Optimized isolation procedure for obtaining strongly actinide binding exopolymeric substances (EPS) from two bacteria (Sagittula stellata and Pseudomonas fluorescens Biovar II)

Bioresource Technology 100 (2009) 6010–6021

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Optimized isolation procedure for obtaining strongly actinide binding exopolymeric substances (EPS) from two bacteria (Sagittula stellata and Pseudomonas fluorescens Biovar II) Chen Xu a,*, Peter H. Santschi a, Kathleen A. Schwehr a, Chin-Chang Hung b a

Laboratory for Oceanographic and Environmental Research (LOER), Departments of Oceanography and Marine Sciences, Texas A&M University, 5007 Avenue U, Galveston, TX 77551, United States b Institute of Marine Environmental Chemistry and Ecology, National Taiwan Ocean University, 2 Pei-Ning Rd., Keelung 202, Taiwan

a r t i c l e

i n f o

Article history: Received 1 May 2008 Received in revised form 9 September 2008 Accepted 2 June 2009 Available online 1 July 2009 Keywords: Actinide binding Isolation Exopolymeric substance (EPS) Growth phases Isoelectric focusing

a b s t r a c t Different chemical extractants (NaCl, EDTA, HCl and NaOH) and physical methods (ultrasonication and heating) were examined by their efficacies of extracting ‘‘attached” exopolymeric substances (EPS) secreted by marine bacterium Sagittula stellata (SS) and terrestrial bacterium Pseudomonas fluorescens Biovar II (PF). Extraction by 0.5 N HCl for 3 h was best for SS while extraction by 0.05 N NaCl for 3–5 h was regarded as optimal for PF. Improvements in EPS purification included a pre-diafiltration step to remove the broth material and reduce the solution volume, thus the usage of ethanol, and time. The EPS harvested at the optimal time and purified by the improved method were enriched in polysaccharides, with smaller amounts of proteins, thus having amphiphilic properties. Isoelectric focusing of 234 Th or 240Pu labeled EPS showed both actinides were strongly bound to macromolecules with low pI, similar to reported marine or soil colloidal natural organic matter (NOM). Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Radionuclides, e.g., plutonium (Pu), are components of nuclear waste, and a certain fraction of it can accidentally end up in the environment. Pu may be leached from sites where it was accidentally (or purposefully) released, and can migrate attached to mobile colloids over long distances from the point of release (Kersting et al., 1999; Novikov et al., 2006; Santschi et al., 2002). Site remediation in the past utilized, at times, caustic reagents which not only caused irreparable damage to the soils or aquatic systems, but also generated large amounts of additional hazardous waste. It has been proposed that exopolymeric substances (EPS) produced by bacteria, algae and fungi could serve as a natural barrier to actinide transport from geologic repositories (Dhami et al., 1998; Runde, 2000), but it is not clear whether and to what extent EPS are able to immobilize Pu in subsurface environments (Gillow et al., 2000). Though the strong binding of EPS with actinides has been demonstrated (Alvarado Quiroz et al., 2006; Guo et al., 2002; Hung et al., 2004; Neu et al., 2005; Quigley et al., 2001, 2002; Santschi et al., 2002, 2003), we still know relatively little about mechanisms of microbial EPS association with Pu due to the low

* Corresponding author. Tel.: +1 409 740 4867; fax: +1 409 740 4786. E-mail address: [email protected] (C. Xu). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.06.008

concentrations and complexity of extractable EPS, thus preventing the application of EPS to bioremediation. Therefore, optimizing the isolation procedures for EPS produced by pure laboratory cultures could provide sufficient amounts of material for in-depth studies. EPS that remain tightly bound to the cell surface by covalent linkages and cannot be easily removed from the cell surface are called the ‘‘attached” fraction. Those EPS that are more loosely bound in the form of slime and can be easily shed into the medium, as well as those in free planktonic state, are called the ‘‘nonattached” fraction. Only a few studies have explicitly differentiated the two fractions produced by diatoms (Staats et al., 1999; Sutherland, 1977; Wolfstein and Stal, 2002) and fewer have explored the differences between two EPS fractions produced by bacteria (Decho and Lopez, 1993). The soil bacterium Pseudomonas fluorescens Biovar II (PF) was isolated from a low-level radioactive waste disposal site at West Valley, New York (Dodge and Francis, 1994) and was demonstrated to produce EPS that could potentially reduce Pu(V) to Pu(IV) and greatly enhance its sorption to silica particles (Roberts et al., 2008). The marine bacterium Sagittula stellata (SS) was isolated from the coastal water of Georgia and identified as a lignin degrading bacterium (Gonzalez et al., 1997). SS cells were shown to form aggregates and produce ‘‘non-attached” EPS that have the strong ability to induce dissolved organic matter (DOM) self-assembly

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into 4 lm microgels (Chin et al., 1998; Ding et al., 2008). Thus SS EPS might act as an efficient filter for radionuclides. Extraction and purification of these EPS from bacterial cultures were time-consuming and expensive (Alvarado Quiroz et al., 2006; Hung et al., 2005) and the yield was not sufficient to allow further characterization (e.g., NMR) or studies of binding properties. Thus, a series of systematic studies were carried out to (1) optimize the isolation methods for both EPS fractions from complex synthetic media; (2) allow the study of changes in the production and composition of both EPS fractions during different growth phases; (3) determine the isoelectric point of ‘‘non-attached” EPS, radiolabeled by 234Th and 240Pu, by isoelectric focusing (IEF) electrophoresis. As far as we know, these studies were carried out for the first time for these two bacteria. The results allowed further molecular level characterization of EPS from these two bacteria as well as exploration of the different functions of EPS components for actinide binding in soils or aquatic environments, the results of which will be reported elsewhere.

