. The ATP/Chl ratio was increased in the zinc-treated seedlings as compared with the respective controls. The ATP concentration and a number of growth parameters were negatively correlated, and thus the high ATP content might contribute to the significantly reduced growth of seedlings.     

Extracellular complexation of Cd in the Hartig net and cytosolic Zn sequestration in the fungal mantle of Picea abies – Hebeloma crustuliniforme ectomycorrhizas

Compartmentation of heavy metals on or within mycorrhizal fungi may serve as a protective function for the roots of forest trees growing in soils containing elevated concentrations of metals such as Cd and Zn. In this paper we present the first quantitative measurements by X‐ray microanalysis of heavy metals in high‐pressure frozen and cryosectioned ectomycorrhizal fungal hyphae. We used this technique to analyse the main sites of Cd and Zn in fungal cells of mantle and Hartig net hyphae and in cortical root cells of symbiotic Picea abies – Hebeloma crustuliniforme associations to gain new insights into the mechanisms of detoxification of these two metals in Norway spruce seedlings. The mycorrhizal seedlings were exposed in growth pouches to either 1 mM Cd or 2 mM Zn for 5 weeks. The microanalytical data revealed that two distinct Cd‐ and Zn‐binding mechanisms are involved in cellular compartmentation of Cd and Zn in the mycobiont. Whereas extracellular complexation of Cd occurred predominantly in the Hartig net hyphae, both extracellular complexation and cytosolic sequestration of Zn occurred in the fungal tissue. The vacuoles were presumed not to be a significant pool for Cd and Zn storage. Cadmium was almost exclusively localized in the cell walls of the Hartig net (up to 161 mmol kg ^{? 1} DW) compared with significantly lower concentrations in the cell walls of mantle hyphae (22 mmol kg ^{? 1} DW) and in the cell walls of cortical cells (15 mmol kg ^{? 1} DW). This suggests that the apoplast of the Hartig net is a primary accumulation site for Cd. Zinc accumulated mainly in the cell walls of the mantle hyphae (111 mmol kg ^{? 1} DW), the Hartig net hyphae (130 mmol kg ^{? 1} DW) and the cortical cells (152 mmol kg ^{? 1} DW). In addition, Zn occurred in high concentrations in the cytoplasm of the fungal mantle hyphae (up to 164 mmol kg ^{? 1} DW) suggesting that both the cell walls and the cytoplasm of fungal tissue are the main accumulation sites for Zn in P. abies resulting in decreased Zn transfer from the fungus to the root.     

Effects of heavy metals on growth and ultrastructure ofChara vulgaris

Summary The toxicity of some heavy metals to the common macrophytic freshwater algaChara vulgaris was studied under laboratory conditions. For experiments, apical tips of algae containing two internodes were cultivated for fourteen days in the presence of various concentrations of cadmium, mercury or lead (as triethyl lead or lead nitrate). Fifty percent growth inhibition occurred with concentrations of 8.5×10^–8 M (9.5 ppb) cadmium, 7.5×10^–7M (150ppb) mercury, 1.6×10^–6 M (330ppb) organic lead or 4× 10^–5 M (8000 ppb) inorganic lead. Sublethal concentrations of these metals caused alterations in the fine structure of internodal cells which turned out to be at least partly metal-specific or in the case of lead, the effects depended on whether the lead was ionic or organically bound. Cadmium and inorganic lead induced disorders of cell wall microfibrils which resulted in local wall protuberances. Mercury affected the chloroplasts which mostly showed considerably increased grana stacks. In addition, mercury caused a dilation of the endoplasmic reticulum and of the mitochondrial tubuli. Organic lead damaged the membrane system of chloroplasts; sheet- or tubule-like thylakoids were disarranged and showed whorl-like structures. At higher concentrations of organic lead, tubular invaginations of the plasmalemma (charasomes) disappeared. The fine structure of nuclei was not altered by any of the metals.     

Zinc ions alter morphology and chitin deposition in an ericoid fungus

A sterile mycelium PS IV, an ascomycete capable of establishing ericoid mycorrhizas, was used to investigate how zinc ions affect the cellular mechanisms of fungal growth. A significant reduction of the fungal biomass was observed in the presence of millimolar zinc concentrations; this mirrored conspicuous changes in hyphal morphology which led to apical swellings and increased branching in the subapical parts. Specific probes for fluorescence and electron microscopy localised chitin, the main cell wall polysaccharide, on the inner part of the fungal wall and on septa in control specimens. In Zn-treated mycelium, hyphal walls were thicker and a more intense chitin labelling was detected on the transverse walls. A quantitative assay showed a significant increase in the amount of chitin in metal-treated hyphae.     

