• 제목/요약/키워드: clone library

검색결과 290건 처리시간 0.028초

Identification of Genes Involved in Primordial-primary Follicle Transition by Suppression Subtractive Hybridization

  • Park, Chang-Eun;Yoon, Se-Jin;Jeon, Eun-Hyun;Kim, Young-Hoon;Lee, Sook-Hwan;Lee, Kyung-Ah
    • 한국수정란이식학회:학술대회논문집
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    • 한국수정란이식학회 2002년도 국제심포지엄
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    • pp.98-98
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    • 2002
  • Recruitment of primordial follicles(PMF) is crucial for female fertility. however, factors and mechanisms that regulate this process is poorly understood. The present study was conducted to obtain an inclusive view of the gene expression and to identify novel factors and their pathways of regulating PMF arrest and/or growth initiation. Ovaries from one-day neonatal(consists of oocyte and PMF) and five-day old(consists of PMF and primary follicles, PRIF) mice were collected, either total RNA or mRNA was isolated, and suppression subtractive hybridization(SSH) was used to isolate and clone genes that differentially expressed in day 1 and day 5 ovaries. Confirmation that some of these genes are differentially expressed in PMF and/or in PRIF was accomplished by using laser captured microdissection(LCM), RT-PCR. in situ hybridization(ISH) and/or immunohistochemistry(IHC). In toto, 357 clones were sequenced and analyzed by BLAST and RIKEN program. Sequences of 330 clones significantly matched database entries while 27 clones were novel. Forty-two and 47 different genes were identified as differentially expressed in day 1 and day 5 ovaries, respectively, while 7 genes were expressed in both stages of ovaries. Day 5-subtracted library included several genes known as markers far growing follicles, such as ZP2, MATER, and fetuin. Among the genes with assigned functions, 23.8% was associated with cell cycle/apoptosis regulation, 7.1% with cellular structure, 11.9% with metabolism, 26.2% with signal transduction, and 31.0% with gene/protein expression in day 1; while 10.6%, 17.0%, 23.5%, 25.5%, and 23.4% in day 5, respectively. Genes such as GDF-8, Lats2, Septin2, and Weel were the highly expressed genes in PMF, while HSP84, Laminin2, MATER, MTi7, PTP, and Wrn were highly expressed genes in PRIF. We have successfully discovered list of genes expressed in day 1 and day 5 ovaries and confirmed that some of them are differentially expressed in PMF and/or PRIF. Gene expression profile from the present study would provide insight for the future study on the mechanism(s) involved in primordial-primary follicular transition. This work was Supported by Korean Health 21 RND Project, Ministry of Health and Welfare, Korea (01-PJ10-PG6-01GN13-0002).

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Molecular characterization and expression pattern of a novel Keratin-associated protein 11.1 gene in the Liaoning cashmere goat (Capra hircus)

  • Jin, Mei;Cao, Qian;Wang, Ruilong;Piao, Jun;Zhao, Fengqin;Piao, Jing'ai
    • Asian-Australasian Journal of Animal Sciences
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    • 제30권3호
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    • pp.328-337
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    • 2017
  • Objective: An experiment was conducted to determine the relationship between the KAP11.1 and the regulation wool fineness. Methods: In previous work, we constructed a skin cDNA library and isolated a full-length cDNA clone termed KAP11.1. On this basis, we conducted a series of bioinformatics analysis. Tissue distribution of KAP11.1 mRNA was performed using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. The expression of KAP11.1 mRNA in primary and secondary hair follicles was performed using real-time PCR (real-time polymerase chain reaction) analysis. The expression location of KAP11.1 mRNA in primary and secondary hair follicles was performed using in situ hybridization. Results: Bioinformatics analysis showed that KAP11.1 gene encodes a putative 158 amino acid protein that exhibited a high content of cysteine, serine, threonine, and valine and has a pubertal mammary gland) structural domain. Secondary structure prediction revealed a high proportion of random coils (76.73%). Semi-quantitative RT-PCR showed that KAP11.1 gene was expressed in heart, skin, and liver, but not expressed in spleen, lung and kidney. Real time PCR results showed that the expression of KAP11.1 has a higher expression in catagen than in anagen in the primary hair follicles. However, in the secondary hair follicles, KAP11.1 has a significantly higher expression in anagen than in catagen. Moreover, KAP11.1 gene has a strong expression in inner root sheath, hair matrix, and a lower expression in hair bulb. Conclusion: We conclude that KAP11.1 gene may play an important role in regulating the fiber diameter.