2. Methods 2.1. Bacterial species Both bacterial species were obtained from the American Type Culture Collection (55241 for P. fluorescens Biovar II and 700073 for S. stellata) and inoculated in complex synthetic soy broth (BD company, 30 g/L) and Marine Broth 2216 (Difco Laboratories, 37.4 g/L), respectively (Alvarado Quiroz et al., 2006; Gonzalez et al., 1997; Hung et al., 2005). At regular time intervals, bacterial growth was monitored by reading the optical density at 600 nm (OD600) using a Turner SP-890 UV–VIS spectrophotometer. For incubation, agitation was performed in a rotary shaker at 60 rev min 1, and the temperature was kept constant at 30 °C by a Precision Incubator (GCA Corporation, USA). 2.2. Experiment 1: optimization of isolation methods 2.2.1. Extraction of ‘‘attached” EPS An optimal extraction method should meet all the three requirements, (1) relatively little cell lysis; (2) high EPS yield, especially the polysaccharides; (3) least chemical modification or denaturing of the extracted polymers. The research strategy was to (1) compare the cell lysis proxy (nucleic acid contents) in the EPS extracted by each method; (2) compare the absolute yield, especially the polysaccharide contents, as well as the TCHO-C to protein-C ratios; (3) compare the MWs distribution of the EPS extracted by each method to that of the control group. For SS, there were six treatments: 3% NaCl, 2% EDTA, 0.5 N HCl, 0.5 N NaOH, 3% NaCl with ultrasonication, and 3% NaCl with heating. Extraction with 3% NaCl solution was used as the control method, since this is the ionic strength in seawater. 0.5 N acidic or alkaline reagents were chosen to give a similar ionic strength as the control method. The EDTA dosage and the heating temperature were chosen according to the literature (Gehr and Henry, 1983; Sheng et al., 2005). The culture was harvested just prior to stationary phase, split into six groups, each with two 200 ml of duplicates, and each sample was centrifuged at approximately 3200g for 30 min (4 °C, the same below). The supernatant was discarded without disrupting the pellet. Two replicate pellets were then re-suspended and extracted with each of the six treatment solutions for 3 h (Sheng et al., 2005). Ultrasonication was carried out in a Bransonic Ultrasonic bath (continuous output: 40 W). During the extraction, all solutions, except the solution using the ultrasonication method, were gently stirred on magnetic stir plates. After extraction, all solutions were centrifuged and the supernatant containing the released

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‘‘attached” EPS was filtered through a 0.22 lm polycarbonate filter (GTTP, Millipore Corporation, USA) to remove bacterial cells. The filtrates were diafiltered against double distilled water (18.2 X) through a 10 kDa regenerated cellulose membrane (PLGC, Millipore Corporation, USA) using an Amicon 8400 stirred cell ultrafiltration system (Millipore Corporation, USA). This step was required in order to remove any remaining chemicals (e.g., EDTA) which might interfere with the later chemical characterization. The yields of TCHO, proteins and nucleic acids that were extracted by different treatments were calculated from the concentration in the original bacterial suspension. For PF, at first, extractants of the same concentrations as those for SS were tested but they were found out to cause severe cell lysis due to the fact that PF is a soil species. Thus, 0.005 N NaCl was chosen as the control group since this is the average ionic strength of soil solutions (Black and Campbell, 1982; Kookana and Naidu, 1998). Accordingly, acid or alkaline solutions having ionic strengths of the same magnitude as the control were used. There were seven treatments: 0.005 N NaCl, 3% NaCl, 2% EDTA, 0.01 N HCl, 0.01 N NaOH, 0.005 N NaCl with ultrasonication, and 0.005 N NaCl with heating. 3% NaCl was also chosen for comparison since Hung et al. (2005) used it to extract the ‘‘attached” EPS from this species. The processing method used was the same as that for SS. To determine the most suitable treatment, the EPS total carbohydrates (TCHO) and protein content were measured (see below). The TCHO-C: protein-C ratio was calculated to indicate the degree of ‘‘likeness” of the EPS extracted by different methods to that of the control group (Comte et al., 2006; Hung et al., 2005; Liu and Fang, 2002). The nucleic acid content was used as an indicator of cell rupture. In order to assess to what extent different chemical or physical extraction methods would hydrolyze or break down selected macromolecules, the EPS molecular weight (MW) distribution was analyzed. The MW distributions of ‘‘attached” EPS extracted by different treatments were measured by a Waters High Performance Liquid Chromatographic (HPLC) system coupled with Tosoh Biosciences G4000 PWxl guard and analytical size exclusion columns and a 2417 refractive index (RI) detector. Millenium 4.00 software was used to operate the HPLC system and to acquire and integrate the chromatograms. The RI detector was set at a temperature of 30 °C and a sensitivity of 4. The mobile phase was a solution of 0.078 M NaNO3 with 10 mM phosphate buffer of pH 6.8, maintained at a flow rate of 0.5 ml min 1. Solutions of EPS were prepared at concentrations of 1 mg ml 1 at least 12 h before analysis to allow uncoiling and solubilization. During this period, the EPS solutions were stored in the dark under refrigeration, and then filtered through 0.2 lm sterile syringe filters (cellulose acetate, VWR international) before injection into the HPLC to remove non-rehydrated aggregates and bacteria (Wilkinson et al., 1997). The injection volume of 150 ll was approximately 1% of the column bed volume (14.3 ml). Polysulfonate standards (8, 35, 100, and 780 kDa) were used for calibration and prepared exactly the same way as the samples. 2.2.2. Purification of EPS EPS were purified with three cycles of ethanol precipitation, followed by two cycles of trichloroacetic acid (TCA) precipitation. To eliminate the possible error caused by batch-to-batch difference, subsamples (200 ml) were taken from the same batch and processed in five different groups (1) one ethanol precipitation; (2) two ethanol precipitation; (3) three ethanol precipitation; (4) three ethanol precipitation plus one TCA precipitation; (5) three ethanol precipitation plus two TCA precipitation. Two replicates (n = 2) for each group were taken. After the purification, sample was extensively diafiltered. The retentate was freezedried to analyze TCHO, protein and URA and calculated as % of the dry weight in order

