Journal of Microbiology

Journal Articles

Regulatory role of cysteines in (2R, 3R)-butanediol dehydrogenase BdhA of Bacillus velezensis strain GH1-13: Yunhee Choi , Yong-Hak Kim; J. Microbiol. 2022;60(4):411-418. Published online March 14, 2022; DOI: https://doi.org/10.1007/s12275-022-2018-y

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Abstract: Bacillus velezensis strain GH1-13 contains a (2R,3R)-butanediol dehydrogenase (R-BDH) BdhA which converts acetoin to R-BD reversibly, however, little is known about its regulatory cysteine and biological significance. We performed sitedirected mutation of three cysteines in BdhA. The C37S mutant had no enzyme activity and the C34S and C177S mutants differed from each other and wild type (WT). After zinc affinity chromatography, 1 mM ZnCl2 treatment resulted in a 3-fold enhancement of the WT activity, but reduced activity of the C34S mutant by more than 2 folds compared to the untreated ones. However, ZnCl2 treatment did not affect the activity of the C177S mutant. Most of the double and triple mutant proteins (C34S/C37S, C34S/C177S, C37S/C177S, and C34S/C37S/C177S) were aggregated in zinc resins, likely due to the decreased protein stability. All of the purified WT and single mutant proteins increased multiple intermolecular disulfide bonds in the presence of H2O2 as the buffer pH decreased from 7.5 to 5.5, whereas an intramolecular disulfide bond of cysteine 177 and another cysteine in the CGIC motif region was likely formed at pH higher than pKa of 7.5. When pH varied, WT and its C34S or C177S mutants reduced acetoin to R-BD at the optimum pH 5.5 and oxidized R-BD to acetoin at the optimum pH 10. This study demonstrated that cysteine residues in BdhA play a regulatory role for the production of acetoin and R-BD depending on pH as well as metal binding and oxidative stress.

Flavobacterium humi sp. nov., a flexirubin-type pigment producing bacterium, isolated from soil: Inhyup Kim , Jiyoun Kim , Geeta Chhetri , Taegun Seo; J. Microbiol. 2019;57(12):1079-1085. Published online November 22, 2019; DOI: https://doi.org/10.1007/s12275-019-9350-x

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29 Citations

Abstract: A yellow pigmented, Gram-stain-negative, strictly aerobic, rod-shaped, motile by means of gliding, catalase and oxidase positive bacterium, designated strain DS2-AT, was isolated from soil. Growth was observed at 4–32°C (optimum, 28°C), pH 6–9 (optimum, 7.0), and with 0–0.25% (w/v) NaCl (optimum, 0%). Phylogenetic analysis of 16S rRNA gene sequence revealed that strain DS2-AT belonged to the genus Flavobacterium and was most closely related to Flavobacterium aquatile LMG 4008T (96.4%), Flavobacterium terrae DSM 18829T (95.6%), Flavobacterium vireti THG-SM1T (95.5%), Flavobacterium inkyongense IMCC27201T (95.4%), Flavobacterium brevivitae TTM-43T (95.2%), and Flavobacterium cucumis DSM 18830T (95.2%). Strain DS2-AT produces flexirubin- type pigments. The major fatty acids were iso-C15:0, iso-C17:0 3-OH, and iso-C15:0 3-OH. The major respiratory quinone was identified as menaquinone-6. The major polar lipid was found to be phosphatidylethanolamine. The average nucleotide identity values between strain DS2-AT and selected taxa, F. aquatile LMG 4008T, F. terrae DSM 18829T, and F. cucumis DSM 18830T, were 72, 72.7, and 71.6%, respectively. The draft genome of strain DS2-AT has a number of 14 contigs, scaffold N50 of 476,310 bp and a total size of 3,563,867 bp. Additionally, strain DS2-AT contains 3,127 of gene, 41 of tRNA, 6 of rRNA, and 3 of ncRNA. The DNA G + C content of stain DS2-AT was 40.7 mol%. Based on phylogenetic and phenotypic analyses, strain DS2-AT is considered as a novel species of the genus Flavobacterium, for which the name Flavobacterium humi sp. nov., (type strain DS2-AT = KACC 19715T = JCM 32786T) has been proposed.

