World J Microbiol Biotechnol (2007) 23:1705–1710 DOI 10.1007/s11274-007-9418-3
ORIGINAL PAPER
Production and characterization of the exopolysaccharide produced by Paenibacillus jamilae grown on olive mill-waste waters ´ guila Æ Jose´ Antonio Morillo Æ Victor Guerra del A Margarita Aguilera Æ Alberto Ramos-Cormenzana Æ Mercedes Monteoliva-Sa´nchez
Received: 17 October 2006 / Accepted: 1 March 2007 / Published online: 17 May 2007 Springer Science+Business Media B.V. 2007
Abstract Paenibacillus jamilae, a strain isolated from compost prepared with olive-mill wastewaters, produced an extracellular polysaccharide (EPS) when it was grown in a culture containing olive-mill waste waters (OMWW) as sole carbon and energy sources. Maximal EPS production in 100 mL batch-culture experiments (5.1 g L–1) was reached with a concentration of 80% of OMWW as fermentation substrate (v/v). Although an inhibitory effect was observed on growth and EPS production when OMWW concentration was increased, an appreciable amount of EPS (2.7 g L–1) was produced with undiluted OMWW. Sepharose CL-2B chromatography showed that the EPS presented two fractions, EPS I (>2000 kDa) and EPS II (500 kDa). Both fractions were characterized by GC-MS as two different acidic heteropolysaccharides containing glucose, galactose and mannose as the major components. The performed study made evident the possibility of using OMWW as substrate for the production of EPS by P. jamilae with a satisfactory yield. Keywords Olive-mill waste waters (OMWW) Paenibacillus jamilae Exopolysaccharide production
Introduction The three-phase olive oil extraction system generates large amounts (about 3 · 107 m3 per year in the Mediterranean
´ guila M. Aguilera J. A. Morillo V. Guerra del A A. Ramos-Cormenzana M. Monteoliva-Sa´nchez (&) Department of Microbiology, Faculty of Pharmacy, University of Granada, Campus Universitario de Cartuja s/n, Granada 18071, Spain e-mail:
[email protected]
area) of a dark liquid effluent usually known as olive-mill wastewater (OMWW). These effluents are major pollutants and cause severe problems in the Mediterranean area. OMWW is characterized by a high contaminating load and by the presence of certain compounds, phenols in particular, with biostatic and phytotoxic activity and resistant to degradation by the natural soil microbiota (Capasso et al. 1995; Ramos-Cormenzana et al. 1995; Casa et al. 2003). The problem concerning to the OMWW is derived from its composition that makes it resistant to degradation. Essentially, the composition of this pollutant is water (80– 83%), organic compounds (15–18%) and inorganic compounds (2%, mainly potassium salts and phosphates), and it varies broadly depending on many parameters such as olive variety, harvesting time, climatic conditions, etc (Niaounakis et al. 2004). OMWW has a high degree of organic load and high carbon/nitrogen ratios (COD values up to 200 g L–1) and pH between 3 and 6. The organic fraction contains large amounts of proteins, lipids and sugars, but unfortunately OMWW also contains phytotoxic and biotoxic substances, which prevent it from being disposed of easily. The phytotoxic and antibacterial effects of OMWW have been attributed to its phenolic content (0.5–24 g L–1). Many references can be found on this subject (Gonza´lez et al. 1990; Moreno et al. 1990; Gonza´lez-Lo´pez et al. 1994; Knupp et al. 1996; Niaounakis et al. 2004). Agricultural wastes can be used as substrates for biotechnological processes, such as the production of microbial exopolysaccharides (EPSs) (Sutherland 1996). EPSs often show clearly identified properties that form the basis for a wide range of applications in food, pharmaceuticals, petroleum, and other industries (Sutherland 1990). The production of these microbial polymers using OMWW as a low-cost fermentation substrate has been proposed, resulting as well, to be environmentally beneficial
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(Ramos-Cormenzana et al. 1995). The idea is supported by the fact that OMWW presents certain similarities with the standard media for microbial polysaccharides production, mainly with respect to the high carbon/nitrogen ratio. This approach could reduce the cost of polymer production because the substrate is often the first limiting factor. Moreover, OMWW contains free sugars, organic acids, proteins and other compounds such as phenolics that could serve as the carbon source for polymer production (Fiorentino et al. 2004), if the chosen microorganism is able to metabolize these compounds. Xanthan gum, an extracellular heteropolysaccharide produced by the bacterium Xanthomonas campestris (the most commercially accepted microbial polysaccharide) has been obtained from OMWW (Lo´pez and Ramos-Cormenzana 1996; Lo´pez et al. 2001). The fungus Botryospheria rhodina has also been used for the production of b-glucan from OMWW (Crognale et al. 2003). These effluents can also be used as fermentation substrates for obtaining other kinds of microbial polymers, such as homo- and copolymers of polyhydroxyalcanoates (PHAs) (Martı´nez-Toledo et al. 1995; Gonza´lez-Lo´pez et al. 1996; Garcı´a-Ribera et al. 2001; Pozo et al. 2002). These reserve polyesters are accumulated as intracellular granules in a variety of bacteria and are a source of new biodegradable plastics. The objective of the present study was to determine the optimal conditions for the production of EPS by Paenibacillus jamilae using OMWW as fermentation substrate, and the characterization of the polymer obtained. The polymer produced by P. jamilae (a Gram-positive sporeforming bacteria that was isolated from compost prepared with OMWW) presents biotechnological interest due to its possible application as heavy metal biosorbent (Morillo et al. 2006).
