World J Microbiol Biotechnol DOI 10.1007/s11274-014-1627-y
ORIGINAL PAPER
Physicochemical properties and membrane biofouling of extra-cellular polysaccharide produced by a Micrococcus luteus strain Lei Feng • Xiufen Li • Ping Song • Guocheng Du Jian Chen
•
Received: 15 May 2013 / Accepted: 21 February 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The physicochemical properties of the extra-cellular polysaccharide (EPS) produced by a Micrococcus luteus strain, a dominating strain isolated from membrane biofouling layer, were determined in this study. The EPS isolated from this strain was measured to have an average molecular weight of 63,540 Da and some typical polysaccharide absorption peaks in Fourier transform infrared spectrum. Monosaccharide components of the EPS contained rhamnose, fucose, arabinose, xylose, mannose, galactose and glucose in a molar ratio of 0.2074:0.0454:0.0262:0.0446:1.7942:1.2086:0.4578. Pseudo plastic properties were also observed for the EPS through the rheological measurement. The EPS was further characterized for its behavior to cause membrane flux decline. The results showed that both flux declines for polyvinylidenefluoride (PVDF) and polypropylene membranes became more severe as EPS feed concentration increased. A higher irreversible fouling for the PVDF membrane suggested that the EPS had the larger fouling potential to this microfiltration membrane. L. Feng (&) College of Resoure and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, People’s Republic of China e-mail: [email protected] X. Li Lab of Environmental Biotechnology, School of Environment and Civil Engineering, Jiangnan University, Wuxi 214122, Jiangsu Province, People’s Republic of China P. Song College of Forestry, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, People’s Republic of China G. Du J. Chen School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu Province, People’s Republic of China
Keywords Micrococcus luteus Extra-cellular polysaccharide Biofouling Microfiltration Irreversible fouling
Introduction In the last decade, membrane bioreactor (MBR) systems have been considered to be an efficient method to wastewater treatment due to their potential advantages, such as superior nutrient and organic removals, low energy consumption, high loading rate capabilities, small land requirements and low/zero excess sludge production. However, one of the critical issues in the successful application of membrane systems for water treatment is biofouling, which causes severe performance loss, requiring costly periodic cleaning or membrane replacement and so increasing operating costs and/or decreasing plant output (Lodge et al. 2004; Kim et al. 2006). Membrane biofouling originates from the biological components [i.e. activated floc(s) (residues) and extracellular polymeric substances]. Compared with other biological components, extra-cellular polysaccharide (EPS), which has higher molecular weight distributions, viscous characteristics and relatively larger amount in the extra-cellular polymeric substances, plays a critical role in biofouling formation (Nagaoka et al. 1996; Le-Clech et al. 2005; Chen et al. 2006). Some previous studies have even identified EPS as one of the most significant biological factors responsible for the biofouling (Danese et al. 2000; Frank 2001). For a better understanding of the EPS fouling, some model EPS have been used to investigate the effect of EPS fouling on the membrane filtration. For example, dynamic filtration experiments of sodium alginate were conducted to determine fouling mechanisms using microfiltration and ultrafiltration
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membranes (Ye et al. 2005a, b, 2006; Katsoufidou et al. 2007). Xanthan, actigum and glucan were chosen as model polysaccharides to identify actual fouling mechanisms through the cross flow filtration mode (Nataraj et al. 2008). Aqueous solutions containing dextran and cellulose were used to test the influence of operating parameters on the ultrafiltration (Garcı´a-Molina et al. 2006a, b). From these studies, some useful references to EPS fouling mechanism were achieved, even though these model EPS can not completely represent real EPS, especially due to several of them being secreted by algae or plants. As a long chain macromolecular polymer, the functional characteristics of microbial EPS are closely related to its physicochemical properties such as sugar composition, glycosidic bond configuration and rheology. Recently, the EPS from several microbes in wastewater treatment systems have been characterized. Allen et al. (2004) reported on the purification and glycosyl composition of the exopolysaccharide isolated from the flocforming wastewater bacterium Thauera sp. MZ1T. The capsular EPS of Pseudomonas putida G7, an aromatic hydrocarbon-degrading bacterium, was also purified and its preliminary structural and chemical properties were further described (Kachlany et al. 2001). However, until now, little is known about the nature of the EPS secreted by microorganisms of membrane biofouling layer. In this work, physicochemical properties of the EPS produced by Micrococcus luteus, a dominant strain extracted from membrane biofouling layer, was firstly investigated. To understand the fouling characteristics of the EPS, filtration flux and fouling resistance in the process of microfiltration (MF) were further examined. The results of this study will provide a basis for further study of the correlation between EPS physicochemical properties and membrane biofouling.
pellet the cells. The supernatant was precipitated by the addition of ethanol to a final concentration of 75 % (v/v), and the precipitates were collected by centrifugation, washed with acetone, dissolved in deionized water, dialyzed and finally lyophilized to yield the crude preparation. This crude polysaccharide was further purified by liquid chromatography (Shanghai Hu Xi Instruments Corp, China) using 1 m 9 2.5 cm column packed with Bio-Gel P 30 and 100 resin (Bio-Rad Laboratories). A detailed procedure was previously described (Allen et al. 2004). Molecular weight EPS molecular weight was determined by size-exclusion gel permeation chromatography in a Waters 600 system equipped with an ultra hydrogel linear column (300 mm 9 7.8 mm ID). The mobile phase (0.9 mL/min) was made of 0.1 M NaNO3. Six dextran standards (Sigma Aldrich) of molecular weight 180, 4,600, 21,400, 41,100, 133,800 and 2,000,000 Da were injected for column calibration, all within the linear range of retention times. The detection was carried out at 25 °C with a double array UV detector (Waters 2487 Series) at 280 nm and, simultaneously, with a refractive index detector (Waters 2410 Series). FT-IR spectral analysis The FT-IR spectra were measured using a Nicolet Nexus 470 FT-IR spectrometer (Thermo Electron Corporation). The obtained EPS sample was pressed into KBr pellets at 1:100 of sample:KBr. The spectra were recorded in transmittance mode over the wave range of 4,000–400 1/cm.
