New Guidelines For Selecting Best Extraction Methods of EPS Using Atomic Force Microscopy Seoktae Kang*, Yongtae Ahn**, Heechul Choi***, Menachem Elimelech**** * Yale University, New Haven, CT, USA, seoktae.kang@yale.edu ** KAIST, Daejeon, Rep. of Korea, ytahn@kaist.ac.kr ***GIST, Gwangju, Rep. of Korea, hcchoi@gist.ac.kr **** Yale University, New Haven, CT, USA, menachem.elimelech@yale.edu ABSTRACT A new guideline with atomic force microscopy (AFM) to select the effective extraction method for extracellular polymeric substances (EPS), was proposed and validated by ‘classical’ biochemical ways. The classical biochemical guidelines showed that efficiencies of four extraction methods were comparable regardless of different microbial species and the extraction using NaOH or Formaldehyde- NaOH was the best. The efficiencies of extraction were further investigated using AFM by analyzing interaction forces between bio-probe, which a single cell of S. cerevisiae was attached at the end of the cantilever, and flat surface. Interaction forces fitted with steric model showed the thickness of residual polymer layer had good agreement with biochemical results. The comparison of results from two guidelines explained the basic mechanisms of extraction methods. Keywords : extracellular polymeric substances, atomic force microscopy, extraction, steric model, cell lysis INTRODUCTION Extracellular polymeric substances (EPS) are metabolic products accumulated on the bacterial cell surface [1]. It has been believed that EPS plays key roles during the floc formation, the adhesion and the maturation of biofilm, and the self-protection of microorganisms [1-7]. There are several physico-chemical methods for extraction of EPS, but they have exhibited wide variation as a function of extraction methods and conditions [8-10]. Also, the evaluation guidelines to select the best extraction method have been limited to indirect bio-chemical assay such as the comparisons of carbohydrate, protein, uronic acid, and DNA contents. However, indirect characterization methods cannot exclude the potential contamination of extracted solution by extracting reagents [8]. In this study, we established a new and potential guideline for the direct evaluation for the effectiveness of EPS extraction methods using atomic force microscopy. MATERIALS AND METHODS Four extraction methods (Heat, EDTA, NaOH, formaldehyde with NaOH) were compared using 2 g VSS/L of four microbial communities (S. cerevisiae, P. aeruginosa, activated sludge from biological denitrification reactor (BNR), activated sludge from DeN process (DeN)). Extractions were performed according to Liu and Fang [9] and Shin et al.[11]. The total amount of extracted EPS was measured by the sum of protein [12] and polysaccharides contents [13]. The DNA in EPS solution was measured by the spectro-photometric method with DNA of E. coli as a standard [14]. A single cell of wild and EPS-extracted S. cerevisiae was attached at the end of the cantilever [15], and interaction forces were measured using atomic force microscopy (AFM). The approach curves were analyzed by the steric force model as equation 1 [16]. L h B St e TrL k F / 2 2 / 3 50 π − Γ = (1) where F st is steric interaction force, k B is Boltzmann constant, T is temperature ( o K), r is the radius of S. cerevisiae, L is the equilibrium height of the polymer layer, 592 NSTI-Nanotech 2006, www.nsti.org, ISBN 0-9767985-6-5 Vol. 1, 2006