Materials and methods
World J Microbiol Biotechnol (2007) 23:1705–1710 Table 1 Characteristics of OMWW used in this work Parameter pH
4.7
Conductivity (W cm–1)
0.035
Density (g L–1)
1.03
Total Phenolics (g L–1)
2.8
Total Solids (g L–1)
55.59
Volatile Solids (g L–1)
44.46
Non-volatile Solids (g L–1)
11.13
COD (g L–1)
46.35
–1
BOD (g L )
38.16
prepared by filtering the stock of OMWW through a No. 40 Whatman membrane filter. The resulting liquid was mixed with distilled water to achieve the desired concentration level, and pH was adjusted from 5 to 8 with 0.1 mol L–1 NaOH or HCl prior to autoclaving at 121 C for 20 min. The same OMWW was used for all experiments and its characteristics are summarized in Table 1. Exopolysaccharide production Batch culture experiments These experiments were carried out in order to study the effect of OMWW concentration, pH and temperature on growth and EPS production. Culture media consisted on 100 mL of the basic medium containing OMWW at the appropriate dilution supplemented with 0.1 g of NH4Cl and 0.1 g of yeast extract (Difco). Media were inoculated with 1 mL of a 24 h Paenibacillus jamilae culture in Yeast Mold Broth (YM Broth, Difco). Cultures were incubated in 250 mL flasks with rotary shaking at 100 rev. min–1 for 5 days at 30 C. After culturing the bacteria, biomass and polymer concentration were estimated as described below.
Microorganism One-liter fermentation experiments Paenibacillus jamilae CECT 5266 was used in this study. This microorganism was isolated from compost samples treated with OMWW and also screened for their capacity to grow in 100% of OMWW and to produce expolysaccharide (Aguilera et al. 2001). Substrate preparation OMWW was obtained from the manufacturing of olive oil with a three-phase system (‘‘Aceites Jimena S.A.’’, Granada, Spain). The effluent was collected and stored in sterile flasks at –20 C until needed. The medium was
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A 2 L jar-fermentor BIOSTAT M (Braun-Biotech) containing 900 mL of medium was also used to study the production of EPS. The composition of the medium was OMWW 80% (vol/vol), NH4Cl (0.2% wt/vol), and yeast extract 0.1% (wt/vol). The medium was inoculated with 100 mL of a 48 h Paenibacillus jamilae culture in the same medium. Fermentation conditions (30C, pH 7 and 150 rev. min–1) were kept constant for 5 days with the addition of 2 mL min–1 of air by bubbling. For biomass and EPS determinations, 20 mL of culture were sampled at 0, 12, 24, 48, 72, 96 and 120 h.
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Exopolysaccharide and biomass separation The production of biomass was calculated from dry weight of cells harvested from media by centrifugation at 10000 rev. min–1 for 30 min in a Sorvall centrifuge (Delaware, USA). The EPS was recovered from cell-free supernatant by precipitation with two volumes of –20 C ethanol with a prior addition of 1% (w/v) KCl as the electrolyte. The solution was kept at –20 C overnight and centrifuged at 5000 rev. min–1 for 10 min. The precipitated polymer was dissolved in distilled water and dialyzed extensively against distilled water for 48 h at 4 C. Dry weight of polymer was obtained by freeze-drying. The reported biomass and EPS were corrected for the mass of the pellet resulting from OMWW itself. All assays were performed in triplicates. Purification of exopolysaccharide and determination of molecular weight The EPS was purified and fractioned by gel filtration chromatography. The freeze-dried polymer was dissolved in phosphate buffer (Na2HPO4-NaH2PO4, pH 7.0) containing 0.5 M NaCl to a concentration of 10 g L–1, and 2 mL were loaded onto a Sepharose CL-2B column (2.6 · 95 cm). The column was eluted with the same buffer at flow rate of 15 mL h–1 with a peristaltic pump. Total carbohydrate content of 5 mL fractions was determined by the phenol-sulphuric acid method (Dubois et al. 1956). Standard dextran with molecular weights of 25, 80, 270, 670 and 2000 kDa (Fluka Chemical Corp.) were used for the estimation of molecular weight of EPS.
