Mikiya Hiroki

Effects of Heavy Metal Contamination on Soil Microbial Population

Abstract In order to estimate the effect of heavy metal pollution on soil microorganisms, a field survey was carried out in a heavy-metal-contaminated (Cd, 1.1-2.7 mg/ kg soil; Zn, 234-571 mg/kg soil; Cu, 310-751 mg/kg soil) fallow paddy field. Although the number of actinomycetes and bacteria decreased significantly with increasing heavy metal content, there was no significant correlation between the number of fungi and heavy metal content. Actinomycetes were affected by heavy metals most strongly. The degree of tolerance to heavy metals appears to be: fungi > bacteria > actinomycetes. These results suggest that the relatively low concentrations of heavy metals in the research area affected the soil microbial population.

Chemotaxonomy of planktonic cyanobacteria based on non-polar and 3-hydroxy fatty acid composition

Twenty‐eight axenio planktonic cyanobacterial strains (10 Microcystis, three Oscillatoria, one Spirulina, one Aphanizomenon, 13 Anabaena) were investigated for their fatty acid composition by measurement of non‐polar and hydroxy fatty acids. No 2‐hydroxy fatty acids were detected in any strain, but 3‐hydroxy fatty acids were detected in minor quantities in 24 strains. The highest portion of total fatty acids were non‐polar fatty acids. Qualitative and quantitative analyses of 3‐hydroxy fatty acids showed no taxonomic value in these strains, while the type of non‐polar fatty acid composition was shown to be consistent within Microcystis and Anabaena strains, distinguishing them as type 4, characterized by the presence of 18:4, and type 2, characterized by 18:3 (α) of the Kenyon‐Murata system. Two Oscillatoria agardhii Gomont strains were also included in the type 2 group due to the presence of 18: 3 (α), but the difference in characteristics of 16:2 and 16:3 between O. agardhii and Anabaena further divided type 2 into two subgroups: type 2A for Anabaena and type 2B for O. agardhii. A simplified unweighted pair group method with arithmetic averages (UPGMA) dendrogram demonstrated that the classification of 28 strains (Microcystis spp., Anabaena spp., Aphanizomenon flos‐aquae (Lemmermann) Ralfs f. gracile (Lemmermann) Elenkin, O. agardhii and Spirullnasubsalsa Oersted ex Gomont based on numerical analysis of non‐polar fatty acids corresponded to morphological species criteria, suggesting that non‐polar fatty acid composition is a valuable chemical marker in the taxonomy of planktonic cyanobacteria. However, the fatty acid composition in Oscillatoria raciborskii is similar to that of Microcystis and very different from that of O. agardhii, suggesting its special position in Oscillatoria and the chemical diversity in the genus Oscillatoria.

Effect of arsenic pollution on soil microbial population

Abstract In order to estimate the effect of arsenic pollution on soil microorganisms, a field survey was carried out in arsenic polluted (391-459 mg/kg dry soil) and unpolluted (32-43 mg/kg dry soil) fields, and the following results were obtained: The ratio of the number of bacteria to the number of fungi in plot I which was polluted and subjected to submerged conditions in summer was significantly lower than that in the other plots. Fungi appeared to be much more tolerant to As(III) than bacteria and actinomycetes. The ratio of the As(III)-tolerant fungi to the whole fungal population was higher in plot I than in the other plots. The fungal group which predominated in plot I belonged to a unique group and was never observed in the other plots. The diversity index of the fungal flora of plot I was lower than the indices of the other plots. In conclusion, the continuous pollution of soil by As promoted the predominance of As-tolerant fungi, and the effect of As pollution on the microbial population was mo...

Biomass, species composition and diversity of epipelic algae in mire pools

The biomass, species composition and diversity of epipelic algae in two small pools of contrasting physicochemical characteristics in Miyatoko Mire were studied in 1992 (pool 3 =site B4 and pool 50 =site D2). A total of 93 species and 67 species of epipelic algae occurred at sites B4 and D2, respectively. Considerable differences were observed between the two sites in the seasonal fluctuations of species number, biomass and dominant species. At site B4, little changed with species number during April–August and markedly increased in October, while biomass was largest in April and gradually decreased during June–October. Diatoms and desmids occupied 33–82% and 15–63% of total algal biomass, respectively. At site D2, species number and biomass were small in April just after the snow-thaw, and increased in June and decreased in August and October. Diatoms occupied 90–98% of total algal biomass. The species diversity was always higher at site D2 than B4. As a result of analyses of water chemistry in the two pools, pool B4 can be recognized as a habitat experiencing high disturbance frequency. It is predicted that pools experiencing frequent disturbance will have less epipelic algal biomass and diversity.

