Available online at www.sciencedirect.com

Bioresource Technology 99 (2008) 5640–5644

Characterisation of Paenibacillus jamilae strains thatproduce exopolysaccharide during growth on and detoxification of

olive mill wastewaters

Margarita Aguilera, Maria Teresa Quesada, Vıctor Guerra del Aguila,Jose Antonio Morillo, Maria Angustias Rivadeneyra, Alberto Ramos-Cormenzana,

Mercedes Monteoliva-Sanchez *

Department of Microbiology, Faculty of Pharmacy, University of Granada, Campus Universitario de Cartuja s/n, 18071 Granada, Spain

Received 11 October 2006; received in revised form 17 October 2007; accepted 18 October 2007Available online 3 December 2007

Abstract

A total of 10 bacterial strains were isolated from a compost of corn treated with olive mill wastewaters (OMW) and selected by theircapacity to synthesize exopolysaccharides (EPS). Morphological, physiological, biochemical and nutritional tests were used for a phe-notypic study. A numerical analysis showed that all strains were 90% similar to each other. A DNA–DNA hybridization assay confirmedthat all the strains belonged to Paenibacillus jamilae species. All the characterized strains were able to produce EPS growing on OMWbatch cultures. The strain which was able to produce the highest EPS yield was chosen to perform an assay for testing its putative detox-ifying activity, and it showed to reduce more than half the toxic capacity of the OMW. The results presented in this study, indicated thepossible perspectives for using these bacterial strains to produce EPS and contribute to the bioremediation of the waste waters that areproduced in the olive oil elaboration process.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Olive mill waste waters; Exopolysaccharides; Paenibacillus jamilae; Bioremediation

1. Introduction

In order to obtain olive oil, the olive industry generateselevated amount of toxic wastes, which are highly pollu-tant. Treatment and disposal of olive mill effluents, partic-ularly olive mill waste-water (OMW), is currently one ofthe most serious environmental problems in the countrieswhere olive oil is produced. OMW is a dark-brown col-oured liquid that contains soluble olive fractions, as wellas water used in the fruit oil extraction processing. OMWcomprises about 15% organic material that is composedof carbohydrates, proteins and lipids as well as a number

0960-8524/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2007.10.032

* Corresponding author. Tel.: +34 958 243875; fax: +34 958 246235.E-mail address: mmonteol@ugr.es (M. Monteoliva-Sanchez).

of other organic compounds including monoaromatic andpolyaromatic molecules (Capasso et al., 1995); the mostrepresentative of these molecules are phenolic compounds,which act as antimicrobial and phytotoxic agents (Ramos-Cormenzana et al., 1995). Traditionally, the disposal ofOMWs has become a great problem in Mediterraneancountries, because of their polluting effects on soil andwater (Sierra et al., 2001; Piotrowska et al., 2006).

The possibility of finding methods to eliminate the phe-nolic content of OMW by different approaches and its fur-ther use as recycled product, is one of the most desirablesolution. Bioremediation, is a valuable tool for the detoxi-fication of OMW by breaking down these phenolic com-pounds (Ramos-Cormenzana et al., 1995). Recentresearch on OMW bioremediation has included differentapproaches, such as aerobic biodegradation (El Hajjouji

M. Aguilera et al. / Bioresource Technology 99 (2008) 5640–5644 5641

et al., 2007), cultivation of fungi (Kalmis et al., 2008) andanaerobic digestion (Filidei et al., 2003). In this sense,one of the objectives of our research is led to eliminatethe toxic part of OMW and select microorganisms thatare tolerant to the phenolic fraction.

The use of agricultural wastes may lead to the produc-tion of microbial exopolysaccharides (EPS) (Sutherland,1996). Microbial polymers offer more advantages in theirapplication than those obtained from seaweeds and plants,since they present a greater range of structures and proper-ties which can be used in different applications. OMW con-stitute an interesting substrate for EPS production. It has ahigh C/N ratio (Alburquerque et al., 2004; Baddi et al.,2004), which could limit cellular growth but favour or stim-ulate EPS production. OMW has been used as organic sub-strate for the production of pullulan by fermentation(Israelides et al., 1994). Furthermore, Lopez and Ramos-Cormenzana (1996) described xanthan production fromOMW, but the process should be optimized dependingon the type and quality of the effluent employed (Lopezet al., 2001). EPS production for different species of Azoto-

bacter (Fiorelli et al., 1996) in OMW has also been shown.The difficulty lies in the antimicrobial character of OMW(Tomati et al., 1996) which limits the concentration ofthe used waste as culture medium, usually not exceeding50%.