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Instruments). A stainless steel SPB 2380 column was used and the temperature program was 125 C for 2 min and 125–280 C at 5 C min–1.
Results Growth and exopolysaccharide production The effect of OMWW concentration, temperature and pH on growth and EPS production is shown in Fig. 1. At low OMWW concentration, EPS production and growth increased with the concentration of the substrate. The maximal EPS production (5.1 g L–1) was reached with a concentration of 80% of OMWW. When more than this concentration of OMWW was used to prepare the culture media, a decrease of microbial growth and EPS production was obtained. However, it is remarkable that a substantial amount of EPS (2.7 g L–1) was produced with 100% OMWW. Optimal values of pH and temperature for EPS production were 7 and 30 C respectively, although growth and polymer production occurred in all the conditions tested in this study (pH from 5 to 8 and temperature from 25 to 40 C). The kinetics of cell growth and polysaccharide production in a 1-liter fermentation vessel containing OMWW 80% (v/v) and NH4Cl 0.2% (w/v) with pH adjusted to 7 and temperature to 30 C is shown in Fig. 2. EPS production started during the log growth phase and reached the maximal amount during the stationary phase of growth. After 72 h of cultivation, cells entered in stationary phase and the concentration of EPS remained fairly constant between 5 and 5.5 g L–1.
Compositional analysis of exopolysaccharide Exopolysaccharide composition and characterization The biochemical composition of EPS was determined by the following colorimetric methods. Total carbohydrates were analyzed by the phenol-sulphuric method (Dubois et al. 1956) with D-glucose as the standard. Uronic acids were measured by the carbazole-sulfuric acid method (Dische 1962) with D-glucuronic acid as standard. Total protein was analyzed by the method of Bradford (1976) with bovine serum albumin as the standard. Hexosamine was determined by the method of Johnson (1971). Pyruvic acid was quantified by the enzymatic assay described by Duckworth and Yaphe (1970). Acetyl groups were assayed using a modified colorimetric procedure of Hestrin (1949). All determinations were performed in triplicates. For the analyses of sugars, the polysaccharide was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 120 C for 120 min and evaporated to dryness under nitrogen stream. Carbohydrate composition was analysed by gas chromatography (Carlo Ebra serie 8000 model 8060) equipped with a spectrometer Platform II (Micromass
A Sepharose CL-2B gel permeation chromatography revealed that the polymer obtained in 1-liter fermentation experiment presented two EPS groups (Fig. 3). The molecular weight of the smaller group was estimated to be about 500 kDa (EPS II), while the weight of the EPS I was more than 2000 kDa. Biochemical characterization (Table 2) confirmed that there were qualitative differences between both fractions of the polymer. EPS I and EPS II represented about 36 and 64% of the total amount of carbohydrates, respectively. EPS II presented a small fraction of proteins (not detected in EPS I), and a higher content in hexosamines and uronic acids. The neutral carbohydrate composition in both EPS groups was similar (Table 3). The major components were glucose, galactose and mannose. No substantial differences in these three major components were observed among EPS I and EPS II. However, it is interesting to note that in the smaller fraction in terms of molecular weight (EPS II),
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World J Microbiol Biotechnol (2007) 23:1705–1710 6
0.6
3 0.4
2 1
0.2
0
0 50
60
70
80
90
100
OMWW (%) 6
1
5
0.8
4
0.6
3 0.4
2
0.2
1 30
35
6
MW 670 MW 270
0.8
EPS I MW 80
5
MW 25
0.4
4 0
20
40
60
80
100
Fraction No. (5ml/tube)
0
0 25
1.2
0.0
biomass (g l -1 )
EPS (g l -1 )
B
7
EPS II MW 2000
Log molecular weight (kDa)
0.8
4
biomass (g l -1 )
EPS (g l -1 )
1.8
1
5
Absorbance at 490nm
A
Fig. 3 Elution profile and determination of molecular weight of Paenibacillus jamilae exopolysaccharide by gel filtration on Sepharose CL-2B. Elutes were analyzed by measuring the absorbances at 490 nm for carbohydrates. Standard dextran with molecular weights of 25, 80, 270, 670 and 2000 kDa were used for estimation of molecular weight of EPS
40
T (°C) 6
1
5
0.8
4
0.6
3
0.4
2 1
0.2
0
0 5
6
7
biomass (g l -1 )
EPS (g l -1 )
C
8
pH
Fig. 1 Effect of OMWW concentration (A), temperature (B), and pH (C) on biomass (d) and EPS (n) production
Table 2 Biochemical composition of the two fractions of the EPS produced from OMWW by Paenibacillus jamilae in 1-liter fermentation experiment EPS I
EPS II
Carbohydrates
45.87
48.29
Acetic acid.
