Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Antimicrobial activities of Burkholderia sp. strains and optimization of culture conditions

Burkholderia sp. OS17의 항균활성 증진을 위한 배양최적화

  • Nam, Young Ho (Culture Techniques Research Division, Nakdonggang National Institute of Biological Resources (NNIBR)) ;
  • Choi, Ahyoung (Culture Techniques Research Division, Nakdonggang National Institute of Biological Resources (NNIBR)) ;
  • Hwang, Buyng Su (Bioresources Industrialization Research Division, Nakdonggang National Institute of Biological Resources (NNIBR)) ;
  • Chung, Eu Jin (Culture Techniques Research Division, Nakdonggang National Institute of Biological Resources (NNIBR))
  • 남영호 (국립낙동강생물자원관 배양기술개발부) ;
  • 최아영 (국립낙동강생물자원관 배양기술개발부) ;
  • 황병수 (국립낙동강생물자원관 산업소재화연구부) ;
  • 정유진 (국립낙동강생물자원관 배양기술개발부)
  • Received : 2018.06.04
  • Accepted : 2018.10.08
  • Published : 2018.12.31
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Abstract

In this study, we isolated and identified bacteria from freshwater and soil collected from Osang reservoir, to screen antimicrobial bacteria against various pathogenic bacteria. 38 strains were isolated and assigned to the class Proteobacteria (22 strains), Actinobacteria (7 strains), Bacteroidets (6 strains), and Firmicutes (3 strains) based on 16S rRNA gene sequence analysis. Among them, strain OS17 showed a good growth inhibition against 5 methicillin-resistant Staphylococcus aureus subsp. aureus strains and Bacillus cereus, Bacillus subtilis, Filobasidium neoformans. As a result of the 16S rRNA gene sequence analysis, strain OS17 show the high similarity with Burkholderia ambifaria $AMMD^T$, B. diffusa $AM747629^T$, B. tettitorii $LK023503^T$ 99.8%, 99.7%, 99.6%, respectively. We investigated cell growth and antimicrobial activity according to commercial culture medium, temperature, pH for culture optimization of strain OS17. Optimal conditions for growth and antimicrobial activity in strain OS17 were found to be: YPD medium, $35^{\circ}C$ and pH 6.5. When the strain was cultured in LB, NB, TSB, R2A media at $20^{\circ}C$ and $25^{\circ}C$, the antimicrobial activity did not show. Culture filtrate of strain OS17 showed antimicrobial activity against 5 MRSA strains, Bacillus cereus, Bacillus subtilis, and Filobasidium neoformans with inhibition zones from 2 to 8 mm. Optimal reaction time was 48 h in YPD medium, 100 rpm and 0.3 vvm in 2 L-scale fed-batch fermentation process for antimicrobial activity. Culture optimization of strain OS17 can be improved on antimicrobial activity. Therefore, the antimicrobial activity of Burkholderia sp. OS17 had potential as antibiotics for pathogens including MRSA.

본 연구는 담수환경에서 항균활성을 보유한 미생물을 발굴하고, 활성 증진을 위해 배양조건을 최적화하는 것이다. 상주시 중동면 오상저수지에서 시료를 채취하여 38종의 미생물을 순수분리하였다. 16S rRNA 염기서열 분석에 근거하여 Proteobacteria강(22종), Actinobacteria강(7종), Bacteroidetes강(6종), Firmicutes강(3종)으로 구성되어있는 것을 확인하였다. 메티실린내성 황색포도상구군 등 10종의 유해미생물에 대한 항균활성을 보유한 Burkholderia sp. OS17 균주를 선발하였다. 항균활성 증진을 위한 상용배지, 온도, 초기 pH별 생육 및 항균활성 비교실험을 수행하였다. OS17 균주는 YPD 배지, $35^{\circ}C$, pH 6.5로 배양했을 때 가장 활성이 높았다. LB, NB, TSB, R2A 배지와 $20^{\circ}C$, $25^{\circ}C$ 배양했을 때는 생장은 가능하나 항균활성이 전혀 없었다. 이전결과를 바탕으로 YPD 배지, $35^{\circ}C$에서 배양하면서 5 L fermenter를 이용하여 생육, 항균활성, pH 확인을 통해 배양 48시간을 최적 배양시간으로 선정하였다. 항균활성을 보유한 미생물의 배양 최적화는 항균물질 생산에 영향을 미치고, 이는 상업적 응용에 이점으로 작용할 수 있다.

Keywords

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Fig. 1. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationships among isolates belonging to the order Burkholderia sp. OS17 and related taxa.

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Fig. 2. Growth, pH (A) and antimicrobial activities (B) of Burkholderia sp. OS17 according to commercial media.

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Fig. 3. Growth, pH (A) and antimicrobial activities (B) of Burkholderia sp. OS17 according to culture temperature.

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Fig. 4. Growth, pH (A) and antimicrobial activities (B) of Burkholderia sp. OS17 according to initial pH.

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Fig. 6. HPLC profiles of Burkholderia sp. OS17 according to commercial media.

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Fig. 5. Growth, pH and antimicrobial activities of Burkholderia sp. OS17 according to culture time by 5 L fermenter

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Fig. 7. HPLC profiles of Burkholderia sp. OS17 according to culture temperature.

Table 1. Strains obtained from Osang reservoir

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