Analytical Biochemistry 446 (2014) 90–95
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Effect of temperature on biofilm formation by Antarctic marine bacteria in a microfluidic device Heon-Ho Jeong a,1, Seong-Geun Jeong a,1, Aeri Park a, Sung-Chan Jang a, Soon Gyu Hong b, Chang-Soo Lee a,⇑ a b
Department of Chemical Engineering, Chungnam National University, Yuseong-gu, Deajeon 305-764, Republic of Korea Division of Polar Life Sciences, Korea Polar Research Insitute, 26 Songdomirae-ro, Yeonsu-gu, Incheon 406-840, Republic Korea
a r t i c l e
i n f o
Article history: Received 5 July 2013 Received in revised form 25 September 2013 Accepted 17 October 2013 Available online 26 October 2013 Keywords: Antarctic marine bacteria Biofilm Temperature gradient Microfluidic device Cryobiology
a b s t r a c t Polar biofilms have become an increasingly popular biological issue because new materials and phenotypes have been discovered in microorganisms in the polar region. Various environmental factors affect the functionality and adaptation of microorganisms. Because the polar region represents an extremely cold environment, polar microorganisms have a functionality different from that of normal microorganisms. Thus, determining the effective temperature for the development of polar biofilms is crucial. Here, we present a simple, novel one-pot assay for analysis of the effect of temperature on formation of Antarctic bacterial biofilm using a microfluidic system where continuous temperature gradients are generated. We find that a specific range of temperature is required for the growth of biofilms. Thus, this microfluidic approach provides precise information regarding the effective temperature for polar biofilm development with a new high-throughput screening format. Ó 2013 Elsevier Inc. All rights reserved.
Microorganisms growing at low temperatures are dominant life forms in Antarctic ecosystems [1]. Change in temperature is the dominant factor in external conditions to influence the biological communities. Especially, the diffusion rate of dissolved gases and the exchange of nutrients are greatly retarded at low-temperature conditions. Physiological and biochemical adaptations of Antarctic microorganisms accommodate subsequent rapid physical and chemical changes [2]. The capacity to survive in an extreme environment is a prerequisite for Antarctic microorganisms forming robust stress resistance toward extreme environments. The most critical metabolic requirement at low temperature is maintenance of functional lipid membranes. They can produce large amounts of extracellular polymeric substance (EPS),2 composed mainly of polysaccharides. Thus, in dense accumulation of bacteria, they can develop biofilms because they are able to alter the immediate physicochemical environment and protect themselves from external harsh environments [1,3]. Polar bacteria residing in biofilms show higher resistance to concentrated salinity and cold shock [4,5]. Despite the importance of the research topic, there is still a lack of knowledge about most Antarctic bacterial biofilms and the response to environmental conditions. In particular, temperature is ⇑ Corresponding author. Fax: +82 42 821 5896. E-mail address: [email protected] (C.-S. Lee). These authors contributed equally to this work. Abbreviations used: EPS, extracellular polymeric substance; KOPRI, Korea Polar Research Institute; MR2A, Marine R2A; PR, photoresist; PDMS, polydimethylsiloxane; UV, ultraviolet; CCD, charge-coupled device. 1 2
0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.10.027
the most critical stressor in the Antarctic environment and affects bacterial growth rate, protein activity, and gene expression. In normal bacteria, heat and cold shock denature proteins or increase the amount of unsaturated fatty acids in the cell membrane [6,7]. However, Antarctic microorganisms with cold-adapted genes are more stable at low temperatures [8]. The marine bacterial environment in Antarctica is largely unexplored, and increasing human pressure necessitates a clear understanding of the dynamics of biofilm formation in this sensitive ecosystem. In fact, conventional biology laboratories are performing labor-intensive and tedious experimental procedures to analyze formation of Antarctic bacterial biofilms under several separate temperature conditions. In considering throughput screening and number of Antarctic marine bacteria, it is one of the major hurdles to analyze the influence of temperature on growth of polar marine bacteria and their bacterial biofilm [2]. Unfortunately, there are few methodologies now available for analyzing the effect of temperature on formation of Antarctic marine bacterial biofilm with parallel data acquisition in a combinatorial analytical manner. Here we present a simple microfluidic approach to generate a wide linear temperature gradient from 4 to 40 °C and to analyze Antarctic marine biofilm formation under the temperature gradient. First, we investigated the ability of seven strains isolated in the Antarctic region with regard to continuous temperature gradients. Unlike conventional methods performed at fixed temperature conditions, this approach can provide quantitative and physiologically relevant data under extreme conditions because the
Effect of temperature on biofilm formation by Antarctic marine bacteria in a microfluidic device / H.-H. Jeong et al. / Anal. Biochem. 446 (2014) 90–95
