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6amp7 5th Street Radhakrishnan Salai Chennai Tamil Nadu India ndash 600004Re Biodegradation DOI101007s10532-009-9259-x

Biodegradation of phenol by immobilized Aspergillus awamori NRRL 3112 on modifiedpolyacrylonitrile membrane

Authors G Yordanova middot D Ivanova middot T Godjevargova middot A Krastanov

Permission to publishI have checked the proofs of my article andq I have no corrections The article is ready to be published without changes

q I have a few corrections I am enclosing the following pagesq I have made many corrections Enclosed is the complete article

Date signature ______________________________________________________________________________

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Metadata of the article that will be visualized in OnlineFirst

Please note Images will appear in color online but will be printed in black and whiteArticleTitle Biodegradation of phenol by immobilized Aspergillus awamori NRRL 3112 on modified polyacrylonitrile

membraneArticle Sub-Title

Article CopyRight - Year Springer Science+Business Media BV 2009(This will be the copyright line in the final PDF)

Journal Name Biodegradation

Corresponding Author Family Name GodjevargovaParticle

Given Name TSuffix

Division Department of Biotechnology

Organization University ldquoProf Dr A ZlatarovrdquoAddress 1 Prof Yakimov Str 8010 Bourgas Bulgaria

Email godjevargovayahoocom

Author Family Name YordanovaParticle

Given Name GSuffix

Division Department of Biotechnology

Organization University ldquoProf Dr A ZlatarovrdquoAddress 1 Prof Yakimov Str 8010 Bourgas Bulgaria

Email

Author Family Name IvanovaParticle

Given Name DSuffix

Division Department of Biotechnology

Organization University ldquoProf Dr A ZlatarovrdquoAddress 1 Prof Yakimov Str 8010 Bourgas Bulgaria

Email

Author Family Name KrastanovParticle

Given Name ASuffix

Division Department of Biotechnology

Organization University of Food Technologies

Address 4002 Plovdiv Bulgaria

Email

ScheduleReceived 22 April 2008

Revised

Accepted 18 March 2009

Abstract Covalent immobilization of Aspergillus awamori NRRL 3112 was conducted onto modified polyacrylonitrilemembrane with glutaraldehyde as a coupling agent The polymer carrier was preliminarily modified in anaqueous solution of NaOH and 12-diaminoethane The content of amino groups was determined to be058 mgeq g minus1 Two ways of immobilization were usedmdashin the presence of 02 g l minus1 phenol and withoutphenol The capability of two immobilized system to degrade phenol (concentrationmdash05 g l minus1) as a solecarbon and energy source was investigated in batch experiments Seven cycles of phenol biodegradation wereconducted Better results were obtained with the immobilized system prepared in the presence of phenolregarding degradation time and phenol biodegradation rate Scanning electron micrographs of thepolyacrylonitrile membraneimmobilized Aspergillus awamori NRRL at the beginning of repeated batchcultivation and after the 7th cycle were compared After the 7th cycle of cultivation the observations showedlarge groups of cells The results from the batch experiments with immobilized system were compared to theresults produced by the free strain Phenol biodegradation experiments were carried out also in a bioreactorwith spirally wound membrane with bound Aspergillus awamori NRRL 3112 in a regime of recirculation10 cycles of 05 g l minus1 phenol biodegradation were run consecutively to determine the degradation time andrate for each cycle The design of the bioreactor appeared to be quite effective providing large membranesurface to bind the strain

Keywords (separated by -) Biodegradation - Phenol - Aspergillus awamori NRRL3112 - Immobilization - Polymer membrane

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Please position each reference in the text or delete it Any reference not dealt with will be retained in this section Queries andor remarks Sectionparagraph Details required Authorrsquos response

References Please provide complete details and confirm the proceedings name for the reference Chitra et al (1995)

References Please provide initials for author name Samardjieva for the reference Dimov et al (1983)

References Please confirm publisher and place for the reference Jose et al (2002)

References Please confirm the journal name for the reference Loh et al (2000)

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References Please note that the reference Yiang et al (2005) has not been cross referred in the text

Journal 10532 Article 9259

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

ORIGINAL PAPER1

2 Biodegradation of phenol by immobilized Aspergillus

3 awamori NRRL 3112 on modified polyacrylonitrile

4 membrane

5 G Yordanova D Ivanova T Godjevargova

6 A Krastanov

7 Received 22 April 2008 Accepted 18 March 20098 Springer Science+Business Media BV 2009

9 Abstract Covalent immobilization of Aspergillus

10 awamori NRRL 3112 was conducted onto modified

11 polyacrylonitrile membrane with glutaraldehyde as a

12 coupling agent The polymer carrier was preliminarily

13 modified in an aqueous solution of NaOH and 12-

14 diaminoethane The content of amino groups was

15 determined to be 058 mgeq g-1 Two ways of immo-

16 bilization were usedmdashin the presence of 02 g l-1

17 phenol and without phenol The capability of two

18 immobilized system to degrade phenol (concentra-

19 tionmdash05 g l-1) as a sole carbon and energy source

20 was investigated in batch experiments Seven cycles of

21 phenol biodegradation were conducted Better results

22 were obtained with the immobilized system prepared

23 in the presence of phenol regarding degradation time

24 and phenol biodegradation rate Scanning electron

25 micrographs of the polyacrylonitrile membrane

26 immobilized Aspergillus awamori NRRL at the

27 beginning of repeated batch cultivation and after the

28 7th cycle were compared After the 7th cycle of

29 cultivation the observations showed large groups of

30 cells The results from the batch experiments with

31 immobilized system were compared to the results

32produced by the free strain Phenol biodegradation

33experiments were carried out also in a bioreactor with

34spirally wound membrane with bound Aspergillus

35awamori NRRL 3112 in a regime of recirculation 10

36cycles of 05 g l-1 phenol biodegradation were run

37consecutively to determine the degradation time and

38rate for each cycle The design of the bioreactor

39appeared to be quite effective providing large mem-

40brane surface to bind the strain

41Keywords Biodegradation Phenol

42Aspergillus awamori NRRL3112

43Immobilization Polymer membrane

44

45

46Introduction

47One of the problems attracting highest attention in

48modern ecology is related to the biodegradation of

49xenobiotics Typical representatives of this group of

50contaminants are phenol and its derivatives The

51environmental clean-up of phenol by adsorption

52solvent extraction chemical oxidation incineration

53and abiotic treatment procedure suffers from serious

54drawbacks such as economic issues and the produc-

55tion of hazardous byproducts Biodegradation is

56generally preferred due to the lower costs and the

57complete mineralization

58Extensive studies revealing the potential of micro-

59organisms in phenol biodegradation have been carried

A1 G Yordanova D Ivanova T Godjevargova (amp)

A2 Department of Biotechnology University lsquolsquoProf Dr A

A3 Zlatarovrsquorsquo 1 Prof Yakimov Str 8010 Bourgas Bulgaria

A4 e-mail godjevargovayahoocom

A5 A Krastanov

A6 Department of Biotechnology University of Food

A7 Technologies 4002 Plovdiv Bulgaria

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Biodegradation

DOI 101007s10532-009-9259-x

Au

tho

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60 out Major part of the research in the field of phenol

61 decomposition have been performed using bacterial

62 strains such as Alcaligenes europhus (Hudges et al

63 1984) and Pseudomonas sp (Allsop et al 1993)

64 Other publications report biodegradation of phenol by

65 yeast strainsCandida (MacGillivray and Shiaris 1993)

66 and Trichosporon (Chtourou et al 2004) Chitra et al

67 (1995) have studied the removal of phenol using a

68 mutant strain of Pseudomonas The use of fungal

69 strains for the degradation is relatively untouched area

70 Mycelial fungi such as Fusarium floccieferum

71 (Anselmo and Novais 1992) Aspergillus fumigatus

72 (Jones et al 1995) and Graphium sp (Santos et al

73 2003) have been cited for their potential of phenol

74 degradation Little is known about phenol metabolism

75 in mycelial fungi They have some advantages

76 because of their resistance to a great number of

77 xenobiotics toxic to the most of the microorganisms

78 The fungal strain Aspergillus awamori NRRL 3112 is

79 capable of assimilating a wide range of carbon

80 substrates including phenol and its derivatives (Sun-

81 dar et al 2005 Yordanova et al 2006) This feature

82 makes them a valuable research object for a future

83 development of industrial-water cleaning technology

84 There has been an increasing interest in the

85 immobilization of microorganisms in recent years

86 (Abd-El-Haleem et al 2003 Bolanos et al 2001

87 Gonzalez et al 2001 Fiest and Hegeman 1969

88 Mordocco et al 1999 Pazarlioglu and Telefoncu

89 2005 Shetty et al 2007a b) The immobilized cells

90 have advantages over the native cells due to the

91 possibility for multiple applications for a extended

92 period of time Different carriers have been used for

93 microbial cell immobilization but there arenrsquot many

94 reports for cell immobilization on polymer membranes

95 (Uzunova et al 2002 Marrot et al 2006 Hua et al

96 2005 Godjevargova et al 2006) Synthetic polymers

97 constitute the largest group of supports in use Poly-

98 acrylonitrile-based ultrafiltration (UF) membranes

99 obviously turn up to be more advantageous over other

100 conventional membranes in various aspects such as

101 thermal stability resistance to most organic solvents

102 commercial availability etc (Wang et al 2007)

103 PAN-based membranes can offer some special prom-

104 ises as cradle for cell immobilization which is mainly

105 because different reactive groups can be easily gen-

106 erated on the PAN membrane surface for the covalent

107 immobilization (Godjevargova et al 2006 Kowalska

108 et al 1998)

109The membranes are very suitable for immobiliza-

110tion of microbial cells when a spiral membrane

111module is used There is no common strategy for

112preparing such integrated systems and opportunities

113for using are still at hand Due to their high surface

114area per unit module volume spiral membrane

115modules serve as better immobilization supports than

116alginate beads activated carbon sintered glass and

117polymer beads (Jose et al 2002 Karel et al 1985)

118On the other hand polymer membranes can act as

119barriers limiting the movement of cell mass thus

120providing restricted andor regulated passage of one

121or more species through the pores From this point of

122view membrane bioreactors are promising for

123practical applications for wastewater treatment (Loh

124et al 2000 Stephenson et al 2000)

125The present paper investigates the possibility of

126immobilizing Asp awamori NRRL 3112 spores on

127modified polyacrylonitrile membrane and their ability

128to biodegrade phenol Spiral membrane module with

129immobilized spores was created and phenol biodeg-

130radation was also investigated

131Materials and methods

132Microorganism and growth medium

133A strain of Aspergillus awamoriNRRL 3112 obtained

134fromUSDepartment of Agriculture Illinois USAwas

135used throughout this study The organism was grown

136on slants immersed in a medium having the following

137composition (g l-1) malt extract 30 yeast extract 30

138peptone 50 glucose 100 and agar 200 The organism

139on the slants was allowed to grow for 72 h at 30C and

140then stored at 4 plusmn 1C for further use

141Medium for degradation studies

142The studies on the biodegradation of phenol were

143carried out in the Czapekdox medium which had the

144following composition (g l-1) sodium nitrate 20

145potassium phosphate (dibasic) 10 potassium chlo-

146ride 05 magnesium sulfate heptahydrate 05 and

147ferrous sulfate heptahydrate 001 The initial pH of

148the medium was adjusted to 55 (the optimal pH for

149this strain) using 1 N NaOH or 1 N HCI (Sundar

150et al 2005) The minimal medium contains phenol as

151a sole carbon source with 05 g l-1 concentration

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

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152 Chemical modification of polyacrylonitrile

153 membrane

154 The ultrafiltration polyacrylonitrile (PAN) membrane

155 (average pore size of selective layer 002 lm) was

156 produced by Ecofilter Co (Bourgas Bulgaria) PAN

157 membrane was treated with 10 aqueous solution of

158 NaOH at 50C for 60 min After thorough washing

159 with distilled water the membrane was treated with a

160 10 aqueous solution of 12-diaminohexane for

161 60 min at 40C The membrane was once again

162 thoroughly washed with distilled water At the end of

163 the procedure the modified carrier was immersed in

164 05 aqueous solution of glutaraldehyde for 60 min

165 at room temperature Excess glutaraldehyde was

166 removed by washing the membranes with distilled

167 water until there was a complete absence of glutar-

168 aldehyde in the rinse waters

169 Phenol biodegradation in batch experiments

170 Spore immobilization for batch experiments

171 The 14-day culture in spore form in concentration of

172 333 9 107 ml-1 were introduced by flushing 5 cm3

173 sterile water in 500 cm3 sterile flask containing

174 50 cm3 Czapekdox medium and sterile modified

175 carrier (40 cm2 PAN flat membrane) Sterilization of

176 the modified membrane was carried out by ethanol

177 Two ways of immobilization were usedmdashin the

178 presence of 02 g l-1 phenol and without phenol

179 The two flasks were placed on a shaker for 24 h at

180 30C Then the liquid medium was decanted and the

181 immobilized systems were washed several times with

182 sterile water to remove the unbound spores After

183 that the immobilized spores were used for phenol

184 biodegradation

185 Batch experiments

186 The immobilized spores onto the polymer mem-

187 brane (single flat piecemdash40 cm2) with concentration

188 0158 g cm-2 were introduced in 500 cm3 sterile

189 flask containing 50 cm3 Czapekdox medium as well

190 as phenol as the only carbon source adjusted to the

191 appropriate concentration Then they were cultivated

192 on a shaker (220 rpm) at 30C until the complete

193 phenol degradation At every 24 h samples were

194 withdrawn and analyzed for phenol concentration

195Thus seven cycles were carried out with the same

196immobilized system

197Simultaneously parallel experiments with free

198cells were also performed for comparison The flasks

199containing 50 cm3 liquid medium as well as 05 g l-1

200phenol as the only carbon source were inoculated with

201equal volume of inoculum (333 9 107 conidia cm-3

202medium) and agitated on a shaker (220 rpm) at 30C

203Samples were taken at every 24 h interval for

204determination of phenol concentration After the

205biodegradation of the entire phenol the cultural

206medium was decanted and the biomass was reculti-

207vated in fresh liquid medium containing 05 g l-1

208phenol Thus four cycles were carried out in the same

209manner

210Each experiment for phenol degradation was

211repeated twice the reproducibility being always within

21225

213Phenol biodegradation in recycle reactor

214with spirally wound membrane

215Spore immobilization for experiments

216with membrane bioreactor

217The bioreactor was 40 mm in diameter and 100 mm

218high it was made of glass and equipped with

219thermostatic bath (Fig 1) The modified and activated

220with glutaraldehyde polyacrylonitrile membrane with

Fig 1 Scheme of recycle system thermostatic bath (1) feed

reservoir (2) peristaltic pump (3) thermostatic reactor (4)

membrane with immobilized cells (5) sampling (6) air outlet

(7) and air inlet (8)

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

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of

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221 surface area of 100 cm2 was spirally wound in the

222 glass bioreactor using supporting polyethylene mesh

223 In this case the immobilization was carried out by

224 flowing spore suspension (547 9 107 conidia cm-3

225 medium) through the membrane module using peri-

226 staltic pump at rate of 1 ml min-1 for 24 h Then the

227 membrane spiral was thoroughly washed out with

228 sterile water fed by peristaltic pump to remove the

229 non-bound spores

230 Experiments with membrane bioreactor

231 Further the liquid medium (300 ml) containing

232 phenol at certain concentration was fed to the mem-

233 brane bioreactor using peristaltic pump at rate of

234 15 ml min-1 The temperature required for the strain

235 growth (30C) was maintained using the thermostat

236 and the heat-exchanging coating After the full

237 degradation of phenol in the nutrient medium the

238 reservoir was charged with fresh nutrient medium

239 containing phenol Samples were taken at every 24 h

240 interval for determination of phenol concentration

241 Ten cycles were carried out in the same manner

242 Each experiment was repeated twice the repro-

243 ducibility being always within 25

244 Analytical methods

245 Phenol concentration was measured spectrophoto-

246 metrically in the presence of p-nitroaniline at 495 nm

247 (Gonzalez et al 2001)

248 The amount of spores immobilized on the support

249 was estimated by the difference between the wet

250 weight of spores with membrane at the beginning

251 (immediately after the immobilization) and the wet

252 membrane weight The amount of free cells was

253 determined by the measuring of the wet weight of the

254 cells and by the counting of the number of conidia in

255 the samples using a standard microscope method

256 Scanning electron microscopy (SEM)

257 The immobilized system Aspergillus awamori NRRL

258 3112polyacrylonitrile membrane was washed with

259 distilled water and then they were dehydrated in

260 30ndash100 water ethanol series The membrane were

261 coated with 120ndash130 Aacuteof gold in argon medium in

262 an Edwards apparatus (model 150 A) The SEM

263 observations were done on a scanning device attached

264to a Zeiss electron microscope (model 10C) at 20 kV

265accelerating voltage with a 5ndash6 nm electron beam

266Results and discussions

267Modification of membranes

268The aim of the present studies is to obtain an efficient

269immobilized system of Aspergillus awamori NRRL

2703112polyacrylonitrile membrane for biodegradation

271of phenol The immobilization of spores was carried

272out with glutaraldehyde The polymer membrane was

273preliminarily treated by using two different modifica-

274tion procedures The first procedure introduced

275ndashCOOH and ndashNH2 groups on the membrane surface

276by using NaOH solution In order to increase the

277amount of amine groups on the surface of the carrier

278the latter was treated with 12-diaminoethane The

279optimal conditions of modification were defined in a

280previous research work (Godjevargova et al 2000)

281The superficial carboxyl groups on the carrier surface

282reacted with one of the amine groups of 12-diami-

283noethane leaving the other one free The results

284showed that the PANmembranes had beenmodified in

285a greater extent and the content of amino groups was

286058 mgeq g-1 The latter was determined titrimetri-

287cally (Dimov et al 1983) Further the modified

288membrane was treated with an aqueous solution of

289glutaraldehyde The bifunctional agent bound the

290strain to the membrane surface

291Batch experiments

292Spores concentration used for the immobilization was

293333 9 107 ml-1 The first task was to determine

294which immobilization method would serve better for

295these experimentsmdashin the presence or in the absence

296of phenol (02 g l-1) in a nutrient medium containing

297spore suspension The amount of spores bound to the

298membranes in both cases was 0158 g cm-2 (wet

299weight) Then the spores immobilized on the polymer

300membrane by both methods were cultivated in a

301nutrient medium containing 05 g l-1 phenol The

302results obtained for phenol biodegradation by both

303systems are presented in Fig 2a b The rate of

304phenol biodegradation was determined after the end

305of each cycle for both immobilization systems

306(Table 1) As can be seen from Table 1 the lowest

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

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307 biodegradation rate is observed at the first cycle then

308 it increases 3 times at the second and the third cycles

309 and from the fourth to the seventh cycle the rate

310 decreases continuously This trend of the biodegra-

311 dation rate is observed for both of the immobilized

312 systems The deceleration of the phenol biodegrada-

313 tion could be explained with the increasing of

314 biomass and mycelia formation on the membrane

315 surface after the fourth cycle which intensified at the

316 final cycles An obvious inhibition of the biodegra-

317 dation process at the final cycles was observed which

318 was due to the hindrance of the substrate diffusion to

319 the cells When comparing the results presented on

320 Fig 2a and b and Table 1 it becomes obvious that the

321 rate of phenol degradation was higher throughout all

322 cycles in the presence of the system where the spore

323 immobilization was conducted with addition of

32402 g l-1 phenol For better comparison the biodeg-

325radation times of each cycle with the two

326immobilized systems are presented in Fig 3 As

0

02

04

06

08

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

0

02

04

06

08

0 200 400 600 800

0 200 400 600 800 1000

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

(a)

(b)

Fig 2 Phenol

biodegradation (05 g l-1)

by immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane obtained by

immobilization in presence

of 02 g l-1 phenol (a) and

without phenol (b)

Table 1 Phenol biodegradation rate (mg h-1) of free and

immobilized Aspergillus awamori NRRL 3112 on polymer

PAN membrane at batch cultivation with 05 g l-1 phenol

Numberof

cycles

Free

cells

Immobilized cells

without 02 g l-1

phenol

Immobilized cells

in the presence of

02 g l-1 phenol

1 297 300 300

2 201 714 694

3 149 806 543

4 116 625 367

5 403 329

6 308 300

7 275 270

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

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UNCORRECTEDPR

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UNCORRECTEDPR

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327 results suggest better degradation times were

328 obtained with the immobilized system prepared in

329 the presence of phenol This can be explained with

330 the fact that the strain could have adapted to the

331 substrate during the immobilization process All

332 further experiments were carried out with the immo-

333 bilized system prepared in presence of phenol

334 The biodegradation ability of the immobilized

335 system onto polyacrylonitrile membrane towards

336 phenol in a concentration range from 02 to

337 08 g l-1 was studied As can be seen from Fig 4

338 the increase of phenol concentration resulted in

339increased biodegradation time For instance the

340biodegradation cycle at concentration of 04 g l-1

341was 66 h while at 07 g l-1 the cycle lasted for 135 h

342The results obtained with the selected immobilized

343system were compared to those with the free strain

344For this purpose the spores (08 9 107 ml-1)

345were cultivated in Czapekdox medium containing

34605 g l-1 phenol (Fig 5) Four cycles were carried

347out in the same manner The amount of non-bound

348spores in the first cycle of cultivation is equivalent

349to the amount of immobilized spores on PAN

350membrane with surface 40 cm2 in the first cycle

0

50

100

150

200

250

1 2 3 4 5 6 7

Cycles

Tim

e

h

Fig 3 Cycles duration of

phenol biodegradation

(05 g l-1) by two

immobilized systems

Aspergillus awamori NRRL

3112modified PAN

membrane obtained by

immobilization in presence

of 02 g l-1 phenol ( ) and

without phenol (j)

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol co

ncen

trati

on

g

l-1

Fig 4 Phenol

biodegradation (from 02 to

08 g l-1) by immobilized

system Aspergillus

awamori NRRL 3112

modified PAN membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

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351 (wet weightmdash632 g) As can be seen from Fig 5 the

352 phenol degradation time was considerably greater for

353 the four cycles in these experiments This time

354 extension was confirmed by the calculated rate of

355 biodegradation (Table 1) When comparing the rates

356 of phenol biodegradation by the free and the immo-

357 bilized cells the greater stability of the immobilized

358 systems over the free cells becomes more prominent

359 after multiple cultivation in media containing phenol

360 with concentration 05 g l-1 Obviously the free cells

361 were inhibited to a greater extent by phenol (biodeg-

362 radation rate at Cycle 4mdash116 mg h-1) in

363 comparison with the immobilized cells (biodegrada-

364 tion rates at Cycle 4 are 625 and 367 mg h-1

365respectively for immobilized system obtained in the

366presence of 02 g l-1 phenol and the system without

367phenol)

368In order to distinguish between phenol adsorption

369on the membrane surface and phenol degradation by

370microorganisms the pure carrier was also studied for

371phenol adsorption For this purpose 40 cm2 pure

372membrane was immersed in 05 g l-1 phenol solution

373for 6 h The results are presented in Fig 6 Since the

374phenol concentration didnrsquot change significantly

375the only possible reason for the decrease of the

376phenol content was the degradation activity of the

377immobilized cells This simplifies the kinetic studies

378when membrane is to be used as a support material

0

02

04

06

08

0 100 200 300 400 500

Time h

Ph

en

ol

bio

de

gra

da

tio

n

gl-1

Fig 5 Phenol

biodegradation with

concentration 05 g l-1 by

strain Aspergillus awamori

NRRL 3112

0

02

04

06

08

0 1 2 3 4 5 6 7

Time h

Ph

en

ol

bio

deg

rad

ati

on

g

l-1

Fig 6 Adsorption of

phenol by modified PAN

membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

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379 for the immobilization of Asp awamori for the