In this paper, we describe the possibility of producingbiopolymers from microbial strains that are able to growin OMW as their sole carbon and energy source and weanalyse its capacity to detoxify these residues.

2. Methods

2.1. Microorganisms

From a total of 60 bacterial strains isolated from com-post treated with OMW in a previous study (Monteoliva-Sanchez et al., 1996), 10 strains were selected because theyproduced extracellular polysaccharide during their growthon OMW as sole carbon and energy sources, in both solidand liquid media. Those strains selected were: CP-3, CP-7,CP-10, CP-14, CP-19, CP-27, CP-38, CP-41, CP-50 andCP-57. Paenibacillus polymyxa CECT 153 and Paenibacil-

lus jamilae CECT 5266 were used as reference strains.

2.2. Oil factory effluents and culture media

OMW was obtained from the manufacturing of olive oilwith a three-phase system (‘‘Aceites Jimena S.A.”, Gra-nada, Spain). The same OMW was used for all experi-ments, and it was characterized as follows: pH, 4.7;conductivity, 0.035 X cm�1; density, 1.03 g l�1, totalorganic carbon, 10.13 g l�1; total nitrogen, 0.3 g l�1; totalphenolics, 2.8 g l�1; total solids, 55.59 g l�1; volatile solids,44.46 g l�1; non-volatile solids, 11.13 g l�1; chemical oxy-gen demand, 46.53 g l�1; biological oxygen demand,38.16 g l�1. The effluent was collected and stored in sterile

plastic containers at �20 �C until needed. Before prepara-tion of the culture medium, the stock was filtered througha No. 40 Whatman membrane filter. The basic media con-tained the effluent diluted to different concentrations (80%and 100%) with saline solution (0.9% (w/v) NaCl). Thediluted substrate was neutralised to pH 6.8–7 with 0.1 MNaOH prior to autoclaving at 121 �C for 20 min.

2.3. Phenotypic tests

Taxonomic identification phenotypic tests were made aspreviously described Aguilera et al. (2001).

2.4. Molecular taxonomy

Long scale DNA was extracted by the method of Mar-mur (1961), and was submitted to the following tests: (a)determination of the mol% G + C content based in thedetermination of the Tm (Marmur and Doty, 1962), and(b) DNA–DNA hybridization, following the method ofLind and Ursing (1986) and the immunoassay non-radio-active modifications of Ziemke et al. (1998).

2.5. EPS production and characterization analysis

The production of biopolymers from 80% and 100%OMW culture media were carried out in triplicate using500 ml Erlenmeyer flasks for every strain assay and 2 l bio-reactor Biostat� B (Braun Biotech, Germany) for the strainCP-38 assay (30 �C, pH 7, 150 rpm).

The biomass was separated from soluble EPS by centri-fugation at 10,000g for 60 min. The cell pellet was re-sus-pended in 1 M HCl solution in order to separate theadhered particles, centrifuged at 5000g for 20 min and thesupernatant was discarded. The cells were finally re-sus-pended in distilled water, centrifuged in the same condi-tions and the supernatant was discarded. The washedpellets were dried at 105 �C in a dry oven for 18 h andthe cells weight was estimated.

EPS was precipitated from the cell-free supernatant bythe addition of 2% (w/v) NaCl and cold 95% ethanol at�20 �C over night. The precipitated polysaccharide wasrecovered by centrifugation at 5000g for 10 min, it wassemi-dried directly in the same container at 40 �C, re-dis-solved in 20 ml of sterile water, recovered and dialysed inspecial dialysis bag against distilled water for 2 days atroom temperature, lyophilized and weighted.

EPS chemical composition was determined by total car-bohydrates assay (Dubois et al., 1956); uronic acids assay(Dische, 1962); hexoxamines assay (Johnson, 1971; Brad-ford Assay (1976)) to determine total proteins. The sameassays were performed over prepared controls media of80% and 100% OMW at time zero, respectively and the val-ues were subtracted to every independent results in order toovercome the background signal due to it complex chemi-cal composition.

Table 1Molecular taxonomic parameters: G + C content and non-radioactiveDNA–DNA hybridization assays among the studied strains

Strain G + C content,mean (mol%)

DNA–DNA hybridization,RBRa (%)

CP-3 40.48 73.50CP-7 42.68 83.00CP-10b 40.21 100CP-14 43.41 78.50CP-19 42.07 91.20CP-27 43.41 86.21CP-38 40.68 78.21CP-41 40.71 79.31CP-50 40.85 85.46CP-57 42.43 78.21P. jamilae 41.85 86.96P. polymyxa 41.21 15.20

a Relative binding ratio.b Digoxigenin labelled strain.

