Abstract: The aim of this study was to compare the ability of commonly used conventional biochemical tests, sequencing analysis of 16S rRNA genes and tDNA-intergenic spacer length polymorphism (tDNA-PCR) to identify species of the genus Bacillus recovered from marine sediments. While biochemical tests were not sufficiently sensitive to distinguish between the 23 marine strains analyzed, partial 16S rRNA gene sequences allowed a correct identification, clustering them into four species belonging to Bacillus licheniformis (n = 6), Bacillus cereus (n = 9), Bacillus subtilis (n = 7) and Bacillus pumilus (n = 1). The identification results obtained with 16S rRNA sequencing were validated by tDNA-PCR analysis of 23 marine isolates that were identified by the similarities of their fingerprints to those of reference strains. tDNA-PCR fingerprinting was as discriminatory as 16S rRNA sequencing analysis. Although it was not able to distinguish among the species of the B. cereus and B. subtilis groups, it should be considered a rapid and easy approach for the reliable identification of unknown Bacillus isolates or at least for the primary differentiation of Bacillus groups.

Abstract: In the present study three mesophilic Bacillus isolates were analyzed for their α-amylase activity in shakeflask cultures. The organisms were capable to produce hydrolysis zone around their colonies on starch agar medium. The effect of various fermentation conditions on α-amylase production was investigated, and in every case it was found that B. subtilis was the best producer of the enzyme, which was followed by the newly isolated Bacillus sp. and B. amyloliquefaciens. The synthesis of extracellular α-amylase by the bacteria was repressed by the presence of readily metabolizable carbon source like glucose in the culture medium. Maximum α-amylase activity by the Bacillus isolates was obtained at 37°C with an initial medium pH 7.0 under agitation at 160-180 rpm for 72 h of growth. Keywords: Bacillus species, α-Amylase, Enzyme production, Shake-flask cultureDOI: http://dx.doi.org/10.3329/bjm.v24i2.1257 Bangladesh J Microbiol, Volume 24, Number 2, December 2007, pp 129-132

Abstract: Twenty isolates of Bacillus species obtained from livestock manure composts and cotton-waste composts were tested for their antagonistic effects in vitro against three green mold pathogens of mushrooms (Trichoderma harzianum, T. koningii, and T. viridescens). However, there exists a possibility Bacillus species may have antagonistic effects against mushrooms themselves, and thus the same 20 isolates were tested in vitro against three species of mushrooms (Flammulina velutipes, Lentinus edodes, and Pleurotus ostreatus). Of the 20 Bacillus species isolates tested, two inhibited mycelial growth of T. harzianum, seven that of T. koningii, and eight that of T. viridescens. Importantly, the bacterial isolates M27 and RM29 strongly inhibited mycelial growth of all the Trichoderma spp. isolates tested. The isolate M27 was subsequently identified as the most effective in inhibiting mycelial growth of all the Trichoderma species. Interesting results of the effect Bacillus isolates had upon the mushroom species followed. It was found that most Bacillus isolates except 5T33 at least somewhat inhibited mycelial growth of the three mushroom species or some of the mushrooms. Furhermore, the antagonistic effects of the bacterial isolates against the three species of mushrooms varied depending on the mushroom species, suggesting a role for mushroom type in the mechanism of inhibition. The bacterial isolates M27 and RM29 were identified as having the most antagonistic activity, inhibiting mycelial growth of all the Trichoderma spp. as well as mycelial growth of the three species of mushrooms. These results suggest that the bacterial isolates and their antagonistic effects on green mold pathogens should be further studied for their practical use for biological control of green mold in the growing room of the mushrooms.

Abstract: Damping-off caused by Rhizoctonia solani kuhn AG-4 or Pythium aphanidermatum Edson is a serious disease of cabbage seedlings, especially in culture medium in Taiwan. Thus, preventing culture medium from contamination of these pathogens should be an important method for controlling the disease. Effective evaluations of the volatile compounds produced by Bacillus mycoides isolates CHT2401 and CHT2402 grown on plates of King’s medium B, Luria-Bertani medium, nutrient agar, potato dextrose agar, soy powder milk agar, and tryptic soy agar for suppression of mycelial growth of R. solani RST-04 and P. aphanidermatum Pa01 were conducted. The two bacterial isolates grown on soy powder milk agar and tryptic soy agar expressed better effectiveness in inhibiting the pathogens. The volatile compounds produced by the two bacterial isolates were identified using ammonia detector tube and Gas Chromatography – Mass Spectrometry (GC-MS). The major volatile compounds produced by both isolates were identified as ammonia and dimethyl disulfide that were much more effective in inhibiting mycelial growth of R. solani RST-04 and P. aphanidermatum Pa01. The mycelial morphology of R. solani RST-04 and P. aphanidermatum Pa01 treated with dimethyl disulfide and the volatile compounds produced by B. mycoides CHT2402 respectively were observed under scanning electron microscope and transmission electron microscope. The hyphal structures of R. solani RST-04 and P. aphanidermatum Pa01 after treatments were malformed and the numbers of the organelles in their hyphal cells significantly decreased. These results demonstrated that dimethyl disulfide, ammonia, and the volatile compounds produced by B. mycoides CHT2402 could be harmful to the mycelia of the pathogens. In the greenhouse, the culture media were individually treated with the culture solutions of B. mycoides CHT2401and CHT2402 grown in soy powder milk. The culture solutions of the Bacillus isolates could increase the plant seedlings biomass of asparagus bean, cabbage, edible rape, lettuce, and tomato compared to the untreated control. Furthermore, the culture solutions of the Bacillus isolates were separately tested for controlling damping-off of cabbage seedlings caused by R. solani RST-04 or P aphanidermatum Pa01. The culture solutions of the two bacterial strains could respectively reduce 28% and 27% disease incidence of cabbage seedlings caused by P. aphanidermatum Pa01, but not significantly reduce the disease incidence of seedlings damping-off caused by R. solani RST-04. The results suggested that B. mycoides isolates CHT2401 and CHT2402 were potential biocontrol agents on controlling damping-off of cabbage seedlings caused by P. aphanidermatum Pa01.