Materials and methods
Monose compositions analysis
Model microorganism
The EPS sample (10 mg) obtained in this work was dissolved in 2 mL of 2 M trifluoroacetic acid (TFA) and hydrolyzed at 120 °C for 2 h. After reduction with 20 mg of sodium borohydride, monosaccharide alditol acetate was prepared using a method previously described (Blakeney et al. 1983). The alditol acetates were analyzed by a gas chromatograph (Shimadzu GC-14A, Japan) with an OV1701 flexible quartz capillary column (30 m 9 0.32 mm) and a flame-ionization detector.
The M. luteus strain used in this work was one of the dominant strains isolated from a MF membrane biofouling layer of a pilot submerged MBR (Li et al. 2008). The strain was cultivated in shake flasks at 25 °C with shaking at 200 rpm. A defined liquid medium (DLM), which had the same composition as the synthetic wastewater in MBR, was used during its cultivation. The MBR parameters and DLM compositions had been described in details in our previous study (Feng et al. 2009, 2011). Isolation and purification of EPS The cultures of M. luteus at the late logarithmic growth phase (48 h old) were centrifuged at 8,0009g for 15 min to
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Measurement of particle size distribution (PSD) Particle size of the EPS was measured using dynamic light scattering (DLS, high performance particle sizer, model HPP5001, Malvern Instruments) equipped with a helium– neon laser at a wavelength of 633 nm. The correlograms of
World J Microbiol Biotechnol
PSD analysis were evaluated using Malvern DTS v3.00 software. Rheological measurement Rheological measurement was carried out with a Brookfield RVDV-3 rheometer fitted with a small sample adapter (Brookfield Engineering Laboratories Inc., Stoughton, MA, USA). The relationship between the shearing rate and the apparent viscosity was characterized by assuming the simple power law model (Moreno et al. 2000):
filtration resistance, Rm is the primary membrane resistance, Rir is the irreversible fouling resistance, which can not be removed by the backwashing step, and Rr is the reversible fouling resistance, which can be removed by the backwashing step. For a fouled membrane, Rm and Rt could be calculated from the pure water flux before and after the filtration experiment. Rir was evaluated by the pure water flux after the backwashing step. Rr could be obtained from the equation: Rr = Rt - Rir - Rm.
Results g ¼ kr n1
ð1Þ Molecular weight of EPS
where g is the viscosity and r is the shearing rate. The constants, k and n represent the consistency index and the flow behavior index, respectively, which can be determined by nonlinear fitting program of SPSS 10.0 software. Filtration set-up of EPS A plexiglas dead-end filtration module, which has a valid volume of 500 mL and 60 mm in diameter, was used as the filtration cell. A feed vessel (500 mL), which contained the EPS solutions, was placed above the cell and continuously stirred. Pressurized N2 was used as the filtration or backwashing pressure controlled by a set of pressure control valves. Two types of membranes, 0.22 lm hydrophilic polyvinylidenefluoride (PVDF) and 0.22 lm hydrophobic polypropylene (PP) membranes, purchased from Shanghai Jingxi Chemistry Engineering Co. Ltd, were adopted in this study. Both MF membranes were commonly used during the wastewater treatment in China. New membranes were required for each new experiment. Filtration analysis
Chromatographic profile of the molecular size distribution of the EPS is illustrated in Fig. 1. A single and symmetrical peak from the biopolymer was presented, which indicated that the EPS was a homogeneous polysaccharide. The average molecular weight was measured to be 63,540 Da. FT-IR spectral analysis of EPS Fourier-transform infrared (FT-IR) transmittance spectrum of the EPS is shown in Fig. 2. Some typical absorption peaks of the polysaccharide occurred. Five bands appearing in 3,409.14, 2,932.47, 1,725.84, 1,554.37 cm-1 and 1,379.63 cm-1 should correspond to the stretching vibrations of hydroxyl, C–H, carboxyl, C=C and C–H bending of aliphatic CH2, respectively (Lee et al. 2004, 2006). Infrared bands at 1,643.05 cm-1 were probably associated with the interference of water peak brought by the KBr compression process. A stretching peak at 1,116.08 cm-1 was observed, indicating the presence of C–O bonds. In addition, an absorption peak at 600 cm-1 was also found, which was possibly attributed to the presence of O–H outof-plane vibration (Chiovitti et al. 1997).