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to track their changes with the progression of the purification steps. 2.3. Experiment 2: EPS in different growth phases Bacterial suspensions collected at various growth phases (200 ml), were immediately centrifuged and the resulting supernatant containing the ‘‘non-attached” EPS was filtered through a 0.22 lm polycarbonate filter. The filtrate was diafiltered to remove the broth media. The retentate was defined as crude ‘‘nonattached” EPS. Both polycarbonate filters and the pellet from the previous centrifugation step were combined and extracted in the optimal extractant for each species, as determined in the previous experiment. After extraction, the solution was centrifuged and the supernatant was collected, filtered through a 0.22 lm polycarbonate filter and the filtrate was diafiltered. The retentate was defined as crude ‘‘attached EPS”. Purification was not performed any further since the purpose of this section was to study the compositional variation of the whole EPS (i.e., 10 kDa–0.22 lm) at different growth phases, thus any protein removal such as TCA precipitation would artificially bias the results. Specific productivity for EPS or their components (TCHO and protein) was estimated as the mass of EPS or polysaccharides or proteins per unit of absorbance of the bacterial culture suspension at 600 nm (initial blank absorbance subtracted), assuming that one unit of OD600 corresponds to a certain amount of dry cell mass (Torino et al., 2005; Vaningelgem et al., 2004). 2.4. Compositional characterization Chemical characterization included the spectrophotometric determination of TCHO (Hung and Santschi, 2001; Myklestad et al., 1997), URA (Filisetti-Cozzi and Carpita, 1991; Hung and Santschi, 2001), and proteins (Smith et al., 1985). Uronic acids are regarded as important components of EPS produced by algae and bacteria as they play a significant role in the extracellular milieu by forming flocs and biofilms, binding extracellular enzymes in their active forms, scavenging trace metals or radionuclides from the water, and altering the surface characteristics of suspended particles, etc. (Bhaskar et al., 2005). The nucleic acid content was quantified by measuring the absorbance at 260 nm with a quartz cuvette having a 1 cm light path length and multiplying the absorbance by the extinction coefficient at 50 lg ml 1 (Manchester, 1996). The ratio of absorption at 260 nm to that at 280 nm was used as an indicator to monitor protein contamination by this method. Total organic carbon (TOC) was measured on a Shimadzu TOC 5000 carbon analyzer (Guo et al., 1994). Neutral sugars and URA in the EPS collected from different growth phases were simultaneously determined following Walters and Hedges (1988). The sample was injected to a gas chromatography (Thermo Finnigan) coupled with an AS3000 autosampler and a Finnigan Polaris Q external ion source mass spectrometer. Xcalibur Data System was used to acquire and integrate the real-time plot of total ion current (TIC) and mass spectrum. TM

2.5. Experiment 3: radiolabeling EPS with isoelectric focusing electrophoresis (IEF)

234

Th and

240

Pu and

Radiolabeling EPS with these two actinides was carried out according to Quigley et al. (2002). IEF is performed by mixing the radiolabeled EPS with the rehydrated solution, which includes urea and detergent, to make sure that each polymer is present only in one configuration and to minimize aggregation and molecular interactions. Two-hundred and forty microliters of this solution (100 ll sample + 140 ll rehydrated solution) was loaded in each IPG strip (Amersham Biosciences, Immobiline Dry Strip, pH 3–10,

11 cm, Cat. No: 18-1016-61). One strip was loaded with 100 ll of D.I. and 140 ll of rehydrated solution and treated as the blank. For each IEF experiment, there were 11 replicate sample strips and one blank strip running in parallel. All strips were placed into the reswelling tray for at least 12 h and then placed in the electrophoresis apparatus for 17.5 h with a programmed current recommended by the manufacturer (Amersham Biosciences, Multiphor II Electrophoresis System). After the program was finished, pH value at every centimeter of each strip was measured immediately, by a bench pH/mv/temperature meter (pH/Ion 510 series, OAKLON Instrument, USA). Strips were cut evenly into 11 fractions and extracted with 1% sodium dodecyl sulfate (SDS) for 24 h. These fractions were further processed, respectively, for 234Th or 240Pu activity analysis. 234 Th activity was counted by a Beckman Model 8100 Liquid Scintillation Counter (LSC) after the Liquid scintillation cocktail (Ecolume, ICN) was added (Alvarado Quiroz et al., 2006; Quigley et al., 2002). 240Pu activity was a-counted, using 242Pu as a yield tracer (Santschi and Roberts, 2002). Recovery of both radionuclides was calculated as sum of activity within all gel fractions to total spiked activity. 2.6. Statistical analysis By using SPSS 11.0 (SPSS Inc., Chicago, IL 60606), data were first tested for normality using Kolmogorov–Smirnov test (a = 0.05). If normally distributed, they were further tested for correlation using two-tailed, bivariate Pearson analysis (a = 0.05). If data were not normally distributed, non-parametric Spearman rank analysis was performed, which was more conservative than the Pearson test against Type I error (Dytham, 1999). To study the correlation between TCHO or protein production and bacterial growth, the natural logarithm of OD600 was used, instead of OD600.