Abstract: Several bacterial isolates were isolated from Dakahlia and Damitta Governorates. The isolates were purified, identified and tested for PHB production. The tested isolates were 17 isolates of Azotobacter chroococcum, 8 isolates of Azospirillum lipoferum, 22 isolates of Alcaligenes eutrophus, 3 isolates of A. latus, 3isolates of Bacillus subtilis, 2isolates of B. cereus, 5 isolates of B. megaterium, 2 isolates of B. coagulans, 3 isolates of B. polymexa, 4 isolates of B. thuringiensis, 2 isolates of Pseudomonas fluorescens, 2 isolates of P. putida, 2 isolates of P. alcaligenes, 2 isolates of P. aeruginosa, 4 isolates of Rhizobium leguminosarim, 2 isolates of R. meliloti, one isolate of R. japonicum and 12 isolates of Streptomyces albus.Also, the occurrence of alginate in different isolates of Azotobacter and Pseudomonas were investigated. All isolated strains proved to be PHB producers. The amount of PHB produced by Bacillus isolates varied from 0.02 to 0.16 g/l, while by Azospirillum isolates the PHB amounts ranged from 0.12 to 0.58 g/l. Using Rhizobium isolates, the highest amount of PHB (0.36g/l) was produced by R. leguminasarim No 3 , while the lowest amount (0.06 g/l) was obtained by R. leguminosarim No 2. Usig two media, Alcaligenus eutrophus No. 20, seemed to be the most active PHB producer among all Alcaligenes isolates (0.52 g/l). By Pseudomonas isolates, Pseudomonas fluorescens No. 2 produced 0.30g/L of PHB while P. aeruginosa No. 2 produced 0.10 g/L. Generally the isolate of Azotobacter chroococcum No. 16 showed the highest values. The cell dry weight, PHB concentration and the yield of PHB were 2.28 g/l, 0.78 g/l, 34.21%, respectively. And the highest amount of alginate concentration (g/l) was 0.44 by Azotobacter chroococcum No. 14.

Abstract: Fermented soybean foods are an important component of the Korean diet. Eoyukjang is a type of traditional fermented soybean source. Microbial analysis of eoyukjang was conducted during the fermentation period in this study. Microorganisms isolated from eoyukjang were identified by biochemical tests and 16S rDNA sequencing. 17 different microorganisms, including bacteria, yeast, and fungi were detected in eoyukjang during the fermentation period. Even though Aspergillus participated in the early stage of fermentation of eoyukjang, Bacillus species and Saccharomyces cerevisiae were the major microzymes in eoyukjang throughout the maturation period. Eoyukjang is generally consumed after the boiling of the final sample. Therefore, the final sample of eoyukjang was boiled and analyzed. Our results showed that no vegetative microorganisms survived under the boiling conditions for eoyukjang. Fermented soybean products in the domestic market were also assessed for comparison with the results from eoyukjang. The total cell number of kanjang (soy sauce) samples was between 0 to 42 CFU/mL. The isolated microorganisms were identified as Bacillus species. All Bacillus isolates were not found to harbor the three enterotoxin-producing and emetic toxin-producing genes.

Abstract: The basic goal of this study is the isolation of some Bacillus isolates from different sources for production of bioactive products such as detection of cellulose(s) and pectinase(s) and identification of these isolates using quantitative real-time PCR (qRT PCR) depending on 16S rRNA gene in each isolate. Nine, six and two isolates were obtained from municipal sewage water, composted rice straw and expired pharmaceutical drugs (Capozide), respectively. Out of nine isolates, only four Bacilli (SW2, SW3, SW6 and SW9) were selected and examined for cellulose(s) and pectinase(s) production, and revealed that only SW2 and SW9 exhibited maximum pectate lyase (PL) and polygalacturonase (PGase) viz. 465.55 and 222.53 (U/mg), respectively. Out of six Bacilli isolates (RS1, RS3 and RS5), only RS3 showed maximum productivity of carboxymethylcellulase (CMCase) viz. 657.21 (U/mg). On the other hand, only one Bacillus isolate out of two was selected ECC2 and exhibited the maximum Avicelase productivity viz. 533.32 (U/mg). TaqMan qRT PCR method was used to detect and quantify the 16S rRNA genes of Bacillus isolates tested. Forward primer Bsub5 F and the reverse primer Bsub3 R were used for the amplification of 16S rRNA of the “Bacillus subtilis group”, it is also successful to demonstrate the most similarities of the Bacillus isolates in this study and they are closely related to “Bacillus subtilis group”.