Microbial transformation of Se oxyanions in cultures of Delftia lacustris grown under aerobic conditions: Shrutika L. Wadgaonkar , Yarlagadda V. Nancharaiah , Claus Jacob , Giovanni Esposito , Piet N. L. Lens; J. Microbiol. 2019;57(5):362-371. Published online March 21, 2019; DOI: https://doi.org/10.1007/s12275-019-8427-x

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Abstract: Delftia lacustris is reported for the first time as a selenate and selenite reducing bacterium, capable of tolerating and growing in the presence of ≥ 100 mM selenate and 25 mM selenite. The selenate reduction profiles of D. lacustris were investigated by varying selenate concentration, inoculum size, concentration and source of organic electron donor in minimal salt medium. Interestingly, the bacterium was able to reduce both selenate and selenite under aerobic conditions. Although considerable removal of selenate was observed at all concentrations investigated, D. lacustris was able to completely reduce 0.1 mM selenate within 96 h using lactate as the carbon source. Around 62.2% unaccounted selenium (unidentified organo-selenium compounds), 10.9% elemental selenium and 26.9% selenite were determined in the medium after complete reduction of selenate. Studies of the enzymatic activity of the cell fractions show that the selenite/selenate reducing enzymes were intracellular and independent of NADPH availability. D. lacustris shows an unique metabolism of selenium oxyanions to form elemental selenium and possibly also selenium ester compounds, thus a potential candidate for the remediation of selenium-contaminated wastewaters in aerobic environments. This novel finding will advance the field of bioremediation of selenium-contaminated sites and selenium bio-recovery and the production of potentially beneficial organic and inorganic reactive selenium species.

Oxygen-mediated growth enhancement of an obligate anaerobic archaeon Thermococcus onnurineus NA1: Seong Hyuk Lee , Hwan Youn , Sung Gyun Kang , Hyun Sook Lee; J. Microbiol. 2019;57(2):138-142. Published online January 31, 2019; DOI: https://doi.org/10.1007/s12275-019-8592-y

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Abstract: Thermococcus onnurineus NA1, an obligate anaerobic hyperthermophilic archaeon, showed variable oxygen (O2) sensitivity depending on the types of substrate employed as an energy source. Unexpectedly, the culture with yeast extract as a sole energy source showed enhanced growth by 2-fold in the presence of O2. Genome-wide transcriptome analysis revealed the upregulation of several antioxidant-related genes encoding thioredoxin peroxidase (TON_0862), rubrerythrin (TON_0864), rubrerythrin-related protein (TON_0873), NAD(P)H rubredoxin oxidoreductase (TON_0865), or thioredoxin reductase (TON_1603), which can couple the detoxification of reactive oxygen species with the regeneration of NAD(P)+ from NAD(P)H. We present a plausible mechanism by which O2 serves to maintain the intracellular redox balance. This study demonstrates an unusual strategy of an obligate anaerobe underlying O2-mediated growth enhancement despite not having heme-based or cytochrome-type proteins.

Microbial diversity in the rumen, reticulum, omasum, and abomasum of yak on a rapid fattening regime in an agro-pastoral transition zone: Dan Xue , Huai Chen , Xiaolin Luo , Jiuqiang Guan , Yixin He , Xinquan Zhao; J. Microbiol. 2018;56(10):734-743. Published online August 22, 2018; DOI: https://doi.org/10.1007/s12275-018-8133-0

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28 Citations

Abstract: The ruminant digestive system harbors a complex gut microbiome, which is poorly understood in the case of the four stomach compartments of yak. High-throughput sequencing and quantitative PCR were used to analyse microbial communities in the rumen, reticulum, omasum, and abomasum of six domesticated yak. The diversity of prokaryotes was higher in reticulum and omasum than in rumen and abomasum. Bacteroidetes predominated in the four stomach compartments, with abundance gradually decreasing in the trend rumen > reticulum > omasum > abomasum. Microorganism composition was different among the four compartments, all of which contained high levels of bacteria, methanogens, protozoa and anaerobic fungi. Some prokaryotic genera were associated with volatile fatty acids and pH. This study provides the first insights into the microorganism composition of four stomach compartments in yak, and may provide a foundation for future studies in this area.

