[국내논문]모델 상수관망에 형성된 초기 생물막에서 분리한 종속영양세균의 생장 동역학 및 염소 내성 Growth kinetics and chlorine resistance of heterotrophic bacteria isolated from young biofilms formed on a model drinking water distribution system원문보기
본 연구에서는 염소 소독제를 함유한 수돗물을 수리학적 체류시간 2시간 수준으로 공급한 모델 상수관망에서 형성된 초기 생물막의 생장에 대해 연구하였다. PVC slide 표면에 형성된 세균 생물막의 비생장률(specific growth rate, ${\mu}$)은 총세균수와 종속영양세균수 기준으로 각각 $0.14{\pm}0.09day^{-1}$와 $0.16{\pm}0.08day^{-1}$로 측정되었으며, 생물막 형성 정도는 실험 개시 10일 후에 각각 $3.1{\times}10^4cells/cm^2$와 $6.6{\times}10^3CFU/cm^2$에 이르렀다. Bulk-phase 세균에 비해 훨씬 높은 생물막 형성 세균의 비생장률(${\mu}$)은 관망내에서 생물막 세균의 증식이 세균 재생장의 주된 요인으로 작용함을 의미하였다. 분리 배양된 생물막 균주들은 acetate 농도를 달리한 생장배지에서 얻어진 Monod 모델에서 특징적인 ${\mu}_{max}$와 $K_S$값을 보여주었다. 가장 낮은 ${\mu}_{max}$값을 보여준 Methylobacterium 균주는 느린 생장을 통해 염소 소독제 처리(0.5 mg/L, 10분간)에 대해 높은 내성을 나타내었다. 반포화상수(half-saturation constant) $K_S$값은 Sphingomonas 균주에서 다른 분리 균주들에 비해 100배 정도 낮게 측정되어 기질친화도가 매우 높게 나타났다. 이는 수돗물과 같이 영양물질의 농도가 매우 낮은 조건에서 생존할 수 있도록 적응된 절대 빈영양성 세균의 특징으로 판단된다. 비록 특징적인 ${\mu}_{max}$와 $K_S$값을 보이는 균주 만을 대상으로 수행되었지만, 이상의 결과는 상수관망에서 초기에 형성되는 복합 세균종으로 구성된 생물막에 대한 이해와 조절에 도움을 줄 수 있을 것으로 기대된다.
본 연구에서는 염소 소독제를 함유한 수돗물을 수리학적 체류시간 2시간 수준으로 공급한 모델 상수관망에서 형성된 초기 생물막의 생장에 대해 연구하였다. PVC slide 표면에 형성된 세균 생물막의 비생장률(specific growth rate, ${\mu}$)은 총세균수와 종속영양세균수 기준으로 각각 $0.14{\pm}0.09day^{-1}$와 $0.16{\pm}0.08day^{-1}$로 측정되었으며, 생물막 형성 정도는 실험 개시 10일 후에 각각 $3.1{\times}10^4cells/cm^2$와 $6.6{\times}10^3CFU/cm^2$에 이르렀다. Bulk-phase 세균에 비해 훨씬 높은 생물막 형성 세균의 비생장률(${\mu}$)은 관망내에서 생물막 세균의 증식이 세균 재생장의 주된 요인으로 작용함을 의미하였다. 분리 배양된 생물막 균주들은 acetate 농도를 달리한 생장배지에서 얻어진 Monod 모델에서 특징적인 ${\mu}_{max}$와 $K_S$값을 보여주었다. 가장 낮은 ${\mu}_{max}$값을 보여준 Methylobacterium 균주는 느린 생장을 통해 염소 소독제 처리(0.5 mg/L, 10분간)에 대해 높은 내성을 나타내었다. 반포화상수(half-saturation constant) $K_S$값은 Sphingomonas 균주에서 다른 분리 균주들에 비해 100배 정도 낮게 측정되어 기질친화도가 매우 높게 나타났다. 이는 수돗물과 같이 영양물질의 농도가 매우 낮은 조건에서 생존할 수 있도록 적응된 절대 빈영양성 세균의 특징으로 판단된다. 비록 특징적인 ${\mu}_{max}$와 $K_S$값을 보이는 균주 만을 대상으로 수행되었지만, 이상의 결과는 상수관망에서 초기에 형성되는 복합 세균종으로 구성된 생물막에 대한 이해와 조절에 도움을 줄 수 있을 것으로 기대된다.