3. Results 3.1. Experiment 1: optimization of isolation methods 3.1.1. Extraction of ‘‘attached” EPS The contents of capsular EPS components extracted by different treatments are shown in Fig. 1a for SS and Fig. 1b for PF, respectively. Ratios of TCHO-C (40% C by weight) to protein-C (33% C by weight) are summarized in Fig. 1c and d. They varied from 0.10 to 0.92 for SS and from 0.05 to 0.34 for PF, depending on different treatments. The extraction of EPS from SS with 3% NaCl, 2% EDTA, 0.5 N HCl, 0.5 N NaOH or ultrasonication did not change the MW distribution of the EPS (single peak at very similar retention time, with a MW of 28.1 ± 1.4 kDa), while EPS extracted with a combination of 3% NaCl solution and heating at 75 °C gave double peaks and very different retention times (Fig. 2a), indicating MWs of 31.9 kDa and 12.8 kDa, respectively. The content of nucleic acid for the six groups was in this order: 3% NaCl 3% NaCl with heating >2% EDTA > 0.5 N NaOH > 0.5 HCl > 3% NaCl with ultrasonication. Results from HPLC-SEC and nucleic acid contents agreed well, suggesting that all these treatments did not cause severe cell lysis, while extraction by 3% NaCl with heating did cause modification of the macromolecules (Fig. 2a). 0.5 N HCl was the most efficient method to extract capsular polysaccharides (Fig. 1a) with the highest yield (1.43 mg glucose equivalents/L-bacterial culture), while having the similar ratio of TCHO-C to protein-C (Fig. 1c) to that of the control group (0.92 ± 0.12 vs. 0.84 ± 0.06). Results for PF showed that extraction with 3% NaCl had double peaks (18 and 167 kDa) (Fig. 2b). EPS extracted by 0.01 N NaOH showed a very broad peak (Fig. 2c) while the 0.005 N NaCl with

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Fig. 1. Chemical composition of the ‘‘attached” exopolymeric substances (EPS) by different extraction methods. (a) Chemical composition of EPS from SS; (b) chemical composition of EPS from PF; (c) TCHO-C/protein-C from SS; (d) TCHO-C/protein-C from PF.

heating also caused double peaks (Fig. 2d), which indicates either the presence of cell membrane material or leakage of intracellular molecules at these conditions. All the other treatments showed similar patterns in their chromatograms to that of the control group, with an average molecular weight of 25.5 ± 3.6 kDa. When 3% NaCl, 0.01 N NaOH and 0.005 N NaCl with heating were used to extract EPS, the nucleic acid content was 10.3, 7.4 and 5.2 times higher than that of the control method (Fig. 1b), indicating that cell lysis was severe and a large amount of intracellular material leaked out. This agrees well with the results of HPLC-SEC. Both 2% EDTA and 0.01 N HCl caused less cell lysis; however, the extracellular polysaccharides yields were not promising. Therefore, 0.005 N NaCl was the most suitable method. In subsequent experiments, both concentrations of the optimal extractants and extraction times were tested for these two bacteria, respectively. 0.5 N HCl was still regarded as the best concentration for SS (Fig. 3a) while 0.05 N NaCl worked better for PF (Fig. 3c), with no significant elevation of nucleic acid content compared to that of the control group. The extraction time did not have significant influence on the extraction efficiency for the EPS of SS (Fig. 3b), while the contents of EPS components were dependent on extraction time for PF (Fig. 3d).

0.22 lm, was added before the alcohol precipitation step. This fractionation step concentrated the culture suspension from a volume of 1–2 L to approximately 100–150 ml and much less alcohol was required in the latter purification steps. The EPS components (% of EPS dry weight) changed with the progression of alcohol and TCA precipitations (Table 1). For SS, TCHO increased by 4% after the first ethanol precipitation, compared to a sample without ethanol precipitation, remained almost constant with three additional ethanol precipitations, and increased by 14% with TCA precipitation. Protein became more enriched with the progression of ethanol precipitation, but decreased dramatically after TCA precipitation. For PF, the TCHO content increased by 4% after the first ethanol precipitation and remained almost constant for each additional precipitation step. The protein content decreased a lot by two ethanol precipitations and one TCA precipitation. The second TCA precipitation for both species produced no visible protein precipitate after centrifugation or filtration through a 0.22 lm polycarbonate membrane (data not shown). Compositional analysis of both ‘‘attached” and ‘‘non-attached” EPS produced by the two bacteria, harvested at their respective optimal growth phases (see below) and purified by the improved methods are shown in Table 2.