Co‐hydrolysis of lignocellulosic biomass for microbial lipid accumulation

The herbaceous perennial energy crops miscanthus, giant reed, and switchgrass, along with the annual crop residue corn stover, were evaluated for their bioconversion potential. A co‐hydrolysis process, which applied dilute acid pretreatment, directly followed by enzymatic saccharification without detoxification and liquid–solid separation between these two steps was implemented to convert lignocellulose into monomeric sugars (glucose and xylose). A factorial experiment in a randomized block design was employed to optimize the co‐hydrolysis process. Under the optimal reaction conditions, corn stover exhibited the greatest total sugar yield (glucose + xylose) at 0.545 g g^?1 dry biomass at 83.3% of the theoretical yield, followed by switch grass (0.44 g g^?1 dry biomass, 65.8% of theoretical yield), giant reed (0.355 g g^?1 dry biomass, 64.7% of theoretical yield), and miscanthus (0.349 g g^?1 dry biomass, 58.1% of theoretical yield). The influence of combined severity factor on the susceptibility of pretreated substrates to enzymatic hydrolysis was clearly discernible, showing that co‐hydrolysis is a technically feasible approach to release sugars from lignocellulosic biomass. The oleaginous fungus Mortierella isabellina was selected and applied to the co‐hydrolysate mediums to accumulate fungal lipids due to its capability of utilizing both C5 and C6 sugars. Fungal cultivations grown on the co‐hydrolysates exhibited comparable cell mass and lipid production to the synthetic medium with pure glucose and xylose. These results elucidated that combining fungal fermentation and co‐hydrolysis to accumulate lipids could have the potential to enhance the utilization efficiency of lignocellulosic biomass for advanced biofuels production. Biotechnol. Bioeng. 2013; 110: 1039–1049. © 2012 Wiley Periodicals, Inc.     

Cation exchange capacity and lead sorption in ectomycorrhizal fungi

Two ectomycorrhizal fungi, Paxillus involutus 533 and Laccaria bicolor S238, differing greatly in their mycelial characteristics, were investigated with regard to their cation exchange capacity and Pb-binding capacity in vitro after growth with either NO₃ ^- or NH₄ ⁺ as N source. The CECs of 800–1200 mol g-1 dry weight for Paxillus involutus 533 and 2000–3000 mol g-1 dry weight for Laccaria bicolor S238, were high compared to plant roots. The fungal mycelium also had a high Pb sorption capacity. It was higher in Laccaria bicolor S238 than in Paxillus involutus 533 and higher after pregrowth in NO₃ ^- compared to NH₄ ⁺. Both the higher CEC and the higher Pb sorption capacity of Laccaria bicolor S238 compared to Paxillus involutus 533 might have been the result of the hydrophilic nature of the of Laccaria bicolor S238 mycelium. It would have absorbed the solutions better than the hydrophobic mycelium of Paxillus involutus 533. X-ray microanalysis of the cell walls revealed that the Pb content of the cell walls was higher in Paxillus involutus 533 than in Laccaria bicolor S238. Nevertheless, electron dense deposits in the cell walls of Laccaria bicolor S238 contained large amounts of Pb, P and S. Thus, while Pb was evenly distributed in the cell walls of Paxillus involutus 533, Pb was accumulated in electron dense deposits in Laccaria bicolor S238. The results are discussed in view of their significance for the mycorrhizal symbiosis.     

Zinc phytotoxicity to oilseed rape grown on zinc-loaded substrates consisting of Fe oxide-coated and calcite sand