380 purpose of phenol degradation

381 The amount of cells (wet weight) on the membrane

382 of 40 cm2 was measured after the sixth cycle of

383 phenol biodegradationmdash1104 g cm-2 The signifi-

384 cant increase of the biomass with the increased

385 number of cycles is obvious (after immobilizationmdash

386 0158 g cm-2) Thus the biomass accumulated on

387 the membrane was approximately ten times higher

388 after six cycles of phenol biodegradation The high

389 operational stability of investigated biocatalysts was

390 confirmed by the scanning electron micrographs of

391 the polyacrylonitrile membraneimmobilized Asper-

392 gillus awamori NRRL at the beginning of repeated

393 batch cultivation and after the 7th cycle After the 7th

394 cycle of cultivation the observations showed large

395 groups of cells (Fig 7a) The significant increase of

396 biomass and mycelia formation on the membrane

397 surface after the 7th cycle of cultivation was inev-

398 itably followed by deceleration of the phenol

399 degradation rate

400Experiments with membrane bioreactor

401The next step of the research work involved exper-

402iments which were carried out in a bioreactor with

403spirally wound membrane in a regime of recircula-

404tion The spores were immobilized onto the

405membrane directly in the system as described in

406lsquolsquoMaterials and methodsrsquorsquo section Phenol biodegra-

407dation was carried out under aerobic conditions The

408operation parameters of the bioreactor are presented

409in Table 2 Fresh nutrient medium containing

41005 g l-1 phenol were added after each cycle Phenol

411concentration was monitored for degradation At the

412end of a cycle the system was washed with sterile

413distilled water and charged with nutrient medium and

414phenol for the next cycle Thus 10 cycles of 05 g l-1

415phenol biodegradation were run consecutively to

416determine the degradation time for each cycle

417(Fig 8) No significant difference was observed

418between the degradation times of the cycles from

419the second to the seventh (about 50 h) and between

420the biodegradation rates (100 mg h-1) After the 7th

421cycle the biodegradation rate began to decrease

422gradually and at 10 cycles it was 40 mg h-1 These

423results confirmed the advantages of the recycled

424reactor with spirally wound membrane The design of

425the bioreactor appeared to be quite effective provid-

426ing large membrane surface to bind the strain cells

427Besides phenol would flow tangentially which pre-

428vents membrane from a decrease of the flow velocity

429Conclusion

430A novel immobilized systemmdashAspergillus awamori

431NRRL 3112polyacrylonitrile membranewas obtained

432for the purpose of phenol biodegradation It was

433established that the immobilized system prepared in

434the presence of 02 g l-1 phenol was more effective

Fig 7 Scanning electron micrographs of immobilized system

Aspergillus awamori NRRL 3112modified PAN membrane

after 7th cycle (a) and before 1st cycle (b)

Table 2 Operation conditions of bioreactor

Parameter Value

Rate flow 15 ml min-1

Volume 300 ml

Initial phenol concentration 05 g l-1

Membrane surface area 100 cm2

Immobilized spores 0174 g cm-2 (wet weight)

Biodegradation

123

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435 than the system prepared in the phenol absence It was

436 revealed that the immobilized systems were more

437 stable andmore effective when biodegrading 05 g l-1

438 phenol in comparison with the free cells A bioreactor

439 was constructed containing spirally wound membrane

440 with immobilized spores of Aspergillus awamori

441 NRRL 3112 in a regime of recirculation which was

442 successfully applied for phenol biodegradation The

443 obtained results confirmed the advantages of the

444 recycled reactor with spirally wound membrane Thus

445 the immobilized microbial technology is an extremely

446 versatile approach that can be used for degradation of

447 toxic pollutants from the industrial effluents

448 Acknowledgments The authors gratefully acknowledge to449 the Bulgarian Ministry for Education and to the National450 Science Fund for their financial support and encouragement of451 the scientific research work in state universities

452 References

453 Abd-El-Haleem D Beshay U Abdelhamid AO Moawad H454 Zaki S (2003) Effects of mixed nitrogen sources on bio-455 degradation of phenol by immobilized Acinetobacter sp456 strain W-17 Afr J Biotechnol 2(1)8ndash12457 Allsop PJ Chisti Y Moi-Youngand M Sullivan GR (1993)458 Dynamics of phenol degradation by Pseudomonas putida459 Biotechnol Bioeng 41572ndash579460 Anselmo AM Novais JM (1992) Degradation of phenol by461 immobilized mycelium of Fusarium floccieferum in con-462 tinuous culture Water Sci Technol 25161ndash168463 Bolanos ML Varesche MBA Zaiat M Foresti E (2001) Phenol464 degradation in horizontal-flow anaerobic immobilized465 biomass (HAIB) reactor under mesophilic conditions466 Water Sci Technol 44(4)167ndash174

467Chitra S Sekaran G Padmavathi S Gowri C (1995) Removal468of phenol from wastewater using a biocatalyst In Pro-469ceedings of the national symposium frontiers in applied470environmental microbiology pp 74ndash77471Chtourou M Ammar E Nasri M Medhioub K (2004) Isolation472of a yeast Trichosporon cutaneum able to use low473molecular weight phenolic compounds application to474olive mill waste water treatment J Chem Technol Bio-475technol 79869ndash878476Dimov K Samardjieva Pavlov P (1983) Laboratory practice477on technology of synthetic fibers Technica Sofia BG478Fiest CF Hegeman GD (1969) Phenol and benzoate metabo-479lism by Pseudomonas putida J Bacteriol 100869ndash877480Godjevargova T Aleksieva Z Ivanova D (2000) Cell immo-481bilization of Trichosporon cutaneum strain with phenol482degradation ability on new modified polymer carries483Process Biochem 35699ndash704484Godjevargova T Ivanova D Aleksieva Z Burdelova G (2006)485Biodegradation of phenol by immobilized Trichosporon

486cutaneum R57 on modified polymer membranes Process487Biochem 412342ndash2346488Gonzalez G Herrera MG Pena MM (2001) Biodegradation of489phenol in a continuous process comparative study of490stirred tank and fluidized-bed bioreactors Bioresour491Technol 76245ndash251492Hua L Sun ZH Leng Y (2005) Continuous biocatalytic reso-493lution of DL-pantolactone by cross-linked cells in494membrane bioreactor Process Biochem 401137ndash1142495Hudges EJL Bayly RC Skurray RA (1984) Evidence for496isofunctional enzymes in the degradation of phenol m-497and p-toluate and p-cresol at catechol meta-cleavage498pathways in Alcaligenes eutrophus J Bacteriol 15879ndash83499Jones KH Trudgill PW Hopper DJ (1995) Evidence of two500pathways for the metabolism of phenol by Aspergillus

501fumigatus Arch Microbiol 163176ndash181502Jose G Sanchez M Theodore T (2002) Membranes and mem-503brane reactors Wiley-VCH Veriag GmbH Weinheim504Karel S Libichi S Robertson C (1985) The immobilization of505whole cells engineering principles Chem Eng Sci506401321ndash1354

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

Fig 8 Concentration

profile of phenol in a

recirculating system with

immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

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of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

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507 Kowalska M Bodzek M Bohdziewicz J (1998) Biodegradation508 of phenols and cyanides using membranes immobilized509 microorganisms Process Biochem 33189ndash197510 Loh K Chung T Ang W (2000) Immobilized-cell membrane511 bioreactor for high-strength phenol wastewater J Environ512 Eng ASCE 12675ndash79513 Marrot B Barrios-Martinez A Moulin P Roche N (2006)514 Biodegradation of high phenol concentration by activated515 sludge in an immersed membrane bioreactor Biochem516 Eng J 30174ndash183517 Mordocco A Kuek C Jenkins R (1999) Continuous degradation518 of phenol at low concentration using immobilized Pseu-

519 domonas putida Enzyme Microb Technol 25530ndash536520 Pazarlioglu NK Telefoncu A (2005) Biodegradation of phenol521 by Pseudomonas putida immobilized on activated pumice522 particles Process Biochem 401807ndash1814523 Santos VL Heilbuth NM Braga T Monteiro AS Linardi VR524 (2003) Phenol degradation by aGraphium sp FIB4 isolated525 from industrial effluents J Basic Microbiol 43238ndash248526 Shetty KV Kalifathulla I Srinikethan G (2007a) Performance527 of pulsed plate bioreactor for biodegradation of phenol528 J Hazard Mater 140346ndash352529 Shetty KV Ramanjaneyulu R Srinikethan G (2007b) Biolog-530 ical phenol removal using immobilized cells in a pulsed

531plate bioreactor effect of dilution rate and influent phenol532concentration J Hazard Mater 149452ndash459533Stephenson T Judd S Jefferson B Brindle K (2000) Mem-534brane bioreactors for wastewater treatment IWA535Publishing London UK pp 129ndash136536Sundar P Daniel D Krastanov A (2005) Biodegradation of537phenol by immobilized Aspergillus awamori cells Bulg J538Agric Sci 1161ndash71539Uzunova K Vassileva A Ivanova V Spasova D Tonkova A540(2002) Thermostable exo-inulinase production by semi-541continuous cultivation of membrane-immobilized Bacillus542sp 11 cells Process Biochem 37863ndash868543Wang Z Wan L Xu Z (2007) Surface engineerings of poly-544acrylonitrile-based asymmetric membranes towards545biomedical applications an overview J Membr Sci5463048ndash23547Yiang Y Wen J Li H Yang S Hu Z (2005) The biodegra-548dation of phenol at high initial concentration by the yeast549Candida tropicalis Biochem Eng J 24243ndash247550Yordanova G Ivanova D Godjevargova T Krastanov A (2006)551Biodegradation of phenol by immobilized Aspergillus

552awamori NRRL3112 cells Food science engineering and553technologies conference Scientific works Plovdiv554pp 260ndash265

555

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

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Accepted 18 March 2009

Abstract Covalent immobilization of Aspergillus awamori NRRL 3112 was conducted onto modified polyacrylonitrilemembrane with glutaraldehyde as a coupling agent The polymer carrier was preliminarily modified in anaqueous solution of NaOH and 12-diaminoethane The content of amino groups was determined to be058 mgeq g minus1 Two ways of immobilization were usedmdashin the presence of 02 g l minus1 phenol and withoutphenol The capability of two immobilized system to degrade phenol (concentrationmdash05 g l minus1) as a solecarbon and energy source was investigated in batch experiments Seven cycles of phenol biodegradation wereconducted Better results were obtained with the immobilized system prepared in the presence of phenolregarding degradation time and phenol biodegradation rate Scanning electron micrographs of thepolyacrylonitrile membraneimmobilized Aspergillus awamori NRRL at the beginning of repeated batchcultivation and after the 7th cycle were compared After the 7th cycle of cultivation the observations showedlarge groups of cells The results from the batch experiments with immobilized system were compared to theresults produced by the free strain Phenol biodegradation experiments were carried out also in a bioreactorwith spirally wound membrane with bound Aspergillus awamori NRRL 3112 in a regime of recirculation10 cycles of 05 g l minus1 phenol biodegradation were run consecutively to determine the degradation time andrate for each cycle The design of the bioreactor appeared to be quite effective providing large membranesurface to bind the strain

Keywords (separated by -) Biodegradation - Phenol - Aspergillus awamori NRRL3112 - Immobilization - Polymer membrane

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Please position each reference in the text or delete it Any reference not dealt with will be retained in this section Queries andor remarks Sectionparagraph Details required Authorrsquos response

References Please provide complete details and confirm the proceedings name for the reference Chitra et al (1995)

References Please provide initials for author name Samardjieva for the reference Dimov et al (1983)

References Please confirm publisher and place for the reference Jose et al (2002)

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Journal 10532 Article 9259

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ORIGINAL PAPER1

2 Biodegradation of phenol by immobilized Aspergillus

3 awamori NRRL 3112 on modified polyacrylonitrile

4 membrane

5 G Yordanova D Ivanova T Godjevargova

6 A Krastanov

7 Received 22 April 2008 Accepted 18 March 20098 Springer Science+Business Media BV 2009

9 Abstract Covalent immobilization of Aspergillus

10 awamori NRRL 3112 was conducted onto modified

11 polyacrylonitrile membrane with glutaraldehyde as a

12 coupling agent The polymer carrier was preliminarily

13 modified in an aqueous solution of NaOH and 12-

14 diaminoethane The content of amino groups was

15 determined to be 058 mgeq g-1 Two ways of immo-

16 bilization were usedmdashin the presence of 02 g l-1

17 phenol and without phenol The capability of two

18 immobilized system to degrade phenol (concentra-

19 tionmdash05 g l-1) as a sole carbon and energy source

20 was investigated in batch experiments Seven cycles of

21 phenol biodegradation were conducted Better results

22 were obtained with the immobilized system prepared

23 in the presence of phenol regarding degradation time

24 and phenol biodegradation rate Scanning electron

25 micrographs of the polyacrylonitrile membrane

26 immobilized Aspergillus awamori NRRL at the

27 beginning of repeated batch cultivation and after the

28 7th cycle were compared After the 7th cycle of

29 cultivation the observations showed large groups of

30 cells The results from the batch experiments with

31 immobilized system were compared to the results

32produced by the free strain Phenol biodegradation

33experiments were carried out also in a bioreactor with

34spirally wound membrane with bound Aspergillus

35awamori NRRL 3112 in a regime of recirculation 10

36cycles of 05 g l-1 phenol biodegradation were run

37consecutively to determine the degradation time and

38rate for each cycle The design of the bioreactor

39appeared to be quite effective providing large mem-

40brane surface to bind the strain

41Keywords Biodegradation Phenol

42Aspergillus awamori NRRL3112

43Immobilization Polymer membrane

44

45

46Introduction

47One of the problems attracting highest attention in

48modern ecology is related to the biodegradation of

49xenobiotics Typical representatives of this group of

50contaminants are phenol and its derivatives The

51environmental clean-up of phenol by adsorption

52solvent extraction chemical oxidation incineration

53and abiotic treatment procedure suffers from serious

54drawbacks such as economic issues and the produc-

55tion of hazardous byproducts Biodegradation is

56generally preferred due to the lower costs and the

57complete mineralization

58Extensive studies revealing the potential of micro-

59organisms in phenol biodegradation have been carried

A1 G Yordanova D Ivanova T Godjevargova (amp)

A2 Department of Biotechnology University lsquolsquoProf Dr A

A3 Zlatarovrsquorsquo 1 Prof Yakimov Str 8010 Bourgas Bulgaria

A4 e-mail godjevargovayahoocom

A5 A Krastanov

A6 Department of Biotechnology University of Food

A7 Technologies 4002 Plovdiv Bulgaria

123

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Biodegradation

DOI 101007s10532-009-9259-x

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60 out Major part of the research in the field of phenol

61 decomposition have been performed using bacterial

62 strains such as Alcaligenes europhus (Hudges et al

63 1984) and Pseudomonas sp (Allsop et al 1993)

64 Other publications report biodegradation of phenol by

65 yeast strainsCandida (MacGillivray and Shiaris 1993)

66 and Trichosporon (Chtourou et al 2004) Chitra et al

67 (1995) have studied the removal of phenol using a

68 mutant strain of Pseudomonas The use of fungal

69 strains for the degradation is relatively untouched area

70 Mycelial fungi such as Fusarium floccieferum

71 (Anselmo and Novais 1992) Aspergillus fumigatus

72 (Jones et al 1995) and Graphium sp (Santos et al

73 2003) have been cited for their potential of phenol

74 degradation Little is known about phenol metabolism

75 in mycelial fungi They have some advantages

76 because of their resistance to a great number of

77 xenobiotics toxic to the most of the microorganisms

78 The fungal strain Aspergillus awamori NRRL 3112 is

79 capable of assimilating a wide range of carbon

80 substrates including phenol and its derivatives (Sun-

81 dar et al 2005 Yordanova et al 2006) This feature

82 makes them a valuable research object for a future

83 development of industrial-water cleaning technology

84 There has been an increasing interest in the

85 immobilization of microorganisms in recent years

86 (Abd-El-Haleem et al 2003 Bolanos et al 2001

87 Gonzalez et al 2001 Fiest and Hegeman 1969

88 Mordocco et al 1999 Pazarlioglu and Telefoncu

89 2005 Shetty et al 2007a b) The immobilized cells

90 have advantages over the native cells due to the

91 possibility for multiple applications for a extended

92 period of time Different carriers have been used for

93 microbial cell immobilization but there arenrsquot many

94 reports for cell immobilization on polymer membranes

95 (Uzunova et al 2002 Marrot et al 2006 Hua et al

96 2005 Godjevargova et al 2006) Synthetic polymers

97 constitute the largest group of supports in use Poly-

98 acrylonitrile-based ultrafiltration (UF) membranes

99 obviously turn up to be more advantageous over other

100 conventional membranes in various aspects such as

101 thermal stability resistance to most organic solvents

102 commercial availability etc (Wang et al 2007)

103 PAN-based membranes can offer some special prom-

104 ises as cradle for cell immobilization which is mainly

105 because different reactive groups can be easily gen-

106 erated on the PAN membrane surface for the covalent

107 immobilization (Godjevargova et al 2006 Kowalska

108 et al 1998)

109The membranes are very suitable for immobiliza-

110tion of microbial cells when a spiral membrane

111module is used There is no common strategy for

112preparing such integrated systems and opportunities

113for using are still at hand Due to their high surface

114area per unit module volume spiral membrane

115modules serve as better immobilization supports than

116alginate beads activated carbon sintered glass and

117polymer beads (Jose et al 2002 Karel et al 1985)

118On the other hand polymer membranes can act as

119barriers limiting the movement of cell mass thus

120providing restricted andor regulated passage of one

121or more species through the pores From this point of

122view membrane bioreactors are promising for

123practical applications for wastewater treatment (Loh

124et al 2000 Stephenson et al 2000)

125The present paper investigates the possibility of

126immobilizing Asp awamori NRRL 3112 spores on

127modified polyacrylonitrile membrane and their ability

128to biodegrade phenol Spiral membrane module with

129immobilized spores was created and phenol biodeg-

130radation was also investigated

131Materials and methods

132Microorganism and growth medium

133A strain of Aspergillus awamoriNRRL 3112 obtained

134fromUSDepartment of Agriculture Illinois USAwas

135used throughout this study The organism was grown

136on slants immersed in a medium having the following

137composition (g l-1) malt extract 30 yeast extract 30

138peptone 50 glucose 100 and agar 200 The organism

139on the slants was allowed to grow for 72 h at 30C and

140then stored at 4 plusmn 1C for further use

141Medium for degradation studies

142The studies on the biodegradation of phenol were

143carried out in the Czapekdox medium which had the

144following composition (g l-1) sodium nitrate 20

145potassium phosphate (dibasic) 10 potassium chlo-

146ride 05 magnesium sulfate heptahydrate 05 and

147ferrous sulfate heptahydrate 001 The initial pH of

148the medium was adjusted to 55 (the optimal pH for

149this strain) using 1 N NaOH or 1 N HCI (Sundar

150et al 2005) The minimal medium contains phenol as

151a sole carbon source with 05 g l-1 concentration

Biodegradation

123

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152 Chemical modification of polyacrylonitrile

153 membrane

154 The ultrafiltration polyacrylonitrile (PAN) membrane

155 (average pore size of selective layer 002 lm) was

156 produced by Ecofilter Co (Bourgas Bulgaria) PAN

157 membrane was treated with 10 aqueous solution of

158 NaOH at 50C for 60 min After thorough washing

159 with distilled water the membrane was treated with a

160 10 aqueous solution of 12-diaminohexane for

161 60 min at 40C The membrane was once again

162 thoroughly washed with distilled water At the end of

163 the procedure the modified carrier was immersed in

164 05 aqueous solution of glutaraldehyde for 60 min

165 at room temperature Excess glutaraldehyde was

166 removed by washing the membranes with distilled

167 water until there was a complete absence of glutar-

168 aldehyde in the rinse waters

169 Phenol biodegradation in batch experiments

170 Spore immobilization for batch experiments

171 The 14-day culture in spore form in concentration of

172 333 9 107 ml-1 were introduced by flushing 5 cm3

173 sterile water in 500 cm3 sterile flask containing

174 50 cm3 Czapekdox medium and sterile modified

175 carrier (40 cm2 PAN flat membrane) Sterilization of

176 the modified membrane was carried out by ethanol

177 Two ways of immobilization were usedmdashin the

178 presence of 02 g l-1 phenol and without phenol

179 The two flasks were placed on a shaker for 24 h at

180 30C Then the liquid medium was decanted and the

181 immobilized systems were washed several times with

182 sterile water to remove the unbound spores After

183 that the immobilized spores were used for phenol

184 biodegradation

185 Batch experiments

186 The immobilized spores onto the polymer mem-

187 brane (single flat piecemdash40 cm2) with concentration

188 0158 g cm-2 were introduced in 500 cm3 sterile

189 flask containing 50 cm3 Czapekdox medium as well

190 as phenol as the only carbon source adjusted to the

191 appropriate concentration Then they were cultivated

192 on a shaker (220 rpm) at 30C until the complete

193 phenol degradation At every 24 h samples were

194 withdrawn and analyzed for phenol concentration

195Thus seven cycles were carried out with the same

196immobilized system

197Simultaneously parallel experiments with free

198cells were also performed for comparison The flasks

199containing 50 cm3 liquid medium as well as 05 g l-1

200phenol as the only carbon source were inoculated with

201equal volume of inoculum (333 9 107 conidia cm-3

202medium) and agitated on a shaker (220 rpm) at 30C

203Samples were taken at every 24 h interval for

204determination of phenol concentration After the

205biodegradation of the entire phenol the cultural

206medium was decanted and the biomass was reculti-

207vated in fresh liquid medium containing 05 g l-1

208phenol Thus four cycles were carried out in the same

209manner

210Each experiment for phenol degradation was

211repeated twice the reproducibility being always within

21225

213Phenol biodegradation in recycle reactor

214with spirally wound membrane

215Spore immobilization for experiments

216with membrane bioreactor

217The bioreactor was 40 mm in diameter and 100 mm

218high it was made of glass and equipped with

219thermostatic bath (Fig 1) The modified and activated

220with glutaraldehyde polyacrylonitrile membrane with

Fig 1 Scheme of recycle system thermostatic bath (1) feed

reservoir (2) peristaltic pump (3) thermostatic reactor (4)

membrane with immobilized cells (5) sampling (6) air outlet

(7) and air inlet (8)

Biodegradation

123

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Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

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221 surface area of 100 cm2 was spirally wound in the

222 glass bioreactor using supporting polyethylene mesh

223 In this case the immobilization was carried out by

224 flowing spore suspension (547 9 107 conidia cm-3

225 medium) through the membrane module using peri-

226 staltic pump at rate of 1 ml min-1 for 24 h Then the

227 membrane spiral was thoroughly washed out with

228 sterile water fed by peristaltic pump to remove the

229 non-bound spores

230 Experiments with membrane bioreactor

231 Further the liquid medium (300 ml) containing

232 phenol at certain concentration was fed to the mem-

233 brane bioreactor using peristaltic pump at rate of

234 15 ml min-1 The temperature required for the strain

235 growth (30C) was maintained using the thermostat

236 and the heat-exchanging coating After the full

237 degradation of phenol in the nutrient medium the

238 reservoir was charged with fresh nutrient medium

239 containing phenol Samples were taken at every 24 h

240 interval for determination of phenol concentration

241 Ten cycles were carried out in the same manner

242 Each experiment was repeated twice the repro-

243 ducibility being always within 25

244 Analytical methods

245 Phenol concentration was measured spectrophoto-

246 metrically in the presence of p-nitroaniline at 495 nm

247 (Gonzalez et al 2001)

248 The amount of spores immobilized on the support

249 was estimated by the difference between the wet

250 weight of spores with membrane at the beginning

251 (immediately after the immobilization) and the wet

252 membrane weight The amount of free cells was

253 determined by the measuring of the wet weight of the

254 cells and by the counting of the number of conidia in

255 the samples using a standard microscope method

256 Scanning electron microscopy (SEM)

257 The immobilized system Aspergillus awamori NRRL

258 3112polyacrylonitrile membrane was washed with

259 distilled water and then they were dehydrated in

260 30ndash100 water ethanol series The membrane were

261 coated with 120ndash130 Aacuteof gold in argon medium in

262 an Edwards apparatus (model 150 A) The SEM

263 observations were done on a scanning device attached

264to a Zeiss electron microscope (model 10C) at 20 kV

265accelerating voltage with a 5ndash6 nm electron beam

266Results and discussions

267Modification of membranes

268The aim of the present studies is to obtain an efficient

269immobilized system of Aspergillus awamori NRRL

2703112polyacrylonitrile membrane for biodegradation

271of phenol The immobilization of spores was carried

272out with glutaraldehyde The polymer membrane was

273preliminarily treated by using two different modifica-

274tion procedures The first procedure introduced

275ndashCOOH and ndashNH2 groups on the membrane surface

276by using NaOH solution In order to increase the

277amount of amine groups on the surface of the carrier

278the latter was treated with 12-diaminoethane The

279optimal conditions of modification were defined in a

280previous research work (Godjevargova et al 2000)

281The superficial carboxyl groups on the carrier surface

282reacted with one of the amine groups of 12-diami-

283noethane leaving the other one free The results

284showed that the PANmembranes had beenmodified in

285a greater extent and the content of amino groups was

286058 mgeq g-1 The latter was determined titrimetri-

287cally (Dimov et al 1983) Further the modified

288membrane was treated with an aqueous solution of

289glutaraldehyde The bifunctional agent bound the

290strain to the membrane surface

291Batch experiments

292Spores concentration used for the immobilization was

293333 9 107 ml-1 The first task was to determine

294which immobilization method would serve better for

295these experimentsmdashin the presence or in the absence

296of phenol (02 g l-1) in a nutrient medium containing

297spore suspension The amount of spores bound to the

298membranes in both cases was 0158 g cm-2 (wet

299weight) Then the spores immobilized on the polymer

300membrane by both methods were cultivated in a

301nutrient medium containing 05 g l-1 phenol The

302results obtained for phenol biodegradation by both

303systems are presented in Fig 2a b The rate of

304phenol biodegradation was determined after the end

305of each cycle for both immobilization systems

306(Table 1) As can be seen from Table 1 the lowest

Biodegradation

123

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307 biodegradation rate is observed at the first cycle then