Prior to each filtration experiments, the clean water flux, J0, of virgin membrane module was determined by filtering deionized water. A series of EPS filtrations (30 min for each) were performed at 0.1 MPa pressure for various feed concentrations. After each filtration, a backwash was carried out for 3 min at 0.1 MPa pressure and then the pure water flux was detected. In this study, the permeate flux can be described by combining the permeate data and Darcy’s law as follows. J¼
DP DP ¼ lRt lðRm þ Rir þ Rr Þ
ð2Þ
where J is the filtrate flux, DP is the trans-membrane pressure drop, l is the feed viscosity, Rt is the total
Fig. 1 Molecular weight of the EPS produced by M. luteus
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40
60
35
1643.05
35 30
20 4000
3409.14
25
3500
600.31
40
30mg/L 50mg/L 100mg/L
30 25 20 15 10
1116.08
45
1554.37 1379.63
%T
50
1725.84
2932.47
55
Intendity (%)
45
65
802.26
70
5 0 1
3000
2500
2000
1500
1000
10
500
Wavenumbers (cm-1)
Fig. 2 FT-IR spectrum of the EPS produced by M. luteus
100
1000
10000
Diameter (nm)
Fig. 4 Particle size distribution characterization of the EPS produced by M. luteus at various concentrations
Monose components of EPS Monosaccharide components of the EPS was determined after acid hydrolysis. The peak in Fig. 3 revealed that the EPS purified was a complex polysaccharide, which was composed of at least seven monoses, rhamnose, fucose, arabinose, xylose, mannose, glucose and galactose in a molar ratio of 0.2074:0.0454:0.0262:0.0446:1.7942:1.2086:0.4578. It was evident that mannose and glucose were the major monoses constructing backbones of the EPS. Particle size distribution of EPS DLS measurements of the various EPS samples are presented in Fig. 4. We could find that the particle size
distributions in the EPS solutions were similar and yielded the hydrodynamic averaged sizes of 162–182 nm range. Rheological studies of EPS Flow curves of aqueous solutions of the EPS with increasing shearing rates are shown in Fig. 5. The rheograms, within the available shearing-rate range, demonstrated that the viscosity of the biopolymer went up with the increasing of feed EPS concentration and went down with the increasing of shearing rate. It could be assumed that the aqueous dispersions of the EPS exhibited nonNewtonian and shearing-thinning behavior with pseudo plastic properties.
Fig. 3 Monose components of the EPS produced by M. luteus
Mannose
Glucose
Galactose
Fucose Arabinose Rhamnose
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Xylose
Myo-inositol (internal standard)
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In Eq. (1), as n tends to 1, the shearing-thinning properties are so less and less, pronouncing that Newtonian behavior is achieved when n is equal to 1 (Moreno et al. 2000). In this study, n values for 30, 50 and 100 mg/L EPS solutions were 0.74, 0.67 and 0.56, respectively. This suggested that pseudo plastic rheological properties could be enhanced with increasing EPS concentration.
filtration processes, the Rir always played a dominant role with the highest percentage (54.62–58.79 %) of the total fouling resistances Rt, followed by the Rr, and then the Rm was the lowest with the percentages of 2.67–4.45 %. Furthermore, at the same feed concentration, the contribution of Rir towards the Rt for the PVDF membrane was relatively higher than that for the PP membrane, although it exhibited a lower Rt value.
Flux behavior of EPS The normalized fluxes of PVDF and PP membranes are shown in Fig. 6 as a function of time for various EPS concentrations. The results showed that all the permeation curves of two MF membranes exhibited an initial sharp drop in the permeate flux at the start of the filtration. Then, after the initial steep decline, the flux declined gradually and eventually attained steady state. It also could be observed from the curve shapes in Fig. 6 that the extent of the flux decline increased with an increase in the bulk polysaccharide concentration. In addition, with respect to the same EPS solution, the flux for the PP membrane showed more significant decline than that for the PVDF membrane.
Discussion Microbial polysaccharide is a major fouling component in MBR systems (Ramesh et al. 2006). Numerous fouling mechanism studies have been performed using various EPS models to describe fouling in MBR (Susanto and Ulbricht 2005; Nataraj et al. 2008). However, there is a general lack of knowledge about the relation between membrane fouling and polysaccharide characteristics. In addition, M. luteus is a common Gram-positive microorganism and has
A
100
30mg/L 50mg/L 100mg/L
The values of Rir, Rm, Rr and Rt and their relative percentages under different EPS feed concentrations and membrane materials are listed in Table 1. It can be observed that various resistance (Rir, Rr and Rt) values for the both membranes increased with EPS feed concentration. Under the same conditions, the Rt of the PP membrane was much larger than that of the PVDF membrane, indicating a relatively higher permeation hinder, which are in line with the above flux analysis. In addition, among all the
Normalized flux (%)
Resistance analysis 80
60
40
20
0 0
300
600
900
1200
1500
1800
Time (s) 14
Viscosity (Pas)
10
Normalized flux (%)
12
-0.4441
y = 14.467x 2 R = 0.9555
8 6 4 2
y = 5.2636x 2 R = 0.9294
B
100
100 mg/L 50 mg/L 30 mg/L
-0.3289
y = 9.2667x 2 R = 0.9512
30mg/L 50mg/L 100mg/L
80
60
40
20
-0.2582
0
0
300
600
900
1200
1500
1800
Shear rate (s-1)
Fig. 5 Effect of shearing rates on dispersion viscosity of the EPS produced by M. luteus at various concentrations
0
300
600
900
1200
1500
1800
Time (s)
Fig. 6 Flux decline profiles for PVDF (a) and PP (b) membranes of the EPS produced by M. luteus at various concentrations
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World J Microbiol Biotechnol Table 1 Various fouling resistances and relative percentages as a function of EPS concentrations for the PVDF and PP membranes EPS concentrations (mg/L)