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2.6. Biotoxicity of OMW

For determination of biotoxicity, the luminescent bacte-rium Photobacterium phosphoreum, was used in the Micro-tox� System M500 (Microbics Corporation, Carlsbad, CA)employing reagents and procedures recommended and fol-lowing the method previously described by Monteoliva-Sanchez et al. (1996). Each sample was tested from effluentsof strain CP-38 culture grown in a 2 l bioreactor Biostat� B(Braun Biotech, Germany) (30 �C, 150 rpm) containingautoclaved OMW at 80% as a culture medium at pH 7.Three replicated assays were performed and every samplewas assayed at 24, 72 and 120 h and were diluted 1:100before analysis. Samples taken at time 0 h were corre-sponding to an initial control assay, where the intrinsic tox-icity of autoclaved 80% OMW medium with a dilution1:100 was determined. The effective relative toxic concen-tration able to decrease the bacterial luminescence by50% (EC50) was determined after 5 min of contact. A tox-icity unit (TU) is obtained by dividing 100 by the EC50.

3. Results and discussion

3.1. Taxonomic characterization of the strains

The results of the phenotypic tests were comparable tothose previously described for P. jamilae (Aguilera et al.,2001) and they were subjected to a numerical analysis thatresulted in the grouping of the 10 analysed strains asunique phenon (data not shown). P. jamilae CECT 5266was included at 90% of similarity in this phenon; however,the P. polymyxa species was related at 85% of similarity.Global values obtained for G + C content were between40.21 and 43.41 mol%. The duplicated results of theDNA–DNA hybridization assay, labelling the CP-10 withdigoxigenin, are shown in Table 1. All 10 RBR values (Rel-ative Binding Ratio) from the strains and that one corre-sponding to P. jamilae were over 70% (established limitfor species) and the value of RBR with P. polymyxa was

Table 2Production of biomass and EPS of the 10 strains of Paenibacillus jamilae grow

Strain OMW 80% (v/v)

Biomass (g l�1) EPS (g l�1) Specific productivity

CP-3 0.39b 2.4b 6.15CP-7 0.55 3.0 5.45CP-10 0.45 2.7 6.00CP-14 0.48 2.9 6.04CP-19 0.48 3.0 6.25CP-27 0.48 3.0 6.25CP-38 0.55 4.2 7.64CP-41 0.47 3.1 6.59CP-50 0.52 3.8 7.31CP-57 0.50 3.6 7.20

a Specific productivity = EPS/biomass.b Mean values between three replicates assays.

clearly inferior. Therefore, the 10 strains studied could beincluded as member of P. jamilae species.

3.2. Study of specific productivity of EPS

Different strains of genus Paenibacillus have beendescribed as EPS producers (Seo et al., 1999; Aguileraet al., 2001; Yoon et al., 2002, 2003). The specific produc-tivity of EPS by P. jamilae isolates cultured in liquid mediaprepared with OMW under optimal conditions (pH 7 and30 �C), varied depending on the waste concentration used(Table 2). Both, growth and EPS production for all thestrains were higher with 80% OMW. The strain CP-38showed a higher yield of EPS production within all theresults (4.2 g l�1). The evident decrease in growth andEPS production using 100% OMW concentration mediacould be associated with an increased inhibition effectbecause of higher concentration of phenolic compoundsand/or a consequence of increased C/N ratio; in any case,both theories should be analysed in detail. Others studies ofpolysaccharide production using OMW as the fermenta-tion substrate had shown the need for dilution to reduce

n on OMW at 80% and 100% after 48 h

OMW 100% (v/v)

a Biomass (g l�1) EPS (g l�1) Specific productivitya

0.20 0.9 4.500.35 1.5 4.280.27 1.3 4.810.35 1.5 4.280.26 1.2 4.610.28 1.3 4.640.30 1.8 6.000.25 1.2 4.800.24 1.4 5.830.32 1.8 5.62

Table 3Toxicity assay performed by Microtox test over CP-38 strain culturegrowing in autoclaved 80% OMW medium

Time of growth (h) Inoculated 80% OMW Non-inoculated 80%OMW

EC50

(%)aToxicity (TU)a EC50

(%)Toxicity (TU)

0b 1.68 59.5 1.68 59.524 8.84 11.3 1.84 54.372 9.13 10.9 1.98 50.5120 21.59 4.6 2.33 42.9

a Mean of triplicate assays.b Culture medium containing autoclaved OMW at 80%.