3.1.2. Purification of EPS Filtered marine broth 2216 (<0.22 lm) had a MW of 5.28 ± 0.70 kDa and filtered soy broth had MWs of 7.21 ± 0.16 kDa and 4.81 ± 0.13 kDa (spectra not shown) as determined by HPLC-SEC. Therefore a 10 kDa regenerated cellulose diafiltration membrane was chosen to effectively ‘‘wash out” the broth materials. A size fractionation step (pre-filtration and diafiltration) collecting EPS from the bacterial culture, with sizes from 10 kDa to

3.2. Experiment 2: EPS production and composition in different growth phases 3.2.1. SS Nucleic acids detected in ‘‘non-attached” EPS were considered an indicator for cell lysis during growth and an indicator for cell lysis during the ‘‘attached” EPS extraction process. Little cell lysis occurred during the extraction operation. ‘‘Non-attached” EPS from

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SS were more enriched in polysaccharides (Fig. 4a), with an average value of TCHO-C to protein-C ratios as 1.04 ± 0.13 and ‘‘attached” EPS contained more protein (Fig. 4b). Not including the bacterial death phase, the TCHO-C to protein-C ratio remained relatively constant in the ‘‘non-attached” EPS; while in the ‘‘attached” EPS, this ratio increased relatively slowly from 0.16 to 0.30 during the lag (0–9 h) and log (9–47 h) phases and then increased dramatically to 1.21 at the stationary phase (47–89 h) (Fig. 4c). URA was quite low in both types of EPS and undetectable by the spectrophotometric method with the subsample aliquots (Hung and Santschi, 2001), thus not reported here. Statistical analysis revealed that the ‘‘non-attached” EPS components (TCHO and proteins) were significantly and positively correlated with bacterial growth (log10(OD600), a = 0.05, p  0.05). The ‘‘attached” EPS components were weakly correlated with bacterial growth (log10(OD600)), with both p values close to the critical level of 0.05 (p = 0.035). The specific productivity of both types of EPS is shown in Fig. 4d and e and the maximal production rate for both occurred at the stationary phase. The monosaccharide composition of ‘‘non-attached” (left) and ‘‘attached” (right) EPS differed (Fig. 5). In the case of ‘‘nonattached” EPS, galactose and mannose were the dominant carbohydrates, with minor quantities of other sugars. Mannose was the most abundant carbohydrate until bacterial growth entered the stationary phase (47–89 h), when galactose became the dominant sugar. For ‘‘attached” EPS, galactose, glucose and mannose were the three main monosaccharides. Higher concentrations of glucose and mannose were detected than those of galactose, when the culture

was in the exponential phase (9–47 h). At the stationary phase (47–89 h), galactose was the dominant sugar. Since the monosaccharide composition was determined for the gross mixtures of EPS, the variations of monosaccharide compositions indicate that various polysaccharide strains were present in both types of EPS and their proportions changed with bacterial growth phases. Interestingly, glucose, which was found in only small amounts in ‘‘nonattached” EPS, was abundant in ‘‘attached” EPS, and in even higher concentration than both mannose and galactose before the culture entered stationary phase. In addition, the two major monosaccharides (galactose and mannose) in the ‘‘non-attached” EPS were significantly correlated with galactose and mannose, respectively, in the ‘‘attached” EPS (a = 0.05, p < 0.05). Results from HPLC-SEC showed that the MW distribution of both types of EPS during different growth phases did not change much throughout the bacterial growth phase. There was only one peak, with MW of 24.2 ± 2.0 kDa for ‘‘attached” EPS. Two peaks were found for ‘‘non-attached” EPS, with MWs of 64.5 ± 0.8 kDa and 24.7 ± 2.4 kDa. 3.2.2. PF For PF, changes of EPS components with growth phase are shown in Fig. 6 for ‘‘non-attached” (a) and ‘‘attached” (b) EPS, respectively. Contrary to SS, the URA content was confidently quantified in both types of PF EPS by the spectrophotometric method. No significant cell lysis was detected during the ‘‘attached” EPS extraction process. Statistical analysis revealed that the ‘‘nonattached” EPS components (TCHO and proteins), were significantly

Fig. 2. Size exclusion chromatograms of ‘‘attached” EPS. (a) Heating for SS EPS; (b) 3% NaCl for PF EPS; (c) 0.01 N NaOH for PF EPS; (d) heating for PF EPS.

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Fig. 3. Effects of extractant concentrations and extraction time on the EPS yield. (a) Effects of HCl concentration on SS; (b) effects of extraction time on SS; (c) Effects of NaCl concentration on PF; (d) effects of extraction time on PF.

Table 1 Chemical composition of both types of exopolymeric substances (EPS) produced by Sagittula stellata (SS) and Pseudomonas fluorescens Biovar II (PF) (in % of EPS dry weight). Sagittula stellata (n = 2)

Without purification 1st ethanol ppt 2nd ethanol ppt 3rd ethanol ppt 1st TCA ppt

Pseudomonas fluorescens Biovar II (n = 2)

TCHO

Protein

URA

TCHO

Protein

URA

14.58 ± 0.58 18.63 ± ± 0.56 20.07 ± 0.60 20.18 ± 1.01 34.23 ± 2.40

30.26 ± 1.21 28.13 ± 0.84 31.80 ± 0.95 42.19 ± 2.11 3.25 ± 0.23

0.95 ± 0.02 1.02 ± 0.03 1.45 ± 0.01 1.21 ± 0.04 2.17 ± 0.04

7.86 ± 0.79 11.80 ± 1.30 12.51 ± 1.00 12.82 ± 1.15 11.60 ± 1.28

32.21 ± 1.29 33.83 ± 1.01 10.58 ± 0.53 18.94 ± 1.33 5.2 ± 0.21

0.81 ± 0.03 1.53 ± 0.06 1.42 ± 0.07 1.14 ± 0.04 2.24 ± 0.20

Note: TCA, trichloroacetic acid; TCHO, total carbohydrates; URA, uronic acid; ppt, precipitation.