Resistances
PVDF
PP
30
Rm (91011 m-1)
1.4594
1.0540
50
100
Percentage
19.42
13.30
Rir (91011 m-1)
3.2872
3.4474
Percentage
43.74
43.50
Rr (91011 m-1)
2.7686
3.4237
Percentage
36.84
43.20
Rt (91011 m-1) Percentage
7.5152 100
7.9252 100
Rm (91011 m-1)
1.4483
1.0629
Percentage
17.62
10.24
Rir (91011 m-1)
4.1664
5.0621
Percentage
50.68
48.79
Rr (91011 m-1)
2.6057
4.2503
Percentage
31.70
40.97
Rt (91011 m-1)
8.2204
10.3753
Percentage
100
100
Rm (91011 m-1)
1.4673
1.0599
Percentage
15.45
9.45
Rir (91011 m-1)
5.4290
5.7389
Percentage
57.16
51.15
Rr (91011 m-1)
2.6008
4.4203
Percentage Rt (91011 m-1)
27.39 9.4972
39.40 11.2191
Percentage
100
100
membrane, which also implied that this biopolymer had the higher fouling potential to PVDF membrane. This is consistent with the analysis results of FT-IR spectra and monose components, which proved that the EPS consisted of various monosaccharides with hydrophilic groups (such as hydroxyl, carboxyl and ether bond etc.) (Allen et al. 2004; Weiner and Langille 1995), which probably made more EPS molecules adhere on the hydrophilic PVDF membrane and resulted in a higher Rir. In conclusion, some physicochemical properties of the EPS had been obtained in the present works. However, as a complicated biological macromolecule, EPS still need further studies on other important characteristics, such as the connection pattern of monosaccharide, the dimensional structure of carbohydrate chain, the binding site of functional groups and so on, which may be more closely correlated with its fouling. Thus, future study needs to be conducted to explore these characteristics for revealing the internal mechanism of EPS membrane fouling. Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 30901131, 31000264), the Excellent Youth Cultivation Program of Fujian Province (Nos. JA10088), the New Century Excellent Talents of Fujian Province (No. K8011025, K8012015A), Key Project of Chinese Ministry of Education (No. 212089) and the Excellent Youth Program of Fujian Agriculture and Forestry University (No. xjq201210).
References been reported in various fields (Perromat et al. 2003; Mauclaire and Egli 2010). To the best of our knowledge, however, there have been no works performed to reveal the fouling properties of this strain. In this study, the pores of MF membranes had a uniform diameter that was larger than EPS particle size (Fig. 4) and thus, many EPS biopolymers might deposit in the membrane pores or plugged the pore entrances resulting in a significant flux decline. The EPS concentrations caused different fouling impacts on the MF membranes, the permeate volumes decreasing with increasing EPS concentrations. Enhanced solution viscosity due to the rise in EPS concentration might have a great contribution to the decrease, which was confirmed by the rheology analysis. Comparing with the PVDF membrane, the PP membrane showed the larger flux decline and Rt value, but the relatively smaller percentage of Rir. This implied that the serious fouling for PP membrane can be attributed to its smaller Rm value, which caused the larger initial flux and could transfer more EPS biopolymer to the PP membrane surface during the filtration time. Furthermore, the higher proportion of Rir for the PVDF membrane indicated that the EPS preferred to absorb to this membrane surface rather than the PP
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Allen MS, Welch KT, Prebyl BS, Baker DC, Meyers AJ, Sayler GS (2004) Analysis and glycosyl composition of the exopolysaccharide isolated from the floc-forming wastewater bacterium Thauera sp. MZ1T. Environ Microbiol 6:780–790 Blakeney AB, Harris PJ, Henry RJ, Stone BA (1983) A simple and rapid preparation of alditol acetates for monosaccharide analysis. Carbohydr Res 113:291–299 Chen MY, Lee DJ, Tay JH (2006) Extracellular polymeric substances in fouling layer. Sep Sci Technol 41:1467–1474 Chiovitti A, Bacic A, Craik DJ, Munro SLA, Kraft GT, Liao ML (1997) Cell-wall polysaccharides from Australian red algae of the family Solieriaceae (Gigartinales, Rhodophyta): novel, highly pyruvated carrageenans from the genus Callophycus. Carbohydr Res 299:229–243 Danese PN, Pratt LA, Kolter R (2000) Exopolysaccharide production is required for development of Escherichia coli K-12 biofilm architecture. J Bacteriol 182:3593–3596 Feng L, Li XF, Chen J (2009) Fouling behavior of extra-cellular polysaccharide produced by Micrococcus luteus during dead-end microfiltration. IEEE, ICEET’09, vol 3, pp 353–356 Feng L, Li XF, Song P, Du GC, Chen J (2011) Surface interactions and fouling properties of micrococcus luteus with microfiltration membranes. Appl Biochem Biotechnol 165:1235–1244 Frank JF (2001) Microbial attachment to food and food contact surfaces. Adv Food Nutr Res 43:319–370 Garcı´a-Molina V, Esplugas S, Wintgens T, Melin T (2006a) Ultrafiltration of aqueous solutions containing dextran. Desalination 188:217–227
World J Microbiol Biotechnol Garcı´a-Molina V, Lyko S, Esplugas S, Wintgens T, Melin T (2006b) Ultrafiltration of aqueous solutions containing organic polymers. Desalination 189:110–118 Kachlany SC, Levery SB, Kim JS, Reuhs BL, Lion LW, Ghiorse WC (2001) Structure and carbohydrate analysis of the exopolysaccharide capsule of Pseudomonas putida G7. Environ Microbiol 3:774–784 Katsoufidou K, Yiantsios SG, Karabelas AJ (2007) Experimental study of ultrafiltration membrane fouling by sodium alginate and flux recovery by backwashing. J Membr Sci 300:137–146 Kim AS, Chen H, Yuan R (2006) EPS biofouling in membrane filtration: an analytic modeling study. J Coll Interf Sci 303:243–249 Le-Clech P, Jefferson B, Judd SJ (2005) A comparison of submerged and sidestream tubular membrane bioreactor configurations. Desalination 173:113–122 Lee NH, Amy G, Croue JP, Buisson H (2004) Identification and understanding of fouling in low-pressure membrane (MF/UF) filtration by natural organic matter (NOM). Water Res 38:4511–4523 Lee NH, Amy G, Croue´ JP (2006) Low-pressure membrane (MF/UF) fouling associated with allochthonous versus autochthonous natural organic matter. Water Res 40:2357–2368 Li XF, Li YJ, Liu H, Hua ZZ, Du GC, Chen J (2008) Correlation between extracellular polymeric substances and aerobic biogranulation in membrane bioreactor. Sep Purif Technol 59:26–33 Lodge B, Judd SJ, Smith AJ (2004) Characterisation of dead-end ultrafiltration of biotreated domestic wastewater. J Membr Sci 231:91–98 Mauclaire L, Egli M (2010) Effect of simulated microgravity on growth and production of exopolymeric substances of Micrococcus luteus space and earth isolates. FEMS Immunol Med Microbiol 59:350–356