Calmodulin of Olive Flounder Paralichthys olivaceus : Cloning and Expression Analysis

  • Hong, Gyeong-Eun;Kong, Hee Jeong;Nam, Bo-Hye;Kim, Young-Ok;Kim, Woo-Jin;Lee, Sang-Jun;Choi, Tae-Jin
    • 한국해양바이오학회지
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    • 제2권4호
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    • pp.234-237
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    • 2007
  • Calmodulin은 $Ca^{2+}$ 결합단백질로서 생체 내에서 $Ca^{2+}$ 의존적 기작을 통하여 다양한 생물학적 기능에 관여한다. 본 연구에서는 넙치 Paralichthys olivaceus의 cDNA library로부터 Calmodulin cDNA를 분리 동정하였다. 염기 서열 및 아미노산 서열을 분석한 결과, 넙치 Calmodulin cDNA는 782개의 nucleotides로 구성되어 있고, 4개의 잘 보존된 $Ca^{2+}$결합 motifs (EF-I, EF-II, EF-III, EF-IV)를 가지는 149개의 아미노산 잔기를 전사할 수 있는 open reading frame을 포함한다. 또한 번역된 아미노산 서열은 인간, 쥐, zebrafish, 개구리의 Calmodulin 아미노산 서열과 100% 동일성을 보이며 보라성게, 침팬지의 Calmodulin 아미노산 서열과 각각 97, 99%의 동일성을 보인다. 넙치 Calmodulin 전사체는 뇌와 장 조직에서 많은 양이 발현되었고, 신장, 아가미, 눈, 근육, 피부, 지느러미에서도 발현이 관찰되었다. 또한 넙치 Calmodulin 전사체는 수정 후 7일째의 발생 초기 단계 시료에서도 발현되어 수정 후 34일째까지 그 발현이 서서히 증가하였다. 이상의 결과들로부터 넙치 Calmodulin은 넙치의 발생 초기 단계에서 필요한 단백질로 생각되며 아마 항상성 유지에 중요한 역할을 할 것으로 예상된다.

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Isolation and Characterization of a Novel Calcium/Calmodulin-Dependent Protein Kinase, AtCK, from Arabidopsis

  • Jeong, Jae Cheol;Shin, Dongjin;Lee, Jiyoung;Kang, Chang Ho;Baek, Dongwon;Cho, Moo Je;Kim, Min Chul;Yun, Dae-Jin
    • Molecules and Cells
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    • 제24권2호
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    • pp.276-282
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    • 2007
  • Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin ($Ca^{2+}/CaM$)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about $Ca^{2+}/CaM$-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative $Ca^{2+}$-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a $Ca^{2+}$-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM $Mn^{2+}$. The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other $Ca^{2+}/CaM$-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and $Ca^{2+}/CaM$-dependent protein kinase), increasing the concentration of calmodulin to more than $3{\mu}M$ suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis $Ca^{2+}/CaM$-dependent protein kinase which is presumably involved in CaM-mediated signaling.