M. Aguilera et al. / Bioresource Technology 99 (2008) 5640–5644 5643

the amount of phenols to prevent and/or to limit theirinhibitory action (Lopez and Ramos-Cormenzana, 1996;Fiorelli et al., 1996). A yield of 4 g l�1 of xanthan has beenobtained, with an optimal concentration of OMW of 50%(Lopez and Ramos-Cormenzana, 1996). However, thefungi Botryosphaeria rhodina was able to grow on undi-luted OMW producing a significant amount of the exo-polysaccharide b-glucan (17.2 g l�1) (Crognale et al., 2003).

A test in 2 l bioreactor was performed with the strainCP-38, in order to investigate the production of biomassand EPS at higher scale. A correlative increase of the twoparameters evaluated was also observed (Fig. 1). If we sub-tract from every sample the background EPS contained inthe media at time zero, we can consider that the specificproductivity rate was notably increased during the loga-rithmic phase period of cellular growth, reaching a maxi-mum value at the beginning of stationary phase wherebeyond it remained constant.

In the work presented here, the OMW was autoclavedbefore preparing the culture media, to avoid the growthof the indigenous microbiota of the waste. In this sense,the majority of previous studies dealing with the biodegra-dation of olive wastes have involved sterilization of the res-idues before inoculation of a specific microorganism orconsortium. However, not only does autoclaving kill theindigenous microbiota, but also the high temperature couldcause physico-chemical modifications in the OMW (Agge-lis et al., 2003); this fact should be considered in the possi-ble industrial use of the process of EPS production fromthe waste.

3.3. Chemical composition of EPS

The results of the study of the chemical analysis showedthat all the EPS samples were very similar to each other.All contained about 4% protein content, and approxi-mately 7% uronic acid value. Hexoxamine content wasabout 20%. Carbohydrates predominated, exceeding 35%of the total composition in all cases. The percentage ofuronic acids, that can be considered high, confers a strong

0

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0 12 24 48 72 96 120

Time (hours)

Bio

mas

s g

/L-1

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8

9

10

EP

S g

/L-1

Sp

ecif

ic P

rod

uct

ivit

y(E

PS

/Bio

mas

s)

Fig. 1. Kinetic of biomass (d), EPS production (h), and specificproductivity (N) of strain CP-38 growing on 80% autoclaved OMW (2 lbioreactor).

polyanionic nature to the EPSs (Ashtaputre and Shah,1995; Novak et al., 1992). This polyanionic feature of EPSsplays an important role in the adhesion of the microorgan-isms to the surfaces, and it is also involved in the uptake ofmetallic ions. EPSs can be used to remove toxic heavy met-als from polluted waters, and the interaction between thesemicrobial polymers and toxic heavy metals has been previ-ously well documented (Corzo et al., 1994; Loaec et al.,1997). The heavy metal biosorption capacity of the EPSproduced by P. jamilae have been recently studied (Morilloet al., 2006).

3.4. Evaluation of biotoxicity of the effluents obtained from

the culture of CP-38 strain on OMW

The results obtained in the evaluation of the biotoxicityof the effluents produced during the growth of the strainCP-38 on OMW are shown in Table 3. It was observedthat, in relation to the autoclaved control of OMW at80%, there was a significant decrease in the toxicity of morethan 75% during the first 24 h, and a progressive decreasethroughout the later period. This effect could be relatedto the interesting properties in the field of bioresource uti-lization that have been previously described in othersstrains of the genus Paenibacillus, like the high phenolic-degrading activity (Raj et al., 2007) and the productionof extracellular polysaccharide-degrading enzymes (Koet al., 2007). The EPS could also have been involved inthe resistance/neutralization of the toxic compounds ofOMW by P. jamilae.

3.5. Conclusion

Different strains of P. jamilae were able to grow andproduce EPS using OMW as the sole nutrient and energysource, with a concomitant reduction in the toxicity ofthe waste. The results obtained in this study, opened aninteresting perspective for using these bacterial strains,not only as EPS producers with possibilities of biotechno-logical uses, but also as a basis for developing methods ofbioremediation of the wastes that are produced in the oliveoil elaboration process.

5644 M. Aguilera et al. / Bioresource Technology 99 (2008) 5640–5644

Acknowledgements

The research was supported by grants from the Ministe-rio de Ciencia y Tecnologıa, Spain (Projects Nos.REN2000-1502 and CTM2004-05302).

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