Moreno J, Vargas MA, Madiedo JM, Mun˜oz J, Rivas J, Guerrero MG (2000) Chemical and rheological properties of an extracellular polysaccharide produced by the cyanobacterium Anabaena sp. ATCC 33047. Biotechnol Bioeng 67:283–290 Nagaoka H, Ueda S, Miya A (1996) Influence of bacterial extracellular polymers on the membrane separation activated sludge process. Water Sci Technol 34:165–172 Nataraj S, Schoma¨cker R, Kraume M, Mishra IM, Drews A (2008) Analyses of polysaccharide fouling mechanisms during cross flow membrane filtration. J Membr Sci 308:152–161 Perromat A, Melin AM, Lorin C, Deleris G (2003) Fourier transform IR spectroscopic appraisal of radiation damage in Micrococcus luteus. Biopolymers 72:207–216 Ramesh A, Lee DJ, Wang ML, Hsu JP, Juang RS, Hwang KJ, Liu JC, Tseng SJ (2006) Biofouling in membrane bioreactor. Sep Purif Technol 41:1345–1370 Susanto H, Ulbricht M (2005) Influence of ultrafiltration membrane characteristics on adsorptive fouling with dextrans. J Membr Sci 266:132–142 Weiner R, Langille E (1995) Structure, function and immunochemistry of bacterial exopolysaccharides. J Ind Microbiol Biotechnol 15:339–346 Ye Y, Clech PL, Chen V, Fane AG (2005a) Evolution of fouling during crossflow filtration of model EPS solutions. J Membr Sci 264:190–199 Ye Y, Le Clech P, Chen V, Fane AG, Jefferson B (2005b) Fouling mechanisms of alginate solutions as model extracellular polymeric substances. Desalination 175:7–20 Ye Y, Chen V, Fane AG (2006) Modeling long-term subcritical filtration of model EPS solutions. Desalination 191:318–327
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ORIGINAL PAPER
Physicochemical properties and membrane biofouling of extra-cellular polysaccharide produced by a Micrococcus luteus strain Lei Feng • Xiufen Li • Ping Song • Guocheng Du Jian Chen
•
Received: 15 May 2013 / Accepted: 21 February 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The physicochemical properties of the extra-cellular polysaccharide (EPS) produced by a Micrococcus luteus strain, a dominating strain isolated from membrane biofouling layer, were determined in this study. The EPS isolated from this strain was measured to have an average molecular weight of 63,540 Da and some typical polysaccharide absorption peaks in Fourier transform infrared spectrum. Monosaccharide components of the EPS contained rhamnose, fucose, arabinose, xylose, mannose, galactose and glucose in a molar ratio of 0.2074:0.0454:0.0262:0.0446:1.7942:1.2086:0.4578. Pseudo plastic properties were also observed for the EPS through the rheological measurement. The EPS was further characterized for its behavior to cause membrane flux decline. The results showed that both flux declines for polyvinylidenefluoride (PVDF) and polypropylene membranes became more severe as EPS feed concentration increased. A higher irreversible fouling for the PVDF membrane suggested that the EPS had the larger fouling potential to this microfiltration membrane. L. Feng (&) College of Resoure and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, People’s Republic of China e-mail: [email protected] X. Li Lab of Environmental Biotechnology, School of Environment and Civil Engineering, Jiangnan University, Wuxi 214122, Jiangsu Province, People’s Republic of China P. Song College of Forestry, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, People’s Republic of China G. Du J. Chen School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu Province, People’s Republic of China
Keywords Micrococcus luteus Extra-cellular polysaccharide Biofouling Microfiltration Irreversible fouling
Introduction In the last decade, membrane bioreactor (MBR) systems have been considered to be an efficient method to wastewater treatment due to their potential advantages, such as superior nutrient and organic removals, low energy consumption, high loading rate capabilities, small land requirements and low/zero excess sludge production. However, one of the critical issues in the successful application of membrane systems for water treatment is biofouling, which causes severe performance loss, requiring costly periodic cleaning or membrane replacement and so increasing operating costs and/or decreasing plant output (Lodge et al. 2004; Kim et al. 2006). Membrane biofouling originates from the biological components [i.e. activated floc(s) (residues) and extracellular polymeric substances]. Compared with other biological components, extra-cellular polysaccharide (EPS), which has higher molecular weight distributions, viscous characteristics and relatively larger amount in the extra-cellular polymeric substances, plays a critical role in biofouling formation (Nagaoka et al. 1996; Le-Clech et al. 2005; Chen et al. 2006). Some previous studies have even identified EPS as one of the most significant biological factors responsible for the biofouling (Danese et al. 2000; Frank 2001). For a better understanding of the EPS fouling, some model EPS have been used to investigate the effect of EPS fouling on the membrane filtration. For example, dynamic filtration experiments of sodium alginate were conducted to determine fouling mechanisms using microfiltration and ultrafiltration