Molecular Analysis of Colonized Bacteria in a Human Newborn Infant Gut

  • Park Hee-Kyung;Shim Sung-Sub;Kim Su-Yung;Park Jae-Hong;Park Su-Eun;Kim Hak-Jung;Kang Byeong-Chul;Kim Cheol-Min
    • Journal of Microbiology
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    • 제43권4호
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    • pp.345-353
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    • 2005
  • The complex ecosystem of intestinal micro flora is estimated to harbor approximately 400 different microbial species, mostly bacteria. However, studies on bacterial colonization have mostly been based on culturing methods, which only detect a small fraction of the whole microbiotic ecosystem of the gut. To clarify the initial acquisition and subsequent colonization of bacteria in an infant within the few days after birth, phylogenetic analysis was performed using 16S rDNA sequences from the DNA iso-lated from feces on the 1st, 3rd, and 6th day. 16S rDNA libraries were constructed with the amplicons of PCR conditions at 30 cycles and $50^{\circ}C$ annealing temperature. Nine independent libraries were produced by the application of three sets of primers (set A, set B, and set C) combined with three fecal samples for day 1, day 3, and day 6 of life. Approximately 220 clones ($76.7\%$) of all 325 isolated clones were characterized as known species, while other 105 clones ($32.3\%$) were characterized as unknown species. The library clone with set A universal primers amplifying 350 bp displayed increased diversity by days. Thus, set A primers were better suited for this type of molecular ecological analysis. On the first day of the life of the infant, Enterobacter, Lactococcus lactis, Leuconostoc citreum, and Streptococcus mitis were present. The largest taxonomic group was L. lactis. On the third day of the life of the infant, Enterobacter, Enterococcus faecalis, Escherichia coli, S. mitis, and Streptococcus salivarius were present. On the sixth day of the life of the infant, Citrobacter, Clostridium difficile, Enterobacter sp., Enterobacter cloacae, and E. coli were present. The largest taxonomic group was E. coli. These results showed that microbiotic diversity changes very rapidly in the few days after birth, and the acquisition of unculturable bacteria expanded rapidly after the third day.

Bacillus subtilis를 이용한 대두 발효식품의 혈전용해능

  • 정영기
    • 한국생명과학회:학술대회논문집
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    • 한국생명과학회 2001년도 제32회 학술심포지움
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    • pp.67-86
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    • 2001
  • A strain producing strongly fibrinolytic enzyme was isolated from soil and was identified to be Bacillus subtilis by biochemical and physiological characterization. The optimal culture conditions for the production of fibrinolytic enzyme was determined to be 1.0% tryptone, 1.5% soluble starch, 0.5% Peptone, 0.5% NaCl, $(NH_{4})_{3}PO_4.3H_{2}O, and MgSO_{4}.7H_{2}O.$ Initial pH and temperature were pH 8.0 and $30^{\circ}C$ , respectively, The highest enzyme production was observed at 30 hours of cultivation at $30^{\circ}C$ The fibrinolytic enzyme was purified to homogeneity by DEAE Sephadex A-50 ion exchange column chromatography, 70% ammonium sulfate precipitation, Sephadex G-200 and G-75 gel filtration column chromatography. The molecular weight of the purified enzyme was 28,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A gene encoding the fibrinolytic enzyme was cloned into a plasmid vector pBluescript, transforming E.coli XL-1 Blue. The clone was able to degrade fibrin, This indicated that the gene could encode a fibrinolytic enzyme. The nucleotide sequence of the 2.7 kb insert was determined in both direction. One open reading frame composed of 1023 nucleotides was found to be a potential protein coding region. There was the putative Shine-Dalgano sequence and TATA box upstream of the open reading frame. The homology search data in the genome database showed that both the 2.7 kb insert and 1 kb open reading frame carried no significance in the nucleotide sequence of known fibrinolytic enzyme from Bacillus serovars. The recombinant cell harboring the novel gene involved in fibrinolysis was subjected to protein purification. The molecular mass of the purified fibrinolytic enzyme was determined to be 31864 Dalton, which was highly in accordance with the molecular mass(33 kDa) of the fibrinolytic gene deduced from the insert. The fibrinolytic enzyme was Purified 50.5 folds to homogeneity in overall yield of 10.7% by DEAE Sephadex A-50 ion exchange, 85% ammonium sulfate precipitation, Sephadex G-50, Superdex 75 HR FPLC gel filtration. In conclusion, a novel fibrinolytic gene from Bacillus subtilis was identified and characterized by cloning a genomic library of Bacillus subtilis into pBleuscript. For the soybean fermented by this strain, it is found that there increased assistant protein about 20% compared to the soybean not fermented and increased about 30% according to amino acid analysis and, in particular, essential amino acid increased about 40%. When keeping this fermented soybean powder at room temperature for about 70days, it showed very high stability maintaining almost perfect activity and, therefore, it gave us great suggestion its possibility of development as a new functional food.