Non-ureolytic calcium carbonate precipitation by Lysinibacillus sp. YS11 isolated from the rhizosphere of Miscanthus sacchariflorus: Yun Suk Lee , Hyun Jung Kim , Woojun Park; J. Microbiol. 2017;55(6):440-447. Published online May 28, 2017; DOI: https://doi.org/10.1007/s12275-017-7086-z

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42 Citations

Abstract: Although microbially induced calcium carbonate precipita-tion (MICP) through ureolysis has been widely studied in en-vironmental engineering fields, urea utilization might cause environmental problems as a result of ammonia and nitrate production. In this study, many non-ureolytic calcium car-bonate-precipitating bacteria that induced an alkaline envi-ronment were isolated from the rhizosphere of Miscanthus sacchariflorus near an artificial stream and their ability to pre-cipitate calcium carbonate minerals with the absence of urea was investigated. MICP was observed using a phase-contrast microscope and ion-selective electrode. Only Lysinibacillus sp. YS11 showed MICP in aerobic conditions. Energy disper-sive X-ray spectrometry and X-ray diffraction confirmed the presence of calcium carbonate. Field emission scanning elec-tron microscopy analysis indicated the formation of morpho-logically distinct minerals around cells under these condi-tions. Monitoring of bacterial growth, pH changes, and Ca2+ concentrations under aerobic, hypoxia, and anaerobic con-ditions suggested that strain YS11 could induce alkaline con-ditions up to a pH of 8.9 and utilize 95% of free Ca2+ only under aerobic conditions. Unusual Ca2+ binding and its re-lease from cells were observed under hypoxia conditions. Bio-film and extracellular polymeric substances (EPS) formation were enhanced during MICP. Strain YS11 has resistance at high pH and in high salt concentrations, as well as its spore- forming ability, which supports its potential application for self-healing concrete.

Metagenomic analysis reveals the contribution of anaerobic methanotroph-1b in the oxidation of methane at the Ulleung Basin, East Sea of Korea: Jin-Woo Lee , Kae Kyoung Kwon , Jang-Jun Bahk , Dong-Hun Lee , Hyun Sook Lee , Sung Gyun Kang , Jung-Hyun Lee; J. Microbiol. 2016;54(12):814-822. Published online November 26, 2016; DOI: https://doi.org/10.1007/s12275-016-6379-y

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Abstract: We have previously identified a sulfate methane transition zone (SMTZ) within the methane hydrate-bearing sediment in the Ulleung Basin, East Sea of Korea, and the presence of ANME-1b group in the sediment has been shown by phylogenetic analysis of a 16S rRNA gene. Herein, we describe taxonomic and functional profiling in the SMTZ sample by metagenomic analysis, comparing with that of surface sediment. Metagenomic sequences of 115 Mbp and 252 Mbp were obtained from SMTZ and surface sediments, respectively. The taxonomic profiling using BLASTX against the SEED within MG-RAST showed the prevalence of methanogens (19.1%), such as Methanosarcinales (12.0%) and Methanomicrobiales (4.1%) predominated within the SMTZ metagenome. A number of 185,200 SMTZ reads (38.9%) and 438,484 surface reads (62.5%) were assigned to functional categories, and methanogenesis-related reads were statistically significantly overrepresented in the SMTZ metagenome. However, the mapping analysis of metagenome reads to the reference genomes, most of the sequences of the SMTZ metagenome were mapped to ANME-1 draft genomes, rather than those of methanogens. Furthermore, the two copies of the methyl-coenzyme M reductase gene (mcrA) segments of the SMTZ metagenome were clustered with ANME-1b in the phylogenetic cluster. These results indicate that ANME- 1b reads were miss-annotated to methanogens due to limitation of database. Many of key genes necessary for reverse methanogenesis were present in the SMTZ metagenome, except for N5,N10-methenyl-H4MPT reductase (mer) and CoBCoM heterodisulfide reductase subunits D and E (hdrDE). These data suggest that the ANME-1b represents the primary player the anaerobic methane oxidation in the SMTZ, of the methane hydrate-bearing sediment at the Ulleung Basin, East Sea of Korea.

Description of a novel pectin-degrading bacterial species Prevotella pectinovora sp. nov., based on its phenotypic and genomic traits: Brigita Nograsek , Tomaz Accetto , Lijana Fanedl , Gorazd Avgustin; J. Microbiol. 2015;53(8):503-510. Published online July 31, 2015; DOI: https://doi.org/10.1007/s12275-015-5142-0