123
World J Microbiol Biotechnol
membranes (Ye et al. 2005a, b, 2006; Katsoufidou et al. 2007). Xanthan, actigum and glucan were chosen as model polysaccharides to identify actual fouling mechanisms through the cross flow filtration mode (Nataraj et al. 2008). Aqueous solutions containing dextran and cellulose were used to test the influence of operating parameters on the ultrafiltration (Garcı´a-Molina et al. 2006a, b). From these studies, some useful references to EPS fouling mechanism were achieved, even though these model EPS can not completely represent real EPS, especially due to several of them being secreted by algae or plants. As a long chain macromolecular polymer, the functional characteristics of microbial EPS are closely related to its physicochemical properties such as sugar composition, glycosidic bond configuration and rheology. Recently, the EPS from several microbes in wastewater treatment systems have been characterized. Allen et al. (2004) reported on the purification and glycosyl composition of the exopolysaccharide isolated from the flocforming wastewater bacterium Thauera sp. MZ1T. The capsular EPS of Pseudomonas putida G7, an aromatic hydrocarbon-degrading bacterium, was also purified and its preliminary structural and chemical properties were further described (Kachlany et al. 2001). However, until now, little is known about the nature of the EPS secreted by microorganisms of membrane biofouling layer. In this work, physicochemical properties of the EPS produced by Micrococcus luteus, a dominant strain extracted from membrane biofouling layer, was firstly investigated. To understand the fouling characteristics of the EPS, filtration flux and fouling resistance in the process of microfiltration (MF) were further examined. The results of this study will provide a basis for further study of the correlation between EPS physicochemical properties and membrane biofouling.
pellet the cells. The supernatant was precipitated by the addition of ethanol to a final concentration of 75 % (v/v), and the precipitates were collected by centrifugation, washed with acetone, dissolved in deionized water, dialyzed and finally lyophilized to yield the crude preparation. This crude polysaccharide was further purified by liquid chromatography (Shanghai Hu Xi Instruments Corp, China) using 1 m 9 2.5 cm column packed with Bio-Gel P 30 and 100 resin (Bio-Rad Laboratories). A detailed procedure was previously described (Allen et al. 2004). Molecular weight EPS molecular weight was determined by size-exclusion gel permeation chromatography in a Waters 600 system equipped with an ultra hydrogel linear column (300 mm 9 7.8 mm ID). The mobile phase (0.9 mL/min) was made of 0.1 M NaNO3. Six dextran standards (Sigma Aldrich) of molecular weight 180, 4,600, 21,400, 41,100, 133,800 and 2,000,000 Da were injected for column calibration, all within the linear range of retention times. The detection was carried out at 25 °C with a double array UV detector (Waters 2487 Series) at 280 nm and, simultaneously, with a refractive index detector (Waters 2410 Series). FT-IR spectral analysis The FT-IR spectra were measured using a Nicolet Nexus 470 FT-IR spectrometer (Thermo Electron Corporation). The obtained EPS sample was pressed into KBr pellets at 1:100 of sample:KBr. The spectra were recorded in transmittance mode over the wave range of 4,000–400 1/cm.
Materials and methods
Monose compositions analysis
Model microorganism
The EPS sample (10 mg) obtained in this work was dissolved in 2 mL of 2 M trifluoroacetic acid (TFA) and hydrolyzed at 120 °C for 2 h. After reduction with 20 mg of sodium borohydride, monosaccharide alditol acetate was prepared using a method previously described (Blakeney et al. 1983). The alditol acetates were analyzed by a gas chromatograph (Shimadzu GC-14A, Japan) with an OV1701 flexible quartz capillary column (30 m 9 0.32 mm) and a flame-ionization detector.
The M. luteus strain used in this work was one of the dominant strains isolated from a MF membrane biofouling layer of a pilot submerged MBR (Li et al. 2008). The strain was cultivated in shake flasks at 25 °C with shaking at 200 rpm. A defined liquid medium (DLM), which had the same composition as the synthetic wastewater in MBR, was used during its cultivation. The MBR parameters and DLM compositions had been described in details in our previous study (Feng et al. 2009, 2011). Isolation and purification of EPS The cultures of M. luteus at the late logarithmic growth phase (48 h old) were centrifuged at 8,0009g for 15 min to
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Measurement of particle size distribution (PSD) Particle size of the EPS was measured using dynamic light scattering (DLS, high performance particle sizer, model HPP5001, Malvern Instruments) equipped with a helium– neon laser at a wavelength of 633 nm. The correlograms of
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PSD analysis were evaluated using Malvern DTS v3.00 software. Rheological measurement Rheological measurement was carried out with a Brookfield RVDV-3 rheometer fitted with a small sample adapter (Brookfield Engineering Laboratories Inc., Stoughton, MA, USA). The relationship between the shearing rate and the apparent viscosity was characterized by assuming the simple power law model (Moreno et al. 2000):
filtration resistance, Rm is the primary membrane resistance, Rir is the irreversible fouling resistance, which can not be removed by the backwashing step, and Rr is the reversible fouling resistance, which can be removed by the backwashing step. For a fouled membrane, Rm and Rt could be calculated from the pure water flux before and after the filtration experiment. Rir was evaluated by the pure water flux after the backwashing step. Rr could be obtained from the equation: Rr = Rt - Rir - Rm.