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송사리 Tyrosine Hydroxylase: cDNA 클로닝 및 생물지표로서의 TH 유전자 발현의 분자생물학적 추적 (Ttrosine Hydroxylase in Japanese Medaka (Oryzias latipes): cDNA Cloning and Molecular Monitoring of TH Gene Expression As a Biomarker)

  • Shin, Sung-Woo;Kim, Jung-Sang;Chon, Tae-Soo;Lee, Sung-Kyu;Koh, Sung-Cheol
    • Environmental Analysis Health and Toxicology
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    • 제15권4호
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    • pp.131-137
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    • 2000
  • 최근 독성 유해물질의 환경으로의 방출로 인해 인간 및 생태계에 대한 위해성 문제가 심각하게 제기되고 있다. 독성화학물질을 포함한 여러 환경 오염물질의 위해성평가는 화학물질의 유해성과 노출량 측정을 동시에 측정함으로써 가능한데 이 경우 생물지표(biomarker)가 최근 각광을 받고 있다. 본 연구에서는 동물의 행동에 관련된 신경전달물질의 생성에 결정적 역할을 하는 tyrosine hydroxylase(TH)및 그 유전자가 생물지표로서 이용 가능성이 있는지를 검토하였다. Ovary cDNA library의 PCR 스크리닝을 통한 송사리 TH유전자를 부분적으로 를론하였으며(327 bp), DNA염기서열 분석 결과 쥐 (rat)의 TH유전자와 동일한 염기서열을 보였다. 그리고 다이아지논 처리구 및 무처리구에서 송사리의 머리부분(head)및 몸통 부분(body)에서 추출된 총RNA에 TH mRNA가 존재함을 RT-PCR를 통하여 확인하였다. 그러나 다이아지논의 처리효과가 송사리의 행동에 미치는 영향을 보기 위해서는 TH의 발현을 보다 정량적으로 검토할 필요가 있을 것으로 판단된다. 생물지표로서 TH의 활성 및 mRNA의 기관별 또는 조직별 검출은 독성물질에 영향을 받는 어류 신경행동 변화를 모니터링 할 수 있는 유용한 수단이 될 것이다. 나아가 환경관리에 있어서 신경화학물질과 분자생물학적 상관관계를 통한 이상반응행동의 분석은 환경 위해성평가에 상당히 기여할 것이다.

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누에나방 수명관련 특이발현 유전자 탐색 (Investigation of lifespan related genes of the silkmoth, Bombyx mori L)