microfluidic system can imitate versatile microenvironmental conditions such as varying sizes of fluids, shapes of microchannels, shear stress, and high-pressure fluid with temperature gradient. Thus, the method would provide more precise information regarding ecologically relevant environment, dependency on temperature, and the rational design of extreme environments in a new high-throughput screening format. Materials and methods Bacterial strain and reagents All seven types of Antarctic marine bacteria shown in Table 1 were obtained from the Polar and Alpine Microbial Collection (PAMC). Marine R2A (MR2A) medium was prepared in our laboratory. SU-8 photoresist (PR) was purchased from MicroChem (USA). Silicon wafer (3 inches) was purchased from Silicone Sense (USA). Sylgard 184 polydimethylsiloxane (PDMS) prepolymer and curing agent were purchased from Dow Corning (USA). Rhodamin B (Sigma–Aldrich, USA) was used as a temperature probe. EPS-specific fluorescent dye (Film Tracer SYPRO Ruby Biofilm Matrix Stain) was purchased from Invitrogen (USA). Cultivation of Antarctic marine bacteria Bacteria were grown in MR2A medium at 25 °C. The cultured bacteria were harvested by centrifugation, washed twice with MR2A medium, and resuspended in 1 ml of fresh MR2A medium with the final concentration of bacteria adjusted to 1 109 cells/ ml. Two-step photolithography The microfluidic device was fabricated by using two-step photolithography and soft lithography, as shown in Fig. 1. The multilayer micromold was fabricated using SU-8 PR on a 3-inch silicon wafer. An SU-8 was first spin-coated on the silicon wafer and then exposed to ultraviolet (UV) light through an aligned photomask (Fig. 1A). We obtained micromold with a height of 240 lm. In the second step, an SU-8 PR was spin-coated on the first SU-8 layer and then exposed to UV light through a second aligned photomask. We selectively obtained micromold (height of 800 lm) for cold and hot sources (Fig. 1B). Finally, the multilayer of SU-8 micromold was generated by developing an uncured SU-8 PR. For replica micromolding based on soft lithography (Fig. 1C), a mixture of a PDMS prepolymer and curing agent (10:1) was thoroughly stirred and then degassed in a vacuum chamber. The degassed PDMS mixture was poured onto the silicon master and cured at 65 °C in an oven. After curing, the PDMS replica was peeled away from the silicon master and inlet holes were punched using a stainless-steel needle. The PDMS replica and glass substrate were bonded by treatment with oxygen plasma (PDC-002, Harrick, USA) for 1 min.
Calibration of temperature measurement To determine the dependence of the fluorescence intensity on temperature, we calibrated the fluorescence intensity at each temperature in the detection microchannel. The procedures were performed according to the recommended protocols from previous studies [9]. Briefly, optical images and fluorescence intensity of a rhodamine B solution (0.1 mM) as a temperature probe were acquired using an inverted fluorescence microscope (TE20000, Nikon, Japan) equipped with a long working distance 20 objective, a fluorescence filter (excitation = 500–550 nm, emission > 565 nm), and a charge-coupled device (CCD) camera (CoolSNAP cf, Photometrics, USA). To evaluate the dependence of the fluorescence intensity on temperature, the rhodamine B solution was stored in an incubator (MCL-20A BOD, Science & Technology, USA) for 2 h at each individual temperature and then filled in the detection microchannel. The temperature of the microfluidic device was maintained by circulating water thermostats at fluorescence microscopy combined with a homemade temperature-controlled incubator. Fluorescence intensities at different temperatures ranging from 4 to 40 °C were measured by line profiling across detection microchannel for calibration of temperature. The intensity at each temperature was determined by averaging the intensity value (after background subtraction) of all the pixels of the corresponding image. The software ImagePro (MediaCybernetics, USA) was used for the fluorescence analysis of each obtained image. For generation of a temperature gradient, two circulating water thermostats were integrated with the microfluidic device containing hot and cold source microchannels. The water in the hot source was circulated at 60 °C, whereas the water in the cold source, which contained 50% antifreezing solution, was circulated at 15 °C. The temperature gradient was identified by a change of fluorescence intensity of rhodamine B across the detection microchannel. Finally, the measured fluorescence intensity was correlated with the calibration curve for determining the temperature. Analysis of polar biofilm on a temperature gradient Planktonic bacteria were introduced from the inlet to the outlet at a low flow rate (0.5 ll min 1). After 5 min, the syringe pump was turned off to allow bacterial attachment for 30 min. Some bacteria were freely floating; therefore, the microchannels were washed with fresh MR2A medium (10 ll/min) for 10 min to remove free-floating bacteria. The temperature gradient established by the hot and cold sources was applied, and polar biofilms were grown in the detection microchannel for 12 h at 3.0 ll/min. The polar biofilms were monitored using an inverted fluorescence microscope (TE2000) equipped with a CCD camera (CoolSNAP cf). After development of Antarctic marine bacterial biofilms, the biofilms were stained with an EPS-specific fluorescent dye (Film Tracer SYPRO Ruby Biofilm Matrix Stain) for 30 min at 0.5 ll/min.
Table 1 List of Antarctic marine bacteria and their temperature dependency of biofilm. Classification
Species
Habitat
Temperature of biofilm formation (°C)
Psychrophile Thermophile
Pseudoalteromonas sp. Pseudoalteromonas tetraodonis Shewanella sp. Shewanella frigidimarina Pseudoalteromonas arctica Cellulophaga sp. Shewanella vesiculosa
South Shetland islands Kara sea Norway Kara sea South Shetland islands South Shetland islands Kara sea
0–18 13–41 15–41 30–41 37–41 0–41 0–41
Independent of temperature
91
92
Effect of temperature on biofilm formation by Antarctic marine bacteria in a microfluidic device / H.-H. Jeong et al. / Anal. Biochem. 446 (2014) 90–95
Fig.1. Schematic diagram of a two-step lithography process to fabricate microchannels with two different heights. (A) Lithography of the first layer for the detection microchannel. (B) Lithography of the second layer for hot and cold source microchannels with increased height. (C) Conventional soft lithography to fabricate final assembled microfluidic device.