The risk of zinc (Zn) phytotoxicity in soils has increased in various regions following application of different anthropogenic materials. In order to assess the relative efficiency of Fe oxide and calcite in sorbing Zn and hence alleviating Zn phytotoxicity, we grew oilseed rape for 28 days in pots containing Zn-loaded model substrates consisting of Fe oxide (ferrihydrite)-coated sand (FOCS, 0.2–0.5 mm, 0.3 m² ferrihydrite g^–1 sand) and calcium carbonate (calcite) sand (CCS, 0.2–0.5 mm, 0.3 m² calcite g^–1 sand). Five substrates containing 5, 10, 20, 40, and 80% FOCS and supplied with ZnSO₄ at a rate of 30, 100, 300, and 1000 mg Zn kg^–1 were used in the cropping experiment and in an in vitro study of Zn desorption for 62 days. Plants exhibited good growth and a similar dry matter yield (DMY) at the 30 and 100 mg Zn kg^–1 rates. On the other hand, DMY was markedly reduced at the 300 and, especially, at the 1000 mg Zn kg^–1 rate, particularly for the substrates with the higher FOCS proportions. Symptoms of phytotoxicity (viz. chlorosis, purple colouration due to P deficiency) were apparent at such rates and were accompanied by high Zn concentrations in both shoot (average values >1000 and >1500 mg Zn kg^–1 dry matter for the 300 and 1000 mg Zn kg^–1 rate, respectively) and root (average values >2500 and >6000 mg Zn kg^–1 dry matter for the 300 and 1000 mg Zn kg^–1 rate, respectively). Total Zn uptake was maximal at 300 mg Zn kg^–1. The results of water extractable Zn in the substrate after cropping and the dissolved Zn concentrations measured in substrate–water systems (desorption experiment) suggest that, on a surface area basis, calcite is more effective than Fe oxide to retain Zn and thus alleviate phytotoxicity at high Zn loadings. However, the Zn-sorption capacity of the Fe oxide cannot be neglected, particularly at low Zn loadings, where Fe oxide seems to exhibit a higher affinity for Zn – but not a higher Zn-sorption capacity – than does calcite.     

Chitin production by arthropods in the hydrosphere

Chitin is widely distributed in nature and its annual production is thought to be huge. However, the chitin production has been rarely estimated in aquatic ecosystems, despite the growing economic interest in this polymer. Arthropods are one of the main chitin producers in the hydrosphere and a correct evaluation of the chitin production by these organisms in the different marine and freshwater ecosystems is of prime interest to understand their importance in the biogeochemical cycles of carbon and nitrogen. Such evaluation is also worth considering to achieve a rational exploitation of crustaceans which are currently the major source of chitin for the industry. Annual chitin production of crustaceans and insects in aquatic ecosystems was estimated on the basis of annual tissue production estimates and body chitin content measurements. About 800 annual tissue production estimates were collected from the literature. Estimates mainly concerned continental fresh waters and neritic ecosystems. Data were almost inexistent for athalassohaline and oceanic ecosystems. On the whole, 60% of the production estimates fell between 0.1 and 10.0 g dry weight m^–2 yr^–1. Published chitin levels in crustaceans and insects ranged from 3 to 16% of the whole body dry weight. Data were, however, lacking for some major groups such as trichopterans or amphipods. Aquatic insects and crustaceans were therefore collected and assayed for chitin using a highly specific enzymatic method. The chitin content of the collected insects (Coleoptera, Diptera, Ephemeroptera, Odonata, Plecoptera, Trichoptera) varied from 3 to 10% of the whole body dry weight; that of the collected crustaceans (Amphipoda, Branchiopoda, Copepoda) from 2.5 to 8.5% of the whole body dry weight. Total annual chitin production by arthropods had been estimated to 28 × 10⁶ T chitin yr^–1 for the freshwater ecosystems, to 6 × 10⁶ T chitin yr^–1 for athalassohaline ecosystems and to 1328 × 10⁶ T chitin yr^–1 for marine ecosystems. The importance of the chitin production corresponding to the formation of exuviae and peritrophic membranes in arthropods and the chitin production by non-arthropod organisms in the chitin budget of aquatic ecosystems was highlighted and discussed.     

Removal characteristics of high load ammonia gas by a biofilter seeded with a marine bacterium, Vibrio alginolyticus

When the cells of the newly isolated marine bacterium, Vibrio alginolyticus, were inoculated on to an inorganic packing material in biofilter, and a load of ammonia of 2.4–22.5 g-N kg^–1dry packing material was introduced continuously under non-sterile conditions, the average amount of NH₃removed exceeded 85% over 61-d operation. The maximum removal capacity and the complete removal capacity were 22.8 g-N kg^–1dry packing material dand 18.6 g-N kg^–1dry packing material d, respectively, which were about four times larger than those obtained in autotrophic nitrifying sludge inoculated on the same packing material.     