  • 최광호;구태원;김성렬;김성완;강석우;강필돈
    • 한국잠사곤충학회지
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    • 제51권2호
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    • pp.211-217
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    • 2013
  • 일반적으로 누에나방의 평균 생존시수는 암컷과 수컷이 각각 8일과 5일로 알려져 있다. 그러나 J037 품종의 성충 수명은 상당히 길며, 반대로 Daizo(sdi) 품종은 상당히 짧은 성충 수명 특성을 갖고 있다. 이러한 누에 품종 간 성충 수명의 차이는 유전적 차이에 따른 현상으로 추정되어 왔다. 본 연구에서는 장단명누에 품종의 발현유전자를 분석함으로써 누에 수명 관련 유전자를 탐색하여 그 특성을 분석하고자 하였다. 우선, 장명 수나방 mRNA를 사용하여 cDNA 유전자은행을 제작하고, 제작된 유전자은행으로부터 2,688개 클론을 무작위 선발한 후 장단명 수나방 cDNA를 각각의 탐침으로 차별화선별을 수행하였다. 본 연구에서 차별화 발현하는 193개의 클론을 선발하여 EST 상동성 분석을 통해 최종적으로 154개의 J037 수나방 수명 관련 독립유전자를 선발할 수 있었는데, 장명 탐침에서 과발현하는 124개와 단명 탐침에서 과발현하는 30개로 구성된다. 154개 독립유전자 중 가장 많은 발현빈도수를 보인 유전자는 cytochrome oxidase subunit-1으로서 모두 9회로 확인되었고, 현재까지 기능에 관해 알려진 바 없는 1-50번 독립유전자가 5회로 두 번째로 높은 발현빈도를 보였다. 154개 선발 독립유전자를 기능별로 분류한 결과, unclassified protein군에 가장 많은 24%의 독립유전자가 포함되어 있었다. 본 연구에서 두 번째로 발현빈도가 높은 클론(ID;1-50)의 염기서열 및 아미노산 서열을 분석하였는데, 1-50번 유전자는 전체 1,523 bp 염기로 구성되어 있으며, 723개 염기쌍이 240개의 아미노산을 coding 하고 있었다. 본 연구에서 선발된 주요 수명 관련 유전자는 국제유전자은행 EST database에 등록하였다.

Schizosaccharomyces bombe 포자형성 유전자(spo5)의 Cloning 및 전사조절 (Cloning and Transcription Analysis of Sporulation Gene (spo5) in Schizosaccharomyces pombe)

  • 김동주
    • 한국식품영양학회지
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    • 제15권2호
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    • pp.112-118
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    • 2002
  • 분열효모 S. pombe의 포자형성은 배지상의 질소원 고갈에 의해 유도되어지며 감수분열로부터 포자형성에 도달하는 과정에는 다수의 특이적인 유전자들이 관여하고 있다. 본 실험에서는 S. pombe genomic library 형질 전환법으로 spo5 유전자를 상보하는 clone을 screening한 후, sport 유전자를 단리하였다$^{8)}$ . 전포자막 구축에 필수적인 sport 유전자를 보유하는 약 5kb의 DNA 단편을 대장균, 효모 shuttle vector pTB248'의 Hind III 부위에 subclonning하였다. 그리고 이 DNA단편으로부터 제한 효소 지도를 작성하여(Fig. 2), spo5 변이체의 상보 능력을 조사하였다 (Fig. 3). 결과에서 서술한 바와 같이 상보능력은 동일하였으며, 이러한 상보성 실험 결과로부터 삽입된 단편상의 유전자 발현은 벡터의 promoter로부터 전사가 일어나는 것이 아니라, 삽입 단편상의 효모 고유의 promoter 에 의해서 전사가 일어나는 것으로 확인되었다. 따라서 clone화 한 DNA 단편 배열상에는 변역영역뿐만 아니라 promoter 영역이 포함된 것으로 판단되었다. 결실변이 도입 해석으로부터, spo5 유전자는 Sma I 부터 Hind m의 3kb 영역에 존재하였고 (Fig. 3), Nor-thern분석에 의해서 spo5 유전자의 전사를 조사한 결과, spo5 -mRNA는 Sma I 부터 Hind III 의 3kb 영 역에서 약2.5kb 크기로 검출되었다. 이 단편의 유전해석으로 부터 약 2.5kb의 전사산물은 최대 800개의 아미노산 잔기를 code하는 단백질로 판단되었다(Fig. 4). 그리고, Northern 분석법에 의해서 spo5 유전자의 전사를 조사한 결과, 서술한 바와 같이, 이 유전자는 질소기아 조건하에서만 유전자가 발현되는 것을 확인하였다(Fig. 4-2.5kb 단편).었다. 그리고 Edman법으로 결정한 PPIase의 39아미노산 잔기가 이 배열내에 완전히 보존되어 있었다. 이 결과로부터 이 ORF는PPIase구조 유전자의 1/3에 해당하는 단편임을 확인하였다. training system to a dangerous work like as "Interruption-free live-line work exchanging COS(Cut-Out-Switch)". In this program, the user works with a instruction on the window and speaker and can't work other tasks until each part of the task completed. The workers using this system can use their hands and viewpoint movement as he is in a real environment but the trainee can't use all parts and senses of a real body with the current VR technology. Despite of this weak point, when we consider the trends of improvement in electrical devices and communication technology, we can say that 3D graphic VR application has a high potentiality.) 야생화 초지(NWP, IWP)는 관행 혼파초지나 하번초 혼파초지에 비하여 동물상이 다양하고 많게 분포되었으며 그중 외국산 야생화초지의 동물 개체수가 가장 많게 나타났다. 이상의 결과를 종합할 때, 야생화 초지는 봄부터 가을까지 야생화가 지속되었고, 양서류 및 곤충의 개체 수가 증가되었던 것으로 보아 야생화 초지의 공익적인 측면에서의 활용 가능성도 클 것으로 기대된다