Results and discussion
Monitoring of biofilm development on temperature gradient
Generation of temperature gradient in the microfluidic device
Among several environmental factors affecting the development of bacterial biofilm, cultivation temperatures critically regulate attachment and affect stages of biofilm formation [19,20]. To evaluate the effect of different cultivation temperatures on the development of Antarctic marine bacteria biofilm, liquid-cultured planktonic bacteria were loaded in the detection microchannel and then incubated for 30 min to induce stable attachment of individual bacteria onto the glass surface. The unattached bacteria were washed away through fluid flow, and fresh medium was introduced into the microchannel to permit development of biofilm under a temperature gradient (0–40 °C). We identified the border line of the biofilm developing based on the fluorescence staining. The corresponding specific temperature at the border of the biofilm was then calculated based on the linear temperature gradient (Figs. 2C and 3). Interestingly, we found that the formation of the pattern of Antarctic biofilms was classified into three main types with the dominant growth range of temperature (Fig. 3). First, the biofilm of Pseudoalteromonas arctica was developed at a relatively high temperature in the range of 37 to 41 °C (Fig. 3A). In contrast, biofilm development by a Pseudoalteromonas species occurred in the range of 0 to 18 °C (Fig. 3B) because it is a psychrotolerant bacterium that is found in both sea ice and the underlying water [21]. This result is consistent with previous reports showing that this bacterium is able to grow at 2 °C, but no growth occurred at 37 °C [22]. We
Recently, microfluidic systems have been developed as efficient tools for biological assays because they can generate linear gradients of temperature and chemical and oxygen concentration [10– 16]. For example, experimental studies in nutrient concentration, quorum sensing molecules, and shear stress have explored formation of bacterial biofilm [17,18], whereas the effect of temperature on formation of biofilm has rarely been analyzed. In this study, we applied a microfluidic device having microchannels with different heights for efficient generation of temperature gradients. The microfluidic system generates temperature gradients by integrating with a circulating water thermostat. To examine the temperature gradients in the microchannels, we used a temperature-dependent fluorescent dye (rhodamine B, 0.1 mM) that is known to have different quantum yields over a temperature range of 0 to 100 °C [9]. For the calibration curve, we first introduced hot and cold water solutions into the source microchannel, and then the solution of fluorescent dye (rhodamine B, 0.1 mM) was introduced as a temperature probe into the main detection microchannel. The fluorescence intensity is determined at various temperatures in the main detection microchannel (Fig. 2B). As expected, the microfluidic device shows a linear temperature gradient in the main detection microchannel from 0 to 40 °C (Fig. 2C).
Effect of temperature on biofilm formation by Antarctic marine bacteria in a microfluidic device / H.-H. Jeong et al. / Anal. Biochem. 446 (2014) 90–95
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Fig.2. Microfluidic device for generation of a linear temperature gradient. (A) Final assembled microfluidic device and a schematic diagram of linear temperature gradient. The microfluidic device consists of a detection microchannel (green: 240 lm [height] 950 lm [width], 4.5 cm length) and hot (red) and cold (blue) source microchannels (800 lm [height] 5000 lm [width], 4.5 cm length). The gap distance between the hot/cold and main detection microchannels is approximately 1 mm. The heat flux is generated from the hot source on the left to the cold one on the right. (B) Fluorescence intensity as a function of temperature in the detection channel. The fluorescence intensity was used for the calibration of the measured temperature. (C) Linear temperature gradient over distance in the detection microchannel. The fluorescence image indicates the temperature gradient in the detection microchannel. All experiments are carried out in triplicate and repeated in three independent sets of experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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Effect of temperature on biofilm formation by Antarctic marine bacteria in a microfluidic device / H.-H. Jeong et al. / Anal. Biochem. 446 (2014) 90–95
Fig.3. Different types of biofilm formation under a temperature gradient. Three types of biofilm formation were observed at a high temperature range (thermophilic biofilm formation) (A), at a low temperature range (psychrophilic biofilm formation) (B), and in a condition independent of the temperature (C).