Results g ¼ kr n1
ð1Þ Molecular weight of EPS
where g is the viscosity and r is the shearing rate. The constants, k and n represent the consistency index and the flow behavior index, respectively, which can be determined by nonlinear fitting program of SPSS 10.0 software. Filtration set-up of EPS A plexiglas dead-end filtration module, which has a valid volume of 500 mL and 60 mm in diameter, was used as the filtration cell. A feed vessel (500 mL), which contained the EPS solutions, was placed above the cell and continuously stirred. Pressurized N2 was used as the filtration or backwashing pressure controlled by a set of pressure control valves. Two types of membranes, 0.22 lm hydrophilic polyvinylidenefluoride (PVDF) and 0.22 lm hydrophobic polypropylene (PP) membranes, purchased from Shanghai Jingxi Chemistry Engineering Co. Ltd, were adopted in this study. Both MF membranes were commonly used during the wastewater treatment in China. New membranes were required for each new experiment. Filtration analysis
Chromatographic profile of the molecular size distribution of the EPS is illustrated in Fig. 1. A single and symmetrical peak from the biopolymer was presented, which indicated that the EPS was a homogeneous polysaccharide. The average molecular weight was measured to be 63,540 Da. FT-IR spectral analysis of EPS Fourier-transform infrared (FT-IR) transmittance spectrum of the EPS is shown in Fig. 2. Some typical absorption peaks of the polysaccharide occurred. Five bands appearing in 3,409.14, 2,932.47, 1,725.84, 1,554.37 cm-1 and 1,379.63 cm-1 should correspond to the stretching vibrations of hydroxyl, C–H, carboxyl, C=C and C–H bending of aliphatic CH2, respectively (Lee et al. 2004, 2006). Infrared bands at 1,643.05 cm-1 were probably associated with the interference of water peak brought by the KBr compression process. A stretching peak at 1,116.08 cm-1 was observed, indicating the presence of C–O bonds. In addition, an absorption peak at 600 cm-1 was also found, which was possibly attributed to the presence of O–H outof-plane vibration (Chiovitti et al. 1997).
Prior to each filtration experiments, the clean water flux, J0, of virgin membrane module was determined by filtering deionized water. A series of EPS filtrations (30 min for each) were performed at 0.1 MPa pressure for various feed concentrations. After each filtration, a backwash was carried out for 3 min at 0.1 MPa pressure and then the pure water flux was detected. In this study, the permeate flux can be described by combining the permeate data and Darcy’s law as follows. J¼
DP DP ¼ lRt lðRm þ Rir þ Rr Þ
ð2Þ
where J is the filtrate flux, DP is the trans-membrane pressure drop, l is the feed viscosity, Rt is the total
Fig. 1 Molecular weight of the EPS produced by M. luteus
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40
60
35
1643.05
35 30
20 4000
3409.14
25
3500
600.31
40
30mg/L 50mg/L 100mg/L
30 25 20 15 10
1116.08
45
1554.37 1379.63
%T
50
1725.84
2932.47
55
Intendity (%)
45
65
802.26
70
5 0 1
3000
2500
2000
1500
1000
10
500
Wavenumbers (cm-1)
Fig. 2 FT-IR spectrum of the EPS produced by M. luteus
100
1000
10000
Diameter (nm)
Fig. 4 Particle size distribution characterization of the EPS produced by M. luteus at various concentrations
Monose components of EPS Monosaccharide components of the EPS was determined after acid hydrolysis. The peak in Fig. 3 revealed that the EPS purified was a complex polysaccharide, which was composed of at least seven monoses, rhamnose, fucose, arabinose, xylose, mannose, glucose and galactose in a molar ratio of 0.2074:0.0454:0.0262:0.0446:1.7942:1.2086:0.4578. It was evident that mannose and glucose were the major monoses constructing backbones of the EPS. Particle size distribution of EPS DLS measurements of the various EPS samples are presented in Fig. 4. We could find that the particle size
distributions in the EPS solutions were similar and yielded the hydrodynamic averaged sizes of 162–182 nm range. Rheological studies of EPS Flow curves of aqueous solutions of the EPS with increasing shearing rates are shown in Fig. 5. The rheograms, within the available shearing-rate range, demonstrated that the viscosity of the biopolymer went up with the increasing of feed EPS concentration and went down with the increasing of shearing rate. It could be assumed that the aqueous dispersions of the EPS exhibited nonNewtonian and shearing-thinning behavior with pseudo plastic properties.
Fig. 3 Monose components of the EPS produced by M. luteus
Mannose
Glucose
Galactose
Fucose Arabinose Rhamnose
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Xylose
Myo-inositol (internal standard)
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In Eq. (1), as n tends to 1, the shearing-thinning properties are so less and less, pronouncing that Newtonian behavior is achieved when n is equal to 1 (Moreno et al. 2000). In this study, n values for 30, 50 and 100 mg/L EPS solutions were 0.74, 0.67 and 0.56, respectively. This suggested that pseudo plastic rheological properties could be enhanced with increasing EPS concentration.
filtration processes, the Rir always played a dominant role with the highest percentage (54.62–58.79 %) of the total fouling resistances Rt, followed by the Rr, and then the Rm was the lowest with the percentages of 2.67–4.45 %. Furthermore, at the same feed concentration, the contribution of Rir towards the Rt for the PVDF membrane was relatively higher than that for the PP membrane, although it exhibited a lower Rt value.