Table 2 Chemical composition of both types of exopolymeric substances (EPS) produced by Sagittula stellata (SS) and Pseudomonas fluorescens Biovar II (PF). Bacterial species

SS

EPS type components

‘‘Non-attached” n = 5

‘‘Attached” n = 5

PF ‘‘Non-attached” n = 5

‘‘Attached” n = 5

OC (%, dry weight) TCHO-C/OC (%) Protein-C/OC (%) URA-C/OC (%) URA/TCHO (%) Molecular weight (kDa) TCHO-C/protein-C Characterized OM (%)

24.2 ± 3.8 39.6 ± 3.6 7.7 ± 3.0 1.3 ± 0.3 3.3 ± 0.8 27.6 ± 0.9 5.1 (vs. 0.6)a 47.3 ± 6.6

31.7 ± 2.3 34.3 ± 3.3 5.1 ± 0.5 1.9 ± 0.1 5.5 ± 0.2 27.5 ± 3.5 6.7 (vs.0.9)a 39.4 ± 5.4

26.3 ± 4.9 29.7 ± 10.4 9.1 ± 4.3 5.9 ± 1.1 19.9 ± 7.9 20.7 ± 0.1 3.3 (vs.0.6)a 39.8 ± 14.7

20.1 ± 1.2 20.5 ± 2.3 2.3 ± 0.1 0.3 ± 0.0 1.5 ± 0.1 20.1 ± 0.0 8.9 (vs.0.6)a 22.8 ± 2.4

Note: OC, organic carbon; TCHO, total carbohydrates; URA, uronic acid; OM, organic matter (TCHO + protein). The calculation of TCHO-C assumes that total carbohydrates contained 40% of carbon, protein-C, protein contained a carbon content of 33%, URA-C, uronic acid contained a carbon content of 37.11%. a Value in the bracket is the TCHO-C to protein-C ratio of the initial ‘‘supernatant” ultrafiltrate (10 kDa–0.22 lm) that was filtered out.

and positively correlated with bacterial growth (log10(OD600), a = 0.05, p  0.05). However, this relationship was not observed for the ‘‘attached” EPS.

The TCHO-C to protein-C ratios were relatively constant in the ‘‘non-attached” EPS produced by PF, with an average value of 1.32 ± 0.21 (Fig. 6c). This observation is similar to that of the

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Fig. 4. Effects of growth phases on the SS EPS production and composition (0–9 h, lag phase; 9–47 h, log or exponential phase; 47–89 h, stationary phase; 89–108 h, death phase). (a) ‘‘Non-attached” EPS components variations with growth; (b) ‘‘attached” EPS components variations with growth; (c) TCHO-C to protein-C ratios with growth in both types of EPS; (d) specific productivity of the ‘‘non-attached” EPS with growth; (e) specific productivity of the ‘‘attached” EPS with growth.

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Fig. 5. Monosaccharide composition of the EPS by SS at different growth phases (left: ‘‘non-attached” EPS; right: ‘‘attached” EPS. Rha: rhamnose; Fuc: fucose; Ara: arabinose; Xyl: xylose; Man: mannose; Gal: galactose; Glu: glucose; GluAc: glucuronic acid).

‘‘non-attached” EPS in SS. Different from SS, these ratios for ‘‘attached” EPS in PF decreased dramatically during the bacterial exponential phase (9–24 h), from 3.07 to 0.57, and then decreased very slightly during the stationary phase (24–89 h) (Fig. 7c). The maximal specific productivity for TCHO of ‘‘non-attached” EPS occurred in the middle of the stationary phase (39 h) and the end of exponential phase (23 h) for proteins (Fig. 6d). The maximal specific productivity for both components of the ‘‘attached” EPS occurred at the end of exponential phase (23 h) (Fig. 6e). 3.3. Isoelectric focusing (IEF) electrophoresis of radiolabeled ‘‘nonattached” EPS Results for IEF showed that 234Th and 240Pu had similar activity distribution in the IEF gel rehydrated with EPS solution, with a significant peak for both radionuclides at a pH of 3 near the beginning of the gel (0–1 cm), indicating a low isoelectric point and negative charge at neutral pH for the carrier molecules (Fig. 7). Recoveries of 234 Th and 240Pu for SS EPS were 86(±5)% and 64(±6)%, respectively, and for PF EPS were 80(±5)% and 60(±8)%, respectively. 4. Discussion This work provides a ‘‘model” for selecting an extraction and purification procedure suitable for routine and quantitative operation as well as the optimal time to harvest both types of EPS from bacterial cultures. IEF study provides abundant information on the 234 Th and 240Pu interaction with bacterial ‘‘non-attached” EPS. 4.1. Optimization of EPS extraction and purification methods The different extraction methods did influence cell lysis, EPS yield and chemical modification. The optimal condition for extracting ‘‘attached” EPS from SS was 0.5 N HCl for three hours and from PF was 0.05 N NaCl for 3–5 h. These conditions gave the best EPS yield, the least cell rupture and minimal disturbance of the extracellular macromolecules. Therefore, no universally applicable method could be found. Although the use of nucleic acid content as a proxy for cell lysis has limitations (Dell’Anno and Corinaldesi, 2004; Dell’Anno and Danovaro, 2007), it was useful when comparing the relative nucleic acid content of each treatment to that of the control group in this experimental system. In addition, samples were extensively diafil-