Effect of temperature on biofilm formation by Antarctic marine bacteria in a microfluidic device / H.-H. Jeong et al. / Anal. Biochem. 446 (2014) 90–95
also found that biofilm development in Shewanella frigidimarina is temperature independent (Fig. 3C), which indicates that the formation of the biofilm is not associated with the cultivation temperature and reflects that the temperature alone is not a sufficient regulation factor for specific bacterial biofilm formation. Table 1 summarizes the temperature range of Antarctic marine bacteria biofilm obtained by seven different origins (Table 1). The relationship between the cultivation temperature and biofilm development in Antarctic marine bacteria does not exhibit same pattern; the result indicates that there are optimal temperature ranges for the biofilm development of Antarctic marine bacteria, although they are harvested from the same region. Table 1 hints that the control of cultivation temperature may have synergistic and antagonistic effects on one strain of bacteria that might not work for other strains. The current study shows that environmental temperature might be one of the important factors for the formation of biofilm, which reflects that the change of weather could produce dramatic change of bacterial physiologies in the Antarctic ecosystem. The observed bacteria response to temperature may be due to the production of EPS, which is known to enhance the adherence capability of bacteria and seeding materials for the biofilm [23]. In particular, EPS production by Pseudoalteromonas species at 2 and 10 °C is higher than that at 20 °C [18]. In addition, temperature regulates many environmental genes in microorganisms, resulting in a cell surface charge that could affect attachment [19,24]. Thus, the dependency of formation of Antarctic marine bacterial biofilm on a specific temperature range suggests that cultivation temperature may change physiological activity of each strain, probably influencing the EPS production and change of cell surface charge. These results confirm that the microfluidic approach is useful to rapidly analyze the effective temperature of biofilm development in a single experiment. Bacteria perform a variety of biological activities to adapt to extreme conditions. Temperature is a critical environmental parameter for the understanding of evolution and biodiversity of microorganisms. Especially, information regarding Antarctic marine biofilm is critical for understanding biofilm-forming processes because polar biofilms are able to be developed at specific depths of sea ice. One key finding of the study is that the biofilm formations are affected by temperature in their naturally occurring ranges. Conclusions To the best of our knowledge, there are few works investigating the effects of temperature on the formation and maturity of Antarctic marine biofilm. This study presents the first attempt to develop an in situ analytical system generating temperature gradients for monitoring of biofilm development of Antarctic marine bacteria. The result obtained from the microfluidic assay reveals that the bacteria are capable of enhanced biofilm formation at a specific temperature range. This is a key issue that needs to be developed further in order to advance the understanding of these complex ecological systems. Elucidation of the impact of each environment factor may uncover the mechanisms that regulate biofilm formation and provide insight into the fundamental ecology of the Antarctic region. Furthermore, the method could be applied to the analysis of various biological events in a single experiment and used to provide a strong basic understanding of the formation of microbial biofilms under the specific conditions. Moreover, it would be useful in resolving the low-throughput limitation of conventional methods.
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Acknowledgments This research was supported by the Polar Academic Program (PAP), KOPRI, and the Converging Research Center Program through the Ministry of Science, ICT and Future Planning, Korea (2013K000339). References [1] L.J. Rothschild, R.L. Mancinelli, Life in extreme environments, Nature 409 (2001) 1092–1101. [2] D.N. Thomas, G.S. Dieckmann, Antarctic sea ice—a habitat for extremophiles, Science 295 (2002) 641–644. [3] J.T. Staley, J.J. Gosink, Poles apart: biodiversity and biogeography of sea ice bacteria, Annu. Rev. Microbiol. 53 (1999) 189–215. [4] A. Goodchild, N.F. Saunders, H. Ertan, M. Raftery, M. Guilhaus, P.M. Curmi, R. Cavicchioli, A proteomic determination of cold adaptation in the Antarctic archaeon, Methanococcoides burtonii, Mol. Microbiol. 53 (2004) 309–321. [5] S. Phadtare, Recent developments in bacterial cold-shock response, Curr. Issues Mol. Biol. 6 (2004) 125–136. [6] N.M. Mesbah, J. Wiegel, Life under multiple extreme conditions: diversity and physiology of the halophilic alkalithermophiles, Appl. Environ. Microbiol. 78 (2012) 4074–4082. [7] F. Rao, S. Pasunooti, Y.L. Ng, W.C. Zhuo, L. Lim, A.W.X. Liu, Z.X. Liang, Enzymatic synthesis of c-di-GMP using a thermophilic diguanylate cyclase, Anal. Biochem. 389 (2009) 138–142. [8] T. Varin, C. Lovejoy, A.D. Jungblut, W.F. Vincent, J. Corbeil, Metagenomic analysis of stress genes in microbial mat communities from Antarctica and the high Arctic, Appl. Environ. Microbiol. 