Shinjiro Kanazawa

Isolation and Characterization of Agar-degrading Paenibacillus spp. Associated with the Rhizosphere of Spinach

Agar-degrading bacteria in spinach plant roots cultivated in five soils were screened, and four strains of Paenibacillus sp. were isolated from roots cultivated in three soils. The agar-degrading bacteria accounted for 1.3% to 2.5% of the total bacteria on the roots. In contrast, no agar-degrading colony was detected in any soil (non-rhizosphere soil samples) by the plate dilution method, and thus these agar-degrading bacteria may specifically inhabit plant roots. All isolates produced extracellular agarase, and could grow using agar in the culture medium as the sole carbon source. Zymogram analyses of agarase showed that all four isolates extracellularly secreted multiple agarases (75-160 kDa). In addition, the isolates degraded not only agar but also various plant polysaccharides, i.e., cellulose, pectin, starch, and xylan.

Effect of fertilizer and manure application on microbial numbers, biomass, and enzyme activities in volcanic ash soils: I. Microbial numbers and biomass carbon

Abstract Fertilizer and manure application affected the microbial numbers and biomass in topsoil and subsoil fields consisting of volcanic ash upland soil. 1. Microbial numbers (bacteria, spore-forming bacteria, actinomycetes, and fungi) were the largest in the farmyard manure plot, followed by the chemical fertilizer plot, and smallest in the no-fertilizer plot in both fields. The numbers of all the microorganisms except fungi in the samples from the topsoil field were larger than in the samples from the subsoil field in the respective plots. Microbial numbers obtained here were several times as large as the mean values of microorganisms in volcanic ash upland soils, presumably due to the use of a Waring blender for dispersing the soil samples. 2. Soil microbial biomass carbon was the largest in the farmyard manure plot, followed by the chemical fertilizer plot, and smallest in the no fertilizer plot in both fields. Microbial biomass carbon in the samples from the subsoil field was larger than that in th...

Distribution of microorganisms, biomass ATP, and enzyme activities in organic and mineral particles of a long-term wastewater irrigated soil.

The aim of the study was to elucidate the spatial distribution of soil microorganisms and enyzme activities in a long-term wastewater treated soil. Soil was sampled from a plough layer of the Ah horizon of a sandy Haplic Luvisol which was either (1) irrigated with municipal wastewater for almost 100 years, or (2) no more irrigated since 20 years, or (3) never received wastewater. The samples were fractionated by wet sieving to obtain seven size fractions of organic and mineral soil particles, and a separate silt+clay fraction. The individual soil samples contained between 1.2% (never irrigated) and 4.1% (long-term irrigated) organic particles by weight, but these particles harboured up to 47.8% of the total soil carbon and 41.7% of nitrogen, and thus represented an important storage of energy and nutrient for microorganisms. In total, however, the highest C and N amounts were accumulated in the silt+clay fraction, whereas coarser mineral particles which dominanted by weight in the Haplic Luvisol were low in C and N. The highest numbers of bacteria, actinomycetes and fungi per gram of the individual soil fractions were found in organic particles of the long-term irrigated soil. Less nutrient-dependent oligotrophic bacteria were for the most part associated with the silt+clay fraction, irrespective of the soil treatment with wastewater. Similar to microbial counts, also the ATP content, as a measure of active microbial biomass, and the activities of β-glucosidase, β-acetylglucosaminidase, and proteinase were higher in the long-term irrigated soil than in that which was never irrigated. In most cases slightly enhanced values of microbiological and biochemical parameters were still detectable 20 years after the wastewater irrigation was terminated. The values of the individual parameters decreased in all soil samples under testing in general gradually with decrease in size of the organic soil particles. In conclusion, the coarse soil organic particles > 5 mm and the silt+clay fraction < 0.05 mm represent the sites with the highest microbial inhabitance, ATP contents and enzyme activities in the Ah horizon of an Haplic Luvisol. Long-term wastewater irrigation resulted in an increase of microbial counts, total biomass and soil enzyme activities. Verteilung von Mikroorganismen, Biomasse-ATP und Enzymaktivitaten in organischen und mineralischen Partikeln eines langzeitig mit Abwasser berieselten Bodens Die Untersuchungen hatten zum Ziel, den Einfluss einer langzeitigen Abwasserverrieselung auf die raumliche Verteilung der Bodenmikroorganismen und enzymatischer Aktivitaten im Boden zu beleuchten. Es wurden Proben aus dem Oberboden (Ah-Horizont) eines sandigen Haplic Luvisol untersucht. Die einzelnen Bodenstandorte wurden (1) seit ca. 100 Jahren mit mechanisch vorgeklartem Kommunalabwasser berieselt, (2) seit ca. 20 Jahren nicht mehr berieselt, (3) niemals berieselt. Die entnommenen Bodenproben wurden einer Nasssiebung unterzogen. Auf diese Weise konnten sieben Fraktionen von organischen und mineralischen Partikeln unterschiedlicher Grose gewonnen werden. Die Bodenproben enthielten zwischen 1,2% (unberieselt) und 4,1% (ca. 100 Jahre berieselt) organische Partikel nach Gewicht. Jedoch waren in diesen Partikeln bis zu 47,8% des Bodengehalts an Kohlenstoff bzw. 41,7% an Stickstoff und somit auch ein bedeutendes Energie- und Nahrstoffreservoir fur Mikroorganismen enthalten. In absoluten Zahlen waren allerdings die hochsten C- und N-Gehalte in der jeweiligen Schluff+Ton-Fraktion akkumuliert. Die groberen mineralischen Partikel, die ihrem Gewicht nach in den Bodenproben dominierten, enthielten nur kleine Mengen an C und N. Die hochsten Zahlen an Bakterien, Actinomyceten und Pilzen per Gramm der gesiebten Bodenfraktionen wurden in den organischen Partikeln der Bodenproben aus dem langzeitig berieselten Standort festgestellt. Die an Nahrstoffkonzentrationen wenig anspruchsvollen oligotrophischen Bakterien waren uberwiegend mit der Schluff+Ton-Fraktion der jeweiligen Bodenprobe assoziiert. Ahnlich wie die Mikrobenzahlen waren auch der ATP-Gehalt als ein Masstab fur aktive mikrobielle Biomasse und die Aktivitaten von β-Glucosidase, β-Acetylglucosamidase und Protease im berieselten Boden hoher als im Boden des unberieselten Standortes. Etwas abgeschwacht waren die Erhohungen der einzelnen Parameter auch noch 20 Jahre nach der Beendigung der Bodenberieselung feststellbar. Die Werte der einzelnen biologischen und biochemischen Parameter zeigten in den untersuchten Bodenproben fast immer eine mit der sinkenden Grose der organischen Partikel einhergehende rucklaufige Tendenz. Man kann insgesamt feststellen, dass die organischen Partikel > 5 mm und die Schluff+Ton-Partikel < 0,05 mm in den untersuchten Bodenproben aus einem Haplic Luvisol die Mikrohabitate mit der hochsten mikrobiellen Besiedlung, dem hochsten ATP-Gehalt und den hochsten enzymatischen Aktivitaten darstellen. Eine langzeitige Abwasserverrieselung bewirkte deutliche Erhohungen der mikrobiellen Besiedlungsdichte und der biochemischen Aktivitaten des Bodens.