308 it increases 3 times at the second and the third cycles

309 and from the fourth to the seventh cycle the rate

310 decreases continuously This trend of the biodegra-

311 dation rate is observed for both of the immobilized

312 systems The deceleration of the phenol biodegrada-

313 tion could be explained with the increasing of

314 biomass and mycelia formation on the membrane

315 surface after the fourth cycle which intensified at the

316 final cycles An obvious inhibition of the biodegra-

317 dation process at the final cycles was observed which

318 was due to the hindrance of the substrate diffusion to

319 the cells When comparing the results presented on

320 Fig 2a and b and Table 1 it becomes obvious that the

321 rate of phenol degradation was higher throughout all

322 cycles in the presence of the system where the spore

323 immobilization was conducted with addition of

32402 g l-1 phenol For better comparison the biodeg-

325radation times of each cycle with the two

326immobilized systems are presented in Fig 3 As

0

02

04

06

08

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

0

02

04

06

08

0 200 400 600 800

0 200 400 600 800 1000

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

(a)

(b)

Fig 2 Phenol

biodegradation (05 g l-1)

by immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane obtained by

immobilization in presence

of 02 g l-1 phenol (a) and

without phenol (b)

Table 1 Phenol biodegradation rate (mg h-1) of free and

immobilized Aspergillus awamori NRRL 3112 on polymer

PAN membrane at batch cultivation with 05 g l-1 phenol

Numberof

cycles

Free

cells

Immobilized cells

without 02 g l-1

phenol

Immobilized cells

in the presence of

02 g l-1 phenol

1 297 300 300

2 201 714 694

3 149 806 543

4 116 625 367

5 403 329

6 308 300

7 275 270

Biodegradation

123

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327 results suggest better degradation times were

328 obtained with the immobilized system prepared in

329 the presence of phenol This can be explained with

330 the fact that the strain could have adapted to the

331 substrate during the immobilization process All

332 further experiments were carried out with the immo-

333 bilized system prepared in presence of phenol

334 The biodegradation ability of the immobilized

335 system onto polyacrylonitrile membrane towards

336 phenol in a concentration range from 02 to

337 08 g l-1 was studied As can be seen from Fig 4

338 the increase of phenol concentration resulted in

339increased biodegradation time For instance the

340biodegradation cycle at concentration of 04 g l-1

341was 66 h while at 07 g l-1 the cycle lasted for 135 h

342The results obtained with the selected immobilized

343system were compared to those with the free strain

344For this purpose the spores (08 9 107 ml-1)

345were cultivated in Czapekdox medium containing

34605 g l-1 phenol (Fig 5) Four cycles were carried

347out in the same manner The amount of non-bound

348spores in the first cycle of cultivation is equivalent

349to the amount of immobilized spores on PAN

350membrane with surface 40 cm2 in the first cycle

0

50

100

150

200

250

1 2 3 4 5 6 7

Cycles

Tim

e

h

Fig 3 Cycles duration of

phenol biodegradation

(05 g l-1) by two

immobilized systems

Aspergillus awamori NRRL

3112modified PAN

membrane obtained by

immobilization in presence

of 02 g l-1 phenol ( ) and

without phenol (j)

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol co

ncen

trati

on

g

l-1

Fig 4 Phenol

biodegradation (from 02 to

08 g l-1) by immobilized

system Aspergillus

awamori NRRL 3112

modified PAN membrane

Biodegradation

123

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351 (wet weightmdash632 g) As can be seen from Fig 5 the

352 phenol degradation time was considerably greater for

353 the four cycles in these experiments This time

354 extension was confirmed by the calculated rate of

355 biodegradation (Table 1) When comparing the rates

356 of phenol biodegradation by the free and the immo-

357 bilized cells the greater stability of the immobilized

358 systems over the free cells becomes more prominent

359 after multiple cultivation in media containing phenol

360 with concentration 05 g l-1 Obviously the free cells

361 were inhibited to a greater extent by phenol (biodeg-

362 radation rate at Cycle 4mdash116 mg h-1) in

363 comparison with the immobilized cells (biodegrada-

364 tion rates at Cycle 4 are 625 and 367 mg h-1

365respectively for immobilized system obtained in the

366presence of 02 g l-1 phenol and the system without

367phenol)

368In order to distinguish between phenol adsorption

369on the membrane surface and phenol degradation by

370microorganisms the pure carrier was also studied for

371phenol adsorption For this purpose 40 cm2 pure

372membrane was immersed in 05 g l-1 phenol solution

373for 6 h The results are presented in Fig 6 Since the

374phenol concentration didnrsquot change significantly

375the only possible reason for the decrease of the

376phenol content was the degradation activity of the

377immobilized cells This simplifies the kinetic studies

378when membrane is to be used as a support material

0

02

04

06

08

0 100 200 300 400 500

Time h

Ph

en

ol

bio

de

gra

da

tio

n

gl-1

Fig 5 Phenol

biodegradation with

concentration 05 g l-1 by

strain Aspergillus awamori

NRRL 3112

0

02

04

06

08

0 1 2 3 4 5 6 7

Time h

Ph

en

ol

bio

deg

rad

ati

on

g

l-1

Fig 6 Adsorption of

phenol by modified PAN

membrane

Biodegradation

123

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379 for the immobilization of Asp awamori for the

380 purpose of phenol degradation

381 The amount of cells (wet weight) on the membrane

382 of 40 cm2 was measured after the sixth cycle of

383 phenol biodegradationmdash1104 g cm-2 The signifi-

384 cant increase of the biomass with the increased

385 number of cycles is obvious (after immobilizationmdash

386 0158 g cm-2) Thus the biomass accumulated on

387 the membrane was approximately ten times higher

388 after six cycles of phenol biodegradation The high

389 operational stability of investigated biocatalysts was

390 confirmed by the scanning electron micrographs of

391 the polyacrylonitrile membraneimmobilized Asper-

392 gillus awamori NRRL at the beginning of repeated

393 batch cultivation and after the 7th cycle After the 7th

394 cycle of cultivation the observations showed large

395 groups of cells (Fig 7a) The significant increase of

396 biomass and mycelia formation on the membrane

397 surface after the 7th cycle of cultivation was inev-

398 itably followed by deceleration of the phenol

399 degradation rate

400Experiments with membrane bioreactor

401The next step of the research work involved exper-

402iments which were carried out in a bioreactor with

403spirally wound membrane in a regime of recircula-

404tion The spores were immobilized onto the

405membrane directly in the system as described in

406lsquolsquoMaterials and methodsrsquorsquo section Phenol biodegra-

407dation was carried out under aerobic conditions The

408operation parameters of the bioreactor are presented

409in Table 2 Fresh nutrient medium containing

41005 g l-1 phenol were added after each cycle Phenol

411concentration was monitored for degradation At the

412end of a cycle the system was washed with sterile

413distilled water and charged with nutrient medium and

414phenol for the next cycle Thus 10 cycles of 05 g l-1

415phenol biodegradation were run consecutively to

416determine the degradation time for each cycle

417(Fig 8) No significant difference was observed

418between the degradation times of the cycles from

419the second to the seventh (about 50 h) and between

420the biodegradation rates (100 mg h-1) After the 7th

421cycle the biodegradation rate began to decrease

422gradually and at 10 cycles it was 40 mg h-1 These

423results confirmed the advantages of the recycled

424reactor with spirally wound membrane The design of

425the bioreactor appeared to be quite effective provid-

426ing large membrane surface to bind the strain cells

427Besides phenol would flow tangentially which pre-

428vents membrane from a decrease of the flow velocity

429Conclusion

430A novel immobilized systemmdashAspergillus awamori

431NRRL 3112polyacrylonitrile membranewas obtained

432for the purpose of phenol biodegradation It was

433established that the immobilized system prepared in

434the presence of 02 g l-1 phenol was more effective

Fig 7 Scanning electron micrographs of immobilized system

Aspergillus awamori NRRL 3112modified PAN membrane

after 7th cycle (a) and before 1st cycle (b)

Table 2 Operation conditions of bioreactor

Parameter Value

Rate flow 15 ml min-1

Volume 300 ml

Initial phenol concentration 05 g l-1

Membrane surface area 100 cm2

Immobilized spores 0174 g cm-2 (wet weight)

Biodegradation

123

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435 than the system prepared in the phenol absence It was

436 revealed that the immobilized systems were more

437 stable andmore effective when biodegrading 05 g l-1

438 phenol in comparison with the free cells A bioreactor

439 was constructed containing spirally wound membrane

440 with immobilized spores of Aspergillus awamori

441 NRRL 3112 in a regime of recirculation which was

442 successfully applied for phenol biodegradation The

443 obtained results confirmed the advantages of the

444 recycled reactor with spirally wound membrane Thus

445 the immobilized microbial technology is an extremely

446 versatile approach that can be used for degradation of

447 toxic pollutants from the industrial effluents

448 Acknowledgments The authors gratefully acknowledge to449 the Bulgarian Ministry for Education and to the National450 Science Fund for their financial support and encouragement of451 the scientific research work in state universities

452 References

453 Abd-El-Haleem D Beshay U Abdelhamid AO Moawad H454 Zaki S (2003) Effects of mixed nitrogen sources on bio-455 degradation of phenol by immobilized Acinetobacter sp456 strain W-17 Afr J Biotechnol 2(1)8ndash12457 Allsop PJ Chisti Y Moi-Youngand M Sullivan GR (1993)458 Dynamics of phenol degradation by Pseudomonas putida459 Biotechnol Bioeng 41572ndash579460 Anselmo AM Novais JM (1992) Degradation of phenol by461 immobilized mycelium of Fusarium floccieferum in con-462 tinuous culture Water Sci Technol 25161ndash168463 Bolanos ML Varesche MBA Zaiat M Foresti E (2001) Phenol464 degradation in horizontal-flow anaerobic immobilized465 biomass (HAIB) reactor under mesophilic conditions466 Water Sci Technol 44(4)167ndash174

467Chitra S Sekaran G Padmavathi S Gowri C (1995) Removal468of phenol from wastewater using a biocatalyst In Pro-469ceedings of the national symposium frontiers in applied470environmental microbiology pp 74ndash77471Chtourou M Ammar E Nasri M Medhioub K (2004) Isolation472of a yeast Trichosporon cutaneum able to use low473molecular weight phenolic compounds application to474olive mill waste water treatment J Chem Technol Bio-475technol 79869ndash878476Dimov K Samardjieva Pavlov P (1983) Laboratory practice477on technology of synthetic fibers Technica Sofia BG478Fiest CF Hegeman GD (1969) Phenol and benzoate metabo-479lism by Pseudomonas putida J Bacteriol 100869ndash877480Godjevargova T Aleksieva Z Ivanova D (2000) Cell immo-481bilization of Trichosporon cutaneum strain with phenol482degradation ability on new modified polymer carries483Process Biochem 35699ndash704484Godjevargova T Ivanova D Aleksieva Z Burdelova G (2006)485Biodegradation of phenol by immobilized Trichosporon

486cutaneum R57 on modified polymer membranes Process487Biochem 412342ndash2346488Gonzalez G Herrera MG Pena MM (2001) Biodegradation of489phenol in a continuous process comparative study of490stirred tank and fluidized-bed bioreactors Bioresour491Technol 76245ndash251492Hua L Sun ZH Leng Y (2005) Continuous biocatalytic reso-493lution of DL-pantolactone by cross-linked cells in494membrane bioreactor Process Biochem 401137ndash1142495Hudges EJL Bayly RC Skurray RA (1984) Evidence for496isofunctional enzymes in the degradation of phenol m-497and p-toluate and p-cresol at catechol meta-cleavage498pathways in Alcaligenes eutrophus J Bacteriol 15879ndash83499Jones KH Trudgill PW Hopper DJ (1995) Evidence of two500pathways for the metabolism of phenol by Aspergillus

501fumigatus Arch Microbiol 163176ndash181502Jose G Sanchez M Theodore T (2002) Membranes and mem-503brane reactors Wiley-VCH Veriag GmbH Weinheim504Karel S Libichi S Robertson C (1985) The immobilization of505whole cells engineering principles Chem Eng Sci506401321ndash1354

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

Fig 8 Concentration

profile of phenol in a

recirculating system with

immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

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of

UNCORRECTEDPR

OOF

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507 Kowalska M Bodzek M Bohdziewicz J (1998) Biodegradation508 of phenols and cyanides using membranes immobilized509 microorganisms Process Biochem 33189ndash197510 Loh K Chung T Ang W (2000) Immobilized-cell membrane511 bioreactor for high-strength phenol wastewater J Environ512 Eng ASCE 12675ndash79513 Marrot B Barrios-Martinez A Moulin P Roche N (2006)514 Biodegradation of high phenol concentration by activated515 sludge in an immersed membrane bioreactor Biochem516 Eng J 30174ndash183517 Mordocco A Kuek C Jenkins R (1999) Continuous degradation518 of phenol at low concentration using immobilized Pseu-

519 domonas putida Enzyme Microb Technol 25530ndash536520 Pazarlioglu NK Telefoncu A (2005) Biodegradation of phenol521 by Pseudomonas putida immobilized on activated pumice522 particles Process Biochem 401807ndash1814523 Santos VL Heilbuth NM Braga T Monteiro AS Linardi VR524 (2003) Phenol degradation by aGraphium sp FIB4 isolated525 from industrial effluents J Basic Microbiol 43238ndash248526 Shetty KV Kalifathulla I Srinikethan G (2007a) Performance527 of pulsed plate bioreactor for biodegradation of phenol528 J Hazard Mater 140346ndash352529 Shetty KV Ramanjaneyulu R Srinikethan G (2007b) Biolog-530 ical phenol removal using immobilized cells in a pulsed

531plate bioreactor effect of dilution rate and influent phenol532concentration J Hazard Mater 149452ndash459533Stephenson T Judd S Jefferson B Brindle K (2000) Mem-534brane bioreactors for wastewater treatment IWA535Publishing London UK pp 129ndash136536Sundar P Daniel D Krastanov A (2005) Biodegradation of537phenol by immobilized Aspergillus awamori cells Bulg J538Agric Sci 1161ndash71539Uzunova K Vassileva A Ivanova V Spasova D Tonkova A540(2002) Thermostable exo-inulinase production by semi-541continuous cultivation of membrane-immobilized Bacillus542sp 11 cells Process Biochem 37863ndash868543Wang Z Wan L Xu Z (2007) Surface engineerings of poly-544acrylonitrile-based asymmetric membranes towards545biomedical applications an overview J Membr Sci5463048ndash23547Yiang Y Wen J Li H Yang S Hu Z (2005) The biodegra-548dation of phenol at high initial concentration by the yeast549Candida tropicalis Biochem Eng J 24243ndash247550Yordanova G Ivanova D Godjevargova T Krastanov A (2006)551Biodegradation of phenol by immobilized Aspergillus

552awamori NRRL3112 cells Food science engineering and553technologies conference Scientific works Plovdiv554pp 260ndash265

555

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

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References Please provide complete details and confirm the proceedings name for the reference Chitra et al (1995)

References Please provide initials for author name Samardjieva for the reference Dimov et al (1983)

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Journal 10532 Article 9259

UNCORRECTEDPR

OOF

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ORIGINAL PAPER1

2 Biodegradation of phenol by immobilized Aspergillus

3 awamori NRRL 3112 on modified polyacrylonitrile

4 membrane

5 G Yordanova D Ivanova T Godjevargova

6 A Krastanov

7 Received 22 April 2008 Accepted 18 March 20098 Springer Science+Business Media BV 2009

9 Abstract Covalent immobilization of Aspergillus

10 awamori NRRL 3112 was conducted onto modified

11 polyacrylonitrile membrane with glutaraldehyde as a

12 coupling agent The polymer carrier was preliminarily

13 modified in an aqueous solution of NaOH and 12-

14 diaminoethane The content of amino groups was

15 determined to be 058 mgeq g-1 Two ways of immo-

16 bilization were usedmdashin the presence of 02 g l-1

17 phenol and without phenol The capability of two

18 immobilized system to degrade phenol (concentra-

19 tionmdash05 g l-1) as a sole carbon and energy source

20 was investigated in batch experiments Seven cycles of

21 phenol biodegradation were conducted Better results

22 were obtained with the immobilized system prepared

23 in the presence of phenol regarding degradation time

24 and phenol biodegradation rate Scanning electron

25 micrographs of the polyacrylonitrile membrane

26 immobilized Aspergillus awamori NRRL at the

27 beginning of repeated batch cultivation and after the

28 7th cycle were compared After the 7th cycle of

29 cultivation the observations showed large groups of

30 cells The results from the batch experiments with

31 immobilized system were compared to the results

32produced by the free strain Phenol biodegradation

33experiments were carried out also in a bioreactor with

34spirally wound membrane with bound Aspergillus

35awamori NRRL 3112 in a regime of recirculation 10

36cycles of 05 g l-1 phenol biodegradation were run

37consecutively to determine the degradation time and

38rate for each cycle The design of the bioreactor

39appeared to be quite effective providing large mem-

40brane surface to bind the strain

41Keywords Biodegradation Phenol

42Aspergillus awamori NRRL3112

43Immobilization Polymer membrane

44

45

46Introduction

47One of the problems attracting highest attention in

48modern ecology is related to the biodegradation of

49xenobiotics Typical representatives of this group of

50contaminants are phenol and its derivatives The

51environmental clean-up of phenol by adsorption

52solvent extraction chemical oxidation incineration

53and abiotic treatment procedure suffers from serious

54drawbacks such as economic issues and the produc-

55tion of hazardous byproducts Biodegradation is

56generally preferred due to the lower costs and the

57complete mineralization

58Extensive studies revealing the potential of micro-

59organisms in phenol biodegradation have been carried

A1 G Yordanova D Ivanova T Godjevargova (amp)

A2 Department of Biotechnology University lsquolsquoProf Dr A

A3 Zlatarovrsquorsquo 1 Prof Yakimov Str 8010 Bourgas Bulgaria

A4 e-mail godjevargovayahoocom

A5 A Krastanov

A6 Department of Biotechnology University of Food

A7 Technologies 4002 Plovdiv Bulgaria

123

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Biodegradation

DOI 101007s10532-009-9259-x

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60 out Major part of the research in the field of phenol

61 decomposition have been performed using bacterial

62 strains such as Alcaligenes europhus (Hudges et al

63 1984) and Pseudomonas sp (Allsop et al 1993)

64 Other publications report biodegradation of phenol by

65 yeast strainsCandida (MacGillivray and Shiaris 1993)

66 and Trichosporon (Chtourou et al 2004) Chitra et al

67 (1995) have studied the removal of phenol using a

68 mutant strain of Pseudomonas The use of fungal

69 strains for the degradation is relatively untouched area

70 Mycelial fungi such as Fusarium floccieferum

71 (Anselmo and Novais 1992) Aspergillus fumigatus

72 (Jones et al 1995) and Graphium sp (Santos et al

73 2003) have been cited for their potential of phenol

74 degradation Little is known about phenol metabolism

75 in mycelial fungi They have some advantages

76 because of their resistance to a great number of

77 xenobiotics toxic to the most of the microorganisms

78 The fungal strain Aspergillus awamori NRRL 3112 is

79 capable of assimilating a wide range of carbon

80 substrates including phenol and its derivatives (Sun-

81 dar et al 2005 Yordanova et al 2006) This feature

82 makes them a valuable research object for a future

83 development of industrial-water cleaning technology

84 There has been an increasing interest in the

85 immobilization of microorganisms in recent years

86 (Abd-El-Haleem et al 2003 Bolanos et al 2001

87 Gonzalez et al 2001 Fiest and Hegeman 1969

88 Mordocco et al 1999 Pazarlioglu and Telefoncu

89 2005 Shetty et al 2007a b) The immobilized cells

90 have advantages over the native cells due to the

91 possibility for multiple applications for a extended

92 period of time Different carriers have been used for

93 microbial cell immobilization but there arenrsquot many

94 reports for cell immobilization on polymer membranes

95 (Uzunova et al 2002 Marrot et al 2006 Hua et al

96 2005 Godjevargova et al 2006) Synthetic polymers

97 constitute the largest group of supports in use Poly-

98 acrylonitrile-based ultrafiltration (UF) membranes

99 obviously turn up to be more advantageous over other

100 conventional membranes in various aspects such as

101 thermal stability resistance to most organic solvents

102 commercial availability etc (Wang et al 2007)

103 PAN-based membranes can offer some special prom-

104 ises as cradle for cell immobilization which is mainly

105 because different reactive groups can be easily gen-

106 erated on the PAN membrane surface for the covalent

107 immobilization (Godjevargova et al 2006 Kowalska

108 et al 1998)

109The membranes are very suitable for immobiliza-

110tion of microbial cells when a spiral membrane

111module is used There is no common strategy for

112preparing such integrated systems and opportunities

113for using are still at hand Due to their high surface

114area per unit module volume spiral membrane

115modules serve as better immobilization supports than

116alginate beads activated carbon sintered glass and

117polymer beads (Jose et al 2002 Karel et al 1985)

118On the other hand polymer membranes can act as

119barriers limiting the movement of cell mass thus

120providing restricted andor regulated passage of one

121or more species through the pores From this point of

122view membrane bioreactors are promising for

123practical applications for wastewater treatment (Loh

124et al 2000 Stephenson et al 2000)

125The present paper investigates the possibility of

126immobilizing Asp awamori NRRL 3112 spores on

127modified polyacrylonitrile membrane and their ability

128to biodegrade phenol Spiral membrane module with

129immobilized spores was created and phenol biodeg-

130radation was also investigated

131Materials and methods

132Microorganism and growth medium

133A strain of Aspergillus awamoriNRRL 3112 obtained

134fromUSDepartment of Agriculture Illinois USAwas

135used throughout this study The organism was grown

136on slants immersed in a medium having the following

137composition (g l-1) malt extract 30 yeast extract 30

138peptone 50 glucose 100 and agar 200 The organism

139on the slants was allowed to grow for 72 h at 30C and

140then stored at 4 plusmn 1C for further use

141Medium for degradation studies

142The studies on the biodegradation of phenol were

143carried out in the Czapekdox medium which had the

144following composition (g l-1) sodium nitrate 20

145potassium phosphate (dibasic) 10 potassium chlo-

146ride 05 magnesium sulfate heptahydrate 05 and

147ferrous sulfate heptahydrate 001 The initial pH of

148the medium was adjusted to 55 (the optimal pH for

149this strain) using 1 N NaOH or 1 N HCI (Sundar

150et al 2005) The minimal medium contains phenol as

151a sole carbon source with 05 g l-1 concentration

Biodegradation

123

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152 Chemical modification of polyacrylonitrile

153 membrane

154 The ultrafiltration polyacrylonitrile (PAN) membrane

155 (average pore size of selective layer 002 lm) was

156 produced by Ecofilter Co (Bourgas Bulgaria) PAN

157 membrane was treated with 10 aqueous solution of

158 NaOH at 50C for 60 min After thorough washing

159 with distilled water the membrane was treated with a

160 10 aqueous solution of 12-diaminohexane for

161 60 min at 40C The membrane was once again

162 thoroughly washed with distilled water At the end of

163 the procedure the modified carrier was immersed in

164 05 aqueous solution of glutaraldehyde for 60 min

165 at room temperature Excess glutaraldehyde was

166 removed by washing the membranes with distilled

167 water until there was a complete absence of glutar-

168 aldehyde in the rinse waters

169 Phenol biodegradation in batch experiments

170 Spore immobilization for batch experiments

171 The 14-day culture in spore form in concentration of

172 333 9 107 ml-1 were introduced by flushing 5 cm3

173 sterile water in 500 cm3 sterile flask containing

174 50 cm3 Czapekdox medium and sterile modified

175 carrier (40 cm2 PAN flat membrane) Sterilization of

176 the modified membrane was carried out by ethanol

177 Two ways of immobilization were usedmdashin the

178 presence of 02 g l-1 phenol and without phenol

179 The two flasks were placed on a shaker for 24 h at

180 30C Then the liquid medium was decanted and the

181 immobilized systems were washed several times with

182 sterile water to remove the unbound spores After

183 that the immobilized spores were used for phenol

184 biodegradation

185 Batch experiments

186 The immobilized spores onto the polymer mem-

187 brane (single flat piecemdash40 cm2) with concentration

188 0158 g cm-2 were introduced in 500 cm3 sterile

189 flask containing 50 cm3 Czapekdox medium as well

190 as phenol as the only carbon source adjusted to the

191 appropriate concentration Then they were cultivated

192 on a shaker (220 rpm) at 30C until the complete

193 phenol degradation At every 24 h samples were

194 withdrawn and analyzed for phenol concentration

195Thus seven cycles were carried out with the same

196immobilized system

197Simultaneously parallel experiments with free

198cells were also performed for comparison The flasks

199containing 50 cm3 liquid medium as well as 05 g l-1

200phenol as the only carbon source were inoculated with

201equal volume of inoculum (333 9 107 conidia cm-3

202medium) and agitated on a shaker (220 rpm) at 30C

203Samples were taken at every 24 h interval for

204determination of phenol concentration After the

205biodegradation of the entire phenol the cultural

206medium was decanted and the biomass was reculti-

207vated in fresh liquid medium containing 05 g l-1

208phenol Thus four cycles were carried out in the same

209manner

210Each experiment for phenol degradation was

211repeated twice the reproducibility being always within

21225

213Phenol biodegradation in recycle reactor

214with spirally wound membrane

215Spore immobilization for experiments

216with membrane bioreactor

217The bioreactor was 40 mm in diameter and 100 mm

218high it was made of glass and equipped with

219thermostatic bath (Fig 1) The modified and activated

220with glutaraldehyde polyacrylonitrile membrane with

Fig 1 Scheme of recycle system thermostatic bath (1) feed

reservoir (2) peristaltic pump (3) thermostatic reactor (4)

membrane with immobilized cells (5) sampling (6) air outlet

(7) and air inlet (8)

