Reduction of Dissolved Fe(III) by As(V)-tolerant Bacteria Isolated from Rhizosphere Soil

Khanal, Anamika,Song, Yoonjin,Cho, Ahyeon,Lee, Ji-Hoon The Korean Society of Environmental Agriculture 2021 한국환경농학회지 Vol.40 No.1

BACKGROUND: Biological iron redox transformation alters iron minerals, which may act as effective adsorbents for arsenate [As(V)] in the environments. In the viewpoint of alleviating arsenate, microbial Fe(III) reduction was sought under high concentration of As(V). In this study, Fe(III)-reducing bacteria were isolated from the wild plant rhizosphere soils collected at abandoned mine areas, which showed tolerance to high concentration of As(V), in pursuit of potential agents for As(V) bioremediation. METHODS AND RESULTS: Bacterial isolation was performed by a series of enrichment, transfer, and dilutions. Among the isolated strains, two strains (JSAR-1 and JSAR-3) with abilities of tolerance to 10 mM As(V) and Fe(III) reduction were selected. Phylogenetic analysis using 16S rRNA genesequences indicated the closest members of Pseudomonas stutzeri DSM 5190 and Paenibacillus selenii W126, respectively for JSAR-1 and JSAR-3. Ferric and ferrous iron concentrations were measured by ferrozine assay, and arsenic concentration was analyzed by ICP-AES, suggesting inability of As(V) reduction whereas ability of Fe(III) reduction. CONCLUSION: Fe(III)-reducing bacteria isolated from the enrichments with arsenate and ferric iron were found to be resistant to a high concentration of As(III) at 10 mM. We suppose that those kinds of microorganisms may suggest good application potentials for As(V) bioremediation, since the bacteria can transform Fe while surviving under As-contaminated environments. The isolated Fe(III)-reducing bacterial strains could contribute to transformations of iron minerals which may act as effective adsorbents for arsenate, and therefore contribute to As(V) immobilization

Direct and Indirect Reduction of Cr(VI) by Fermentative Fe(III)-Reducing Cellulomonas sp. Strain Cellu-2a

( Anamika Khanal ),( Hor-gil Hur ),( James K. Fredrickson ),( Ji-hoon Lee ) 한국미생물 · 생명공학회 2021 Journal of microbiology and biotechnology Vol.31 No.11

Hexavalent chromium (Cr(VI)) is recognized to be carcinogenic and toxic and registered as a contaminant in many drinking water regulations. It occurs naturally and is also produced by industrial processes. The reduction of Cr(VI) to Cr(III) has been a central topic for chromium remediation since Cr(III) is less toxic and less mobile. In this study, fermentative Fe(III)-reducing bacterial strains (Cellu-2a, Cellu-5a, and Cellu-5b) were isolated from a groundwater sample and were phylogenetically related to species of Cellulomonas by 16S rRNA gene analysis. One selected strain, Cellu-2a showed its capacity of reduction of both soluble iron (ferric citrate) and solid iron (hydrous ferric oxide, HFO), as well as aqueous Cr(VI). The strain Cellu-2a was able to reduce 15 μM Cr(VI) directly with glucose or sucrose as a sole carbon source under the anaerobic condition and indirectly with one of the substrates and HFO in the same incubations. The heterogeneous reduction of Cr(VI) by the surface-associated reduced iron from HFO by Cellu-2a likely assisted the Cr(VI) reduction. Fermentative features such as large-scale cell growth may impose advantages on the application of bacterial Cr(VI) reduction over anaerobic respiratory reduction.

Detection and Potential Abundances of Anammox Bacteria in the Paddy Soil

( Anamika Khanal ),( Seul Lee ),( Ji-hoon Lee ) 한국환경농학회 2020 한국환경농학회지 Vol.39 No.1

BACKGROUND: Microbes that govern a unique biochemical process of oxidizing ammonia into dinitrogen gas, such as anaerobic ammonium oxidation (anammox) have been reported to play a pivotal role in agricultural soils and in oceanic environments. However, limited information for anammox bacterial abundance and distribution in the terrestrial habitats has been known. METHODS AND RESULTS: Phylogenetic and next-generation sequencing analyses of bacterial 16S rRNA gene were performed to examine potential anammox bacteria in paddy soils. Through clone libraries constructed by using the anammox bacteria-specific primers, some clones showed sequence similarities with Planctomycetes (87% to 99%) and anammox bacteria (94% to 95%). Microbial community analysis for the paddy soils by using Illumina Miseq sequencing of 16S rRNA gene at phylum level was dominated by unclassified Bacteria at 33.2 ± 7.6%, followed by Chloroflexi at 20.4 ± 2.0% and Acidobacteria at 17.0 ± 6.5%. Planctomycetes that anammox bacteria are belonged to was 1.5% (± 0.3) on average from the two paddy soils. CONCLUSION: We suggest evidence of anammox bacteria in the paddy soil. In addition to the relatively well-known microbial processes for nitrogen-cycle, anammox can be a potential contributor on the cycle in terrestrial environments such as paddy soils.