Flux behavior of EPS The normalized fluxes of PVDF and PP membranes are shown in Fig. 6 as a function of time for various EPS concentrations. The results showed that all the permeation curves of two MF membranes exhibited an initial sharp drop in the permeate flux at the start of the filtration. Then, after the initial steep decline, the flux declined gradually and eventually attained steady state. It also could be observed from the curve shapes in Fig. 6 that the extent of the flux decline increased with an increase in the bulk polysaccharide concentration. In addition, with respect to the same EPS solution, the flux for the PP membrane showed more significant decline than that for the PVDF membrane.
Discussion Microbial polysaccharide is a major fouling component in MBR systems (Ramesh et al. 2006). Numerous fouling mechanism studies have been performed using various EPS models to describe fouling in MBR (Susanto and Ulbricht 2005; Nataraj et al. 2008). However, there is a general lack of knowledge about the relation between membrane fouling and polysaccharide characteristics. In addition, M. luteus is a common Gram-positive microorganism and has
A
100
30mg/L 50mg/L 100mg/L
The values of Rir, Rm, Rr and Rt and their relative percentages under different EPS feed concentrations and membrane materials are listed in Table 1. It can be observed that various resistance (Rir, Rr and Rt) values for the both membranes increased with EPS feed concentration. Under the same conditions, the Rt of the PP membrane was much larger than that of the PVDF membrane, indicating a relatively higher permeation hinder, which are in line with the above flux analysis. In addition, among all the
Normalized flux (%)
Resistance analysis 80
60
40
20
0 0
300
600
900
1200
1500
1800
Time (s) 14
Viscosity (Pas)
10
Normalized flux (%)
12
-0.4441
y = 14.467x 2 R = 0.9555
8 6 4 2
y = 5.2636x 2 R = 0.9294
B
100
100 mg/L 50 mg/L 30 mg/L
-0.3289
y = 9.2667x 2 R = 0.9512
30mg/L 50mg/L 100mg/L
80
60
40
20
-0.2582
0
0
300
600
900
1200
1500
1800
Shear rate (s-1)
Fig. 5 Effect of shearing rates on dispersion viscosity of the EPS produced by M. luteus at various concentrations
0
300
600
900
1200
1500
1800
Time (s)
Fig. 6 Flux decline profiles for PVDF (a) and PP (b) membranes of the EPS produced by M. luteus at various concentrations
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World J Microbiol Biotechnol Table 1 Various fouling resistances and relative percentages as a function of EPS concentrations for the PVDF and PP membranes EPS concentrations (mg/L)
Resistances
PVDF
PP
30
Rm (91011 m-1)
1.4594
1.0540
50
100
Percentage
19.42
13.30
Rir (91011 m-1)
3.2872
3.4474
Percentage
43.74
43.50
Rr (91011 m-1)
2.7686
3.4237
Percentage
36.84
43.20
Rt (91011 m-1) Percentage
7.5152 100
7.9252 100
Rm (91011 m-1)
1.4483
1.0629
Percentage
17.62
10.24
Rir (91011 m-1)
4.1664
5.0621
Percentage
50.68
48.79
Rr (91011 m-1)
2.6057
4.2503
Percentage
31.70
40.97
Rt (91011 m-1)
8.2204
10.3753
Percentage
100
100
Rm (91011 m-1)
1.4673
1.0599
Percentage
15.45
9.45
Rir (91011 m-1)
5.4290
5.7389
Percentage
57.16
51.15
Rr (91011 m-1)
2.6008
4.4203
Percentage Rt (91011 m-1)
27.39 9.4972
39.40 11.2191
Percentage
100
100
membrane, which also implied that this biopolymer had the higher fouling potential to PVDF membrane. This is consistent with the analysis results of FT-IR spectra and monose components, which proved that the EPS consisted of various monosaccharides with hydrophilic groups (such as hydroxyl, carboxyl and ether bond etc.) (Allen et al. 2004; Weiner and Langille 1995), which probably made more EPS molecules adhere on the hydrophilic PVDF membrane and resulted in a higher Rir. In conclusion, some physicochemical properties of the EPS had been obtained in the present works. However, as a complicated biological macromolecule, EPS still need further studies on other important characteristics, such as the connection pattern of monosaccharide, the dimensional structure of carbohydrate chain, the binding site of functional groups and so on, which may be more closely correlated with its fouling. Thus, future study needs to be conducted to explore these characteristics for revealing the internal mechanism of EPS membrane fouling. Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 30901131, 31000264), the Excellent Youth Cultivation Program of Fujian Province (Nos. JA10088), the New Century Excellent Talents of Fujian Province (No. K8011025, K8012015A), Key Project of Chinese Ministry of Education (No. 212089) and the Excellent Youth Program of Fujian Agriculture and Forestry University (No. xjq201210).
References been reported in various fields (Perromat et al. 2003; Mauclaire and Egli 2010). To the best of our knowledge, however, there have been no works performed to reveal the fouling properties of this strain. In this study, the pores of MF membranes had a uniform diameter that was larger than EPS particle size (Fig. 4) and thus, many EPS biopolymers might deposit in the membrane pores or plugged the pore entrances resulting in a significant flux decline. The EPS concentrations caused different fouling impacts on the MF membranes, the permeate volumes decreasing with increasing EPS concentrations. Enhanced solution viscosity due to the rise in EPS concentration might have a great contribution to the decrease, which was confirmed by the rheology analysis. Comparing with the PVDF membrane, the PP membrane showed the larger flux decline and Rt value, but the relatively smaller percentage of Rir. This implied that the serious fouling for PP membrane can be attributed to its smaller Rm value, which caused the larger initial flux and could transfer more EPS biopolymer to the PP membrane surface during the filtration time. Furthermore, the higher proportion of Rir for the PVDF membrane indicated that the EPS preferred to absorb to this membrane surface rather than the PP
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