Biodegradation

123

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221 surface area of 100 cm2 was spirally wound in the

222 glass bioreactor using supporting polyethylene mesh

223 In this case the immobilization was carried out by

224 flowing spore suspension (547 9 107 conidia cm-3

225 medium) through the membrane module using peri-

226 staltic pump at rate of 1 ml min-1 for 24 h Then the

227 membrane spiral was thoroughly washed out with

228 sterile water fed by peristaltic pump to remove the

229 non-bound spores

230 Experiments with membrane bioreactor

231 Further the liquid medium (300 ml) containing

232 phenol at certain concentration was fed to the mem-

233 brane bioreactor using peristaltic pump at rate of

234 15 ml min-1 The temperature required for the strain

235 growth (30C) was maintained using the thermostat

236 and the heat-exchanging coating After the full

237 degradation of phenol in the nutrient medium the

238 reservoir was charged with fresh nutrient medium

239 containing phenol Samples were taken at every 24 h

240 interval for determination of phenol concentration

241 Ten cycles were carried out in the same manner

242 Each experiment was repeated twice the repro-

243 ducibility being always within 25

244 Analytical methods

245 Phenol concentration was measured spectrophoto-

246 metrically in the presence of p-nitroaniline at 495 nm

247 (Gonzalez et al 2001)

248 The amount of spores immobilized on the support

249 was estimated by the difference between the wet

250 weight of spores with membrane at the beginning

251 (immediately after the immobilization) and the wet

252 membrane weight The amount of free cells was

253 determined by the measuring of the wet weight of the

254 cells and by the counting of the number of conidia in

255 the samples using a standard microscope method

256 Scanning electron microscopy (SEM)

257 The immobilized system Aspergillus awamori NRRL

258 3112polyacrylonitrile membrane was washed with

259 distilled water and then they were dehydrated in

260 30ndash100 water ethanol series The membrane were

261 coated with 120ndash130 Aacuteof gold in argon medium in

262 an Edwards apparatus (model 150 A) The SEM

263 observations were done on a scanning device attached

264to a Zeiss electron microscope (model 10C) at 20 kV

265accelerating voltage with a 5ndash6 nm electron beam

266Results and discussions

267Modification of membranes

268The aim of the present studies is to obtain an efficient

269immobilized system of Aspergillus awamori NRRL

2703112polyacrylonitrile membrane for biodegradation

271of phenol The immobilization of spores was carried

272out with glutaraldehyde The polymer membrane was

273preliminarily treated by using two different modifica-

274tion procedures The first procedure introduced

275ndashCOOH and ndashNH2 groups on the membrane surface

276by using NaOH solution In order to increase the

277amount of amine groups on the surface of the carrier

278the latter was treated with 12-diaminoethane The

279optimal conditions of modification were defined in a

280previous research work (Godjevargova et al 2000)

281The superficial carboxyl groups on the carrier surface

282reacted with one of the amine groups of 12-diami-

283noethane leaving the other one free The results

284showed that the PANmembranes had beenmodified in

285a greater extent and the content of amino groups was

286058 mgeq g-1 The latter was determined titrimetri-

287cally (Dimov et al 1983) Further the modified

288membrane was treated with an aqueous solution of

289glutaraldehyde The bifunctional agent bound the

290strain to the membrane surface

291Batch experiments

292Spores concentration used for the immobilization was

293333 9 107 ml-1 The first task was to determine

294which immobilization method would serve better for

295these experimentsmdashin the presence or in the absence

296of phenol (02 g l-1) in a nutrient medium containing

297spore suspension The amount of spores bound to the

298membranes in both cases was 0158 g cm-2 (wet

299weight) Then the spores immobilized on the polymer

300membrane by both methods were cultivated in a

301nutrient medium containing 05 g l-1 phenol The

302results obtained for phenol biodegradation by both

303systems are presented in Fig 2a b The rate of

304phenol biodegradation was determined after the end

305of each cycle for both immobilization systems

306(Table 1) As can be seen from Table 1 the lowest

Biodegradation

123

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307 biodegradation rate is observed at the first cycle then

308 it increases 3 times at the second and the third cycles

309 and from the fourth to the seventh cycle the rate

310 decreases continuously This trend of the biodegra-

311 dation rate is observed for both of the immobilized

312 systems The deceleration of the phenol biodegrada-

313 tion could be explained with the increasing of

314 biomass and mycelia formation on the membrane

315 surface after the fourth cycle which intensified at the

316 final cycles An obvious inhibition of the biodegra-

317 dation process at the final cycles was observed which

318 was due to the hindrance of the substrate diffusion to

319 the cells When comparing the results presented on

320 Fig 2a and b and Table 1 it becomes obvious that the

321 rate of phenol degradation was higher throughout all

322 cycles in the presence of the system where the spore

323 immobilization was conducted with addition of

32402 g l-1 phenol For better comparison the biodeg-

325radation times of each cycle with the two

326immobilized systems are presented in Fig 3 As

0

02

04

06

08

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

0

02

04

06

08

0 200 400 600 800

0 200 400 600 800 1000

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

(a)

(b)

Fig 2 Phenol

biodegradation (05 g l-1)

by immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane obtained by

immobilization in presence

of 02 g l-1 phenol (a) and

without phenol (b)

Table 1 Phenol biodegradation rate (mg h-1) of free and

immobilized Aspergillus awamori NRRL 3112 on polymer

PAN membrane at batch cultivation with 05 g l-1 phenol

Numberof

cycles

Free

cells

Immobilized cells

without 02 g l-1

phenol

Immobilized cells

in the presence of

02 g l-1 phenol

1 297 300 300

2 201 714 694

3 149 806 543

4 116 625 367

5 403 329

6 308 300

7 275 270

Biodegradation

123

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327 results suggest better degradation times were

328 obtained with the immobilized system prepared in

329 the presence of phenol This can be explained with

330 the fact that the strain could have adapted to the

331 substrate during the immobilization process All

332 further experiments were carried out with the immo-

333 bilized system prepared in presence of phenol

334 The biodegradation ability of the immobilized

335 system onto polyacrylonitrile membrane towards

336 phenol in a concentration range from 02 to

337 08 g l-1 was studied As can be seen from Fig 4

338 the increase of phenol concentration resulted in

339increased biodegradation time For instance the

340biodegradation cycle at concentration of 04 g l-1

341was 66 h while at 07 g l-1 the cycle lasted for 135 h

342The results obtained with the selected immobilized

343system were compared to those with the free strain

344For this purpose the spores (08 9 107 ml-1)

345were cultivated in Czapekdox medium containing

34605 g l-1 phenol (Fig 5) Four cycles were carried

347out in the same manner The amount of non-bound

348spores in the first cycle of cultivation is equivalent

349to the amount of immobilized spores on PAN

350membrane with surface 40 cm2 in the first cycle

0

50

100

150

200

250

1 2 3 4 5 6 7

Cycles

Tim

e

h

Fig 3 Cycles duration of

phenol biodegradation

(05 g l-1) by two

immobilized systems

Aspergillus awamori NRRL

3112modified PAN

membrane obtained by

immobilization in presence

of 02 g l-1 phenol ( ) and

without phenol (j)

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol co

ncen

trati

on

g

l-1

Fig 4 Phenol

biodegradation (from 02 to

08 g l-1) by immobilized

system Aspergillus

awamori NRRL 3112

modified PAN membrane

Biodegradation

123

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351 (wet weightmdash632 g) As can be seen from Fig 5 the

352 phenol degradation time was considerably greater for

353 the four cycles in these experiments This time

354 extension was confirmed by the calculated rate of

355 biodegradation (Table 1) When comparing the rates

356 of phenol biodegradation by the free and the immo-

357 bilized cells the greater stability of the immobilized

358 systems over the free cells becomes more prominent

359 after multiple cultivation in media containing phenol

360 with concentration 05 g l-1 Obviously the free cells

361 were inhibited to a greater extent by phenol (biodeg-

362 radation rate at Cycle 4mdash116 mg h-1) in

363 comparison with the immobilized cells (biodegrada-

364 tion rates at Cycle 4 are 625 and 367 mg h-1

365respectively for immobilized system obtained in the

366presence of 02 g l-1 phenol and the system without

367phenol)

368In order to distinguish between phenol adsorption

369on the membrane surface and phenol degradation by

370microorganisms the pure carrier was also studied for

371phenol adsorption For this purpose 40 cm2 pure

372membrane was immersed in 05 g l-1 phenol solution

373for 6 h The results are presented in Fig 6 Since the

374phenol concentration didnrsquot change significantly

375the only possible reason for the decrease of the

376phenol content was the degradation activity of the

377immobilized cells This simplifies the kinetic studies

378when membrane is to be used as a support material

0

02

04

06

08

0 100 200 300 400 500

Time h

Ph

en

ol

bio

de

gra

da

tio

n

gl-1

Fig 5 Phenol

biodegradation with

concentration 05 g l-1 by

strain Aspergillus awamori

NRRL 3112

0

02

04

06

08

0 1 2 3 4 5 6 7

Time h

Ph

en

ol

bio

deg

rad

ati

on

g

l-1

Fig 6 Adsorption of

phenol by modified PAN

membrane

Biodegradation

123

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379 for the immobilization of Asp awamori for the

380 purpose of phenol degradation

381 The amount of cells (wet weight) on the membrane

382 of 40 cm2 was measured after the sixth cycle of

383 phenol biodegradationmdash1104 g cm-2 The signifi-

384 cant increase of the biomass with the increased

385 number of cycles is obvious (after immobilizationmdash

386 0158 g cm-2) Thus the biomass accumulated on

387 the membrane was approximately ten times higher

388 after six cycles of phenol biodegradation The high

389 operational stability of investigated biocatalysts was

390 confirmed by the scanning electron micrographs of

391 the polyacrylonitrile membraneimmobilized Asper-

392 gillus awamori NRRL at the beginning of repeated

393 batch cultivation and after the 7th cycle After the 7th

394 cycle of cultivation the observations showed large

395 groups of cells (Fig 7a) The significant increase of

396 biomass and mycelia formation on the membrane

397 surface after the 7th cycle of cultivation was inev-

398 itably followed by deceleration of the phenol

399 degradation rate

400Experiments with membrane bioreactor

401The next step of the research work involved exper-

402iments which were carried out in a bioreactor with

403spirally wound membrane in a regime of recircula-

404tion The spores were immobilized onto the

405membrane directly in the system as described in

406lsquolsquoMaterials and methodsrsquorsquo section Phenol biodegra-

407dation was carried out under aerobic conditions The

408operation parameters of the bioreactor are presented

409in Table 2 Fresh nutrient medium containing

41005 g l-1 phenol were added after each cycle Phenol

411concentration was monitored for degradation At the

412end of a cycle the system was washed with sterile

413distilled water and charged with nutrient medium and

414phenol for the next cycle Thus 10 cycles of 05 g l-1

415phenol biodegradation were run consecutively to

416determine the degradation time for each cycle

417(Fig 8) No significant difference was observed

418between the degradation times of the cycles from

419the second to the seventh (about 50 h) and between

420the biodegradation rates (100 mg h-1) After the 7th

421cycle the biodegradation rate began to decrease

422gradually and at 10 cycles it was 40 mg h-1 These

423results confirmed the advantages of the recycled

424reactor with spirally wound membrane The design of

425the bioreactor appeared to be quite effective provid-

426ing large membrane surface to bind the strain cells

427Besides phenol would flow tangentially which pre-

428vents membrane from a decrease of the flow velocity

429Conclusion

430A novel immobilized systemmdashAspergillus awamori

431NRRL 3112polyacrylonitrile membranewas obtained

432for the purpose of phenol biodegradation It was

433established that the immobilized system prepared in

434the presence of 02 g l-1 phenol was more effective

Fig 7 Scanning electron micrographs of immobilized system

Aspergillus awamori NRRL 3112modified PAN membrane

after 7th cycle (a) and before 1st cycle (b)

Table 2 Operation conditions of bioreactor

Parameter Value

Rate flow 15 ml min-1

Volume 300 ml

Initial phenol concentration 05 g l-1

Membrane surface area 100 cm2

Immobilized spores 0174 g cm-2 (wet weight)

Biodegradation

123

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435 than the system prepared in the phenol absence It was

436 revealed that the immobilized systems were more

437 stable andmore effective when biodegrading 05 g l-1

438 phenol in comparison with the free cells A bioreactor

439 was constructed containing spirally wound membrane

440 with immobilized spores of Aspergillus awamori

441 NRRL 3112 in a regime of recirculation which was

442 successfully applied for phenol biodegradation The

443 obtained results confirmed the advantages of the

444 recycled reactor with spirally wound membrane Thus

445 the immobilized microbial technology is an extremely

446 versatile approach that can be used for degradation of

447 toxic pollutants from the industrial effluents

448 Acknowledgments The authors gratefully acknowledge to449 the Bulgarian Ministry for Education and to the National450 Science Fund for their financial support and encouragement of451 the scientific research work in state universities

452 References

453 Abd-El-Haleem D Beshay U Abdelhamid AO Moawad H454 Zaki S (2003) Effects of mixed nitrogen sources on bio-455 degradation of phenol by immobilized Acinetobacter sp456 strain W-17 Afr J Biotechnol 2(1)8ndash12457 Allsop PJ Chisti Y Moi-Youngand M Sullivan GR (1993)458 Dynamics of phenol degradation by Pseudomonas putida459 Biotechnol Bioeng 41572ndash579460 Anselmo AM Novais JM (1992) Degradation of phenol by461 immobilized mycelium of Fusarium floccieferum in con-462 tinuous culture Water Sci Technol 25161ndash168463 Bolanos ML Varesche MBA Zaiat M Foresti E (2001) Phenol464 degradation in horizontal-flow anaerobic immobilized465 biomass (HAIB) reactor under mesophilic conditions466 Water Sci Technol 44(4)167ndash174

467Chitra S Sekaran G Padmavathi S Gowri C (1995) Removal468of phenol from wastewater using a biocatalyst In Pro-469ceedings of the national symposium frontiers in applied470environmental microbiology pp 74ndash77471Chtourou M Ammar E Nasri M Medhioub K (2004) Isolation472of a yeast Trichosporon cutaneum able to use low473molecular weight phenolic compounds application to474olive mill waste water treatment J Chem Technol Bio-475technol 79869ndash878476Dimov K Samardjieva Pavlov P (1983) Laboratory practice477on technology of synthetic fibers Technica Sofia BG478Fiest CF Hegeman GD (1969) Phenol and benzoate metabo-479lism by Pseudomonas putida J Bacteriol 100869ndash877480Godjevargova T Aleksieva Z Ivanova D (2000) Cell immo-481bilization of Trichosporon cutaneum strain with phenol482degradation ability on new modified polymer carries483Process Biochem 35699ndash704484Godjevargova T Ivanova D Aleksieva Z Burdelova G (2006)485Biodegradation of phenol by immobilized Trichosporon

486cutaneum R57 on modified polymer membranes Process487Biochem 412342ndash2346488Gonzalez G Herrera MG Pena MM (2001) Biodegradation of489phenol in a continuous process comparative study of490stirred tank and fluidized-bed bioreactors Bioresour491Technol 76245ndash251492Hua L Sun ZH Leng Y (2005) Continuous biocatalytic reso-493lution of DL-pantolactone by cross-linked cells in494membrane bioreactor Process Biochem 401137ndash1142495Hudges EJL Bayly RC Skurray RA (1984) Evidence for496isofunctional enzymes in the degradation of phenol m-497and p-toluate and p-cresol at catechol meta-cleavage498pathways in Alcaligenes eutrophus J Bacteriol 15879ndash83499Jones KH Trudgill PW Hopper DJ (1995) Evidence of two500pathways for the metabolism of phenol by Aspergillus

501fumigatus Arch Microbiol 163176ndash181502Jose G Sanchez M Theodore T (2002) Membranes and mem-503brane reactors Wiley-VCH Veriag GmbH Weinheim504Karel S Libichi S Robertson C (1985) The immobilization of505whole cells engineering principles Chem Eng Sci506401321ndash1354

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

Fig 8 Concentration

profile of phenol in a

recirculating system with

immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

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UNCORRECTEDPR

OOF

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OOF

507 Kowalska M Bodzek M Bohdziewicz J (1998) Biodegradation508 of phenols and cyanides using membranes immobilized509 microorganisms Process Biochem 33189ndash197510 Loh K Chung T Ang W (2000) Immobilized-cell membrane511 bioreactor for high-strength phenol wastewater J Environ512 Eng ASCE 12675ndash79513 Marrot B Barrios-Martinez A Moulin P Roche N (2006)514 Biodegradation of high phenol concentration by activated515 sludge in an immersed membrane bioreactor Biochem516 Eng J 30174ndash183517 Mordocco A Kuek C Jenkins R (1999) Continuous degradation518 of phenol at low concentration using immobilized Pseu-

519 domonas putida Enzyme Microb Technol 25530ndash536520 Pazarlioglu NK Telefoncu A (2005) Biodegradation of phenol521 by Pseudomonas putida immobilized on activated pumice522 particles Process Biochem 401807ndash1814523 Santos VL Heilbuth NM Braga T Monteiro AS Linardi VR524 (2003) Phenol degradation by aGraphium sp FIB4 isolated525 from industrial effluents J Basic Microbiol 43238ndash248526 Shetty KV Kalifathulla I Srinikethan G (2007a) Performance527 of pulsed plate bioreactor for biodegradation of phenol528 J Hazard Mater 140346ndash352529 Shetty KV Ramanjaneyulu R Srinikethan G (2007b) Biolog-530 ical phenol removal using immobilized cells in a pulsed

531plate bioreactor effect of dilution rate and influent phenol532concentration J Hazard Mater 149452ndash459533Stephenson T Judd S Jefferson B Brindle K (2000) Mem-534brane bioreactors for wastewater treatment IWA535Publishing London UK pp 129ndash136536Sundar P Daniel D Krastanov A (2005) Biodegradation of537phenol by immobilized Aspergillus awamori cells Bulg J538Agric Sci 1161ndash71539Uzunova K Vassileva A Ivanova V Spasova D Tonkova A540(2002) Thermostable exo-inulinase production by semi-541continuous cultivation of membrane-immobilized Bacillus542sp 11 cells Process Biochem 37863ndash868543Wang Z Wan L Xu Z (2007) Surface engineerings of poly-544acrylonitrile-based asymmetric membranes towards545biomedical applications an overview J Membr Sci5463048ndash23547Yiang Y Wen J Li H Yang S Hu Z (2005) The biodegra-548dation of phenol at high initial concentration by the yeast549Candida tropicalis Biochem Eng J 24243ndash247550Yordanova G Ivanova D Godjevargova T Krastanov A (2006)551Biodegradation of phenol by immobilized Aspergillus

552awamori NRRL3112 cells Food science engineering and553technologies conference Scientific works Plovdiv554pp 260ndash265

555

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

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ORIGINAL PAPER1

2 Biodegradation of phenol by immobilized Aspergillus

3 awamori NRRL 3112 on modified polyacrylonitrile

4 membrane

5 G Yordanova D Ivanova T Godjevargova

6 A Krastanov

7 Received 22 April 2008 Accepted 18 March 20098 Springer Science+Business Media BV 2009

9 Abstract Covalent immobilization of Aspergillus

10 awamori NRRL 3112 was conducted onto modified

11 polyacrylonitrile membrane with glutaraldehyde as a

12 coupling agent The polymer carrier was preliminarily

13 modified in an aqueous solution of NaOH and 12-

14 diaminoethane The content of amino groups was

15 determined to be 058 mgeq g-1 Two ways of immo-

16 bilization were usedmdashin the presence of 02 g l-1

17 phenol and without phenol The capability of two

18 immobilized system to degrade phenol (concentra-

19 tionmdash05 g l-1) as a sole carbon and energy source

20 was investigated in batch experiments Seven cycles of

21 phenol biodegradation were conducted Better results

22 were obtained with the immobilized system prepared

23 in the presence of phenol regarding degradation time

24 and phenol biodegradation rate Scanning electron

25 micrographs of the polyacrylonitrile membrane

26 immobilized Aspergillus awamori NRRL at the

27 beginning of repeated batch cultivation and after the

28 7th cycle were compared After the 7th cycle of

29 cultivation the observations showed large groups of

30 cells The results from the batch experiments with

31 immobilized system were compared to the results

32produced by the free strain Phenol biodegradation

33experiments were carried out also in a bioreactor with

34spirally wound membrane with bound Aspergillus

35awamori NRRL 3112 in a regime of recirculation 10

36cycles of 05 g l-1 phenol biodegradation were run

37consecutively to determine the degradation time and

38rate for each cycle The design of the bioreactor

39appeared to be quite effective providing large mem-

40brane surface to bind the strain

41Keywords Biodegradation Phenol

42Aspergillus awamori NRRL3112

43Immobilization Polymer membrane

44

45

46Introduction

47One of the problems attracting highest attention in

48modern ecology is related to the biodegradation of

49xenobiotics Typical representatives of this group of

50contaminants are phenol and its derivatives The

51environmental clean-up of phenol by adsorption

52solvent extraction chemical oxidation incineration

53and abiotic treatment procedure suffers from serious

54drawbacks such as economic issues and the produc-

55tion of hazardous byproducts Biodegradation is

56generally preferred due to the lower costs and the

57complete mineralization

58Extensive studies revealing the potential of micro-

59organisms in phenol biodegradation have been carried

A1 G Yordanova D Ivanova T Godjevargova (amp)

A2 Department of Biotechnology University lsquolsquoProf Dr A

A3 Zlatarovrsquorsquo 1 Prof Yakimov Str 8010 Bourgas Bulgaria

A4 e-mail godjevargovayahoocom

A5 A Krastanov

A6 Department of Biotechnology University of Food

A7 Technologies 4002 Plovdiv Bulgaria

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Biodegradation

DOI 101007s10532-009-9259-x

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60 out Major part of the research in the field of phenol

61 decomposition have been performed using bacterial

62 strains such as Alcaligenes europhus (Hudges et al

63 1984) and Pseudomonas sp (Allsop et al 1993)

64 Other publications report biodegradation of phenol by

65 yeast strainsCandida (MacGillivray and Shiaris 1993)

66 and Trichosporon (Chtourou et al 2004) Chitra et al

67 (1995) have studied the removal of phenol using a

68 mutant strain of Pseudomonas The use of fungal

69 strains for the degradation is relatively untouched area

70 Mycelial fungi such as Fusarium floccieferum

71 (Anselmo and Novais 1992) Aspergillus fumigatus

72 (Jones et al 1995) and Graphium sp (Santos et al

73 2003) have been cited for their potential of phenol

74 degradation Little is known about phenol metabolism

75 in mycelial fungi They have some advantages

76 because of their resistance to a great number of

77 xenobiotics toxic to the most of the microorganisms

78 The fungal strain Aspergillus awamori NRRL 3112 is

79 capable of assimilating a wide range of carbon

80 substrates including phenol and its derivatives (Sun-

81 dar et al 2005 Yordanova et al 2006) This feature

82 makes them a valuable research object for a future

83 development of industrial-water cleaning technology

84 There has been an increasing interest in the

85 immobilization of microorganisms in recent years

86 (Abd-El-Haleem et al 2003 Bolanos et al 2001

87 Gonzalez et al 2001 Fiest and Hegeman 1969

88 Mordocco et al 1999 Pazarlioglu and Telefoncu

89 2005 Shetty et al 2007a b) The immobilized cells

90 have advantages over the native cells due to the

91 possibility for multiple applications for a extended

92 period of time Different carriers have been used for

93 microbial cell immobilization but there arenrsquot many

94 reports for cell immobilization on polymer membranes

95 (Uzunova et al 2002 Marrot et al 2006 Hua et al

96 2005 Godjevargova et al 2006) Synthetic polymers

97 constitute the largest group of supports in use Poly-

98 acrylonitrile-based ultrafiltration (UF) membranes

99 obviously turn up to be more advantageous over other

100 conventional membranes in various aspects such as

101 thermal stability resistance to most organic solvents

102 commercial availability etc (Wang et al 2007)

103 PAN-based membranes can offer some special prom-

104 ises as cradle for cell immobilization which is mainly

105 because different reactive groups can be easily gen-

106 erated on the PAN membrane surface for the covalent

107 immobilization (Godjevargova et al 2006 Kowalska

108 et al 1998)