Preparation of pH 3.0 agar plate, enumeration of acid-tolerant, and Al-resistant microorganisms in acid soils

Abstract We attempted to prepare an agar plate containing Al under the acid conditions (pH 3.0) to count the number of colonies of Al-resistant microorganisms and isolate them. As a result, Al-resistant microorganisms were isolated from the tea garden soils and identified. 1. Agar could be solidified when the culture medium solution (pH 3.0), Al2(SO4)3 solution (pH 3.0), and agar solution were mixed after autoclaving separately. 2. The number of Al-resistant microorganisms was largest in the strong acid tea garden soil, followed by the forest soil and smallest in the upland soil. In particular, the number of Al-resistant microorganisms (Al 100 mM in agar medium) accounted for 12.5% of the number of total microorganisms, suggesting that the accumulation of Al-resistant microorganisms increased when the soil pH decreased. 3. Fungi accounted for most of the high Al-resistant microorganisms. 4. About half of the acid-tolerant fungi (grown at pH 3.0) from the tea garden soils consisted of high Al-resistant mic...

Microbial Ecology in Tea Soils : I. Soil Properties and Microbial Populations

Abstract The soil chemical properties and microbial numbers in three volcanic ash soils and two non-volcanic ash soils, which had been continuously subjected to the same tea cultivation practices (21 y), were investigated. The results obtained were as follows. 1) pH values of all the soils gradually decreased from the original pH value (near neutral or mildly acid pH) to strongly acid values of about 4 or lower. In contrast, long-term tea cultivation practices resulted in the increase of the total C and N contents in the surface layers (0–20 cm) while the contents remained stable in the subsurface layers (20–40 cm). The increase in the organic matter content in non-volcanic ash soils was presumably due to the accumulation of microbial residues. The availability of P increased markedly. 2) Numbers of bacteria, actinomycetes, fungi, and denitrifiers were higher in volcanic ash soils than in non-volcanic ash soils, and also higher in surface layers than in subsurface layers. The results suggest that in spite...