tered to remove those contaminants of smaller size (<10 kDa) which might have absorbance at 260 nm. The ratios of absorption at 260 nm to that at 280 nm was very constant within each experimental system (coefficient of variations less than 10, data not shown), which means the contamination from protein, though not negligible, could be regarded as constant and make the use of 260 nm as an indicator of cell lysis reasonable. Moreover, the HPLC-SEC was also used as a supplementary approach to identify cell lysis or EPS disturbance, which was successful in showing differences and similarities (MWs as fingerprints), of EPS extracted by different methods. Alcohol precipitation has often been used to isolate EPS from a culture medium (Alvarado Quiroz et al., 2006; Hung et al., 2005; Staats et al., 1999; Tuinier et al., 1999; Yang et al., 1999). In this study, bacteria were grown in complex media, which would probably interfere with subsequent quantification and characterization of EPS, if the media were not effectively eliminated. In the previous studies on purification of the EPS produced by SS and PF (Alvarado Quiroz et al., 2006; Hung et al. 2005), direct alcohol precipitation was applied to the crude culture ‘‘supernatant” separated by centrifugation. It took approximately six liters of alcohol (95% ethanol and 5% methanol) to obtain purified EPS from one liter of bacterial culture. The processing time was 2–3 weeks due to the required repetitive precipitations. In addition, the behavior of broth materials during the purification process was not explored. In a preliminary study, alcohol precipitation carried out on axenic synthetic broths showed that significant amounts of precipitate formed in the case of marine broth 2216, but not for Tryticase Soy Broth. This precipitate from the broth could not be removed, even after several alcohol precipitations. Since this experiment was conducted with ‘‘blank” broth media, whether broth material and EPS would coprecipitate by ethanol precipitation was not certain but likely. In addition, both broths were mostly composed of protein, as determined by spectrophotometric methods. Therefore, the pre-filtration and diafiltration steps carried out in this work were necessary not only to distinguish EPS, especially exoprotein, from the components of the broth material, but also to concentrate the initial EPS solution (1 L) to a final volume as small as 100– 150 ml. Based on the ‘‘tracking” of the purification steps, one ethanol and one TCA precipitation was enough for SS while two ethanol and one TCA precipitation was required for PF, which finally reduced the total usage of alcohol from 6 L to less than 1 L. It is noticeable that after the alcohol precipitation and TCA precipitation, TCHO-C/protein-C ratios in both types of EPS (‘‘attached”

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Fig. 6. Effects of growth phases on the PF EPS production and composition (0–9 h, lag phase; 9–24 h, log or exponential phase; 24–86 h, stationary phase; 86–107 h, death phase). (a) ‘‘Non-attached” EPS components variations with growth; (b) ‘‘attached” EPS components variations with growth; (c) TCHO-C to protein-C ratios with growth in both types of EPS; (d) specific productivity of ‘‘non-attached” EPS with growth; (e) specific productivity of ‘‘attached” EPS with growth.

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Fig. 7. Isoelectric focusing of

234

Th(IV) and

240

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Pu labeled ‘‘non-attached” EPS produced by (a) SS (b) PF.

and ‘‘non-attached”) obtained from the two bacteria were greatly enhanced (Table 2). A systematic tracking of TCHO, protein and URA in the following purification steps helped to achieve a cost-effective purification design for large scale EPS production. The processing time for purification was shortened from 2–3 weeks to 1–2 weeks, since the repetitive precipitation was not necessary. 4.2. EPS production and composition during different growth phases TCHO, protein and URA of EPS as well as the yield varied a lot at different growth stages. The death phase is not considered here, since contaminants from the cell interior might be present at this stage and could make EPS components hard to quantify and interpret. By comparing the ratios of TCHO-C to protein-C in both types of EPS produced by both species, some common characteristics were observed: (1) the ‘‘non-attached” EPS produced by both bacterial species were significantly coupled to growth, while the ‘‘attached” EPS were either weakly linked to growth (SS) or ‘‘non-growth-associated” (PF); (2) ‘‘non-attached” EPS produced by both bacterial species were relatively enriched in polysaccharides, while ‘‘attached” EPS were relatively enriched in proteins. It was found that the capsular EPS were high in protein content, while the ‘‘slime” EPS were largely composed of polysaccharides for the marine bacterium, Pseudomonas atlantica (Decho and Lopez, 1993). It has been suggested that ‘‘non-attached” EPS were secreted in large amounts resulting from a ‘‘metabolic excess response” (Sutherland, 1977). This usually happens when one kind of nutrient is relatively in excess, usually carbon, because of the depletion of