109The membranes are very suitable for immobiliza-

110tion of microbial cells when a spiral membrane

111module is used There is no common strategy for

112preparing such integrated systems and opportunities

113for using are still at hand Due to their high surface

114area per unit module volume spiral membrane

115modules serve as better immobilization supports than

116alginate beads activated carbon sintered glass and

117polymer beads (Jose et al 2002 Karel et al 1985)

118On the other hand polymer membranes can act as

119barriers limiting the movement of cell mass thus

120providing restricted andor regulated passage of one

121or more species through the pores From this point of

122view membrane bioreactors are promising for

123practical applications for wastewater treatment (Loh

124et al 2000 Stephenson et al 2000)

125The present paper investigates the possibility of

126immobilizing Asp awamori NRRL 3112 spores on

127modified polyacrylonitrile membrane and their ability

128to biodegrade phenol Spiral membrane module with

129immobilized spores was created and phenol biodeg-

130radation was also investigated

131Materials and methods

132Microorganism and growth medium

133A strain of Aspergillus awamoriNRRL 3112 obtained

134fromUSDepartment of Agriculture Illinois USAwas

135used throughout this study The organism was grown

136on slants immersed in a medium having the following

137composition (g l-1) malt extract 30 yeast extract 30

138peptone 50 glucose 100 and agar 200 The organism

139on the slants was allowed to grow for 72 h at 30C and

140then stored at 4 plusmn 1C for further use

141Medium for degradation studies

142The studies on the biodegradation of phenol were

143carried out in the Czapekdox medium which had the

144following composition (g l-1) sodium nitrate 20

145potassium phosphate (dibasic) 10 potassium chlo-

146ride 05 magnesium sulfate heptahydrate 05 and

147ferrous sulfate heptahydrate 001 The initial pH of

148the medium was adjusted to 55 (the optimal pH for

149this strain) using 1 N NaOH or 1 N HCI (Sundar

150et al 2005) The minimal medium contains phenol as

151a sole carbon source with 05 g l-1 concentration

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

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152 Chemical modification of polyacrylonitrile

153 membrane

154 The ultrafiltration polyacrylonitrile (PAN) membrane

155 (average pore size of selective layer 002 lm) was

156 produced by Ecofilter Co (Bourgas Bulgaria) PAN

157 membrane was treated with 10 aqueous solution of

158 NaOH at 50C for 60 min After thorough washing

159 with distilled water the membrane was treated with a

160 10 aqueous solution of 12-diaminohexane for

161 60 min at 40C The membrane was once again

162 thoroughly washed with distilled water At the end of

163 the procedure the modified carrier was immersed in

164 05 aqueous solution of glutaraldehyde for 60 min

165 at room temperature Excess glutaraldehyde was

166 removed by washing the membranes with distilled

167 water until there was a complete absence of glutar-

168 aldehyde in the rinse waters

169 Phenol biodegradation in batch experiments

170 Spore immobilization for batch experiments

171 The 14-day culture in spore form in concentration of

172 333 9 107 ml-1 were introduced by flushing 5 cm3

173 sterile water in 500 cm3 sterile flask containing

174 50 cm3 Czapekdox medium and sterile modified

175 carrier (40 cm2 PAN flat membrane) Sterilization of

176 the modified membrane was carried out by ethanol

177 Two ways of immobilization were usedmdashin the

178 presence of 02 g l-1 phenol and without phenol

179 The two flasks were placed on a shaker for 24 h at

180 30C Then the liquid medium was decanted and the

181 immobilized systems were washed several times with

182 sterile water to remove the unbound spores After

183 that the immobilized spores were used for phenol

184 biodegradation

185 Batch experiments

186 The immobilized spores onto the polymer mem-

187 brane (single flat piecemdash40 cm2) with concentration

188 0158 g cm-2 were introduced in 500 cm3 sterile

189 flask containing 50 cm3 Czapekdox medium as well

190 as phenol as the only carbon source adjusted to the

191 appropriate concentration Then they were cultivated

192 on a shaker (220 rpm) at 30C until the complete

193 phenol degradation At every 24 h samples were

194 withdrawn and analyzed for phenol concentration

195Thus seven cycles were carried out with the same

196immobilized system

197Simultaneously parallel experiments with free

198cells were also performed for comparison The flasks

199containing 50 cm3 liquid medium as well as 05 g l-1

200phenol as the only carbon source were inoculated with

201equal volume of inoculum (333 9 107 conidia cm-3

202medium) and agitated on a shaker (220 rpm) at 30C

203Samples were taken at every 24 h interval for

204determination of phenol concentration After the

205biodegradation of the entire phenol the cultural

206medium was decanted and the biomass was reculti-

207vated in fresh liquid medium containing 05 g l-1

208phenol Thus four cycles were carried out in the same

209manner

210Each experiment for phenol degradation was

211repeated twice the reproducibility being always within

21225

213Phenol biodegradation in recycle reactor

214with spirally wound membrane

215Spore immobilization for experiments

216with membrane bioreactor

217The bioreactor was 40 mm in diameter and 100 mm

218high it was made of glass and equipped with

219thermostatic bath (Fig 1) The modified and activated

220with glutaraldehyde polyacrylonitrile membrane with

Fig 1 Scheme of recycle system thermostatic bath (1) feed

reservoir (2) peristaltic pump (3) thermostatic reactor (4)

membrane with immobilized cells (5) sampling (6) air outlet

(7) and air inlet (8)

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

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221 surface area of 100 cm2 was spirally wound in the

222 glass bioreactor using supporting polyethylene mesh

223 In this case the immobilization was carried out by

224 flowing spore suspension (547 9 107 conidia cm-3

225 medium) through the membrane module using peri-

226 staltic pump at rate of 1 ml min-1 for 24 h Then the

227 membrane spiral was thoroughly washed out with

228 sterile water fed by peristaltic pump to remove the

229 non-bound spores

230 Experiments with membrane bioreactor

231 Further the liquid medium (300 ml) containing

232 phenol at certain concentration was fed to the mem-

233 brane bioreactor using peristaltic pump at rate of

234 15 ml min-1 The temperature required for the strain

235 growth (30C) was maintained using the thermostat

236 and the heat-exchanging coating After the full

237 degradation of phenol in the nutrient medium the

238 reservoir was charged with fresh nutrient medium

239 containing phenol Samples were taken at every 24 h

240 interval for determination of phenol concentration

241 Ten cycles were carried out in the same manner

242 Each experiment was repeated twice the repro-

243 ducibility being always within 25

244 Analytical methods

245 Phenol concentration was measured spectrophoto-

246 metrically in the presence of p-nitroaniline at 495 nm

247 (Gonzalez et al 2001)

248 The amount of spores immobilized on the support

249 was estimated by the difference between the wet

250 weight of spores with membrane at the beginning

251 (immediately after the immobilization) and the wet

252 membrane weight The amount of free cells was

253 determined by the measuring of the wet weight of the

254 cells and by the counting of the number of conidia in

255 the samples using a standard microscope method

256 Scanning electron microscopy (SEM)

257 The immobilized system Aspergillus awamori NRRL

258 3112polyacrylonitrile membrane was washed with

259 distilled water and then they were dehydrated in

260 30ndash100 water ethanol series The membrane were

261 coated with 120ndash130 Aacuteof gold in argon medium in

262 an Edwards apparatus (model 150 A) The SEM

263 observations were done on a scanning device attached

264to a Zeiss electron microscope (model 10C) at 20 kV

265accelerating voltage with a 5ndash6 nm electron beam

266Results and discussions

267Modification of membranes

268The aim of the present studies is to obtain an efficient

269immobilized system of Aspergillus awamori NRRL

2703112polyacrylonitrile membrane for biodegradation

271of phenol The immobilization of spores was carried

272out with glutaraldehyde The polymer membrane was

273preliminarily treated by using two different modifica-

274tion procedures The first procedure introduced

275ndashCOOH and ndashNH2 groups on the membrane surface

276by using NaOH solution In order to increase the

277amount of amine groups on the surface of the carrier

278the latter was treated with 12-diaminoethane The

279optimal conditions of modification were defined in a

280previous research work (Godjevargova et al 2000)

281The superficial carboxyl groups on the carrier surface

282reacted with one of the amine groups of 12-diami-

283noethane leaving the other one free The results

284showed that the PANmembranes had beenmodified in

285a greater extent and the content of amino groups was

286058 mgeq g-1 The latter was determined titrimetri-

287cally (Dimov et al 1983) Further the modified

288membrane was treated with an aqueous solution of

289glutaraldehyde The bifunctional agent bound the

290strain to the membrane surface

291Batch experiments

292Spores concentration used for the immobilization was

293333 9 107 ml-1 The first task was to determine

294which immobilization method would serve better for

295these experimentsmdashin the presence or in the absence

296of phenol (02 g l-1) in a nutrient medium containing

297spore suspension The amount of spores bound to the

298membranes in both cases was 0158 g cm-2 (wet

299weight) Then the spores immobilized on the polymer

300membrane by both methods were cultivated in a

301nutrient medium containing 05 g l-1 phenol The

302results obtained for phenol biodegradation by both

303systems are presented in Fig 2a b The rate of

304phenol biodegradation was determined after the end

305of each cycle for both immobilization systems

306(Table 1) As can be seen from Table 1 the lowest

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

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307 biodegradation rate is observed at the first cycle then

308 it increases 3 times at the second and the third cycles

309 and from the fourth to the seventh cycle the rate

310 decreases continuously This trend of the biodegra-

311 dation rate is observed for both of the immobilized

312 systems The deceleration of the phenol biodegrada-

313 tion could be explained with the increasing of

314 biomass and mycelia formation on the membrane

315 surface after the fourth cycle which intensified at the

316 final cycles An obvious inhibition of the biodegra-

317 dation process at the final cycles was observed which

318 was due to the hindrance of the substrate diffusion to

319 the cells When comparing the results presented on

320 Fig 2a and b and Table 1 it becomes obvious that the

321 rate of phenol degradation was higher throughout all

322 cycles in the presence of the system where the spore

323 immobilization was conducted with addition of

32402 g l-1 phenol For better comparison the biodeg-

325radation times of each cycle with the two

326immobilized systems are presented in Fig 3 As

0

02

04

06

08

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

0

02

04

06

08

0 200 400 600 800

0 200 400 600 800 1000

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

(a)

(b)

Fig 2 Phenol

biodegradation (05 g l-1)

by immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane obtained by

immobilization in presence

of 02 g l-1 phenol (a) and

without phenol (b)

Table 1 Phenol biodegradation rate (mg h-1) of free and

immobilized Aspergillus awamori NRRL 3112 on polymer

PAN membrane at batch cultivation with 05 g l-1 phenol

Numberof

cycles

Free

cells

Immobilized cells

without 02 g l-1

phenol

Immobilized cells

in the presence of

02 g l-1 phenol

1 297 300 300

2 201 714 694

3 149 806 543

4 116 625 367

5 403 329

6 308 300

7 275 270

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

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327 results suggest better degradation times were

328 obtained with the immobilized system prepared in

329 the presence of phenol This can be explained with

330 the fact that the strain could have adapted to the

331 substrate during the immobilization process All

332 further experiments were carried out with the immo-

333 bilized system prepared in presence of phenol

334 The biodegradation ability of the immobilized

335 system onto polyacrylonitrile membrane towards

336 phenol in a concentration range from 02 to

337 08 g l-1 was studied As can be seen from Fig 4

338 the increase of phenol concentration resulted in

339increased biodegradation time For instance the

340biodegradation cycle at concentration of 04 g l-1

341was 66 h while at 07 g l-1 the cycle lasted for 135 h

342The results obtained with the selected immobilized

343system were compared to those with the free strain

344For this purpose the spores (08 9 107 ml-1)

345were cultivated in Czapekdox medium containing

34605 g l-1 phenol (Fig 5) Four cycles were carried

347out in the same manner The amount of non-bound

348spores in the first cycle of cultivation is equivalent

349to the amount of immobilized spores on PAN

350membrane with surface 40 cm2 in the first cycle

0

50

100

150

200

250

1 2 3 4 5 6 7

Cycles

Tim

e

h

Fig 3 Cycles duration of

phenol biodegradation

(05 g l-1) by two

immobilized systems

Aspergillus awamori NRRL

3112modified PAN

membrane obtained by

immobilization in presence

of 02 g l-1 phenol ( ) and

without phenol (j)

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol co

ncen

trati

on

g

l-1

Fig 4 Phenol

biodegradation (from 02 to

08 g l-1) by immobilized

system Aspergillus

awamori NRRL 3112

modified PAN membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

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351 (wet weightmdash632 g) As can be seen from Fig 5 the

352 phenol degradation time was considerably greater for

353 the four cycles in these experiments This time

354 extension was confirmed by the calculated rate of

355 biodegradation (Table 1) When comparing the rates

356 of phenol biodegradation by the free and the immo-

357 bilized cells the greater stability of the immobilized

358 systems over the free cells becomes more prominent

359 after multiple cultivation in media containing phenol

360 with concentration 05 g l-1 Obviously the free cells

361 were inhibited to a greater extent by phenol (biodeg-

362 radation rate at Cycle 4mdash116 mg h-1) in

363 comparison with the immobilized cells (biodegrada-

364 tion rates at Cycle 4 are 625 and 367 mg h-1

365respectively for immobilized system obtained in the

366presence of 02 g l-1 phenol and the system without

367phenol)

368In order to distinguish between phenol adsorption

369on the membrane surface and phenol degradation by

370microorganisms the pure carrier was also studied for

371phenol adsorption For this purpose 40 cm2 pure

372membrane was immersed in 05 g l-1 phenol solution

373for 6 h The results are presented in Fig 6 Since the

374phenol concentration didnrsquot change significantly

375the only possible reason for the decrease of the

376phenol content was the degradation activity of the

377immobilized cells This simplifies the kinetic studies

378when membrane is to be used as a support material

0

02

04

06

08

0 100 200 300 400 500

Time h

Ph

en

ol

bio

de

gra

da

tio

n

gl-1

Fig 5 Phenol

biodegradation with

concentration 05 g l-1 by

strain Aspergillus awamori

NRRL 3112

0

02

04

06

08

0 1 2 3 4 5 6 7

Time h

Ph

en

ol

bio

deg

rad

ati

on

g

l-1

Fig 6 Adsorption of

phenol by modified PAN

membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

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379 for the immobilization of Asp awamori for the

380 purpose of phenol degradation

381 The amount of cells (wet weight) on the membrane

382 of 40 cm2 was measured after the sixth cycle of

383 phenol biodegradationmdash1104 g cm-2 The signifi-

384 cant increase of the biomass with the increased

385 number of cycles is obvious (after immobilizationmdash

386 0158 g cm-2) Thus the biomass accumulated on

387 the membrane was approximately ten times higher

388 after six cycles of phenol biodegradation The high

389 operational stability of investigated biocatalysts was

390 confirmed by the scanning electron micrographs of

391 the polyacrylonitrile membraneimmobilized Asper-

392 gillus awamori NRRL at the beginning of repeated

393 batch cultivation and after the 7th cycle After the 7th

394 cycle of cultivation the observations showed large

395 groups of cells (Fig 7a) The significant increase of

396 biomass and mycelia formation on the membrane

397 surface after the 7th cycle of cultivation was inev-

398 itably followed by deceleration of the phenol

399 degradation rate

400Experiments with membrane bioreactor

401The next step of the research work involved exper-

402iments which were carried out in a bioreactor with

403spirally wound membrane in a regime of recircula-

404tion The spores were immobilized onto the

405membrane directly in the system as described in

406lsquolsquoMaterials and methodsrsquorsquo section Phenol biodegra-

407dation was carried out under aerobic conditions The

408operation parameters of the bioreactor are presented

409in Table 2 Fresh nutrient medium containing

41005 g l-1 phenol were added after each cycle Phenol

411concentration was monitored for degradation At the

412end of a cycle the system was washed with sterile

413distilled water and charged with nutrient medium and

414phenol for the next cycle Thus 10 cycles of 05 g l-1

415phenol biodegradation were run consecutively to

416determine the degradation time for each cycle

417(Fig 8) No significant difference was observed

418between the degradation times of the cycles from

419the second to the seventh (about 50 h) and between

420the biodegradation rates (100 mg h-1) After the 7th

421cycle the biodegradation rate began to decrease

422gradually and at 10 cycles it was 40 mg h-1 These

423results confirmed the advantages of the recycled

424reactor with spirally wound membrane The design of

425the bioreactor appeared to be quite effective provid-

426ing large membrane surface to bind the strain cells

427Besides phenol would flow tangentially which pre-

428vents membrane from a decrease of the flow velocity

429Conclusion

430A novel immobilized systemmdashAspergillus awamori

431NRRL 3112polyacrylonitrile membranewas obtained

432for the purpose of phenol biodegradation It was

433established that the immobilized system prepared in

434the presence of 02 g l-1 phenol was more effective

Fig 7 Scanning electron micrographs of immobilized system

Aspergillus awamori NRRL 3112modified PAN membrane

after 7th cycle (a) and before 1st cycle (b)

Table 2 Operation conditions of bioreactor

Parameter Value

Rate flow 15 ml min-1

Volume 300 ml

Initial phenol concentration 05 g l-1

Membrane surface area 100 cm2

Immobilized spores 0174 g cm-2 (wet weight)

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

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435 than the system prepared in the phenol absence It was

436 revealed that the immobilized systems were more

437 stable andmore effective when biodegrading 05 g l-1

438 phenol in comparison with the free cells A bioreactor

439 was constructed containing spirally wound membrane

440 with immobilized spores of Aspergillus awamori

441 NRRL 3112 in a regime of recirculation which was

442 successfully applied for phenol biodegradation The

443 obtained results confirmed the advantages of the

444 recycled reactor with spirally wound membrane Thus

445 the immobilized microbial technology is an extremely

446 versatile approach that can be used for degradation of

447 toxic pollutants from the industrial effluents

448 Acknowledgments The authors gratefully acknowledge to449 the Bulgarian Ministry for Education and to the National450 Science Fund for their financial support and encouragement of451 the scientific research work in state universities

452 References

453 Abd-El-Haleem D Beshay U Abdelhamid AO Moawad H454 Zaki S (2003) Effects of mixed nitrogen sources on bio-455 degradation of phenol by immobilized Acinetobacter sp456 strain W-17 Afr J Biotechnol 2(1)8ndash12457 Allsop PJ Chisti Y Moi-Youngand M Sullivan GR (1993)458 Dynamics of phenol degradation by Pseudomonas putida459 Biotechnol Bioeng 41572ndash579460 Anselmo AM Novais JM (1992) Degradation of phenol by461 immobilized mycelium of Fusarium floccieferum in con-462 tinuous culture Water Sci Technol 25161ndash168463 Bolanos ML Varesche MBA Zaiat M Foresti E (2001) Phenol464 degradation in horizontal-flow anaerobic immobilized465 biomass (HAIB) reactor under mesophilic conditions466 Water Sci Technol 44(4)167ndash174

467Chitra S Sekaran G Padmavathi S Gowri C (1995) Removal468of phenol from wastewater using a biocatalyst In Pro-469ceedings of the national symposium frontiers in applied470environmental microbiology pp 74ndash77471Chtourou M Ammar E Nasri M Medhioub K (2004) Isolation472of a yeast Trichosporon cutaneum able to use low473molecular weight phenolic compounds application to474olive mill waste water treatment J Chem Technol Bio-475technol 79869ndash878476Dimov K Samardjieva Pavlov P (1983) Laboratory practice477on technology of synthetic fibers Technica Sofia BG478Fiest CF Hegeman GD (1969) Phenol and benzoate metabo-479lism by Pseudomonas putida J Bacteriol 100869ndash877480Godjevargova T Aleksieva Z Ivanova D (2000) Cell immo-481bilization of Trichosporon cutaneum strain with phenol482degradation ability on new modified polymer carries483Process Biochem 35699ndash704484Godjevargova T Ivanova D Aleksieva Z Burdelova G (2006)485Biodegradation of phenol by immobilized Trichosporon

486cutaneum R57 on modified polymer membranes Process487Biochem 412342ndash2346488Gonzalez G Herrera MG Pena MM (2001) Biodegradation of489phenol in a continuous process comparative study of490stirred tank and fluidized-bed bioreactors Bioresour491Technol 76245ndash251492Hua L Sun ZH Leng Y (2005) Continuous biocatalytic reso-493lution of DL-pantolactone by cross-linked cells in494membrane bioreactor Process Biochem 401137ndash1142495Hudges EJL Bayly RC Skurray RA (1984) Evidence for496isofunctional enzymes in the degradation of phenol m-497and p-toluate and p-cresol at catechol meta-cleavage498pathways in Alcaligenes eutrophus J Bacteriol 15879ndash83499Jones KH Trudgill PW Hopper DJ (1995) Evidence of two500pathways for the metabolism of phenol by Aspergillus

501fumigatus Arch Microbiol 163176ndash181502Jose G Sanchez M Theodore T (2002) Membranes and mem-503brane reactors Wiley-VCH Veriag GmbH Weinheim504Karel S Libichi S Robertson C (1985) The immobilization of505whole cells engineering principles Chem Eng Sci506401321ndash1354

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

Fig 8 Concentration

profile of phenol in a

recirculating system with

immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

507 Kowalska M Bodzek M Bohdziewicz J (1998) Biodegradation508 of phenols and cyanides using membranes immobilized509 microorganisms Process Biochem 33189ndash197510 Loh K Chung T Ang W (2000) Immobilized-cell membrane511 bioreactor for high-strength phenol wastewater J Environ512 Eng ASCE 12675ndash79513 Marrot B Barrios-Martinez A Moulin P Roche N (2006)514 Biodegradation of high phenol concentration by activated515 sludge in an immersed membrane bioreactor Biochem516 Eng J 30174ndash183517 Mordocco A Kuek C Jenkins R (1999) Continuous degradation518 of phenol at low concentration using immobilized Pseu-

519 domonas putida Enzyme Microb Technol 25530ndash536520 Pazarlioglu NK Telefoncu A (2005) Biodegradation of phenol521 by Pseudomonas putida immobilized on activated pumice522 particles Process Biochem 401807ndash1814523 Santos VL Heilbuth NM Braga T Monteiro AS Linardi VR524 (2003) Phenol degradation by aGraphium sp FIB4 isolated525 from industrial effluents J Basic Microbiol 43238ndash248526 Shetty KV Kalifathulla I Srinikethan G (2007a) Performance527 of pulsed plate bioreactor for biodegradation of phenol528 J Hazard Mater 140346ndash352529 Shetty KV Ramanjaneyulu R Srinikethan G (2007b) Biolog-530 ical phenol removal using immobilized cells in a pulsed

531plate bioreactor effect of dilution rate and influent phenol532concentration J Hazard Mater 149452ndash459533Stephenson T Judd S Jefferson B Brindle K (2000) Mem-534brane bioreactors for wastewater treatment IWA535Publishing London UK pp 129ndash136536Sundar P Daniel D Krastanov A (2005) Biodegradation of537phenol by immobilized Aspergillus awamori cells Bulg J538Agric Sci 1161ndash71539Uzunova K Vassileva A Ivanova V Spasova D Tonkova A540(2002) Thermostable exo-inulinase production by semi-541continuous cultivation of membrane-immobilized Bacillus542sp 11 cells Process Biochem 37863ndash868543Wang Z Wan L Xu Z (2007) Surface engineerings of poly-544acrylonitrile-based asymmetric membranes towards545biomedical applications an overview J Membr Sci5463048ndash23547Yiang Y Wen J Li H Yang S Hu Z (2005) The biodegra-548dation of phenol at high initial concentration by the yeast549Candida tropicalis Biochem Eng J 24243ndash247550Yordanova G Ivanova D Godjevargova T Krastanov A (2006)551Biodegradation of phenol by immobilized Aspergillus

552awamori NRRL3112 cells Food science engineering and553technologies conference Scientific works Plovdiv554pp 260ndash265

555

Biodegradation

123

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60 out Major part of the research in the field of phenol

61 decomposition have been performed using bacterial

62 strains such as Alcaligenes europhus (Hudges et al

63 1984) and Pseudomonas sp (Allsop et al 1993)

64 Other publications report biodegradation of phenol by

65 yeast strainsCandida (MacGillivray and Shiaris 1993)