78 (2012) 549–559. [9] D. Ross, M. Gaitan, L.E. Locascio, Temperature measurement in microfluidic systems using a temperature-dependent fluorescent dye, Anal. Chem. 73 (2001) 4117–4123. [10] H. Mao, T. Yang, P.S. Cremer, A microfluidic device with a linear temperature gradient for parallel and combinatorial measurements, J. Am. Chem. Soc. 124 (2002) 4432–4435. [11] K.P. Kim, Y.G. Kim, C.H. Choi, H.E. Kim, S.H. Lee, W.S. Chang, C.S. Lee, In situ monitoring of antibiotic susceptibility of bacterial biofilms in a microfluidic device, Lab Chip 10 (2010) 3296–3299. [12] M. Skolimowski, M.W. Nielsen, J. Emneus, S. Molin, R. Taboryski, C. Sternberg, M. Dufva, O. Geschke, Microfluidic dissolved oxygen gradient generator biochip as a useful tool in bacterial biofilm studies, Lab Chip 10 (2010) 2162–2169. [13] D.A. Markov, S. Manuel, L.M. Shor, S.R. Opalenik, J.P. Wikswo, P.C. Samson, Tape underlayment rotary-node (TURN) valves for simple on-chip microfluidic flow control, Biomed. Microdevices 12 (2010) 135–144. [14] H.H. Jeong, S.H. Lee, J.M. Kim, H.E. Kim, Y.G. Kim, J.Y. Yoo, W.S. Chang, C.S. Lee, Microfluidic monitoring of Pseudomonas aeruginosa chemotaxis under the continuous chemical gradient, Biosens. Bioelectron. 26 (2010) 351–356. [15] H.H. Jeong, S.H. Lee, C.S. Lee, Pump-less static microfluidic device for analysis of chemotaxis of Pseudomonas aeruginosa using wetting and capillary action, Biosens. Bioelectron. 47 (2013) 278–284. [16] C. Zhang, D. Xing, Parallel DNA amplification by convective polymerase chain reaction with various annealing temperatures on a thermal gradient device, Anal. Biochem. 387 (2009) 102–112. [17] S.T. Flickinger, M.F. Copeland, E.M. Downes, A.T. Braasch, H.H. Tuson, Y.J. Eun, D.B. Weibel, Quorum sensing between Pseudomonas aeruginosa biofilms accelerates cell growth, J. Am. Chem. Soc. 133 (2011) 5966–5975. [18] C.M. Nichols, J.P. Bowman, J. Guezennec, Effects of incubation temperature on growth and production of exopolysaccharides by an Antarctic sea ice bacterium grown in batch culture, Appl. Environ. Microbiol. 71 (2005) 3519– 3523. [19] G. Di Bonaventura, R. Piccolomini, D. Paludi, V. D’Orio, A. Vergara, M. Conter, A. Ianieri, Influence of temperature on biofilm formation by Listeria monocytogenes on various food-contact surfaces: relationship with motility and cell surface hydrophobicity, J. Appl. Microbiol. 104 (2008) 1552–1561. [20] M. Nevot, V. Deroncele, M.J. Montes, E. Mercade, Effect of incubation temperature on growth parameters of Pseudoalteromonas antarctica NF3 and its production of extracellular polymeric substances, J. Appl. Microbiol. 105 (2008) 255–263. [21] M.V. Brown, J.P. Bowman, A molecular phylogenetic survey of sea-ice microbial communities (SIMCO), FEMS Microbiol. Ecol. 35 (2001) 267–275. [22] C.A. Mancuso Nichols, S. Garon, J.P. Bowman, G. Raguenes, J. Guezennec, Production of exopolysaccharides by Antarctic marine bacterial isolates, J. Appl. Microbiol. 96 (2004) 1057–1066. [23] N. Mangwani, H.R. Dash, A. Chauhan, S. Das, Bacterial quorum sensing: functional features and potential applications in biotechnology, J. Mol. Microbiol. Biotechnol. 22 (2012) 215–227. [24] C.A. White-Ziegler, S. Um, N.M. Perez, A.L. Berns, A.J. Malhowski, S. Young, Low temperature (23 °C) increases expression of biofilm-, cold-shock-, and RpoSdependent genes in Escherichia coli K-12, Microbiology 154 (2008) 148–166.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Effect of temperature on biofilm formation by Antarctic marine bacteria in a microfluidic device Heon-Ho Jeong a,1, Seong-Geun Jeong a,1, Aeri Park a, Sung-Chan Jang a, Soon Gyu Hong b, Chang-Soo Lee a,⇑ a b
Department of Chemical Engineering, Chungnam National University, Yuseong-gu, Deajeon 305-764, Republic of Korea Division of Polar Life Sciences, Korea Polar Research Insitute, 26 Songdomirae-ro, Yeonsu-gu, Incheon 406-840, Republic Korea
a r t i c l e
i n f o
Article history: Received 5 July 2013 Received in revised form 25 September 2013 Accepted 17 October 2013 Available online 26 October 2013 Keywords: Antarctic marine bacteria Biofilm Temperature gradient Microfluidic device Cryobiology
a b s t r a c t Polar biofilms have become an increasingly popular biological issue because new materials and phenotypes have been discovered in microorganisms in the polar region. Various environmental factors affect the functionality and adaptation of microorganisms. Because the polar region represents an extremely cold environment, polar microorganisms have a functionality different from that of normal microorganisms. Thus, determining the effective temperature for the development of polar biofilms is crucial. Here, we present a simple, novel one-pot assay for analysis of the effect of temperature on formation of Antarctic bacterial biofilm using a microfluidic system where continuous temperature gradients are generated. We find that a specific range of temperature is required for the growth of biofilms. Thus, this microfluidic approach provides precise information regarding the effective temperature for polar biofilm development with a new high-throughput screening format. Ó 2013 Elsevier Inc. All rights reserved.