2.70
3.22
Pyruvic acid
5.40
1.61
Uronic acid
13.49
11.27
Hexosamines
24.28
25.75
Proteins
nd
1.61
Data are expressed as relative amounts (%) of total dry weight of the polymer
1
5
0.8
4
0.6
3 0.4
2 1
0.2
0
0
0
24
48
72
96
120
biomass (g l -1 )
EPS (g l -1 )
nd: not detected 6
Time (h)
Fig. 2 Kinetic of biomass (d) and EPS (n) production of Paenibacillus jamilae in a fermentation vessel containing 1-liter of OMWW 80% (v/v) and NH4Cl 0.2% (w/v) with pH adjusted to 7 and temperature to 30 C
the content of arabinose was higher than in the EPS I, and fucose and xylose were not detected.
Table 3 Composition of uncharged monosaccharide residues in the two fractions of the EPS produced from OMWW by Paenibacillus jamilae in 1-liter fermentation experiment EPS I
EPS II
Glucose
36.1
35.3
Galactose
16.4
20.6
Mannose
14.8
14.7
Xylose
8.2
nd
Arabinose
9.8
17.6
Rhamnose
9.8
11.8
Fucose
4.9
nd
Data are expressed as relative amounts (%) of uncharged monosaccharide residues occurring in the two fractions nd: not detected
Discussion Although a large amount of reliable fermentative processes for EPS production have been described, there is a growing
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interest in the search for economical raw substrates. In this respect, the economics of EPS production using inexpensive raw materials like OMWW can make the industrial
World J Microbiol Biotechnol (2007) 23:1705–1710
production of microbial polymers more competitive. This study shows that olive-mill wastewater can be a valuable liquid growth medium for the production of EPS by P. jamilae. The main interest of this polymer is related to its potential application as heavy metal biosorbent (Morillo et al. 2006). Results of the present work indicated that the production of EPS by P. jamilae greatly varied depending on the concentration of OMWW utilized. Maximum EPS production of 5.5 g L–1 was obtained in media with 80% of OMWW. Concentrations between 70 and 90% of OMWW could be considered as an optimal range, taking into account the variability of this waste in composition depending on several parameters (Niaounakis et al. 2004). This effect of OMWW concentration on growth and EPS production could be explained as a consequence of a balance between the nutrients and other components with antimicrobial activity typically found in olive-mill wastes (mainly phenolics). At low OMWW concentration (50–60%), there was also a dilution of nutrients, and hence, less carbon was available for EPS production. As OMWW concentration increased, growth and EPS production rose, but with concentrations higher than 80%, an inhibitory effect was observed. To the best of our knowledge, xanthan gum is the unique bacterial EPS that has been previously obtained with the use of OMWW as the fermentation substrate. A production of 4 g L–1 of xanthan was obtained in optimal conditions with 50% OMWW as the medium culture (Lo´pez and Ramos-Cormenzana 1996; Lo´pez et al. 2001). In this work, we have demonstrated that P. jamilae can produced a significant amount of EPS with 80% OMWW as the medium culture. This difference between the optimal concentration of OMWW for EPS production by Xanthomonas campestris and P. jamilae, could be explained by the fact that the latter was originally isolated from a compost amended with OMWW. Thus, it is not strange that P. jamilae could resist higher concentrations of phenolic compounds than X. campestris. The Sepharose CL-2B gel permeation chromatography revealed that the polymer obtained in the 1-liter fermentation experiment presented two EPS groups, with different molecular weights. The production of two different polysaccharides in pure cultures has been observed for other organisms, for instance, Bacillus thermoantarcticus (Manca et al. 1996) and Paecilomyces tenuipes (Xu et al. 2006). Some exopolysaccharides produced by endospore forming rods belonging to the genus Bacillus have been reported (Kawai et al. 1992; Manca et al. 1996; Isobe et al. 1997; Corsaro et al. 1999; Larpin et al. 2002), but thus far only two description about production of EPS by other species belonging to Paenibacillus has been published
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(Yoon et al. 2002; Prado Acosta et al. 2005). Thus, the present work provides useful information about this new source of biopolymers. The number of species of Paenibacillus is rapidly increasing, making available new resources for the exploitation of molecules with interesting properties. OMWW production in Mediterranean countries is one of the most serious environmental concerns. This study reported the possibility of using OMWW as substrate for the production of EPS by P. jamilae with a satisfactory yield. Optimal conditions for the production of the EPS and its chemical characterization were determined. Acknowledgements The research was supported by grants from the Ministerio de Educacio´n y Ciencia, Spain (projects no OLI96–2189 and no REN2000–1502). We also thank our colleague Lia Wrightsmith for revising the English text.
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