Field Studios of In-situ Aerobic Cometabolism of Chlorinated Aliphatic Hydrocarbons

  • Semprini, Lewts
    • 한국지하수토양환경학회:학술대회논문집
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    • 한국지하수토양환경학회 2004년도 총회 및 춘계학술발표회
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    • pp.3-4
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    • 2004
  • Results will be presented from two field studies that evaluated the in-situ treatment of chlorinated aliphatic hydrocarbons (CAHs) using aerobic cometabolism. In the first study, a cometabolic air sparging (CAS) demonstration was conducted at McClellan Air Force Base (AFB), California, to treat chlorinated aliphatic hydrocarbons (CAHs) in groundwater using propane as the cometabolic substrate. A propane-biostimulated zone was sparged with a propane/air mixture and a control zone was sparged with air alone. Propane-utilizers were effectively stimulated in the saturated zone with repeated intermediate sparging of propane and air. Propane delivery, however, was not uniform, with propane mainly observed in down-gradient observation wells. Trichloroethene (TCE), cis-1, 2-dichloroethene (c-DCE), and dissolved oxygen (DO) concentration levels decreased in proportion with propane usage, with c-DCE decreasing more rapidly than TCE. The more rapid removal of c-DCE indicated biotransformation and not just physical removal by stripping. Propane utilization rates and rates of CAH removal slowed after three to four months of repeated propane additions, which coincided with tile depletion of nitrogen (as nitrate). Ammonia was then added to the propane/air mixture as a nitrogen source. After a six-month period between propane additions, rapid propane-utilization was observed. Nitrate was present due to groundwater flow into the treatment zone and/or by the oxidation of tile previously injected ammonia. In the propane-stimulated zone, c-DCE concentrations decreased below tile detection limit (1 $\mu$g/L), and TCE concentrations ranged from less than 5 $\mu$g/L to 30 $\mu$g/L, representing removals of 90 to 97%. In the air sparged control zone, TCE was removed at only two monitoring locations nearest the sparge-well, to concentrations of 15 $\mu$g/L and 60 $\mu$g/L. The responses indicate that stripping as well as biological treatment were responsible for the removal of contaminants in the biostimulated zone, with biostimulation enhancing removals to lower contaminant levels. As part of that study bacterial population shifts that occurred in the groundwater during CAS and air sparging control were evaluated by length heterogeneity polymerase chain reaction (LH-PCR) fragment analysis. The results showed that an organism(5) that had a fragment size of 385 base pairs (385 bp) was positively correlated with propane removal rates. The 385 bp fragment consisted of up to 83% of the total fragments in the analysis when propane removal rates peaked. A 16S rRNA clone library made from the bacteria sampled in propane sparged groundwater included clones of a TM7 division bacterium that had a 385bp LH-PCR fragment; no other bacterial species with this fragment size were detected. Both propane removal rates and the 385bp LH-PCR fragment decreased as nitrate levels in the groundwater decreased. In the second study the potential for bioaugmentation of a butane culture was evaluated in a series of field tests conducted at the Moffett Field Air Station in California. A butane-utilizing mixed culture that was effective in transforming 1, 1-dichloroethene (1, 1-DCE), 1, 1, 1-trichloroethane (1, 1, 1-TCA), and 1, 1-dichloroethane (1, 1-DCA) was added to the saturated zone at the test site. This