66 and Trichosporon (Chtourou et al 2004) Chitra et al

67 (1995) have studied the removal of phenol using a

68 mutant strain of Pseudomonas The use of fungal

69 strains for the degradation is relatively untouched area

70 Mycelial fungi such as Fusarium floccieferum

71 (Anselmo and Novais 1992) Aspergillus fumigatus

72 (Jones et al 1995) and Graphium sp (Santos et al

73 2003) have been cited for their potential of phenol

74 degradation Little is known about phenol metabolism

75 in mycelial fungi They have some advantages

76 because of their resistance to a great number of

77 xenobiotics toxic to the most of the microorganisms

78 The fungal strain Aspergillus awamori NRRL 3112 is

79 capable of assimilating a wide range of carbon

80 substrates including phenol and its derivatives (Sun-

81 dar et al 2005 Yordanova et al 2006) This feature

82 makes them a valuable research object for a future

83 development of industrial-water cleaning technology

84 There has been an increasing interest in the

85 immobilization of microorganisms in recent years

86 (Abd-El-Haleem et al 2003 Bolanos et al 2001

87 Gonzalez et al 2001 Fiest and Hegeman 1969

88 Mordocco et al 1999 Pazarlioglu and Telefoncu

89 2005 Shetty et al 2007a b) The immobilized cells

90 have advantages over the native cells due to the

91 possibility for multiple applications for a extended

92 period of time Different carriers have been used for

93 microbial cell immobilization but there arenrsquot many

94 reports for cell immobilization on polymer membranes

95 (Uzunova et al 2002 Marrot et al 2006 Hua et al

96 2005 Godjevargova et al 2006) Synthetic polymers

97 constitute the largest group of supports in use Poly-

98 acrylonitrile-based ultrafiltration (UF) membranes

99 obviously turn up to be more advantageous over other

100 conventional membranes in various aspects such as

101 thermal stability resistance to most organic solvents

102 commercial availability etc (Wang et al 2007)

103 PAN-based membranes can offer some special prom-

104 ises as cradle for cell immobilization which is mainly

105 because different reactive groups can be easily gen-

106 erated on the PAN membrane surface for the covalent

107 immobilization (Godjevargova et al 2006 Kowalska

108 et al 1998)

109The membranes are very suitable for immobiliza-

110tion of microbial cells when a spiral membrane

111module is used There is no common strategy for

112preparing such integrated systems and opportunities

113for using are still at hand Due to their high surface

114area per unit module volume spiral membrane

115modules serve as better immobilization supports than

116alginate beads activated carbon sintered glass and

117polymer beads (Jose et al 2002 Karel et al 1985)

118On the other hand polymer membranes can act as

119barriers limiting the movement of cell mass thus

120providing restricted andor regulated passage of one

121or more species through the pores From this point of

122view membrane bioreactors are promising for

123practical applications for wastewater treatment (Loh

124et al 2000 Stephenson et al 2000)

125The present paper investigates the possibility of

126immobilizing Asp awamori NRRL 3112 spores on

127modified polyacrylonitrile membrane and their ability

128to biodegrade phenol Spiral membrane module with

129immobilized spores was created and phenol biodeg-

130radation was also investigated

131Materials and methods

132Microorganism and growth medium

133A strain of Aspergillus awamoriNRRL 3112 obtained

134fromUSDepartment of Agriculture Illinois USAwas

135used throughout this study The organism was grown

136on slants immersed in a medium having the following

137composition (g l-1) malt extract 30 yeast extract 30

138peptone 50 glucose 100 and agar 200 The organism

139on the slants was allowed to grow for 72 h at 30C and

140then stored at 4 plusmn 1C for further use

141Medium for degradation studies

142The studies on the biodegradation of phenol were

143carried out in the Czapekdox medium which had the

144following composition (g l-1) sodium nitrate 20

145potassium phosphate (dibasic) 10 potassium chlo-

146ride 05 magnesium sulfate heptahydrate 05 and

147ferrous sulfate heptahydrate 001 The initial pH of

148the medium was adjusted to 55 (the optimal pH for

149this strain) using 1 N NaOH or 1 N HCI (Sundar

150et al 2005) The minimal medium contains phenol as

151a sole carbon source with 05 g l-1 concentration

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

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152 Chemical modification of polyacrylonitrile

153 membrane

154 The ultrafiltration polyacrylonitrile (PAN) membrane

155 (average pore size of selective layer 002 lm) was

156 produced by Ecofilter Co (Bourgas Bulgaria) PAN

157 membrane was treated with 10 aqueous solution of

158 NaOH at 50C for 60 min After thorough washing

159 with distilled water the membrane was treated with a

160 10 aqueous solution of 12-diaminohexane for

161 60 min at 40C The membrane was once again

162 thoroughly washed with distilled water At the end of

163 the procedure the modified carrier was immersed in

164 05 aqueous solution of glutaraldehyde for 60 min

165 at room temperature Excess glutaraldehyde was

166 removed by washing the membranes with distilled

167 water until there was a complete absence of glutar-

168 aldehyde in the rinse waters

169 Phenol biodegradation in batch experiments

170 Spore immobilization for batch experiments

171 The 14-day culture in spore form in concentration of

172 333 9 107 ml-1 were introduced by flushing 5 cm3

173 sterile water in 500 cm3 sterile flask containing

174 50 cm3 Czapekdox medium and sterile modified

175 carrier (40 cm2 PAN flat membrane) Sterilization of

176 the modified membrane was carried out by ethanol

177 Two ways of immobilization were usedmdashin the

178 presence of 02 g l-1 phenol and without phenol

179 The two flasks were placed on a shaker for 24 h at

180 30C Then the liquid medium was decanted and the

181 immobilized systems were washed several times with

182 sterile water to remove the unbound spores After

183 that the immobilized spores were used for phenol

184 biodegradation

185 Batch experiments

186 The immobilized spores onto the polymer mem-

187 brane (single flat piecemdash40 cm2) with concentration

188 0158 g cm-2 were introduced in 500 cm3 sterile

189 flask containing 50 cm3 Czapekdox medium as well

190 as phenol as the only carbon source adjusted to the

191 appropriate concentration Then they were cultivated

192 on a shaker (220 rpm) at 30C until the complete

193 phenol degradation At every 24 h samples were

194 withdrawn and analyzed for phenol concentration

195Thus seven cycles were carried out with the same

196immobilized system

197Simultaneously parallel experiments with free

198cells were also performed for comparison The flasks

199containing 50 cm3 liquid medium as well as 05 g l-1

200phenol as the only carbon source were inoculated with

201equal volume of inoculum (333 9 107 conidia cm-3

202medium) and agitated on a shaker (220 rpm) at 30C

203Samples were taken at every 24 h interval for

204determination of phenol concentration After the

205biodegradation of the entire phenol the cultural

206medium was decanted and the biomass was reculti-

207vated in fresh liquid medium containing 05 g l-1

208phenol Thus four cycles were carried out in the same

209manner

210Each experiment for phenol degradation was

211repeated twice the reproducibility being always within

21225

213Phenol biodegradation in recycle reactor

214with spirally wound membrane

215Spore immobilization for experiments

216with membrane bioreactor

217The bioreactor was 40 mm in diameter and 100 mm

218high it was made of glass and equipped with

219thermostatic bath (Fig 1) The modified and activated

220with glutaraldehyde polyacrylonitrile membrane with

Fig 1 Scheme of recycle system thermostatic bath (1) feed

reservoir (2) peristaltic pump (3) thermostatic reactor (4)

membrane with immobilized cells (5) sampling (6) air outlet

(7) and air inlet (8)

Biodegradation

123

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221 surface area of 100 cm2 was spirally wound in the

222 glass bioreactor using supporting polyethylene mesh

223 In this case the immobilization was carried out by

224 flowing spore suspension (547 9 107 conidia cm-3

225 medium) through the membrane module using peri-

226 staltic pump at rate of 1 ml min-1 for 24 h Then the

227 membrane spiral was thoroughly washed out with

228 sterile water fed by peristaltic pump to remove the

229 non-bound spores

230 Experiments with membrane bioreactor

231 Further the liquid medium (300 ml) containing

232 phenol at certain concentration was fed to the mem-

233 brane bioreactor using peristaltic pump at rate of

234 15 ml min-1 The temperature required for the strain

235 growth (30C) was maintained using the thermostat

236 and the heat-exchanging coating After the full

237 degradation of phenol in the nutrient medium the

238 reservoir was charged with fresh nutrient medium

239 containing phenol Samples were taken at every 24 h

240 interval for determination of phenol concentration

241 Ten cycles were carried out in the same manner

242 Each experiment was repeated twice the repro-

243 ducibility being always within 25

244 Analytical methods

245 Phenol concentration was measured spectrophoto-

246 metrically in the presence of p-nitroaniline at 495 nm

247 (Gonzalez et al 2001)

248 The amount of spores immobilized on the support

249 was estimated by the difference between the wet

250 weight of spores with membrane at the beginning

251 (immediately after the immobilization) and the wet

252 membrane weight The amount of free cells was

253 determined by the measuring of the wet weight of the

254 cells and by the counting of the number of conidia in

255 the samples using a standard microscope method

256 Scanning electron microscopy (SEM)

257 The immobilized system Aspergillus awamori NRRL

258 3112polyacrylonitrile membrane was washed with

259 distilled water and then they were dehydrated in

260 30ndash100 water ethanol series The membrane were

261 coated with 120ndash130 Aacuteof gold in argon medium in

262 an Edwards apparatus (model 150 A) The SEM

263 observations were done on a scanning device attached

264to a Zeiss electron microscope (model 10C) at 20 kV

265accelerating voltage with a 5ndash6 nm electron beam

266Results and discussions

267Modification of membranes

268The aim of the present studies is to obtain an efficient

269immobilized system of Aspergillus awamori NRRL

2703112polyacrylonitrile membrane for biodegradation

271of phenol The immobilization of spores was carried

272out with glutaraldehyde The polymer membrane was

273preliminarily treated by using two different modifica-

274tion procedures The first procedure introduced

275ndashCOOH and ndashNH2 groups on the membrane surface

276by using NaOH solution In order to increase the

277amount of amine groups on the surface of the carrier

278the latter was treated with 12-diaminoethane The

279optimal conditions of modification were defined in a

280previous research work (Godjevargova et al 2000)

281The superficial carboxyl groups on the carrier surface

282reacted with one of the amine groups of 12-diami-

283noethane leaving the other one free The results

284showed that the PANmembranes had beenmodified in

285a greater extent and the content of amino groups was

286058 mgeq g-1 The latter was determined titrimetri-

287cally (Dimov et al 1983) Further the modified

288membrane was treated with an aqueous solution of

289glutaraldehyde The bifunctional agent bound the

290strain to the membrane surface

291Batch experiments

292Spores concentration used for the immobilization was

293333 9 107 ml-1 The first task was to determine

294which immobilization method would serve better for

295these experimentsmdashin the presence or in the absence

296of phenol (02 g l-1) in a nutrient medium containing

297spore suspension The amount of spores bound to the

298membranes in both cases was 0158 g cm-2 (wet

299weight) Then the spores immobilized on the polymer

300membrane by both methods were cultivated in a

301nutrient medium containing 05 g l-1 phenol The

302results obtained for phenol biodegradation by both

303systems are presented in Fig 2a b The rate of

304phenol biodegradation was determined after the end

305of each cycle for both immobilization systems

306(Table 1) As can be seen from Table 1 the lowest

Biodegradation

123

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307 biodegradation rate is observed at the first cycle then

308 it increases 3 times at the second and the third cycles

309 and from the fourth to the seventh cycle the rate

310 decreases continuously This trend of the biodegra-

311 dation rate is observed for both of the immobilized

312 systems The deceleration of the phenol biodegrada-

313 tion could be explained with the increasing of

314 biomass and mycelia formation on the membrane

315 surface after the fourth cycle which intensified at the

316 final cycles An obvious inhibition of the biodegra-

317 dation process at the final cycles was observed which

318 was due to the hindrance of the substrate diffusion to

319 the cells When comparing the results presented on

320 Fig 2a and b and Table 1 it becomes obvious that the

321 rate of phenol degradation was higher throughout all

322 cycles in the presence of the system where the spore

323 immobilization was conducted with addition of

32402 g l-1 phenol For better comparison the biodeg-

325radation times of each cycle with the two

326immobilized systems are presented in Fig 3 As

0

02

04

06

08

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

0

02

04

06

08

0 200 400 600 800

0 200 400 600 800 1000

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

(a)

(b)

Fig 2 Phenol

biodegradation (05 g l-1)

by immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane obtained by

immobilization in presence

of 02 g l-1 phenol (a) and

without phenol (b)

Table 1 Phenol biodegradation rate (mg h-1) of free and

immobilized Aspergillus awamori NRRL 3112 on polymer

PAN membrane at batch cultivation with 05 g l-1 phenol

Numberof

cycles

Free

cells

Immobilized cells

without 02 g l-1

phenol

Immobilized cells

in the presence of

02 g l-1 phenol

1 297 300 300

2 201 714 694

3 149 806 543

4 116 625 367

5 403 329

6 308 300

7 275 270

Biodegradation

123

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327 results suggest better degradation times were

328 obtained with the immobilized system prepared in

329 the presence of phenol This can be explained with

330 the fact that the strain could have adapted to the

331 substrate during the immobilization process All

332 further experiments were carried out with the immo-

333 bilized system prepared in presence of phenol

334 The biodegradation ability of the immobilized

335 system onto polyacrylonitrile membrane towards

336 phenol in a concentration range from 02 to

337 08 g l-1 was studied As can be seen from Fig 4

338 the increase of phenol concentration resulted in

339increased biodegradation time For instance the

340biodegradation cycle at concentration of 04 g l-1

341was 66 h while at 07 g l-1 the cycle lasted for 135 h

342The results obtained with the selected immobilized

343system were compared to those with the free strain

344For this purpose the spores (08 9 107 ml-1)

345were cultivated in Czapekdox medium containing

34605 g l-1 phenol (Fig 5) Four cycles were carried

347out in the same manner The amount of non-bound

348spores in the first cycle of cultivation is equivalent

349to the amount of immobilized spores on PAN

350membrane with surface 40 cm2 in the first cycle

0

50

100

150

200

250

1 2 3 4 5 6 7

Cycles

Tim

e

h

Fig 3 Cycles duration of

phenol biodegradation

(05 g l-1) by two

immobilized systems

Aspergillus awamori NRRL

3112modified PAN

membrane obtained by

immobilization in presence

of 02 g l-1 phenol ( ) and

without phenol (j)

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol co

ncen

trati

on

g

l-1

Fig 4 Phenol

biodegradation (from 02 to

08 g l-1) by immobilized

system Aspergillus

awamori NRRL 3112

modified PAN membrane

Biodegradation

123

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351 (wet weightmdash632 g) As can be seen from Fig 5 the

352 phenol degradation time was considerably greater for

353 the four cycles in these experiments This time

354 extension was confirmed by the calculated rate of

355 biodegradation (Table 1) When comparing the rates

356 of phenol biodegradation by the free and the immo-

357 bilized cells the greater stability of the immobilized

358 systems over the free cells becomes more prominent

359 after multiple cultivation in media containing phenol

360 with concentration 05 g l-1 Obviously the free cells

361 were inhibited to a greater extent by phenol (biodeg-

362 radation rate at Cycle 4mdash116 mg h-1) in

363 comparison with the immobilized cells (biodegrada-

364 tion rates at Cycle 4 are 625 and 367 mg h-1

365respectively for immobilized system obtained in the

366presence of 02 g l-1 phenol and the system without

367phenol)

368In order to distinguish between phenol adsorption

369on the membrane surface and phenol degradation by

370microorganisms the pure carrier was also studied for

371phenol adsorption For this purpose 40 cm2 pure

372membrane was immersed in 05 g l-1 phenol solution

373for 6 h The results are presented in Fig 6 Since the

374phenol concentration didnrsquot change significantly

375the only possible reason for the decrease of the

376phenol content was the degradation activity of the

377immobilized cells This simplifies the kinetic studies

378when membrane is to be used as a support material

0

02

04

06

08

0 100 200 300 400 500

Time h

Ph

en

ol

bio

de

gra

da

tio

n

gl-1

Fig 5 Phenol

biodegradation with

concentration 05 g l-1 by

strain Aspergillus awamori

NRRL 3112

0

02

04

06

08

0 1 2 3 4 5 6 7

Time h

Ph

en

ol

bio

deg

rad

ati

on

g

l-1

Fig 6 Adsorption of

phenol by modified PAN

membrane

Biodegradation

123

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379 for the immobilization of Asp awamori for the

380 purpose of phenol degradation

381 The amount of cells (wet weight) on the membrane

382 of 40 cm2 was measured after the sixth cycle of

383 phenol biodegradationmdash1104 g cm-2 The signifi-

384 cant increase of the biomass with the increased

385 number of cycles is obvious (after immobilizationmdash

386 0158 g cm-2) Thus the biomass accumulated on

387 the membrane was approximately ten times higher

388 after six cycles of phenol biodegradation The high

389 operational stability of investigated biocatalysts was

390 confirmed by the scanning electron micrographs of

391 the polyacrylonitrile membraneimmobilized Asper-

392 gillus awamori NRRL at the beginning of repeated

393 batch cultivation and after the 7th cycle After the 7th

394 cycle of cultivation the observations showed large

395 groups of cells (Fig 7a) The significant increase of

396 biomass and mycelia formation on the membrane

397 surface after the 7th cycle of cultivation was inev-

398 itably followed by deceleration of the phenol

399 degradation rate

400Experiments with membrane bioreactor

401The next step of the research work involved exper-

402iments which were carried out in a bioreactor with

403spirally wound membrane in a regime of recircula-

404tion The spores were immobilized onto the

405membrane directly in the system as described in

406lsquolsquoMaterials and methodsrsquorsquo section Phenol biodegra-

407dation was carried out under aerobic conditions The

408operation parameters of the bioreactor are presented

409in Table 2 Fresh nutrient medium containing

41005 g l-1 phenol were added after each cycle Phenol

411concentration was monitored for degradation At the

412end of a cycle the system was washed with sterile

413distilled water and charged with nutrient medium and

414phenol for the next cycle Thus 10 cycles of 05 g l-1

415phenol biodegradation were run consecutively to

416determine the degradation time for each cycle

417(Fig 8) No significant difference was observed

418between the degradation times of the cycles from

419the second to the seventh (about 50 h) and between

420the biodegradation rates (100 mg h-1) After the 7th

421cycle the biodegradation rate began to decrease

422gradually and at 10 cycles it was 40 mg h-1 These

423results confirmed the advantages of the recycled

424reactor with spirally wound membrane The design of

425the bioreactor appeared to be quite effective provid-

426ing large membrane surface to bind the strain cells

427Besides phenol would flow tangentially which pre-

428vents membrane from a decrease of the flow velocity

429Conclusion

430A novel immobilized systemmdashAspergillus awamori

431NRRL 3112polyacrylonitrile membranewas obtained

432for the purpose of phenol biodegradation It was

433established that the immobilized system prepared in

434the presence of 02 g l-1 phenol was more effective

Fig 7 Scanning electron micrographs of immobilized system

Aspergillus awamori NRRL 3112modified PAN membrane

after 7th cycle (a) and before 1st cycle (b)

Table 2 Operation conditions of bioreactor

Parameter Value

Rate flow 15 ml min-1

Volume 300 ml

Initial phenol concentration 05 g l-1

Membrane surface area 100 cm2

Immobilized spores 0174 g cm-2 (wet weight)

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

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435 than the system prepared in the phenol absence It was

436 revealed that the immobilized systems were more

437 stable andmore effective when biodegrading 05 g l-1

438 phenol in comparison with the free cells A bioreactor

439 was constructed containing spirally wound membrane

440 with immobilized spores of Aspergillus awamori

441 NRRL 3112 in a regime of recirculation which was

442 successfully applied for phenol biodegradation The

443 obtained results confirmed the advantages of the

444 recycled reactor with spirally wound membrane Thus

445 the immobilized microbial technology is an extremely

446 versatile approach that can be used for degradation of

447 toxic pollutants from the industrial effluents

448 Acknowledgments The authors gratefully acknowledge to449 the Bulgarian Ministry for Education and to the National450 Science Fund for their financial support and encouragement of451 the scientific research work in state universities

452 References

453 Abd-El-Haleem D Beshay U Abdelhamid AO Moawad H454 Zaki S (2003) Effects of mixed nitrogen sources on bio-455 degradation of phenol by immobilized Acinetobacter sp456 strain W-17 Afr J Biotechnol 2(1)8ndash12457 Allsop PJ Chisti Y Moi-Youngand M Sullivan GR (1993)458 Dynamics of phenol degradation by Pseudomonas putida459 Biotechnol Bioeng 41572ndash579460 Anselmo AM Novais JM (1992) Degradation of phenol by461 immobilized mycelium of Fusarium floccieferum in con-462 tinuous culture Water Sci Technol 25161ndash168463 Bolanos ML Varesche MBA Zaiat M Foresti E (2001) Phenol464 degradation in horizontal-flow anaerobic immobilized465 biomass (HAIB) reactor under mesophilic conditions466 Water Sci Technol 44(4)167ndash174

467Chitra S Sekaran G Padmavathi S Gowri C (1995) Removal468of phenol from wastewater using a biocatalyst In Pro-469ceedings of the national symposium frontiers in applied470environmental microbiology pp 74ndash77471Chtourou M Ammar E Nasri M Medhioub K (2004) Isolation472of a yeast Trichosporon cutaneum able to use low473molecular weight phenolic compounds application to474olive mill waste water treatment J Chem Technol Bio-475technol 79869ndash878476Dimov K Samardjieva Pavlov P (1983) Laboratory practice477on technology of synthetic fibers Technica Sofia BG478Fiest CF Hegeman GD (1969) Phenol and benzoate metabo-479lism by Pseudomonas putida J Bacteriol 100869ndash877480Godjevargova T Aleksieva Z Ivanova D (2000) Cell immo-481bilization of Trichosporon cutaneum strain with phenol482degradation ability on new modified polymer carries483Process Biochem 35699ndash704484Godjevargova T Ivanova D Aleksieva Z Burdelova G (2006)485Biodegradation of phenol by immobilized Trichosporon

486cutaneum R57 on modified polymer membranes Process487Biochem 412342ndash2346488Gonzalez G Herrera MG Pena MM (2001) Biodegradation of489phenol in a continuous process comparative study of490stirred tank and fluidized-bed bioreactors Bioresour491Technol 76245ndash251492Hua L Sun ZH Leng Y (2005) Continuous biocatalytic reso-493lution of DL-pantolactone by cross-linked cells in494membrane bioreactor Process Biochem 401137ndash1142495Hudges EJL Bayly RC Skurray RA (1984) Evidence for496isofunctional enzymes in the degradation of phenol m-497and p-toluate and p-cresol at catechol meta-cleavage498pathways in Alcaligenes eutrophus J Bacteriol 15879ndash83499Jones KH Trudgill PW Hopper DJ (1995) Evidence of two500pathways for the metabolism of phenol by Aspergillus

501fumigatus Arch Microbiol 163176ndash181502Jose G Sanchez M Theodore T (2002) Membranes and mem-503brane reactors Wiley-VCH Veriag GmbH Weinheim504Karel S Libichi S Robertson C (1985) The immobilization of505whole cells engineering principles Chem Eng Sci506401321ndash1354

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

Fig 8 Concentration

profile of phenol in a

recirculating system with

immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

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507 Kowalska M Bodzek M Bohdziewicz J (1998) Biodegradation508 of phenols and cyanides using membranes immobilized509 microorganisms Process Biochem 33189ndash197510 Loh K Chung T Ang W (2000) Immobilized-cell membrane511 bioreactor for high-strength phenol wastewater J Environ512 Eng ASCE 12675ndash79513 Marrot B Barrios-Martinez A Moulin P Roche N (2006)514 Biodegradation of high phenol concentration by activated515 sludge in an immersed membrane bioreactor Biochem516 Eng J 30174ndash183517 Mordocco A Kuek C Jenkins R (1999) Continuous degradation518 of phenol at low concentration using immobilized Pseu-

519 domonas putida Enzyme Microb Technol 25530ndash536520 Pazarlioglu NK Telefoncu A (2005) Biodegradation of phenol521 by Pseudomonas putida immobilized on activated pumice522 particles Process Biochem 401807ndash1814523 Santos VL Heilbuth NM Braga T Monteiro AS Linardi VR524 (2003) Phenol degradation by aGraphium sp FIB4 isolated525 from industrial effluents J Basic Microbiol 43238ndash248526 Shetty KV Kalifathulla I Srinikethan G (2007a) Performance527 of pulsed plate bioreactor for biodegradation of phenol528 J Hazard Mater 140346ndash352529 Shetty KV Ramanjaneyulu R Srinikethan G (2007b) Biolog-530 ical phenol removal using immobilized cells in a pulsed

531plate bioreactor effect of dilution rate and influent phenol532concentration J Hazard Mater 149452ndash459533Stephenson T Judd S Jefferson B Brindle K (2000) Mem-534brane bioreactors for wastewater treatment IWA535Publishing London UK pp 129ndash136536Sundar P Daniel D Krastanov A (2005) Biodegradation of537phenol by immobilized Aspergillus awamori cells Bulg J538Agric Sci 1161ndash71539Uzunova K Vassileva A Ivanova V Spasova D Tonkova A540(2002) Thermostable exo-inulinase production by semi-541continuous cultivation of membrane-immobilized Bacillus542sp 11 cells Process Biochem 37863ndash868543Wang Z Wan L Xu Z (2007) Surface engineerings of poly-544acrylonitrile-based asymmetric membranes towards545biomedical applications an overview J Membr Sci5463048ndash23547Yiang Y Wen J Li H Yang S Hu Z (2005) The biodegra-548dation of phenol at high initial concentration by the yeast549Candida tropicalis Biochem Eng J 24243ndash247550Yordanova G Ivanova D Godjevargova T Krastanov A (2006)551Biodegradation of phenol by immobilized Aspergillus