Microorganisms growing at low temperatures are dominant life forms in Antarctic ecosystems [1]. Change in temperature is the dominant factor in external conditions to influence the biological communities. Especially, the diffusion rate of dissolved gases and the exchange of nutrients are greatly retarded at low-temperature conditions. Physiological and biochemical adaptations of Antarctic microorganisms accommodate subsequent rapid physical and chemical changes [2]. The capacity to survive in an extreme environment is a prerequisite for Antarctic microorganisms forming robust stress resistance toward extreme environments. The most critical metabolic requirement at low temperature is maintenance of functional lipid membranes. They can produce large amounts of extracellular polymeric substance (EPS),2 composed mainly of polysaccharides. Thus, in dense accumulation of bacteria, they can develop biofilms because they are able to alter the immediate physicochemical environment and protect themselves from external harsh environments [1,3]. Polar bacteria residing in biofilms show higher resistance to concentrated salinity and cold shock [4,5]. Despite the importance of the research topic, there is still a lack of knowledge about most Antarctic bacterial biofilms and the response to environmental conditions. In particular, temperature is ⇑ Corresponding author. Fax: +82 42 821 5896. E-mail address: [email protected] (C.-S. Lee). These authors contributed equally to this work. Abbreviations used: EPS, extracellular polymeric substance; KOPRI, Korea Polar Research Institute; MR2A, Marine R2A; PR, photoresist; PDMS, polydimethylsiloxane; UV, ultraviolet; CCD, charge-coupled device. 1 2
0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.10.027
the most critical stressor in the Antarctic environment and affects bacterial growth rate, protein activity, and gene expression. In normal bacteria, heat and cold shock denature proteins or increase the amount of unsaturated fatty acids in the cell membrane [6,7]. However, Antarctic microorganisms with cold-adapted genes are more stable at low temperatures [8]. The marine bacterial environment in Antarctica is largely unexplored, and increasing human pressure necessitates a clear understanding of the dynamics of biofilm formation in this sensitive ecosystem. In fact, conventional biology laboratories are performing labor-intensive and tedious experimental procedures to analyze formation of Antarctic bacterial biofilms under several separate temperature conditions. In considering throughput screening and number of Antarctic marine bacteria, it is one of the major hurdles to analyze the influence of temperature on growth of polar marine bacteria and their bacterial biofilm [2]. Unfortunately, there are few methodologies now available for analyzing the effect of temperature on formation of Antarctic marine bacterial biofilm with parallel data acquisition in a combinatorial analytical manner. Here we present a simple microfluidic approach to generate a wide linear temperature gradient from 4 to 40 °C and to analyze Antarctic marine biofilm formation under the temperature gradient. First, we investigated the ability of seven strains isolated in the Antarctic region with regard to continuous temperature gradients. Unlike conventional methods performed at fixed temperature conditions, this approach can provide quantitative and physiologically relevant data under extreme conditions because the
Effect of temperature on biofilm formation by Antarctic marine bacteria in a microfluidic device / H.-H. Jeong et al. / Anal. Biochem. 446 (2014) 90–95
microfluidic system can imitate versatile microenvironmental conditions such as varying sizes of fluids, shapes of microchannels, shear stress, and high-pressure fluid with temperature gradient. Thus, the method would provide more precise information regarding ecologically relevant environment, dependency on temperature, and the rational design of extreme environments in a new high-throughput screening format. Materials and methods Bacterial strain and reagents All seven types of Antarctic marine bacteria shown in Table 1 were obtained from the Polar and Alpine Microbial Collection (PAMC). Marine R2A (MR2A) medium was prepared in our laboratory. SU-8 photoresist (PR) was purchased from MicroChem (USA). Silicon wafer (3 inches) was purchased from Silicone Sense (USA). Sylgard 184 polydimethylsiloxane (PDMS) prepolymer and curing agent were purchased from Dow Corning (USA). Rhodamin B (Sigma–Aldrich, USA) was used as a temperature probe. EPS-specific fluorescent dye (Film Tracer SYPRO Ruby Biofilm Matrix Stain) was purchased from Invitrogen (USA). Cultivation of Antarctic marine bacteria Bacteria were grown in MR2A medium at 25 °C. The cultured bacteria were harvested by centrifugation, washed twice with MR2A medium, and resuspended in 1 ml of fresh MR2A medium with the final concentration of bacteria adjusted to 1 109 cells/ ml. Two-step photolithography The microfluidic device was fabricated by using two-step photolithography and soft lithography, as shown in Fig. 1. The multilayer micromold was fabricated using SU-8 PR on a 3-inch silicon wafer. An SU-8 was first spin-coated on the silicon wafer and then exposed to ultraviolet (UV) light through an aligned photomask (Fig. 1A). We obtained micromold with a height of 240 lm. In the second step, an SU-8 PR was spin-coated on the first SU-8 layer and then exposed to UV light through a second aligned photomask. We selectively obtained micromold (height of 800 lm) for cold and hot sources (Fig. 1B). Finally, the multilayer of SU-8 micromold was generated by developing an uncured SU-8 PR. For replica micromolding based on soft lithography (Fig. 1C), a mixture of a PDMS prepolymer and curing agent (10:1) was thoroughly stirred and then degassed in a vacuum chamber. The degassed PDMS mixture was poured onto the silicon master and cured at 65 °C in an oven. After curing, the PDMS replica was peeled away from the silicon master and inlet holes were punched using a stainless-steel needle. The PDMS replica and glass substrate were bonded by treatment with oxygen plasma (PDC-002, Harrick, USA) for 1 min.