Identification of Key Functional Genes Related with Nitrogen Cycle in the Terrestrial Habitat

Anamika Khanal,Junkoo Yeo(여준구),Kathyleen Nogrado,Seul Lee(이슬),Ahyeon Cho(조아현),Yoonjin Song(송윤진),Miji Kwon(권미지),Ji-Hoon Lee(이지훈) 한국토양비료학회 2018 한국토양비료학회 학술발표회 초록집 Vol.2018 No.5

실시간 중합효소연쇄반응을 이용하여 논 토양 내 질소순환 관련 기능 유전자의 평가

어나미까커날 ( Anamika Khanal ),이슬 ( Seul Lee ),여준구 ( Junkoo Yeo ),노그라도캐시린 ( Kathyleen Nogrado ),조아현 ( Ahyeon Cho ),송윤진 ( Yoonjin Song ),권미지 ( Miji Kwon ),이지훈 ( Ji-hoon Lee ) 한국환경농학회 2018 한국환경농학회 학술대회집 Vol.2018 No.-

The nitrogen cycle and the microbes responsible for mediating this process have an important role in natural ecosystems, terrestrial habitats and also have a major impact on climate change. In this study, we use molecular techniques such as conventional PCR, clone library construction, sequencing, phylogenetic analysis and real-time PCR to explore microbial communities in a rice paddy soil by detecting and quantifying some of the key functional genes that are involved in the process of nitrogen cycle i.e., nitrogen fixation (nifH), hydrazine synthase (hzsA), nitrous oxide reductase (nosZ), copper-containing (nirK), cytochrome cd1- containing nitrite reductase (nirS), nitrite oxidoreductase (nxrB), and ammonium monoxygenase (amoA). The sequence assessment from the clone library targeting these different genes i.e., nifH, hzsA, nosZ, nirK, nirS, nxrB, and amoA showed a high diversity and dominance of bacterial community. Besides that, Real-time PCR using SYBR green dye and some old and recently developed primers specific for each individual gene revealed the high abundance of nxrB gene and low abundance of hzsA gene. This finding will be very helpful in exploring and exploiting microorganism in terrestrial habitat.

Fe(lll) 환원능을 가진 Cellulomonas(Cellu-2a) 균주에 의한 Cr(VI)의 직 · 간접적 환원

어나미까커날 ( Anamika Khanal ),이지훈 ( Ji-hoon Lee ) 한국환경농학회 2020 한국환경농학회 학술대회집 Vol.2020 No.-

Heavy metals contamination and health risk assessment issues have been raised as one of the global concerns. Even though there are various strategies ongoing for the remediation of such pollution, the use of direct and indirect microbial reduction of heavy metals offers a potential mechanism for immobilizing, transforming, or reducing some soluble form of heavy metals to insoluble forms. Besides that these methods are cost-effective and environmentally friendly too. In this study, fermentative Fe(III)-reducing bacterial strains (Cellu-2a, Cellu-5a, and Cellu-5b) from the groundwater in an anaerobic condition has been isolated and characterized by an emphasis on iron reduction and iron biomineralization. The isolated colonies were found to have 99 % similarities with Cellulomonas species. Biochemical characterization of these colonies revealed to utilize L-arabinose, D-xylose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetylglucosamine, Esculine ferric citrate, Salicin, D-saccharose, D-xylose, D-galactose, D-cellobiose, Amidon (starch), Glycogen, D-trehalose within 24 hr to 48 hr. Among these three strains, Cellu-2a was used for further analysis. The isolated strain Cellu-2a showed the reduction of ferric citrate (2 mM) and HFO (2 mM) as well as direct and indirect reduction of Cr(VI) (15 uM) in a time-dependent manner using sucrose as a carbon source.

살코스키 시약과 Indole-3-acetic Acid의 반응 중 트립토판의 간섭

이슬 ( Seul Lee ),어나미까커날 ( Anamika Khanal ),여준구 ( Junkoo Yeo ),노그라도캐시린 ( Kathyleen Nogrado ),조아현 ( Ahyeon Cho ),송윤진 ( Yoonjin Song ),권미지 ( Miji Kwon ),이지훈 ( Ji-hoon Lee ) 한국환경농학회 2018 한국환경농학회 학술대회집 Vol.2018 No.-

Indole-3-acetic acid (IAA) is the most common auxin hormone produced in the plant and is very important because it regulates various aspects of plant growth and development. Tryptophan is the main precursor for IAA while IAA can be synthesized by two pathways i.e., tryptophan-dependent pathway and tryptophan-independent pathway. There have been many studies using Salkowski regent to measure the IAA production by bacteria. In this study, we have found that there was the interference of tryptophan on the colorimetric estimations of indole-3-acetic acid (IAA) by using Salkowski reagent (35% HClO4 and 10 mM FeCl3), which developed a pink color complex of tris-(indole-3-acetate) iron(III) with IAA. Moreover, we detected the effect of different concentration of tryptophan (100, 200, and 300 μM) on different concentration of IAA (100, 200, and 300 μM). The absorbance on IAA for the tested concentrations was found to be increased with the increasing concentration of tryptophan. It seems that both the IAA and tryptophan show the maximum absorbance at the similar wavelengths. It was also observed that the difference between the predicted and measured concentrations of IAA became larger as the predicted IAA concentration decreased. From this study, it is expected that tryptophan would interfere with accurate measurements of IAA, and therefore care should be taken for the measurement of IAA generated by the precursor, tryptophan.