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Abstract: Five strictly anaerobic Gram-negative bacterial strains, P4-65, P4-76T, P5-60, P5-119, and P5-125, presumably belonging to the genus Prevotella were isolated from pig fecal samples. Strains were tested for various phenotypic traits and nearcomplete genome sequences were obtained and analyzed. Phylogenetic analysis based on 16S rRNA gene sequences and multilocus sequence analysis based on five conserved genes confirmed that the strains belong to the genus Prevotella, revealing that they represent a novel and discrete lineage distinct from other known species of this genus. The size of the genome of the isolated strains is 3?.3 Mbp, and the DNA G+C content is 47.5?8.1 mol%. The isolates are strictly anaerobic, rod-shaped with rounded ends, non-motile and non-spore-forming. The main fermentation products are succinate and acetate, with minor concentrations of isovalerate, propionate and isobutyrate. Hydrogen is also produced. Major cellular fatty acids consist of anteiso-C15:0 and iso-C15:0, and a number of additional acids are present in lower concentrations. A substantial portion of genes involved in carbohydrate utilization is devoted to pectin degradation and utilization, while those supporting growth on xylan in ruminal Prevotella could not have been revealed. On the basis of the presented results, a novel species, Prevotella pectinovora sp. nov. is proposed. The type strain is P4-76T (=DSM 29996T =ZIM B1020T).

Research Support, Non-U.S. Gov'ts

Changes in Gene Expression of Actinobacillus pleuropneumoniae in Response to Anaerobic Stress Reveal Induction of Central Metabolism and Biofilm Formation: Lu Li , Jiawen Zhu , Kui Yang , Zhuofei Xu , Ziduo Liu , Rui Zhou; J. Microbiol. 2014;52(6):473-481. Published online April 11, 2014; DOI: https://doi.org/10.1007/s12275-014-3456-y

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Abstract: Actinobacillus pleuropneumoniae is an important porcine respiratory pathogen causing great economic losses in the pig industry worldwide. Oxygen deprivation is a stress that A. pleuropneumoniae will encounter during both early infection and the later, persistent stage. To understand modulation of A. pleuropneumoniae gene expression in response to the stress caused by anaerobic conditions, gene expression profiles under anaerobic and aerobic conditions were compared in this study. The microarray results showed that 631 genes (27.7% of the total ORFs) were differentially expressed in anaerobic conditions. Many genes encoding proteins involved in glycolysis, carbon source uptake systems, pyruvate metabolism, fermentation and the electron respiration transport chain were up-regulated. These changes led to an increased amount of pyruvate, lactate, ethanol and acetate in the bacterial cells as confirmed by metabolite detection. Genes encoding proteins involved in cell surface structures, especially biofilm formation, peptidoglycan biosynthesis and lipopolysaccharide biosynthesis were up-regulated as well. Biofilm formation was significantly enhanced under anaerobic conditions. These results indicate that induction of central metabolism is important for basic survival of A. pleuropneumoniae after a shift to an anaerobic environment. Enhanced biofilm formation may contribute to the persistence of this pathogen in the damaged anaerobic host tissue and also in the early colonization stage. These discoveries give new insights into adaptation mechanisms of A. pleuropneumoniae in response to environmental stress.

Paenibacillus xylaniclasticus sp. nov., a Xylanolytic-Cellulolytic Bacterium Isolated from Sludge in an Anaerobic Digester: Chakrit Tachaapaikoon , Somboon Tanasupawat , Patthra Pason , Somphit Sornyotha , Rattiya Waeonukul , Khin Lay Kyu , Khanok Ratanakhanokchai; J. Microbiol. 2012;50(3):394-400. Published online June 30, 2012; DOI: https://doi.org/10.1007/s12275-012-1480-3

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Abstract: A mesophilic, facultative, anaerobic, xylanolytic-cellulolytic bacterium, TW1T, was isolated from sludge in an anaerobic digester fed with pineapple waste. Cells stained Gram-positive, were spore-forming, and had the morphology of straight to slightly curved rods. Growth was observed in the temperature range of 30 to 50°C (optimum 37°C) and the pH range of 6.0 to 7.5 (optimum pH 7.0) under aerobic and anaerobic conditions. The strain contained meso-diaminopimelic acid in the cell-wall peptidoglycan. The predominant isoprenoid quinone was menaquinone with seven isoprene units (MK-7). Anteiso-C15:0, iso-C16:0, anteiso-C17:0, and C16:0 were the predominant cellular fatty acids. The G+C content of the DNA was 49.5 mol%. A phylogenetic analysis based on 16S rRNA showed that strain TW1T belonged within the genus Paenibacillus and was closely related to Paenibacillus cellulosilyticus LMG 22232T, P. curdlanolyticus KCTC 3759T, and P. kobensis KCTC 3761T with 97.7, 97.5, and 97.3% sequence similarity, respectively. The DNA-DNA hybridization values between the isolate and type strains of P. cellulosilyticus LMG 22232T, P. curdlanolyticus KCTC 3759T, and P. kobensis KCTC 3761T were found to be 18.6, 18.3, and 18.0%, respectively. The protein and xylanase patterns of strain TW1T were quite different from those of the type strains of closely related Paenibacillus species. On the basis of DNA-DNA relatedness and phenotypic analyses, phylogenetic data and the enzymatic pattern presented in this study, strain TW1T should be classified as a novel species of the genus Paenibacillus, for which the name Paenibacillus xylaniclasticus sp. nov. is proposed. The type strain is TW1T (=NBRC 106381T =KCTC 13719T =TISTR 1914T).