Calibration of temperature measurement To determine the dependence of the fluorescence intensity on temperature, we calibrated the fluorescence intensity at each temperature in the detection microchannel. The procedures were performed according to the recommended protocols from previous studies [9]. Briefly, optical images and fluorescence intensity of a rhodamine B solution (0.1 mM) as a temperature probe were acquired using an inverted fluorescence microscope (TE20000, Nikon, Japan) equipped with a long working distance 20 objective, a fluorescence filter (excitation = 500–550 nm, emission > 565 nm), and a charge-coupled device (CCD) camera (CoolSNAP cf, Photometrics, USA). To evaluate the dependence of the fluorescence intensity on temperature, the rhodamine B solution was stored in an incubator (MCL-20A BOD, Science & Technology, USA) for 2 h at each individual temperature and then filled in the detection microchannel. The temperature of the microfluidic device was maintained by circulating water thermostats at fluorescence microscopy combined with a homemade temperature-controlled incubator. Fluorescence intensities at different temperatures ranging from 4 to 40 °C were measured by line profiling across detection microchannel for calibration of temperature. The intensity at each temperature was determined by averaging the intensity value (after background subtraction) of all the pixels of the corresponding image. The software ImagePro (MediaCybernetics, USA) was used for the fluorescence analysis of each obtained image. For generation of a temperature gradient, two circulating water thermostats were integrated with the microfluidic device containing hot and cold source microchannels. The water in the hot source was circulated at 60 °C, whereas the water in the cold source, which contained 50% antifreezing solution, was circulated at 15 °C. The temperature gradient was identified by a change of fluorescence intensity of rhodamine B across the detection microchannel. Finally, the measured fluorescence intensity was correlated with the calibration curve for determining the temperature. Analysis of polar biofilm on a temperature gradient Planktonic bacteria were introduced from the inlet to the outlet at a low flow rate (0.5 ll min 1). After 5 min, the syringe pump was turned off to allow bacterial attachment for 30 min. Some bacteria were freely floating; therefore, the microchannels were washed with fresh MR2A medium (10 ll/min) for 10 min to remove free-floating bacteria. The temperature gradient established by the hot and cold sources was applied, and polar biofilms were grown in the detection microchannel for 12 h at 3.0 ll/min. The polar biofilms were monitored using an inverted fluorescence microscope (TE2000) equipped with a CCD camera (CoolSNAP cf). After development of Antarctic marine bacterial biofilms, the biofilms were stained with an EPS-specific fluorescent dye (Film Tracer SYPRO Ruby Biofilm Matrix Stain) for 30 min at 0.5 ll/min.
Table 1 List of Antarctic marine bacteria and their temperature dependency of biofilm. Classification
Species
Habitat
Temperature of biofilm formation (°C)
Psychrophile Thermophile
Pseudoalteromonas sp. Pseudoalteromonas tetraodonis Shewanella sp. Shewanella frigidimarina Pseudoalteromonas arctica Cellulophaga sp. Shewanella vesiculosa
South Shetland islands Kara sea Norway Kara sea South Shetland islands South Shetland islands Kara sea
0–18 13–41 15–41 30–41 37–41 0–41 0–41
Independent of temperature
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Effect of temperature on biofilm formation by Antarctic marine bacteria in a microfluidic device / H.-H. Jeong et al. / Anal. Biochem. 446 (2014) 90–95
Fig.1. Schematic diagram of a two-step lithography process to fabricate microchannels with two different heights. (A) Lithography of the first layer for the detection microchannel. (B) Lithography of the second layer for hot and cold source microchannels with increased height. (C) Conventional soft lithography to fabricate final assembled microfluidic device.
Results and discussion
Monitoring of biofilm development on temperature gradient
Generation of temperature gradient in the microfluidic device
Among several environmental factors affecting the development of bacterial biofilm, cultivation temperatures critically regulate attachment and affect stages of biofilm formation [19,20]. To evaluate the effect of different cultivation temperatures on the development of Antarctic marine bacteria biofilm, liquid-cultured planktonic bacteria were loaded in the detection microchannel and then incubated for 30 min to induce stable attachment of individual bacteria onto the glass surface. The unattached bacteria were washed away through fluid flow, and fresh medium was introduced into the microchannel to permit development of biofilm under a temperature gradient (0–40 °C). We identified the border line of the biofilm developing based on the fluorescence staining. The corresponding specific temperature at the border of the biofilm was then calculated based on the linear temperature gradient (Figs. 2C and 3). Interestingly, we found that the formation of the pattern of Antarctic biofilms was classified into three main types with the dominant growth range of temperature (Fig. 3). First, the biofilm of Pseudoalteromonas arctica was developed at a relatively high temperature in the range of 37 to 41 °C (Fig. 3A). In contrast, biofilm development by a Pseudoalteromonas species occurred in the range of 0 to 18 °C (Fig. 3B) because it is a psychrotolerant bacterium that is found in both sea ice and the underlying water [21]. This result is consistent with previous reports showing that this bacterium is able to grow at 2 °C, but no growth occurred at 37 °C [22]. We
Recently, microfluidic systems have been developed as efficient tools for biological assays because they can generate linear gradients of temperature and chemical and oxygen concentration [10– 16]. For example, experimental studies in nutrient concentration, quorum sensing molecules, and shear stress have explored formation of bacterial biofilm [17,18], whereas the effect of temperature on formation of biofilm has rarely been analyzed. In this study, we applied a microfluidic device having microchannels with different heights for efficient generation of temperature gradients. The microfluidic system generates temperature gradients by integrating with a circulating water thermostat. To examine the temperature gradients in the microchannels, we used a temperature-dependent fluorescent dye (rhodamine B, 0.1 mM) that is known to have different quantum yields over a temperature range of 0 to 100 °C [9]. For the calibration curve, we first introduced hot and cold water solutions into the source microchannel, and then the solution of fluorescent dye (rhodamine B, 0.1 mM) was introduced as a temperature probe into the main detection microchannel. The fluorescence intensity is determined at various temperatures in the main detection microchannel (Fig. 2B). As expected, the microfluidic device shows a linear temperature gradient in the main detection microchannel from 0 to 40 °C (Fig. 2C).