552awamori NRRL3112 cells Food science engineering and553technologies conference Scientific works Plovdiv554pp 260ndash265

555

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

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152 Chemical modification of polyacrylonitrile

153 membrane

154 The ultrafiltration polyacrylonitrile (PAN) membrane

155 (average pore size of selective layer 002 lm) was

156 produced by Ecofilter Co (Bourgas Bulgaria) PAN

157 membrane was treated with 10 aqueous solution of

158 NaOH at 50C for 60 min After thorough washing

159 with distilled water the membrane was treated with a

160 10 aqueous solution of 12-diaminohexane for

161 60 min at 40C The membrane was once again

162 thoroughly washed with distilled water At the end of

163 the procedure the modified carrier was immersed in

164 05 aqueous solution of glutaraldehyde for 60 min

165 at room temperature Excess glutaraldehyde was

166 removed by washing the membranes with distilled

167 water until there was a complete absence of glutar-

168 aldehyde in the rinse waters

169 Phenol biodegradation in batch experiments

170 Spore immobilization for batch experiments

171 The 14-day culture in spore form in concentration of

172 333 9 107 ml-1 were introduced by flushing 5 cm3

173 sterile water in 500 cm3 sterile flask containing

174 50 cm3 Czapekdox medium and sterile modified

175 carrier (40 cm2 PAN flat membrane) Sterilization of

176 the modified membrane was carried out by ethanol

177 Two ways of immobilization were usedmdashin the

178 presence of 02 g l-1 phenol and without phenol

179 The two flasks were placed on a shaker for 24 h at

180 30C Then the liquid medium was decanted and the

181 immobilized systems were washed several times with

182 sterile water to remove the unbound spores After

183 that the immobilized spores were used for phenol

184 biodegradation

185 Batch experiments

186 The immobilized spores onto the polymer mem-

187 brane (single flat piecemdash40 cm2) with concentration

188 0158 g cm-2 were introduced in 500 cm3 sterile

189 flask containing 50 cm3 Czapekdox medium as well

190 as phenol as the only carbon source adjusted to the

191 appropriate concentration Then they were cultivated

192 on a shaker (220 rpm) at 30C until the complete

193 phenol degradation At every 24 h samples were

194 withdrawn and analyzed for phenol concentration

195Thus seven cycles were carried out with the same

196immobilized system

197Simultaneously parallel experiments with free

198cells were also performed for comparison The flasks

199containing 50 cm3 liquid medium as well as 05 g l-1

200phenol as the only carbon source were inoculated with

201equal volume of inoculum (333 9 107 conidia cm-3

202medium) and agitated on a shaker (220 rpm) at 30C

203Samples were taken at every 24 h interval for

204determination of phenol concentration After the

205biodegradation of the entire phenol the cultural

206medium was decanted and the biomass was reculti-

207vated in fresh liquid medium containing 05 g l-1

208phenol Thus four cycles were carried out in the same

209manner

210Each experiment for phenol degradation was

211repeated twice the reproducibility being always within

21225

213Phenol biodegradation in recycle reactor

214with spirally wound membrane

215Spore immobilization for experiments

216with membrane bioreactor

217The bioreactor was 40 mm in diameter and 100 mm

218high it was made of glass and equipped with

219thermostatic bath (Fig 1) The modified and activated

220with glutaraldehyde polyacrylonitrile membrane with

Fig 1 Scheme of recycle system thermostatic bath (1) feed

reservoir (2) peristaltic pump (3) thermostatic reactor (4)

membrane with immobilized cells (5) sampling (6) air outlet

(7) and air inlet (8)

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

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221 surface area of 100 cm2 was spirally wound in the

222 glass bioreactor using supporting polyethylene mesh

223 In this case the immobilization was carried out by

224 flowing spore suspension (547 9 107 conidia cm-3

225 medium) through the membrane module using peri-

226 staltic pump at rate of 1 ml min-1 for 24 h Then the

227 membrane spiral was thoroughly washed out with

228 sterile water fed by peristaltic pump to remove the

229 non-bound spores

230 Experiments with membrane bioreactor

231 Further the liquid medium (300 ml) containing

232 phenol at certain concentration was fed to the mem-

233 brane bioreactor using peristaltic pump at rate of

234 15 ml min-1 The temperature required for the strain

235 growth (30C) was maintained using the thermostat

236 and the heat-exchanging coating After the full

237 degradation of phenol in the nutrient medium the

238 reservoir was charged with fresh nutrient medium

239 containing phenol Samples were taken at every 24 h

240 interval for determination of phenol concentration

241 Ten cycles were carried out in the same manner

242 Each experiment was repeated twice the repro-

243 ducibility being always within 25

244 Analytical methods

245 Phenol concentration was measured spectrophoto-

246 metrically in the presence of p-nitroaniline at 495 nm

247 (Gonzalez et al 2001)

248 The amount of spores immobilized on the support

249 was estimated by the difference between the wet

250 weight of spores with membrane at the beginning

251 (immediately after the immobilization) and the wet

252 membrane weight The amount of free cells was

253 determined by the measuring of the wet weight of the

254 cells and by the counting of the number of conidia in

255 the samples using a standard microscope method

256 Scanning electron microscopy (SEM)

257 The immobilized system Aspergillus awamori NRRL

258 3112polyacrylonitrile membrane was washed with

259 distilled water and then they were dehydrated in

260 30ndash100 water ethanol series The membrane were

261 coated with 120ndash130 Aacuteof gold in argon medium in

262 an Edwards apparatus (model 150 A) The SEM

263 observations were done on a scanning device attached

264to a Zeiss electron microscope (model 10C) at 20 kV

265accelerating voltage with a 5ndash6 nm electron beam

266Results and discussions

267Modification of membranes

268The aim of the present studies is to obtain an efficient

269immobilized system of Aspergillus awamori NRRL

2703112polyacrylonitrile membrane for biodegradation

271of phenol The immobilization of spores was carried

272out with glutaraldehyde The polymer membrane was

273preliminarily treated by using two different modifica-

274tion procedures The first procedure introduced

275ndashCOOH and ndashNH2 groups on the membrane surface

276by using NaOH solution In order to increase the

277amount of amine groups on the surface of the carrier

278the latter was treated with 12-diaminoethane The

279optimal conditions of modification were defined in a

280previous research work (Godjevargova et al 2000)

281The superficial carboxyl groups on the carrier surface

282reacted with one of the amine groups of 12-diami-

283noethane leaving the other one free The results

284showed that the PANmembranes had beenmodified in

285a greater extent and the content of amino groups was

286058 mgeq g-1 The latter was determined titrimetri-

287cally (Dimov et al 1983) Further the modified

288membrane was treated with an aqueous solution of

289glutaraldehyde The bifunctional agent bound the

290strain to the membrane surface

291Batch experiments

292Spores concentration used for the immobilization was

293333 9 107 ml-1 The first task was to determine

294which immobilization method would serve better for

295these experimentsmdashin the presence or in the absence

296of phenol (02 g l-1) in a nutrient medium containing

297spore suspension The amount of spores bound to the

298membranes in both cases was 0158 g cm-2 (wet

299weight) Then the spores immobilized on the polymer

300membrane by both methods were cultivated in a

301nutrient medium containing 05 g l-1 phenol The

302results obtained for phenol biodegradation by both

303systems are presented in Fig 2a b The rate of

304phenol biodegradation was determined after the end

305of each cycle for both immobilization systems

306(Table 1) As can be seen from Table 1 the lowest

Biodegradation

123

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307 biodegradation rate is observed at the first cycle then

308 it increases 3 times at the second and the third cycles

309 and from the fourth to the seventh cycle the rate

310 decreases continuously This trend of the biodegra-

311 dation rate is observed for both of the immobilized

312 systems The deceleration of the phenol biodegrada-

313 tion could be explained with the increasing of

314 biomass and mycelia formation on the membrane

315 surface after the fourth cycle which intensified at the

316 final cycles An obvious inhibition of the biodegra-

317 dation process at the final cycles was observed which

318 was due to the hindrance of the substrate diffusion to

319 the cells When comparing the results presented on

320 Fig 2a and b and Table 1 it becomes obvious that the

321 rate of phenol degradation was higher throughout all

322 cycles in the presence of the system where the spore

323 immobilization was conducted with addition of

32402 g l-1 phenol For better comparison the biodeg-

325radation times of each cycle with the two

326immobilized systems are presented in Fig 3 As

0

02

04

06

08

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

0

02

04

06

08

0 200 400 600 800

0 200 400 600 800 1000

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

(a)

(b)

Fig 2 Phenol

biodegradation (05 g l-1)

by immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane obtained by

immobilization in presence

of 02 g l-1 phenol (a) and

without phenol (b)

Table 1 Phenol biodegradation rate (mg h-1) of free and

immobilized Aspergillus awamori NRRL 3112 on polymer

PAN membrane at batch cultivation with 05 g l-1 phenol

Numberof

cycles

Free

cells

Immobilized cells

without 02 g l-1

phenol

Immobilized cells

in the presence of

02 g l-1 phenol

1 297 300 300

2 201 714 694

3 149 806 543

4 116 625 367

5 403 329

6 308 300

7 275 270

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

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327 results suggest better degradation times were

328 obtained with the immobilized system prepared in

329 the presence of phenol This can be explained with

330 the fact that the strain could have adapted to the

331 substrate during the immobilization process All

332 further experiments were carried out with the immo-

333 bilized system prepared in presence of phenol

334 The biodegradation ability of the immobilized

335 system onto polyacrylonitrile membrane towards

336 phenol in a concentration range from 02 to

337 08 g l-1 was studied As can be seen from Fig 4

338 the increase of phenol concentration resulted in

339increased biodegradation time For instance the

340biodegradation cycle at concentration of 04 g l-1

341was 66 h while at 07 g l-1 the cycle lasted for 135 h

342The results obtained with the selected immobilized

343system were compared to those with the free strain

344For this purpose the spores (08 9 107 ml-1)

345were cultivated in Czapekdox medium containing

34605 g l-1 phenol (Fig 5) Four cycles were carried

347out in the same manner The amount of non-bound

348spores in the first cycle of cultivation is equivalent

349to the amount of immobilized spores on PAN

350membrane with surface 40 cm2 in the first cycle

0

50

100

150

200

250

1 2 3 4 5 6 7

Cycles

Tim

e

h

Fig 3 Cycles duration of

phenol biodegradation

(05 g l-1) by two

immobilized systems

Aspergillus awamori NRRL

3112modified PAN

membrane obtained by

immobilization in presence

of 02 g l-1 phenol ( ) and

without phenol (j)

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol co

ncen

trati

on

g

l-1

Fig 4 Phenol

biodegradation (from 02 to

08 g l-1) by immobilized

system Aspergillus

awamori NRRL 3112

modified PAN membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

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351 (wet weightmdash632 g) As can be seen from Fig 5 the

352 phenol degradation time was considerably greater for

353 the four cycles in these experiments This time

354 extension was confirmed by the calculated rate of

355 biodegradation (Table 1) When comparing the rates

356 of phenol biodegradation by the free and the immo-

357 bilized cells the greater stability of the immobilized

358 systems over the free cells becomes more prominent

359 after multiple cultivation in media containing phenol

360 with concentration 05 g l-1 Obviously the free cells

361 were inhibited to a greater extent by phenol (biodeg-

362 radation rate at Cycle 4mdash116 mg h-1) in

363 comparison with the immobilized cells (biodegrada-

364 tion rates at Cycle 4 are 625 and 367 mg h-1

365respectively for immobilized system obtained in the

366presence of 02 g l-1 phenol and the system without

367phenol)

368In order to distinguish between phenol adsorption

369on the membrane surface and phenol degradation by

370microorganisms the pure carrier was also studied for

371phenol adsorption For this purpose 40 cm2 pure

372membrane was immersed in 05 g l-1 phenol solution

373for 6 h The results are presented in Fig 6 Since the

374phenol concentration didnrsquot change significantly

375the only possible reason for the decrease of the

376phenol content was the degradation activity of the

377immobilized cells This simplifies the kinetic studies

378when membrane is to be used as a support material

0

02

04

06

08

0 100 200 300 400 500

Time h

Ph

en

ol

bio

de

gra

da

tio

n

gl-1

Fig 5 Phenol

biodegradation with

concentration 05 g l-1 by

strain Aspergillus awamori

NRRL 3112

0

02

04

06

08

0 1 2 3 4 5 6 7

Time h

Ph

en

ol

bio

deg

rad

ati

on

g

l-1

Fig 6 Adsorption of

phenol by modified PAN

membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

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379 for the immobilization of Asp awamori for the

380 purpose of phenol degradation

381 The amount of cells (wet weight) on the membrane

382 of 40 cm2 was measured after the sixth cycle of

383 phenol biodegradationmdash1104 g cm-2 The signifi-

384 cant increase of the biomass with the increased

385 number of cycles is obvious (after immobilizationmdash

386 0158 g cm-2) Thus the biomass accumulated on

387 the membrane was approximately ten times higher

388 after six cycles of phenol biodegradation The high

389 operational stability of investigated biocatalysts was

390 confirmed by the scanning electron micrographs of

391 the polyacrylonitrile membraneimmobilized Asper-

392 gillus awamori NRRL at the beginning of repeated

393 batch cultivation and after the 7th cycle After the 7th

394 cycle of cultivation the observations showed large

395 groups of cells (Fig 7a) The significant increase of

396 biomass and mycelia formation on the membrane

397 surface after the 7th cycle of cultivation was inev-

398 itably followed by deceleration of the phenol

399 degradation rate

400Experiments with membrane bioreactor

401The next step of the research work involved exper-

402iments which were carried out in a bioreactor with

403spirally wound membrane in a regime of recircula-

404tion The spores were immobilized onto the

405membrane directly in the system as described in

406lsquolsquoMaterials and methodsrsquorsquo section Phenol biodegra-

407dation was carried out under aerobic conditions The

408operation parameters of the bioreactor are presented

409in Table 2 Fresh nutrient medium containing

41005 g l-1 phenol were added after each cycle Phenol

411concentration was monitored for degradation At the

412end of a cycle the system was washed with sterile

413distilled water and charged with nutrient medium and

414phenol for the next cycle Thus 10 cycles of 05 g l-1

415phenol biodegradation were run consecutively to

416determine the degradation time for each cycle

417(Fig 8) No significant difference was observed

418between the degradation times of the cycles from

419the second to the seventh (about 50 h) and between

420the biodegradation rates (100 mg h-1) After the 7th

421cycle the biodegradation rate began to decrease

422gradually and at 10 cycles it was 40 mg h-1 These

423results confirmed the advantages of the recycled

424reactor with spirally wound membrane The design of

425the bioreactor appeared to be quite effective provid-

426ing large membrane surface to bind the strain cells

427Besides phenol would flow tangentially which pre-

428vents membrane from a decrease of the flow velocity

429Conclusion

430A novel immobilized systemmdashAspergillus awamori

431NRRL 3112polyacrylonitrile membranewas obtained

432for the purpose of phenol biodegradation It was

433established that the immobilized system prepared in

434the presence of 02 g l-1 phenol was more effective

Fig 7 Scanning electron micrographs of immobilized system

Aspergillus awamori NRRL 3112modified PAN membrane

after 7th cycle (a) and before 1st cycle (b)

Table 2 Operation conditions of bioreactor

Parameter Value

Rate flow 15 ml min-1

Volume 300 ml

Initial phenol concentration 05 g l-1

Membrane surface area 100 cm2

Immobilized spores 0174 g cm-2 (wet weight)

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

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435 than the system prepared in the phenol absence It was

436 revealed that the immobilized systems were more

437 stable andmore effective when biodegrading 05 g l-1

438 phenol in comparison with the free cells A bioreactor

439 was constructed containing spirally wound membrane

440 with immobilized spores of Aspergillus awamori

441 NRRL 3112 in a regime of recirculation which was

442 successfully applied for phenol biodegradation The

443 obtained results confirmed the advantages of the

444 recycled reactor with spirally wound membrane Thus

445 the immobilized microbial technology is an extremely

446 versatile approach that can be used for degradation of

447 toxic pollutants from the industrial effluents

448 Acknowledgments The authors gratefully acknowledge to449 the Bulgarian Ministry for Education and to the National450 Science Fund for their financial support and encouragement of451 the scientific research work in state universities

452 References

453 Abd-El-Haleem D Beshay U Abdelhamid AO Moawad H454 Zaki S (2003) Effects of mixed nitrogen sources on bio-455 degradation of phenol by immobilized Acinetobacter sp456 strain W-17 Afr J Biotechnol 2(1)8ndash12457 Allsop PJ Chisti Y Moi-Youngand M Sullivan GR (1993)458 Dynamics of phenol degradation by Pseudomonas putida459 Biotechnol Bioeng 41572ndash579460 Anselmo AM Novais JM (1992) Degradation of phenol by461 immobilized mycelium of Fusarium floccieferum in con-462 tinuous culture Water Sci Technol 25161ndash168463 Bolanos ML Varesche MBA Zaiat M Foresti E (2001) Phenol464 degradation in horizontal-flow anaerobic immobilized465 biomass (HAIB) reactor under mesophilic conditions466 Water Sci Technol 44(4)167ndash174

467Chitra S Sekaran G Padmavathi S Gowri C (1995) Removal468of phenol from wastewater using a biocatalyst In Pro-469ceedings of the national symposium frontiers in applied470environmental microbiology pp 74ndash77471Chtourou M Ammar E Nasri M Medhioub K (2004) Isolation472of a yeast Trichosporon cutaneum able to use low473molecular weight phenolic compounds application to474olive mill waste water treatment J Chem Technol Bio-475technol 79869ndash878476Dimov K Samardjieva Pavlov P (1983) Laboratory practice477on technology of synthetic fibers Technica Sofia BG478Fiest CF Hegeman GD (1969) Phenol and benzoate metabo-479lism by Pseudomonas putida J Bacteriol 100869ndash877480Godjevargova T Aleksieva Z Ivanova D (2000) Cell immo-481bilization of Trichosporon cutaneum strain with phenol482degradation ability on new modified polymer carries483Process Biochem 35699ndash704484Godjevargova T Ivanova D Aleksieva Z Burdelova G (2006)485Biodegradation of phenol by immobilized Trichosporon

486cutaneum R57 on modified polymer membranes Process487Biochem 412342ndash2346488Gonzalez G Herrera MG Pena MM (2001) Biodegradation of489phenol in a continuous process comparative study of490stirred tank and fluidized-bed bioreactors Bioresour491Technol 76245ndash251492Hua L Sun ZH Leng Y (2005) Continuous biocatalytic reso-493lution of DL-pantolactone by cross-linked cells in494membrane bioreactor Process Biochem 401137ndash1142495Hudges EJL Bayly RC Skurray RA (1984) Evidence for496isofunctional enzymes in the degradation of phenol m-497and p-toluate and p-cresol at catechol meta-cleavage498pathways in Alcaligenes eutrophus J Bacteriol 15879ndash83499Jones KH Trudgill PW Hopper DJ (1995) Evidence of two500pathways for the metabolism of phenol by Aspergillus

501fumigatus Arch Microbiol 163176ndash181502Jose G Sanchez M Theodore T (2002) Membranes and mem-503brane reactors Wiley-VCH Veriag GmbH Weinheim504Karel S Libichi S Robertson C (1985) The immobilization of505whole cells engineering principles Chem Eng Sci506401321ndash1354

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

Fig 8 Concentration

profile of phenol in a

recirculating system with

immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

507 Kowalska M Bodzek M Bohdziewicz J (1998) Biodegradation508 of phenols and cyanides using membranes immobilized509 microorganisms Process Biochem 33189ndash197510 Loh K Chung T Ang W (2000) Immobilized-cell membrane511 bioreactor for high-strength phenol wastewater J Environ512 Eng ASCE 12675ndash79513 Marrot B Barrios-Martinez A Moulin P Roche N (2006)514 Biodegradation of high phenol concentration by activated515 sludge in an immersed membrane bioreactor Biochem516 Eng J 30174ndash183517 Mordocco A Kuek C Jenkins R (1999) Continuous degradation518 of phenol at low concentration using immobilized Pseu-

519 domonas putida Enzyme Microb Technol 25530ndash536520 Pazarlioglu NK Telefoncu A (2005) Biodegradation of phenol521 by Pseudomonas putida immobilized on activated pumice522 particles Process Biochem 401807ndash1814523 Santos VL Heilbuth NM Braga T Monteiro AS Linardi VR524 (2003) Phenol degradation by aGraphium sp FIB4 isolated525 from industrial effluents J Basic Microbiol 43238ndash248526 Shetty KV Kalifathulla I Srinikethan G (2007a) Performance527 of pulsed plate bioreactor for biodegradation of phenol528 J Hazard Mater 140346ndash352529 Shetty KV Ramanjaneyulu R Srinikethan G (2007b) Biolog-530 ical phenol removal using immobilized cells in a pulsed

531plate bioreactor effect of dilution rate and influent phenol532concentration J Hazard Mater 149452ndash459533Stephenson T Judd S Jefferson B Brindle K (2000) Mem-534brane bioreactors for wastewater treatment IWA535Publishing London UK pp 129ndash136536Sundar P Daniel D Krastanov A (2005) Biodegradation of537phenol by immobilized Aspergillus awamori cells Bulg J538Agric Sci 1161ndash71539Uzunova K Vassileva A Ivanova V Spasova D Tonkova A540(2002) Thermostable exo-inulinase production by semi-541continuous cultivation of membrane-immobilized Bacillus542sp 11 cells Process Biochem 37863ndash868543Wang Z Wan L Xu Z (2007) Surface engineerings of poly-544acrylonitrile-based asymmetric membranes towards545biomedical applications an overview J Membr Sci5463048ndash23547Yiang Y Wen J Li H Yang S Hu Z (2005) The biodegra-548dation of phenol at high initial concentration by the yeast549Candida tropicalis Biochem Eng J 24243ndash247550Yordanova G Ivanova D Godjevargova T Krastanov A (2006)551Biodegradation of phenol by immobilized Aspergillus

552awamori NRRL3112 cells Food science engineering and553technologies conference Scientific works Plovdiv554pp 260ndash265

555

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

221 surface area of 100 cm2 was spirally wound in the

222 glass bioreactor using supporting polyethylene mesh

223 In this case the immobilization was carried out by

224 flowing spore suspension (547 9 107 conidia cm-3

225 medium) through the membrane module using peri-

226 staltic pump at rate of 1 ml min-1 for 24 h Then the

227 membrane spiral was thoroughly washed out with

228 sterile water fed by peristaltic pump to remove the

229 non-bound spores

230 Experiments with membrane bioreactor

231 Further the liquid medium (300 ml) containing

232 phenol at certain concentration was fed to the mem-

233 brane bioreactor using peristaltic pump at rate of

234 15 ml min-1 The temperature required for the strain

235 growth (30C) was maintained using the thermostat

236 and the heat-exchanging coating After the full

237 degradation of phenol in the nutrient medium the

238 reservoir was charged with fresh nutrient medium

239 containing phenol Samples were taken at every 24 h

240 interval for determination of phenol concentration

241 Ten cycles were carried out in the same manner

242 Each experiment was repeated twice the repro-

243 ducibility being always within 25

244 Analytical methods

245 Phenol concentration was measured spectrophoto-

246 metrically in the presence of p-nitroaniline at 495 nm

247 (Gonzalez et al 2001)

248 The amount of spores immobilized on the support

249 was estimated by the difference between the wet

250 weight of spores with membrane at the beginning

251 (immediately after the immobilization) and the wet

252 membrane weight The amount of free cells was

253 determined by the measuring of the wet weight of the

254 cells and by the counting of the number of conidia in

255 the samples using a standard microscope method

256 Scanning electron microscopy (SEM)

257 The immobilized system Aspergillus awamori NRRL

258 3112polyacrylonitrile membrane was washed with

259 distilled water and then they were dehydrated in

260 30ndash100 water ethanol series The membrane were

261 coated with 120ndash130 Aacuteof gold in argon medium in

262 an Edwards apparatus (model 150 A) The SEM

263 observations were done on a scanning device attached

264to a Zeiss electron microscope (model 10C) at 20 kV

265accelerating voltage with a 5ndash6 nm electron beam

266Results and discussions

267Modification of membranes

268The aim of the present studies is to obtain an efficient

269immobilized system of Aspergillus awamori NRRL

2703112polyacrylonitrile membrane for biodegradation

271of phenol The immobilization of spores was carried

272out with glutaraldehyde The polymer membrane was

273preliminarily treated by using two different modifica-

274tion procedures The first procedure introduced

275ndashCOOH and ndashNH2 groups on the membrane surface

276by using NaOH solution In order to increase the

277amount of amine groups on the surface of the carrier

278the latter was treated with 12-diaminoethane The

279optimal conditions of modification were defined in a

280previous research work (Godjevargova et al 2000)