Microbial biomass in paddy soil: I. Microbial biomass calculated from direct count using fluorescence microscope

The authors investigated the role of microorganisms in paddy soil ecosystems and the influence of fertilizer and manure applications on the soil microbial biomass in paddy soils based on direct count using a fluorescence microscope. The results are summarized as follows. 1) The number of bacteria in the plough layer was 6.6 × 10l0-7.6x 1010/g oven dry soil in the Konosu paddy field, 2.2 × 10l0-2.8 × 1010/g oven dry soil in the Aizu paddy field, and 1.1 × 1010-1.3 × 1010/g oven dry soil in the Shizuoka paddy field. The different fertilizer managements did not influence significantly the number of bacteria in the three experimental fields. 2) In all the experimental fields, the number of cocci predominated accounting for 90% of the total number of bacteria, and the number of rods was less than 10%. Among the cocci, the number of small cocci (diameter=0.5/µm) accounted for 60–70% of the total cocci bacteria. 3) Hyphal length increased in response to long term application of inorganic fertilizer and organic m...

Microbial Biomass in Paddy Soil: II. “Microbial Biomass Carbon” Measured by Jenkinson’s Fumigation Method

The authors investigated the role of microorganisms in the paddy soil ecosystem and the influence of fertilizer and manure application on the soil “microbial biomass” in paddy soils using Jenkinson’s fumigation method. The results obtained are summarized as follows. 1) “Microbial biomass-C” was greatest in the organic manure plot, followed by the inorganic fertilizer plot, and smallest in the no fertilizer plot in each field. These results indicate that the different fertilizer managements remarkably influenced the “microbial biomass-C” measured by Jenkinson’s method. 2) The proportion of “microbial biomass-C” in total soil organic carbon was 3.6–6.9% in the no fertilizer plot, 4.3–7.5z% in the inorganic fertilizer plot, and 5.2–8.7% in the organic manure plot. These results show that fertilizer and manure application in paddy soils increased the proportion of “microbial biomass-C” in total soil organic carbon compared with that in grassland and forest soils. 3) There was no significant relationship betwe...

A Modified Method for Determination of Cellulase Activity in Forest Soil

The optimum conditions for the determination of the cellulase activity of forest soil were defined as follows: One g of wet soil is placed into a test tube (20 x 150 mm), and 0.2 ml of toluene is added. After the mixture has been allowed to stand for 10 min, 10 ml of 0.2 M sodium acetate buffer solution containing 10 mg of CMC per ml is added. The mixture is stirred by a Thermo-Mixer, covered with a “Parafilm,” and is incubated at 50°C for 4 h in a shaking incubator. The reaction solution is separated by centrifugation, and 0.5 g of Polychlal AT is added, to remove colored humus. Centrifugation is repeated, to remove Polychlal AT. The glucose in the decolorized reaction solution is determined according to the glucostat method.

Isolation of cadmium-resistant bacteria and their resistance mechanisms. Part 1. Isolation of Cd-resistant bacteria from soils contaminated with heavy metals

In the first series of studies aimed at addressing the environmental problems of soils and drainage systems, we attempted to isolate cadmium (Cd)-resistant bacteria from soils heavily contaminated with Cd and determine their growth rate in media with cadmium. In soils contaminated with heavy metals, there was on increase in the number of Cd-resistant bacteria. Among 14 Cd-resistant bacteria isolated from soils contaminated with heavy metals, only six strains (Flavobacterium sp., Comamonas testosteronii, Methylobacterium fujisawaense, Alcaligenes piechaudii, Alcaligenes xylosoxidans) were identified as new types of Cd-resistant bacteria. All the isolated resistant bacteria grew fast, and reached the maximum growth rates within 24 to 48 h (Growth conditions were as follows. Culture medium: nutrient broth, pH: 6.0, temperature: 30°C and of concentration of added Cd: 0.1, 1.0, 10.0, and 100.0 mg kg-1).

Isolation of cadmium-resistant bacteria and their resistance mechanisms. Part 2. Cadmium biosorption by Cd-resistant and sensitive bacteria

Abstract In the first series of studies aimed at addressing the environmental problems of soils and drainage systems, we attempted to isolate cadmium (Cd)-resistant bacteria from soils heavily contaminated with Cd and determine their growth rate in media with cadmium. In soils contaminated with heavy metals, there was on increase in the number of Cd-resistant bacteria. Among 14 Cd-resistant bacteria isolated from soils contaminated with heavy metals, only six strains (Flavobacterium sp., Comamonas testosteronii, Methylobacterium fujisawaense, Alcaligenes piechaudii, Alcaligenes xylosoxidans) were identified as new types of Cd-resistant bacteria. All the isolated resistant bacteria grew fast, and reached the maximum growth rates within 24 to 48 h (Growth conditions were as follows. Culture medium: nutrient broth, pH: 6.0, temperature: 30°C and of concentration of added Cd: 0.1, 1.0, 10.0, and 100.0 mg kg-1).