Effect of temperature on biofilm formation by Antarctic marine bacteria in a microfluidic device / H.-H. Jeong et al. / Anal. Biochem. 446 (2014) 90–95
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Fig.2. Microfluidic device for generation of a linear temperature gradient. (A) Final assembled microfluidic device and a schematic diagram of linear temperature gradient. The microfluidic device consists of a detection microchannel (green: 240 lm [height] 950 lm [width], 4.5 cm length) and hot (red) and cold (blue) source microchannels (800 lm [height] 5000 lm [width], 4.5 cm length). The gap distance between the hot/cold and main detection microchannels is approximately 1 mm. The heat flux is generated from the hot source on the left to the cold one on the right. (B) Fluorescence intensity as a function of temperature in the detection channel. The fluorescence intensity was used for the calibration of the measured temperature. (C) Linear temperature gradient over distance in the detection microchannel. The fluorescence image indicates the temperature gradient in the detection microchannel. All experiments are carried out in triplicate and repeated in three independent sets of experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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Effect of temperature on biofilm formation by Antarctic marine bacteria in a microfluidic device / H.-H. Jeong et al. / Anal. Biochem. 446 (2014) 90–95
Fig.3. Different types of biofilm formation under a temperature gradient. Three types of biofilm formation were observed at a high temperature range (thermophilic biofilm formation) (A), at a low temperature range (psychrophilic biofilm formation) (B), and in a condition independent of the temperature (C).
Effect of temperature on biofilm formation by Antarctic marine bacteria in a microfluidic device / H.-H. Jeong et al. / Anal. Biochem. 446 (2014) 90–95
also found that biofilm development in Shewanella frigidimarina is temperature independent (Fig. 3C), which indicates that the formation of the biofilm is not associated with the cultivation temperature and reflects that the temperature alone is not a sufficient regulation factor for specific bacterial biofilm formation. Table 1 summarizes the temperature range of Antarctic marine bacteria biofilm obtained by seven different origins (Table 1). The relationship between the cultivation temperature and biofilm development in Antarctic marine bacteria does not exhibit same pattern; the result indicates that there are optimal temperature ranges for the biofilm development of Antarctic marine bacteria, although they are harvested from the same region. Table 1 hints that the control of cultivation temperature may have synergistic and antagonistic effects on one strain of bacteria that might not work for other strains. The current study shows that environmental temperature might be one of the important factors for the formation of biofilm, which reflects that the change of weather could produce dramatic change of bacterial physiologies in the Antarctic ecosystem. The observed bacteria response to temperature may be due to the production of EPS, which is known to enhance the adherence capability of bacteria and seeding materials for the biofilm [23]. In particular, EPS production by Pseudoalteromonas species at 2 and 10 °C is higher than that at 20 °C [18]. In addition, temperature regulates many environmental genes in microorganisms, resulting in a cell surface charge that could affect attachment [19,24]. Thus, the dependency of formation of Antarctic marine bacterial biofilm on a specific temperature range suggests that cultivation temperature may change physiological activity of each strain, probably influencing the EPS production and change of cell surface charge. These results confirm that the microfluidic approach is useful to rapidly analyze the effective temperature of biofilm development in a single experiment. Bacteria perform a variety of biological activities to adapt to extreme conditions. Temperature is a critical environmental parameter for the understanding of evolution and biodiversity of microorganisms. Especially, information regarding Antarctic marine biofilm is critical for understanding biofilm-forming processes because polar biofilms are able to be developed at specific depths of sea ice. One key finding of the study is that the biofilm formations are affected by temperature in their naturally occurring ranges. Conclusions To the best of our knowledge, there are few works investigating the effects of temperature on the formation and maturity of Antarctic marine biofilm. This study presents the first attempt to develop an in situ analytical system generating temperature gradients for monitoring of biofilm development of Antarctic marine bacteria. The result obtained from the microfluidic assay reveals that the bacteria are capable of enhanced biofilm formation at a specific temperature range. This is a key issue that needs to be developed further in order to advance the understanding of these complex ecological systems. Elucidation of the impact of each environment factor may uncover the mechanisms that regulate biofilm formation and provide insight into the fundamental ecology of the Antarctic region. Furthermore, the method could be applied to the analysis of various biological events in a single experiment and used to provide a strong basic understanding of the formation of microbial biofilms under the specific conditions. Moreover, it would be useful in resolving the low-throughput limitation of conventional methods.
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