mixture of contaminants was evaluated since they are often present as together as the result of 1, 1, 1-TCA contamination and the abiotic and biotic transformation of 1, 1, 1-TCA to 1, 1-DCE and 1, 1-DCA. Model simulations were performed prior to the initiation of the field study. The simulations were performed with a transport code that included processes for in-situ cometabolism, including microbial growth and decay, substrate and oxygen utilization, and the cometabolism of dual contaminants (1, 1-DCE and 1, 1, 1-TCA). Based on the results of detailed kinetic studies with the culture, cometabolic transformation kinetics were incorporated that butane mixed-inhibition on 1, 1-DCE and 1, 1, 1-TCA transformation, and competitive inhibition of 1, 1-DCE and 1, 1, 1-TCA on butane utilization. A transformation capacity term was also included in the model formation that results in cell loss due to contaminant transformation. Parameters for the model simulations were determined independently in kinetic studies with the butane-utilizing culture and through batch microcosm tests with groundwater and aquifer solids from the field test zone with the butane-utilizing culture added. In microcosm tests, the model simulated well the repetitive utilization of butane and cometabolism of 1.1, 1-TCA and 1, 1-DCE, as well as the transformation of 1, 1-DCE as it was repeatedly transformed at increased aqueous concentrations. Model simulations were then performed under the transport conditions of the field test to explore the effects of the bioaugmentation dose and the response of the system to tile biostimulation with alternating pulses of dissolved butane and oxygen in the presence of 1, 1-DCE (50 $\mu$g/L) and 1, 1, 1-TCA (250 $\mu$g/L). A uniform aquifer bioaugmentation dose of 0.5 mg/L of cells resulted in complete utilization of the butane 2-meters downgradient of the injection well within 200-hrs of bioaugmentation and butane addition. 1, 1-DCE was much more rapidly transformed than 1, 1, 1-TCA, and efficient 1, 1, 1-TCA removal occurred only after 1, 1-DCE and butane were decreased in concentration. The simulations demonstrated the strong inhibition of both 1, 1-DCE and butane on 1, 1, 1-TCA transformation, and the more rapid 1, 1-DCE transformation kinetics. Results of tile field demonstration indicated that bioaugmentation was successfully implemented; however it was difficult to maintain effective treatment for long periods of time (50 days or more). The demonstration showed that the bioaugmented experimental leg effectively transformed 1, 1-DCE and 1, 1-DCA, and was somewhat effective in transforming 1, 1, 1-TCA. The indigenous experimental leg treated in the same way as the bioaugmented leg was much less effective in treating the contaminant mixture. The best operating performance was achieved in the bioaugmented leg with about over 90%, 80%, 60 % removal for 1, 1-DCE, 1, 1-DCA, and 1, 1, 1-TCA, respectively. Molecular methods were used to track and enumerate the bioaugmented culture in the test zone. Real Time PCR analysis was used to on enumerate the bioaugmented culture. The results show higher numbers of the bioaugmented microorganisms were present in the treatment zone groundwater when the contaminants were being effective transformed. A decrease in these numbers was associated with a reduction in treatment performance. The results of the field tests indicated that although bioaugmentation can be successfully implemented, competition for the growth substrate (butane) by the indigenous microorganisms likely lead to the decrease in long-term performance.

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