Microbial Community and Rate of Cellulose Decomposition in Peat Soils in a Mire

Abstract A field survey was carried out from April to October, 1992 in the Miyatoko Mire in Fukushima Prefecture, Japan, to determine the characteristics of the microbial community and cellulose decomposition rates in the peat soil. A total of 14 study sites were selected, including three types; hummocks (type I), hollows covered with Sphagnum (type II), hollows and streams without Sphagnum (type III). The numbers of fungi (2-1,000×104 CFU g-1) and bacteria (8.5-9,000 ×105 CFU g-1) varied with the sites and sampling dates: seasonal fluctuations were especially high in hummocks. The numbers of cellulolytic fungi (4.7-300×;104 CFU g-1) and cellulolytic bacteria (1.5-9.2×105 CFU g-1) also differed between sites. Cellulolytic fungi were predominant in the Sphagnum peat of type I, while cellulolytic bacteria were predominant in the peat soil of type III. Decomposition rates of cellulose filter paper for the 6 month period ranged from 0.01 to 0.83, and tended to be higher in the peat of type II than type I.

Phycoerythrin-containing Microcystis isolated from P.R. China and Thailand

Four strains of the unicellular, colonial cyanobacteria, Microcystis were isolated from P.R. China and Thailand, and showed greyish‐brown to dark brown color of cell mass. Their cells were almost spherical, 3.8–5.5 μm in diameter and had gas vesicles. Their colony forms have some differences among the strains but still have common characteristics including being irregular, reticulate, elongated and lobated. These characteristics are remarkably similar to Microcystis aeruginosa (Kutz.) Kutz. In addition, three of the strains also have the characteristics of Microcystis ichthyoblabe Kutz at times, and another sometimes shows characteristics similar to Microcystis novacekii (Kom.) Comp. The absorption spectra of both intact cells and extracted phy‐cobiliproteins showed an absorption peak at 560–570 nm in wavelength, which is the absorption range of phycoerythrin. Since no species of Microcystis has ever been reported to have phycoerythrin, the existence of phycoerythrin separates these four strains from the known species of Microcystis, and might enable them to be classified as a new species. Phycoerythrin might give these organisms the advantage to live underwater where the light is predominantly green in color.

Populations of Cd-tolerant microorganisms in soils polluted with heavy metals

Abstract In order to estimate the effect of heavy metal pollution on soil microorganisms, field surveys were carried out in paddy fields, upland fields, and fallow paddy fields located in two areas with heavy metal pollution in Japan (Izuhara area, where the soil samples contained 0.2–15.0 mg Cd kg-1, 11–696 mg Zn kg-1, 0.8–16.2 mg Cu kg-1; Toyama area, where the soil samples contained 0.2–3.9 mg Cd kg-1, 4.7–146 mg Zn kg-1, 0.5–21 mg Cu kg-1). There was no significant correlation between the viable counts of microorganisms and heavy metal content of the soils. The dominance value of the bacteria tolerant to 10 mg kg-1 Cd (D 10 value; D c =log(VC c /VC0), where VC c is the viable count on the plates containing c mg L-1 of Cd, VC0 is the viable count on the plates without Cd) increased significantly with the increase of the heavy metal content in the paddy fields. However, there was no significant correlation between the D100 value of the bacteria or the D10 value of the fungi and the heavy metal content o...

Purification of freshwater picoplanktonic cyanobacteria by pour‐plating in ‘ultra‐low‐gelling‐temperature agarose’