other nutrients (N and P). The excessive carbon is ‘‘discarded” by bacteria into the formation of ‘‘non-attached” EPS, which is usually high in polysaccharides and low in proteins. Though nutrient limitation was not purposely tested in this study, it is still reasonable that it could happen at certain growth phases. This could also explain the significant correlation between the production of ‘‘nonattached” EPS and bacterial growth (log10(OD600)). In contrast, the ‘‘attached” EPS were produced in response to very different causes. Decho and Lopez (1993) used 14C radiolabeling and fluorescent dye techniques to demonstrate that high-protein capsular EPS were less digestible than the ‘‘slime” EPS, thus protecting the bacteria against the digestive enzymes (hydrolases). Although in this study, no grazers were added to the pure culture, it is still likely that under most conditions, ‘‘attached” EPS probably represent an adaptation to protect cells from harsh microenvironments besides resisting enzymatic digestion (Decho and Lopez, 1993; Sutherland, 1977). Another explanation for the higher protein content in ‘‘attached” EPS is that high-protein content could enhance the hydrophobic features of the bacterial surface, thus facilitating cell aggregation and transparent exopolymeric particle (TEP) formation (Leppard, 1995, 1997), which is assumed as a life strategy for microorganisms (Giroldo et al., 2003). Therefore, it is not surprising that EPS (‘‘non-attached” and ‘‘attached”) were produced in different ‘‘patterns” (‘‘growth-associated” versus ‘‘non-growth-associated”) and different proportions of components (polysaccharides versus proteins). The synthesis of bacterial EPS involves a large amount of enzymes, which might also be needed for the formation of cell wall polymers, and glycosyl carrier lipid, which might be identical to that involved in sysnthesis of cell wall polymer (Cerning, 1995;

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Looijesteijn et al., 1999). If cells are growing more slowly, the cell wall polymer formation rate would also be reduced, thus providing more enzymes and lipid carriers available for EPS synthesis. This could explain why the highest specific EPS production occurred during the stationary (SS) or late exponential growth phase (PF). 4.3. Implication of IEF results In general, the colloidal macromolecular actinide carriers have two functions: (a) strongly binding of metals, including actinide ions, to the chelating functional groups, and (b) possible attachment of the carrier itself to mineral surfaces. Potential carriers with a pI of 7–8, e.g., iron oxy hydroxides and proteins, would likely strongly sorb to mineral surfaces due to their electroneutrality in the neutral pH range, and thus, would not be mobile, and therefore, of a lesser concern. The IEF results suggest that both actinides likely have a similar complexation behavior to specific clustered functional groups of the EPS. The actinide carrier molecules would have a negative surface charge at neutral pH, and thus, would be highly mobile in the natural environment, as most of the 234Th and 240Pu tracers were found at a low pI of about 3, similar to colloidal organic matter (COM) collected from the surface soil of Rocky Flats Environmental Technology Site (RFETS; Santschi et al., 2002; Santschi and Roberts, 2002; Xu et al., 2008), one of actinide contaminated Department of Energy (DOE) sites, and COM collected from Gulf of Mexico (GOM; Alvarado Quiroz et al., 2006; Guo et al., 2002; Hung et al., 2004; Quigley et al., 2002). The presence of hydrophobic ‘‘moieties” (e.g., lipids, proteins, etc.) in the actinide carriers gives the macromolecules amphiphilic properties, which might be responsible for regulation of particle attachment (Ahimou et al., 2001; Reinhardt, 2004; Wilkinson and Reinhardt, 2004), thus rendering them immobile. Since bacteria are ubiquitous in aquatic and terrestrial environments, using EPS to bioremediate sites with radionuclide contamination has been proposed as one of the most sustainable and economic ways. Therefore, we suggest that these bacterial EPS can play an important role in the mobilization/immobilization of the actinides. Acknowledgements We thank two anonymous reviewers and Editor-in-Chief, Dr. Steve C. Ricke for constructive comments, which improved the manuscript. This work was, in part, supported by the National Science Foundation Chemical Oceanography Program (OCE-0351559), Department of Energy – Office of Science, BER (DE-FG0204ER63899), Welch Foundation (Grant BD-0046) and the Texas Institute of Oceanography. References Ahimou, F., Paquot, M., Jacques, P., Thonart, P., Rouxhet, P.G., 2001. Influence of electrical properties on the evaluation of the surface hydrophobicity of Bacillus subtilis. Journal of Microbiological Methods 45, 119–126. Alvarado Quiroz, N.A., Hung, C.-C., Santschi, P.H., 2006. Binding of thorium(IV) to carboxylate, phosphate and sulfate functional groups from marine exopolymeric substances (EPS). Marine Chemistry 100, 337–353. Black, A.S., Campbell, A.S., 1982. Ionic strength of soil solution and its effect on charge properties of some New Zealand soils. European Journal of Soil Science 33, 249–262. Bhaskar, P.V., Grossart, H.-P., Bhosle, N.B., Simon, M., 2005. Production of macroaggregates from dissolved exopolymeric substances (EPS) of bacterial and diatom origin. FEMS Microbiology 53 (2), 255–264. Cerning, J., 1995. Production of exocellular polysaccharides by lactic acid bacteria and dairy propionibacteria. Lait 75, 463–472. Chin, W.-C., Orellana, M.V., Verdugo, P., 1998. Spontaneous assembly of marine dissolved organic matter into polymer gels. Nature 391, 568–572. Comte, S., Guibaud, G., Baudu, M., 2006. Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and EPS complexation properties Part I. Comparison of the efficiency of eight EPS extraction methods. Enzyme and Microbial Technology 38, 237–245.

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