The present work quantified the growth of young biofilm in a model distribution system that was fed with chlorinated drinking water at a hydraulic retention time of 2 h. Bacterial biofilms grew on the surface of polyvinyl chloride (PVC) slides at a specific growth rate of $0.14{\pm}0.09day^{-1}...
The present work quantified the growth of young biofilm in a model distribution system that was fed with chlorinated drinking water at a hydraulic retention time of 2 h. Bacterial biofilms grew on the surface of polyvinyl chloride (PVC) slides at a specific growth rate of $0.14{\pm}0.09day^{-1}$ for total bacteria and $0.16{\pm}0.08day^{-1}$ for heterotrophic bacteria, reaching $3.1{\times}10^4cells/cm^2$ and $6.6{\times}10^3CFU/cm^2$ after 10 days, respectively. The specific growth rates of biofilm-forming bacteria were found to be much higher than those of bulk-phase bacteria, suggesting that biofilm bacteria account for a major part of the bacterial production in this model system. Biofilm isolates exhibited characteristic kinetic properties, as determined by ${\mu}_{max}$ and $K_S$ values using the Monod model, in a defined growth medium containing various amounts of acetate. The lowest ${\mu}_{max}$ value was observed in bacterial species belonging to the genus Methylobacterium, and their slow growth seemed to confer high resistance to chlorine treatment (0.5 mg/L for 10 min). $K_S$ values (inversely related to substrate affinity) of Sphingomonas were two orders of magnitude lower for acetate carbon than those of other isolates. The Sphingomonas isolates may have obligate-oligotrophic characteristics, since the lower $K_S$ values allow them to thrive under nutrient-deficient conditions. These results provide a better understanding and control of multi-species bacterial biofilms that develop within days in a drinking water distribution system.
The present work quantified the growth of young biofilm in a model distribution system that was fed with chlorinated drinking water at a hydraulic retention time of 2 h. Bacterial biofilms grew on the surface of polyvinyl chloride (PVC) slides at a specific growth rate of $0.14{\pm}0.09day^{-1}$ for total bacteria and $0.16{\pm}0.08day^{-1}$ for heterotrophic bacteria, reaching $3.1{\times}10^4cells/cm^2$ and $6.6{\times}10^3CFU/cm^2$ after 10 days, respectively. The specific growth rates of biofilm-forming bacteria were found to be much higher than those of bulk-phase bacteria, suggesting that biofilm bacteria account for a major part of the bacterial production in this model system. Biofilm isolates exhibited characteristic kinetic properties, as determined by ${\mu}_{max}$ and $K_S$ values using the Monod model, in a defined growth medium containing various amounts of acetate. The lowest ${\mu}_{max}$ value was observed in bacterial species belonging to the genus Methylobacterium, and their slow growth seemed to confer high resistance to chlorine treatment (0.5 mg/L for 10 min). $K_S$ values (inversely related to substrate affinity) of Sphingomonas were two orders of magnitude lower for acetate carbon than those of other isolates. The Sphingomonas isolates may have obligate-oligotrophic characteristics, since the lower $K_S$ values allow them to thrive under nutrient-deficient conditions. These results provide a better understanding and control of multi-species bacterial biofilms that develop within days in a drinking water distribution system.
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문제 정의
The importance of young biofilms lies in the fact that it involves pioneering colonizers, which are able to initiate biofilm formation and influence the subsequent growth and maturation of the biofilm. Therefore, research on young drinking water biofilms will contribute to a better understanding of the dynamics of biofilm formation.
This study was performed to quantify the growth of young biofilm on the surfaces of pipe material exposed to chlorinated drinking water and to identify the heterotrophic bacteria that grow within the young biofilm. In addition, the kinetic properties and chlorine resistance of biofilm isolates were characterized for a better understanding and control of earlystage biofilm formation in drinking water distribution systems.
본 연구에서는 염소 소독제를 함유한 수돗물을 수리학적 체류시간 2시간 수준으로 공급한 모델 상수관망에서 형성된 초기 생물막의 생장에 대해 연구하였다. PVC slide 표면에 형성된 세균 생물막의 비생장률(specific growth rate, μ)은 총세균수와 종속영양세균수 기준으로 각각 0.