Journal Article

Effects of Nicotine on the Growth and Protein Expression of Porphyromonas gingivalis: Orson Baek , Weidong Zhu , Hyeong C. Kim Kim , Seok-Woo Lee Lee; J. Microbiol. 2012;50(1):143-148. Published online February 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1212-8

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16 Citations

Abstract: Tobacco smoking is considered one of the most significant environmental risk factors for destructive periodontal disease. The effect of smoking on periodontopathic microbiota has not yet been elucidated, as previous studies failed to identify a concrete relationship between periodontopathic microorganisms and smoking. However, it is likely that smoking, as an environmental stress factor, may affect the behavior of dental plaque microorganisms, ultimately leading to alteration of the host-parasite interaction. The goal of this study was to examine the effect of nicotine, a major component of tobacco, on the growth and protein expression of the crucial periodontal pathogen Porphyromonas gingivalis. The growth of P. gingivalis 381 was measured after bacterial cells were cultivated in liquid broth containing various nicotine concentrations. First, P. gingivalis cells were allowed to grow in the presence of a single dose of nicotine (the single exposure protocol) at 0, 1, 2, 4, and 8 mg/L, respectively. Second, P. gingivalis cells were exposed to five consecutive doses of nicotine (the multiple exposure protocol) at 0, 1, 2, and 4 mg/L, respectively. Bacterial growth was measured by optical density and protein expression was analyzed by SDS-PAGE and 2-D gel electrophoresis. In the single nicotine exposure protocol, it was observed that the growth of P. gingivalis 381 was inhibited by nicotine in a dose-dependent manner. In the multiple nicotine exposure protocol, the growth rate of P. gingivalis increased with each subsequent nicotine exposure, even though bacterial growth was also inhibited in a dose dependent fashion. SDS-PAGE and 2-D gel electrophoresis analyses revealed a minor change in the pattern of protein expression, showing differences in proteins with low molecular weights (around 20 kDa) on exposure to nicotine. The results of this study suggest that nicotine exerts an inhibitory effect on the growth of P. gingivalis, and has a potential to modulate protein expression in P. gingivalis.

Research Support, Non-U.S. Gov't

Isolation and Characterization of Ethylbenzene Degrading Pseudomonas putida E41: Lan-Hee Kim , Sang-Seob Lee; J. Microbiol. 2011;49(4):575-584. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-0399-4

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Abstract: Pseudomonas putida E41 was isolated from oil-contaminated soil and showed its ability to grow on ethylbenzene as the sole carbon and energy source. Moreover, P. putida E41 show the activity of biodegradation of ethylbenzene in the batch culture. E41 showed high efficiency of biodegradation of ethylbenzene with the optimum conditions (a cell concentration of 0.1 g wet cell weight/L, pH 7.0, 25°C, and ethylbenzene concentration of 50 mg/L) from the results of the batch culture. The maximum degradation rate and specific growth rate (μmax) under the optimum conditions were 0.19±0.03 mg/mg-DCW (Dry Cell Weight)/h and 0.87±0.13 h-1, respectively. Benzene, toluene and ethylbenzene were degraded when these compounds were provided together; however, xylene isomers persisted during degradation by P. putida E41. When using a bioreactor batch system with a binary culture with P. putida BJ10, which was isolated previously in our lab, the degradation rate for benzene and toluene was improved in BTE mixed medium (each initial concentration: 50 mg/L). Almost all of the BTE was degraded within 4 h and 70-80% of m-, p-, and o-xylenes within 11 h in a BTEX mixture (initial concentration: 50 mg/L each). In summary, we found a valuable new strain of P. putida, determined the optimal degradation conditions for this isolate and tested a mixed culture of E41 and BJ10 for its ability to degrade a common sample of mixed contaminants containing benzene, toluene, and xylene.