Sedimentation properties of chitosomes fromMucor rouxii

Summary This study was undertaken to assess the distribution and localization of chitin synthetase in a fungal cell and to evaluate the sedimentation behavior of chitosomes (microvesicular containers of chitin synthetase). Chitosomes were isolated from cell-free extracts of yeast cells ofMucor rouxii by rate-zonal and isopycnic sedimentation in sucrose density gradients. Because of their small size and low density, chitosomes were effectively separated from other subcellular particles. Rate-zonal sedimentation was a suitable final step for isolating chitosomes as long as ribosomes had been eliminated by enzymic digestion. By isopycnic centrifugation, chitosomes could be separated directly from a crude cell-free extract; they cosedimented with a sharp symmetrical peak of chitin synthetase at a buoyant density of d=1.14–1.15g/cm³; the only significant contaminants were particles of fatty acid synthetase complex. From such sedimentations, we estimated that 80–85% of the chitin synthetase activity in the cell-free extract was associated with chitosomes; the rest was found in two smaller peaks sedimenting at d=1.19–1.20 and d=1.21–1.22 (5–10%), and in the cell wall fraction (5–10%). By consecutive rate-zonal and isopycnic sedimentations, chitosome preparations with relatively few contaminating particles were obtained. Potassium/sodium phosphate buffer (pH 6.5)+MgCl₂ was the most effective isolation medium for chitosomes. Other buffers such as TRIS-MES+MgCl₂ led to massive aggregation of chitosomes and a change in sedimentation properties. This tendency of chitosomes to aggregate could explain why most of the chitin synthetase activity of a fungus is sometimes found associated with other subcellular structures,e.g., plasma membrane.     

Accumulation,regulation and toxicity of copper,zinc, lead and mercury in Hyalella azteca

Zinc, lead and mercury accumulation in the amphipod Hyalella azteca increases with increasing exposure to metals. During 10 week chronic toxicity tests, metal accumulated at the highest non-toxic/lowest toxic concentration was 126/136 µg Zn g^–1, 7.1/16 µg Pb g^–1 and 56/90 µg Hg g^–1 dry weight. Concentrations of lead and mercyry in control animals were substantially lower (1.3 µg Pb g^–1 and 0.4 µg Hg g^–1), but concentrations of zinc in controls (74 µg g^–1) were about one half those of the lowest toxic concentration. Copper was completely regulated. Accumulated copper concentrations after 10 weeks exposure to all waterborne copper concentrations resulting in less than 100% mortality were not significantly different from controls (79 µg g^–1). Lead and mercury concentrations in wild H. azteca should be useful indicators of potential toxicity. Zinc accumulation may also be a useful indicator of zinc toxicity, but careful comparison with control or reference animals is necessary because of the small differences between toxic and control concentrations. Copper is not accumulated by H. azteca under chronic exposure conditions and body burdens of field animals cannot be used as an indicator of exposure or potential toxic effects. Short term exposures to copper, however, result in elevated copper concentrations in H. azteca, even at concentrations below those causing chronic toxicity. Short term bioaccumulation studies might, therefore, provide a useful indication of potential chronic copper toxicity.     

Effect of zinc on cadmium toxicity to the amphipod Gammarus pulex

The freshwater amphipod Gammarus pulex was sampled from several sites in Northumberland, England and analysed for total body zinc and cadmium concentrations.The toxicity of cadmium to animals from the different sites was determined and a positive relationship was found between the initial total body zinc concentration and the ability to survive in an artificial medium containing 0.5 mg 1^–1 cadmium as cadmium sulphate.Exposure in the laboratory to a low concentration of zinc (0.01 mg 1^–1) as zinc sulphate for two weeks resulted in a decreased susceptibility to cadmium poisoning.     

Comparative Study of the Cell Walls of the Yeastlike and Mycelial Phases of Histoplasma capsulatum

Cell walls were prepared from the yeastlike and mycelial phases (YP and MP) of Histoplasma capsulatum and from Saccharomyces cerevisiae by mechanical disruption and washing. Lipids were extracted with methanol-ether, chloroform, and acidified methanol:ether; a final extraction was made with ethylenediamine. The lipid contents of H. capsulatum YP and MP walls were about the same. Qualitative and quantitative analyses were made of the products obtained from treatment of the cell walls, or fractions from them, with weak acid or with enzymatic preparations containing glucanase and chitinase activities. YP walls contained much larger quantities of chitin and smaller quantities of mannose and amino acids than the MP walls. H. capsulatum MP was shown to resemble S. cerevisiae by low chitin content and by the presence of a mannose polymer, soluble in ethylenediamine and water. H. capsulatum MP chitin appeared to be intimately associated with glucose in the wall, since enzymatic hydrolysis of the residue after mild acid hydrolysis of cell walls or fractions from them resulted in the release of glucose and acetylglucosamine; only acetylglucosamine was released from YP walls with such treatment. By electron microscopic observations, the unextracted MP cell walls were much thinner than the YP, and neither wall appeared laminated.