제안 방법
The purified templates were sequenced using a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and were analyzed on an ABI Prism® 3730xl DNA Analyzer (Applied Biosystems).
대상 데이터
An annular reactor (AR) (model 1120 LS; Biosurface Technologies Corporation), consisting of a stationary outer cylinder and a rotating inner drum, was used as a model distribution system (Fig. 1). Rotation of the inner drum was controlled by a motor up to 380 rpm.
Water moves in the region between the outer cylinder and inner drum. In this study, a total of 20 polyvinyl chloride (PVC) slides (BST 503-11; Biosurface Technologies Corp.), each of which have an exposed surface area of 19.5 cm2, were analyzed for biofilm accumulation in the AR. Prior to use, the disassembled AR system, slides, and related accessories were cleaned thoroughly and autoclaved at 121°C and 15 psi for 15 min and then reassembled carefully on a clean bench.
The 16S rDNA fragments were amplified with 27F (5′-AGA GTT TGA TCM TGG CTC AG-3′) and 1518R (5′-AAG GAG GTG ATC CAN CCR CA-3′) primers.
Eleven phenotypic colonies of biofilm bacteria (denoted by the symbol B, namely B1, B2, B3, etc.) grown on a randomly selected R2A plate were isolated. Figure 3 shows the kinetic properties (μmax and KS as determined by the Monod equation) of the heterotrophic biofilm-isolated bacterial strains (B1–B11) that were cultured in defined growth media containing various concentrations of acetate.
Three isolates (B2, B5, and B10) were chosen for chlorine exposure experiments. This selection was based on variable kinetic properties: high μmax and KS values (B2), high μmax and low KS values (B5), and low μmax and high KS values (B10) (Fig.
이론/모형
Heterotrophic plate counts (HPC) of the biofilm samples (CFU/cm) were analyzed by the spread plate method (9215 C) or the membrane filter method (9215 D) of Standard Methods (Eaton et al., 2005) on R2A agar (Difco Laboratories) and incubation at 25°C for 7 days.
strain NOX. Ammonia and phosphorus were analyzed with the phenate method (4500-NH3 F) and ascorbic acid method (4500-P E) of Standard Methods (Eaton et al., 2005), respectively. TCC and HPC levels present in water samples were determined by the same method used for biofilm TCC and HPC enumeration.
성능/효과
The KS values of the isolates (B5–B9) that belong to the genus Sphingomonas were two orders of magnitude lower for acetate carbon than the KS values of other isolates.
, 2012). In this study, based on KS values (not just presence in a low carbon environment), the five strains affiliated with the genus Sphingomonas can be considered as obligate-oligotrophic bacteria. In low-nutrient environments, obligate-oligotrophic bacteria might be the first colonizers, followed by copiotrophs as second colonizers.
PVC slide 표면에 형성된 세균 생물막의 비생장률(specific growth rate, μ)은 총세균수와 종속영양세균수 기준으로 각각 0.14 ± 0.09 day–1와 0.16 ± 0.08 day–1로 측정되었으며, 생물막 형성 정도는 실험 개시 10일 후에 각각 3.1 × 104 cells/cm2와 6.6 × 103 CFU/cm2에 이르렀다.
분리 배양된 생물막 균주들은 acetate 농도를 달리한 생장배지에서 얻어진 Monod 모델에서 특징적인 μmax와 KS값을 보여주었다.
5 mg/L, 10분간)에 대해 높은 내성을 나타내었다. 반포화상수(half-saturation constant) KS값은 Sphingomonas 균주에서 다른 분리 균주들에 비해 100배 정도 낮게 측정되어 기질친화도가 매우 높게 나타났다. 이는 수돗물과 같이 영양물질의 농도가 매우 낮은 조건에서 생존할 수 있도록 적응된 절대 빈영양성 세균의 특징으로 판단된다.
후속연구
비록 특징적인 μmax와 KS값을 보이는 균주 만을 대상으로 수행되었지만, 이상의 결과는 상수관망에서 초기에 형성되는 복합 세균종으로 구성된 생물막에 대한 이해와 조절에 도움을 줄 수 있을 것으로 기대된다.
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