Journal Article

Monitoring Nutrient Impact on Bacterial Community Composition during Bioremediation of Anoxic PAH-Contaminated Sediment: Myungsu Kim , Seung Seob Bae , Mijin Seol , Jung-Hyun Lee , Young-Sook Oh; J. Microbiol. 2008;46(6):615-623. Published online December 24, 2008; DOI: https://doi.org/10.1007/s12275-008-0097-z

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Abstract: Marine harbor sediments are frequently polluted with significant amount of polycyclic aromatic hydrocarbons (PAHs) some of which are naturally toxic, recalcitrant, mutagenic, and carcinogenic. To stimulate biodegradation of PAHs in PAH-contaminated sediments collected from near Gwangyang Bay, Korea, lactate was chosen as a supplementary carbonaceous substrate. Sediment packed into 600 ml air-tight jar was either under no treatment condition or lactate amended condition (1%, w/v). Microbial community composition was monitored by bacteria-specific and archaea-specific PCR-terminal restriction fragment length polymorphism (T-RFLP), in addition to measuring the residual PAH concentration. Results showed that lactate amendment enhanced biodegradation rate of PAHs in the sediment by 4 to 8 times, and caused a significant shift in archaebacterial community in terms of structure and diversity with time. Phylogenetic analysis of 23 archaeal clones with distinctive RFLP patterns among 288 archaeal clones indicated that majority of the archaeal members were closest to unculturable environmental rDNA clones from hydrocarbon-contaminated and/or methanogenesis-bearing sediments. Lactate amendment led to the enrichment of some clones that were most closely related to PAH-degrading Methanosarcina species. These results suggest a possible contribution of methanogenic community to PAH degradation and give us more insights on how to effectively remediate PAH-contaminated sediments.

Research Support, Non-U.S. Gov'ts

Effects of Sulfate Concentration on the Anaerobic Dechlorination of Polychlorinated Biphenyls in Estuarine Sediments: Young-Cheol Cho , Kyoung-Hee Oh; J. Microbiol. 2005;43(2):166-171.; DOI: https://doi.org/2167 [pii]

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Abstract: In order to determine the effects of sulfate concentration on the anaerobic dechlorination of polychlorinated biphenyls, sediments spiked with Aroclor 1242 were made into slurries using media which had various sulfate concentrations ranging from 3 to 23 mM. The time course of dechlorination clearly demonstrated that dechlorination was inhibited at high concentration of sulfate due to less dechlorination of meta-substituted congeners. When the dechlorination patterns were analyzed by the calculation of Euclidean distance, the dechlorination pathway in the 3 mM sulfate samples was found to be different from that observed in the 13 mM samples, although the extent of dechlorination in these two samples was similar. It is possible that the dechlorination in the high sulfate concentration samples is inhibited by the suppression of growth of methanogen, which have been shown to be meta-dechlorinating microorganisms.

Development of a Bottle-Free Multipurpose Incubator for Generating Various Bacterial Culture Conditions: Nam Woong Yang , Yong Lim; J. Microbiol. 2005;43(1):28-33.; DOI: https://doi.org/2142 [pii]

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Abstract: The purpose of this study was to develop a multipurpose incubator, without the gas cylinders (bottles) which are required for H_2 and CO_2 supplementation. In our bottle-free multipurpose incubator, the H_2 and CO_2 were generated by chemical reactions induced within the chamber. The reaction between sodium borohydride and acetic acid at a molar ratio of 1:1 was used to generate H_2, according to the following formula: 4NaBH_4 + 2CH_3COOH + 7H_2O → 2CH_3COONa + Na_2B_4O_7 + 16H_2, whereas the other reaction, citric acid and sodium bicarbonate at a 1:1 molar ratio, was used to generate CO_2, according to the following formula: C_6H_8O_7 + 3NaHCO_3 → Na_3(C_6H_5O_7) + 3H_2O + 3CO_2. Five species of obligate anaerobic bacteria, one strain of capnophilic bacterium, and one strain of microaerophilic bacterium were successfully cultured in the presence of their respective suitable conditions, all of which were successfully generated by our bottle-free multipurpose incubator. We conclude that, due to its greater safety, versatility, and significantly lower operating costs, this bottle-free multipurpose incubator can be used for the production of fastidious bacterial cultures, and constitutes a favorable step above existing anaerobic incubators. <br>

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