Seven unialgal cultures of picoplanktonic cyanobacteria isolated from freshwater lakes in Japan were examined in the present study. They are assignable to the Cyanobium cluster of the Synechococcus group sensu Waterbury and Rippka (1989) based on morphology, GC content, fatty acid and quinon compositions. They include one blue‐green colored strain (NIES‐680) with only phycocyanin as well as the PCC strains of Cyanobium cluster (Waterbury and Rippka 1989) and six reddish colored strains (NIES‐715, ‐717, ‐718, ‐721, ‐722 and ‐723) with both phycocyanin and phycoerythrin. All the strains examined developed colonies in pour‐plates I of 1.4%‘ultra‐low‐gel‐ling‐temperature agarose’within 1 month. The colony numbers were 12–20 × 102 c.f.u. mL‐1. Most clones derived from the colonies were axenic by the bacteria‐free check. The length of this procedure, preculture excepted, was 4–6 weeks. Colony formation in pour‐plate II of 0.6% agarose was observed in the blue‐green strain (NIES‐680) and the three reddish strains (NIES‐717,‐721 and ‐723) after 1–2 months. The number of colonies formed on pour‐plate II were 1–2 × 102 c.f.u. mL‐1, much lower than those on pour‐plate I. Only a blue‐green strain formed colonies on spread plates of 0.6% agarose. The colony numbers developed were 10–15 × 102 c.f.u. mL‐1 and similar to those on pour‐plate I. The other reddish strains did not develop colonies on spread plates. The pour‐plating method developed provides a highly efficient and successful method for obtaining axenic clonal cultures of planktonic cyanobacteria.

Development of a database system useful for identification of Anabaena Spp. (Cyanobacteria)

A database system useful for the identification of plank‐tonic Anabaena species based on morphology was designed. The system consisted of two database files (sample database and reference database) and three subsystems (data editing, similarity calculation, and identification). In the reference database file, 19 plank‐tonic Anabaena species were registered with their morphological characteristics of 26 attributes. The sample database of the test strain was compared with the reference one by calculating the average (St) of the similarity values estimated by peculiar methods according to the attributes. The test strain was recognized to be the same as the reference species with the highest St value. Strains of Anabaena spp. which had previously been identified were examined to test the effectiveness of the identification system. Using a non‐weighted identification system, where each morphological attribute has equal importance for identification, St (total similarity) values for the reference species previously identified were more than 0.76; however, they were not always the highest value obtained. Using a weighted identification system, where trichome form, trichome shape and akinete morphology were given greater importance in the identification subsystem, St values for the reference species to which the test strains had previously been assigned were highest. This system proved useful for identification of Anabaena species, and has the additional advantage that there are no individual differences in the diagnosis when different users interrogate the system.

Morphological, biochemical and physiological characteristics of Lyngbya hieronymusii var. hieronymusii (Oscillatoriales, Cyanobacteria)

An axenic clonal strain, CN4–3, of Lyngbya was obtained from a water sample collected from Lake Dalai, Inner Mongolia, P.R. China. The filaments were solitary, usually straight, sometimes a little bent and twisted. They sometimes made loose bundles. A filament was composed of a clear and film sheath and the trichome had no polarity and false branching. The sheath was approximately 0.5 μm in thickness and had no coloration in standard culture conditions, but the sheath was observed to be yellow in some filaments in seawater medium. The trichome had no constriction at the cross‐walls. Many small gas vacuoles were scattered in the cell. The end cells of the trichomes were broadly rounded and sometimes had calyptra. The presence or absence of granulation at the cross‐walls and calyptra was thought to be variable due to the range of environmental conditions or developmental stages of individual filaments. Trichomes were brownish‐green to olive‐green. Cell dimensions were 12.6–14.0 μm wide and 1.7–2.6 μm long. These characteristics are similar to those of Lyngbya hieronymusii Lemmermann var. hieronymusii. DNA base composition was 49.14 ± 0.34 mol% GC. Fatty acids were composed of four major components, 14:0, 16:0, 18:2 and 18:3α and six minor components, 16:1*, 16:1cis, 16:2, 16:3, 18:0 and 18:1 showing characteristics of group 2 (Kenyon et al. 1972; Murata et al. 1992). More than 20% of fatty acids were 14:0. This kind of fatty acid composition has never been reported in the other filamentous cyanobacteria of group 2. In vivo and phycobilin pigment absorption spectra showed that the strain contained phycoerythrin. Growth at different temperatures showed that the strain could grow from 10 to 40°C with the optimum growth at 20 and 30°C. Growth at different salinities showed that the strain could grow from standard (CT) medium to half strength (1.78%) of seawater medium. Even in the seawater medium (3.58%), the strain survived during experiments. These results suggest that L. hieronymusii var. hieronymusii has a peculiar taxonomic position within the filamentous cyanobacteria belonging to group 2 and inhabits various habitats.