The role of acyl-coenzyme A carboxylase complex in lipstatin biosynthesis of <i>Streptomyces toxytricini</i>

Demirev, Atanas V.,Khanal, Anamika,Sedai, Bhishma R.,Lim, Si Kyu,Na, Min Kyun,Nam, Doo Hyun Springer-Verlag 2010 Applied microbiology and biotechnology Vol.87 No.3

<P><I>Streptomyces toxytricini</I> produces lipstatin, a specific inhibitor of pancreatic lipase, which is derived from two fatty acid moieties with eight and 14 carbon atoms. The <I>pccB</I> gene locus in 10.6 kb fragment of <I>S. toxytricini</I> chromosomal DNA contains three genes for acyl-coenzyme A carboxylase (ACCase) complex <I>accA3</I>, <I>pccB</I>, and <I>pccE</I> that are presumed to be involved in secondary metabolism. The <I>pccB</I> gene encoding a β subunit of ACCase [carboxyltransferase (CT)] was identified upstream of <I>pccE</I> gene for a small protein of ε subunit. The <I>accA3</I> encoding the α subunit of ACCase [biotin carboxylase (BC)] was also identified downstream of <I>pccB</I> gene. When the <I>pccB</I> and <I>pccE</I> genes were inactivated by homologous recombination, the lipstatin production was reduced as much as 80%. In contrast, the accumulation of another compound, tetradeca-5.8-dienoic acid (the major lipstatin precursor), was 4.5-fold increased in disruptant compared with wild-type. It implies that PccB of <I>S. toxytricini</I> is involved in the activation of octanoic acid to hexylmalonic acid for lipstatin biosynthesis.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (doi:10.1007/s00253-010-2587-2) contains supplementary material, which is available to authorized users.</P>

Biochemical Characteristization of Propionyl-Coenzyme A Carboxylase Complex of Streptomyces toxytricini

Atanas V. Demirev,Anamika Khanal,Nguyen Phan Kieu Hanh,Kyung Tae Nam,남두현 한국미생물학회 2011 The journal of microbiology Vol.49 No.3

Acyl-CoA carboxylases (ACC) are involved in important primary or secondary metabolic pathways such as fatty acid and/or polyketides synthesis. In the 6.2 kb fragment of pccB gene locus of Streptomyces toxytricini producing a pancreatic inhibitor lipstatin, 3 distinct subunit genes of presumable propionyl-CoA carboxylase (PCCase) complex, assumed to be one of ACC responsible for the secondary metabolism, were identified along with gene for a biotin protein ligase (Bpl). The subunits of PCCase complex were α subunit (AccA3),β subunit (PccB), and auxiliary ε subunit (PccE). In order to disclose the involvement of the PCCase complex in secondary metabolism, some biochemical characteristics of each subunit as well as their complex were examined. In the test of substrate specificity of the PCCase complex, it was confirmed that this complex showed much higher conversion of propionyl-CoA rather than acetyl-CoA. It implies the enzyme complex could play a main role in the production of methylmalonyl-CoA from propionyl-CoA, which is a precursor of secondary polyketide biosynthesis.

중금속 오염 토양에서 분리한 니켈 내성 균주의 동정과 유전체 분석을 통한 잠재적 기작 연구

이슬 ( Seul Lee ),( Anamika Khanal ),여준구 ( Junkoo Yeo ),( Kathyleen Nogrado ),조아현 ( Ahyeon Cho ),송윤진 ( Yoonjin Song ),권미지 ( Miji Kwon ),이지훈 ( Ji-hoon Lee ) 한국환경농학회 2018 한국환경농학회 학술대회집 Vol.2018 No.-

The widespread use of metals influenced many researchers to examine the relationship between heavy metal toxicity and bacterial resistance. In this study, we have inoculated heavy metal contaminated soil from Jang-hang region of South Korea in the nickel-containing media (20 mM Ni) for the enrichment. Among dozens of the colonies acquired from the several transfers and serial dilutions with the same concentrations of Ni, 3 colonies with morphological differences were chosen for further studies, which were named as strains Ni-a, Ni-2 and Ni-3. The isolates were identified for their phylogenetic affiliations by using 16S rRNA gene analysis. The strains Ni-2 and Ni-3 were close to Cupriavidus metallidurans and were found to be resistant to antibiotics of vancomycin, erythromycin, chloramphenicol, ampicillin, gentamicin, streptomycin and kanamycin by disk diffusion method. Of the isolated strains, Ni-2 was sequenced for the whole genome, since the Ni-resistance seemed to be better than the other two strains. From the genome sequence we have found that were a total of 89 metal-resistance-related genes including 11 Ni-resistance genes, 41 heavy metal (As, Cd, Zn, Hg, Cu, and Co)-resistance genes, 22 cation efflux genes, 4 metal pumping ATPase genes, and 11 metal transporter genes.