281The superficial carboxyl groups on the carrier surface

282reacted with one of the amine groups of 12-diami-

283noethane leaving the other one free The results

284showed that the PANmembranes had beenmodified in

285a greater extent and the content of amino groups was

286058 mgeq g-1 The latter was determined titrimetri-

287cally (Dimov et al 1983) Further the modified

288membrane was treated with an aqueous solution of

289glutaraldehyde The bifunctional agent bound the

290strain to the membrane surface

291Batch experiments

292Spores concentration used for the immobilization was

293333 9 107 ml-1 The first task was to determine

294which immobilization method would serve better for

295these experimentsmdashin the presence or in the absence

296of phenol (02 g l-1) in a nutrient medium containing

297spore suspension The amount of spores bound to the

298membranes in both cases was 0158 g cm-2 (wet

299weight) Then the spores immobilized on the polymer

300membrane by both methods were cultivated in a

301nutrient medium containing 05 g l-1 phenol The

302results obtained for phenol biodegradation by both

303systems are presented in Fig 2a b The rate of

304phenol biodegradation was determined after the end

305of each cycle for both immobilization systems

306(Table 1) As can be seen from Table 1 the lowest

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

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OOF

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OOF

307 biodegradation rate is observed at the first cycle then

308 it increases 3 times at the second and the third cycles

309 and from the fourth to the seventh cycle the rate

310 decreases continuously This trend of the biodegra-

311 dation rate is observed for both of the immobilized

312 systems The deceleration of the phenol biodegrada-

313 tion could be explained with the increasing of

314 biomass and mycelia formation on the membrane

315 surface after the fourth cycle which intensified at the

316 final cycles An obvious inhibition of the biodegra-

317 dation process at the final cycles was observed which

318 was due to the hindrance of the substrate diffusion to

319 the cells When comparing the results presented on

320 Fig 2a and b and Table 1 it becomes obvious that the

321 rate of phenol degradation was higher throughout all

322 cycles in the presence of the system where the spore

323 immobilization was conducted with addition of

32402 g l-1 phenol For better comparison the biodeg-

325radation times of each cycle with the two

326immobilized systems are presented in Fig 3 As

0

02

04

06

08

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

0

02

04

06

08

0 200 400 600 800

0 200 400 600 800 1000

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

(a)

(b)

Fig 2 Phenol

biodegradation (05 g l-1)

by immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane obtained by

immobilization in presence

of 02 g l-1 phenol (a) and

without phenol (b)

Table 1 Phenol biodegradation rate (mg h-1) of free and

immobilized Aspergillus awamori NRRL 3112 on polymer

PAN membrane at batch cultivation with 05 g l-1 phenol

Numberof

cycles

Free

cells

Immobilized cells

without 02 g l-1

phenol

Immobilized cells

in the presence of

02 g l-1 phenol

1 297 300 300

2 201 714 694

3 149 806 543

4 116 625 367

5 403 329

6 308 300

7 275 270

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

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of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

327 results suggest better degradation times were

328 obtained with the immobilized system prepared in

329 the presence of phenol This can be explained with

330 the fact that the strain could have adapted to the

331 substrate during the immobilization process All

332 further experiments were carried out with the immo-

333 bilized system prepared in presence of phenol

334 The biodegradation ability of the immobilized

335 system onto polyacrylonitrile membrane towards

336 phenol in a concentration range from 02 to

337 08 g l-1 was studied As can be seen from Fig 4

338 the increase of phenol concentration resulted in

339increased biodegradation time For instance the

340biodegradation cycle at concentration of 04 g l-1

341was 66 h while at 07 g l-1 the cycle lasted for 135 h

342The results obtained with the selected immobilized

343system were compared to those with the free strain

344For this purpose the spores (08 9 107 ml-1)

345were cultivated in Czapekdox medium containing

34605 g l-1 phenol (Fig 5) Four cycles were carried

347out in the same manner The amount of non-bound

348spores in the first cycle of cultivation is equivalent

349to the amount of immobilized spores on PAN

350membrane with surface 40 cm2 in the first cycle

0

50

100

150

200

250

1 2 3 4 5 6 7

Cycles

Tim

e

h

Fig 3 Cycles duration of

phenol biodegradation

(05 g l-1) by two

immobilized systems

Aspergillus awamori NRRL

3112modified PAN

membrane obtained by

immobilization in presence

of 02 g l-1 phenol ( ) and

without phenol (j)

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol co

ncen

trati

on

g

l-1

Fig 4 Phenol

biodegradation (from 02 to

08 g l-1) by immobilized

system Aspergillus

awamori NRRL 3112

modified PAN membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

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r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

351 (wet weightmdash632 g) As can be seen from Fig 5 the

352 phenol degradation time was considerably greater for

353 the four cycles in these experiments This time

354 extension was confirmed by the calculated rate of

355 biodegradation (Table 1) When comparing the rates

356 of phenol biodegradation by the free and the immo-

357 bilized cells the greater stability of the immobilized

358 systems over the free cells becomes more prominent

359 after multiple cultivation in media containing phenol

360 with concentration 05 g l-1 Obviously the free cells

361 were inhibited to a greater extent by phenol (biodeg-

362 radation rate at Cycle 4mdash116 mg h-1) in

363 comparison with the immobilized cells (biodegrada-

364 tion rates at Cycle 4 are 625 and 367 mg h-1

365respectively for immobilized system obtained in the

366presence of 02 g l-1 phenol and the system without

367phenol)

368In order to distinguish between phenol adsorption

369on the membrane surface and phenol degradation by

370microorganisms the pure carrier was also studied for

371phenol adsorption For this purpose 40 cm2 pure

372membrane was immersed in 05 g l-1 phenol solution

373for 6 h The results are presented in Fig 6 Since the

374phenol concentration didnrsquot change significantly

375the only possible reason for the decrease of the

376phenol content was the degradation activity of the

377immobilized cells This simplifies the kinetic studies

378when membrane is to be used as a support material

0

02

04

06

08

0 100 200 300 400 500

Time h

Ph

en

ol

bio

de

gra

da

tio

n

gl-1

Fig 5 Phenol

biodegradation with

concentration 05 g l-1 by

strain Aspergillus awamori

NRRL 3112

0

02

04

06

08

0 1 2 3 4 5 6 7

Time h

Ph

en

ol

bio

deg

rad

ati

on

g

l-1

Fig 6 Adsorption of

phenol by modified PAN

membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

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r P

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UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

379 for the immobilization of Asp awamori for the

380 purpose of phenol degradation

381 The amount of cells (wet weight) on the membrane

382 of 40 cm2 was measured after the sixth cycle of

383 phenol biodegradationmdash1104 g cm-2 The signifi-

384 cant increase of the biomass with the increased

385 number of cycles is obvious (after immobilizationmdash

386 0158 g cm-2) Thus the biomass accumulated on

387 the membrane was approximately ten times higher

388 after six cycles of phenol biodegradation The high

389 operational stability of investigated biocatalysts was

390 confirmed by the scanning electron micrographs of

391 the polyacrylonitrile membraneimmobilized Asper-

392 gillus awamori NRRL at the beginning of repeated

393 batch cultivation and after the 7th cycle After the 7th

394 cycle of cultivation the observations showed large

395 groups of cells (Fig 7a) The significant increase of

396 biomass and mycelia formation on the membrane

397 surface after the 7th cycle of cultivation was inev-

398 itably followed by deceleration of the phenol

399 degradation rate

400Experiments with membrane bioreactor

401The next step of the research work involved exper-

402iments which were carried out in a bioreactor with

403spirally wound membrane in a regime of recircula-

404tion The spores were immobilized onto the

405membrane directly in the system as described in

406lsquolsquoMaterials and methodsrsquorsquo section Phenol biodegra-

407dation was carried out under aerobic conditions The

408operation parameters of the bioreactor are presented

409in Table 2 Fresh nutrient medium containing

41005 g l-1 phenol were added after each cycle Phenol

411concentration was monitored for degradation At the

412end of a cycle the system was washed with sterile

413distilled water and charged with nutrient medium and

414phenol for the next cycle Thus 10 cycles of 05 g l-1

415phenol biodegradation were run consecutively to

416determine the degradation time for each cycle

417(Fig 8) No significant difference was observed

418between the degradation times of the cycles from

419the second to the seventh (about 50 h) and between

420the biodegradation rates (100 mg h-1) After the 7th

421cycle the biodegradation rate began to decrease

422gradually and at 10 cycles it was 40 mg h-1 These

423results confirmed the advantages of the recycled

424reactor with spirally wound membrane The design of

425the bioreactor appeared to be quite effective provid-

426ing large membrane surface to bind the strain cells

427Besides phenol would flow tangentially which pre-

428vents membrane from a decrease of the flow velocity

429Conclusion

430A novel immobilized systemmdashAspergillus awamori

431NRRL 3112polyacrylonitrile membranewas obtained

432for the purpose of phenol biodegradation It was

433established that the immobilized system prepared in

434the presence of 02 g l-1 phenol was more effective

Fig 7 Scanning electron micrographs of immobilized system

Aspergillus awamori NRRL 3112modified PAN membrane

after 7th cycle (a) and before 1st cycle (b)

Table 2 Operation conditions of bioreactor

Parameter Value

Rate flow 15 ml min-1

Volume 300 ml

Initial phenol concentration 05 g l-1

Membrane surface area 100 cm2

Immobilized spores 0174 g cm-2 (wet weight)

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

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UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

435 than the system prepared in the phenol absence It was

436 revealed that the immobilized systems were more

437 stable andmore effective when biodegrading 05 g l-1

438 phenol in comparison with the free cells A bioreactor

439 was constructed containing spirally wound membrane

440 with immobilized spores of Aspergillus awamori

441 NRRL 3112 in a regime of recirculation which was

442 successfully applied for phenol biodegradation The

443 obtained results confirmed the advantages of the

444 recycled reactor with spirally wound membrane Thus

445 the immobilized microbial technology is an extremely

446 versatile approach that can be used for degradation of

447 toxic pollutants from the industrial effluents

448 Acknowledgments The authors gratefully acknowledge to449 the Bulgarian Ministry for Education and to the National450 Science Fund for their financial support and encouragement of451 the scientific research work in state universities

452 References

453 Abd-El-Haleem D Beshay U Abdelhamid AO Moawad H454 Zaki S (2003) Effects of mixed nitrogen sources on bio-455 degradation of phenol by immobilized Acinetobacter sp456 strain W-17 Afr J Biotechnol 2(1)8ndash12457 Allsop PJ Chisti Y Moi-Youngand M Sullivan GR (1993)458 Dynamics of phenol degradation by Pseudomonas putida459 Biotechnol Bioeng 41572ndash579460 Anselmo AM Novais JM (1992) Degradation of phenol by461 immobilized mycelium of Fusarium floccieferum in con-462 tinuous culture Water Sci Technol 25161ndash168463 Bolanos ML Varesche MBA Zaiat M Foresti E (2001) Phenol464 degradation in horizontal-flow anaerobic immobilized465 biomass (HAIB) reactor under mesophilic conditions466 Water Sci Technol 44(4)167ndash174

467Chitra S Sekaran G Padmavathi S Gowri C (1995) Removal468of phenol from wastewater using a biocatalyst In Pro-469ceedings of the national symposium frontiers in applied470environmental microbiology pp 74ndash77471Chtourou M Ammar E Nasri M Medhioub K (2004) Isolation472of a yeast Trichosporon cutaneum able to use low473molecular weight phenolic compounds application to474olive mill waste water treatment J Chem Technol Bio-475technol 79869ndash878476Dimov K Samardjieva Pavlov P (1983) Laboratory practice477on technology of synthetic fibers Technica Sofia BG478Fiest CF Hegeman GD (1969) Phenol and benzoate metabo-479lism by Pseudomonas putida J Bacteriol 100869ndash877480Godjevargova T Aleksieva Z Ivanova D (2000) Cell immo-481bilization of Trichosporon cutaneum strain with phenol482degradation ability on new modified polymer carries483Process Biochem 35699ndash704484Godjevargova T Ivanova D Aleksieva Z Burdelova G (2006)485Biodegradation of phenol by immobilized Trichosporon

486cutaneum R57 on modified polymer membranes Process487Biochem 412342ndash2346488Gonzalez G Herrera MG Pena MM (2001) Biodegradation of489phenol in a continuous process comparative study of490stirred tank and fluidized-bed bioreactors Bioresour491Technol 76245ndash251492Hua L Sun ZH Leng Y (2005) Continuous biocatalytic reso-493lution of DL-pantolactone by cross-linked cells in494membrane bioreactor Process Biochem 401137ndash1142495Hudges EJL Bayly RC Skurray RA (1984) Evidence for496isofunctional enzymes in the degradation of phenol m-497and p-toluate and p-cresol at catechol meta-cleavage498pathways in Alcaligenes eutrophus J Bacteriol 15879ndash83499Jones KH Trudgill PW Hopper DJ (1995) Evidence of two500pathways for the metabolism of phenol by Aspergillus

501fumigatus Arch Microbiol 163176ndash181502Jose G Sanchez M Theodore T (2002) Membranes and mem-503brane reactors Wiley-VCH Veriag GmbH Weinheim504Karel S Libichi S Robertson C (1985) The immobilization of505whole cells engineering principles Chem Eng Sci506401321ndash1354

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

Fig 8 Concentration

profile of phenol in a

recirculating system with

immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

507 Kowalska M Bodzek M Bohdziewicz J (1998) Biodegradation508 of phenols and cyanides using membranes immobilized509 microorganisms Process Biochem 33189ndash197510 Loh K Chung T Ang W (2000) Immobilized-cell membrane511 bioreactor for high-strength phenol wastewater J Environ512 Eng ASCE 12675ndash79513 Marrot B Barrios-Martinez A Moulin P Roche N (2006)514 Biodegradation of high phenol concentration by activated515 sludge in an immersed membrane bioreactor Biochem516 Eng J 30174ndash183517 Mordocco A Kuek C Jenkins R (1999) Continuous degradation518 of phenol at low concentration using immobilized Pseu-

519 domonas putida Enzyme Microb Technol 25530ndash536520 Pazarlioglu NK Telefoncu A (2005) Biodegradation of phenol521 by Pseudomonas putida immobilized on activated pumice522 particles Process Biochem 401807ndash1814523 Santos VL Heilbuth NM Braga T Monteiro AS Linardi VR524 (2003) Phenol degradation by aGraphium sp FIB4 isolated525 from industrial effluents J Basic Microbiol 43238ndash248526 Shetty KV Kalifathulla I Srinikethan G (2007a) Performance527 of pulsed plate bioreactor for biodegradation of phenol528 J Hazard Mater 140346ndash352529 Shetty KV Ramanjaneyulu R Srinikethan G (2007b) Biolog-530 ical phenol removal using immobilized cells in a pulsed

531plate bioreactor effect of dilution rate and influent phenol532concentration J Hazard Mater 149452ndash459533Stephenson T Judd S Jefferson B Brindle K (2000) Mem-534brane bioreactors for wastewater treatment IWA535Publishing London UK pp 129ndash136536Sundar P Daniel D Krastanov A (2005) Biodegradation of537phenol by immobilized Aspergillus awamori cells Bulg J538Agric Sci 1161ndash71539Uzunova K Vassileva A Ivanova V Spasova D Tonkova A540(2002) Thermostable exo-inulinase production by semi-541continuous cultivation of membrane-immobilized Bacillus542sp 11 cells Process Biochem 37863ndash868543Wang Z Wan L Xu Z (2007) Surface engineerings of poly-544acrylonitrile-based asymmetric membranes towards545biomedical applications an overview J Membr Sci5463048ndash23547Yiang Y Wen J Li H Yang S Hu Z (2005) The biodegra-548dation of phenol at high initial concentration by the yeast549Candida tropicalis Biochem Eng J 24243ndash247550Yordanova G Ivanova D Godjevargova T Krastanov A (2006)551Biodegradation of phenol by immobilized Aspergillus

552awamori NRRL3112 cells Food science engineering and553technologies conference Scientific works Plovdiv554pp 260ndash265

555

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

307 biodegradation rate is observed at the first cycle then

308 it increases 3 times at the second and the third cycles

309 and from the fourth to the seventh cycle the rate

310 decreases continuously This trend of the biodegra-

311 dation rate is observed for both of the immobilized

312 systems The deceleration of the phenol biodegrada-

313 tion could be explained with the increasing of

314 biomass and mycelia formation on the membrane

315 surface after the fourth cycle which intensified at the

316 final cycles An obvious inhibition of the biodegra-

317 dation process at the final cycles was observed which

318 was due to the hindrance of the substrate diffusion to

319 the cells When comparing the results presented on

320 Fig 2a and b and Table 1 it becomes obvious that the

321 rate of phenol degradation was higher throughout all

322 cycles in the presence of the system where the spore

323 immobilization was conducted with addition of

32402 g l-1 phenol For better comparison the biodeg-

325radation times of each cycle with the two

326immobilized systems are presented in Fig 3 As

0

02

04

06

08

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

0

02

04

06

08

0 200 400 600 800

0 200 400 600 800 1000

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

(a)

(b)

Fig 2 Phenol

biodegradation (05 g l-1)

by immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane obtained by

immobilization in presence

of 02 g l-1 phenol (a) and

without phenol (b)

Table 1 Phenol biodegradation rate (mg h-1) of free and

immobilized Aspergillus awamori NRRL 3112 on polymer

PAN membrane at batch cultivation with 05 g l-1 phenol

Numberof

cycles

Free

cells

Immobilized cells

without 02 g l-1

phenol

Immobilized cells

in the presence of

02 g l-1 phenol

1 297 300 300

2 201 714 694

3 149 806 543

4 116 625 367

5 403 329

6 308 300

7 275 270

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

327 results suggest better degradation times were

328 obtained with the immobilized system prepared in

329 the presence of phenol This can be explained with

330 the fact that the strain could have adapted to the

331 substrate during the immobilization process All

332 further experiments were carried out with the immo-

333 bilized system prepared in presence of phenol

334 The biodegradation ability of the immobilized

335 system onto polyacrylonitrile membrane towards

336 phenol in a concentration range from 02 to

337 08 g l-1 was studied As can be seen from Fig 4

338 the increase of phenol concentration resulted in

339increased biodegradation time For instance the

340biodegradation cycle at concentration of 04 g l-1

341was 66 h while at 07 g l-1 the cycle lasted for 135 h

342The results obtained with the selected immobilized

343system were compared to those with the free strain

344For this purpose the spores (08 9 107 ml-1)

345were cultivated in Czapekdox medium containing

34605 g l-1 phenol (Fig 5) Four cycles were carried

347out in the same manner The amount of non-bound

348spores in the first cycle of cultivation is equivalent

349to the amount of immobilized spores on PAN

350membrane with surface 40 cm2 in the first cycle

0

50

100

150

200

250

1 2 3 4 5 6 7

Cycles

Tim

e

h

Fig 3 Cycles duration of

phenol biodegradation

(05 g l-1) by two

immobilized systems

Aspergillus awamori NRRL

3112modified PAN

membrane obtained by

immobilization in presence

of 02 g l-1 phenol ( ) and

without phenol (j)

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol co

ncen

trati

on

g

l-1

Fig 4 Phenol

biodegradation (from 02 to

08 g l-1) by immobilized

system Aspergillus

awamori NRRL 3112

modified PAN membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

351 (wet weightmdash632 g) As can be seen from Fig 5 the

352 phenol degradation time was considerably greater for

353 the four cycles in these experiments This time

354 extension was confirmed by the calculated rate of

355 biodegradation (Table 1) When comparing the rates

356 of phenol biodegradation by the free and the immo-

357 bilized cells the greater stability of the immobilized

358 systems over the free cells becomes more prominent

359 after multiple cultivation in media containing phenol

360 with concentration 05 g l-1 Obviously the free cells

361 were inhibited to a greater extent by phenol (biodeg-

362 radation rate at Cycle 4mdash116 mg h-1) in

363 comparison with the immobilized cells (biodegrada-

364 tion rates at Cycle 4 are 625 and 367 mg h-1

365respectively for immobilized system obtained in the

366presence of 02 g l-1 phenol and the system without

367phenol)

368In order to distinguish between phenol adsorption

369on the membrane surface and phenol degradation by

370microorganisms the pure carrier was also studied for

371phenol adsorption For this purpose 40 cm2 pure

372membrane was immersed in 05 g l-1 phenol solution

373for 6 h The results are presented in Fig 6 Since the

374phenol concentration didnrsquot change significantly

375the only possible reason for the decrease of the

376phenol content was the degradation activity of the

377immobilized cells This simplifies the kinetic studies

378when membrane is to be used as a support material

0

02

04

06

08

0 100 200 300 400 500

Time h

Ph

en

ol

bio

de

gra

da

tio

n

gl-1

Fig 5 Phenol

biodegradation with

concentration 05 g l-1 by

strain Aspergillus awamori

NRRL 3112

0

02

04

06

08

0 1 2 3 4 5 6 7

Time h

Ph

en

ol

bio

deg

rad

ati

on

g

l-1

Fig 6 Adsorption of

phenol by modified PAN

membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

379 for the immobilization of Asp awamori for the

380 purpose of phenol degradation

381 The amount of cells (wet weight) on the membrane

382 of 40 cm2 was measured after the sixth cycle of

383 phenol biodegradationmdash1104 g cm-2 The signifi-

384 cant increase of the biomass with the increased

385 number of cycles is obvious (after immobilizationmdash

386 0158 g cm-2) Thus the biomass accumulated on

387 the membrane was approximately ten times higher

388 after six cycles of phenol biodegradation The high

389 operational stability of investigated biocatalysts was

390 confirmed by the scanning electron micrographs of

391 the polyacrylonitrile membraneimmobilized Asper-

392 gillus awamori NRRL at the beginning of repeated

393 batch cultivation and after the 7th cycle After the 7th

394 cycle of cultivation the observations showed large

395 groups of cells (Fig 7a) The significant increase of

396 biomass and mycelia formation on the membrane

397 surface after the 7th cycle of cultivation was inev-

398 itably followed by deceleration of the phenol

399 degradation rate

400Experiments with membrane bioreactor

401The next step of the research work involved exper-

402iments which were carried out in a bioreactor with

403spirally wound membrane in a regime of recircula-

404tion The spores were immobilized onto the

405membrane directly in the system as described in

406lsquolsquoMaterials and methodsrsquorsquo section Phenol biodegra-

407dation was carried out under aerobic conditions The

408operation parameters of the bioreactor are presented

409in Table 2 Fresh nutrient medium containing

41005 g l-1 phenol were added after each cycle Phenol

411concentration was monitored for degradation At the

412end of a cycle the system was washed with sterile

413distilled water and charged with nutrient medium and

414phenol for the next cycle Thus 10 cycles of 05 g l-1

415phenol biodegradation were run consecutively to

416determine the degradation time for each cycle

417(Fig 8) No significant difference was observed

418between the degradation times of the cycles from

419the second to the seventh (about 50 h) and between

420the biodegradation rates (100 mg h-1) After the 7th

421cycle the biodegradation rate began to decrease

422gradually and at 10 cycles it was 40 mg h-1 These

423results confirmed the advantages of the recycled

424reactor with spirally wound membrane The design of

425the bioreactor appeared to be quite effective provid-

426ing large membrane surface to bind the strain cells

427Besides phenol would flow tangentially which pre-

428vents membrane from a decrease of the flow velocity

429Conclusion

430A novel immobilized systemmdashAspergillus awamori

431NRRL 3112polyacrylonitrile membranewas obtained

432for the purpose of phenol biodegradation It was

433established that the immobilized system prepared in

434the presence of 02 g l-1 phenol was more effective

Fig 7 Scanning electron micrographs of immobilized system

Aspergillus awamori NRRL 3112modified PAN membrane

after 7th cycle (a) and before 1st cycle (b)

Table 2 Operation conditions of bioreactor

Parameter Value

Rate flow 15 ml min-1

Volume 300 ml

Initial phenol concentration 05 g l-1

Membrane surface area 100 cm2

Immobilized spores 0174 g cm-2 (wet weight)

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

435 than the system prepared in the phenol absence It was

436 revealed that the immobilized systems were more

437 stable andmore effective when biodegrading 05 g l-1

438 phenol in comparison with the free cells A bioreactor

439 was constructed containing spirally wound membrane

440 with immobilized spores of Aspergillus awamori

441 NRRL 3112 in a regime of recirculation which was

442 successfully applied for phenol biodegradation The

443 obtained results confirmed the advantages of the

444 recycled reactor with spirally wound membrane Thus

445 the immobilized microbial technology is an extremely

446 versatile approach that can be used for degradation of

447 toxic pollutants from the industrial effluents

448 Acknowledgments The authors gratefully acknowledge to449 the Bulgarian Ministry for Education and to the National450 Science Fund for their financial support and encouragement of451 the scientific research work in state universities

452 References

453 Abd-El-Haleem D Beshay U Abdelhamid AO Moawad H454 Zaki S (2003) Effects of mixed nitrogen sources on bio-455 degradation of phenol by immobilized Acinetobacter sp456 strain W-17 Afr J Biotechnol 2(1)8ndash12457 Allsop PJ Chisti Y Moi-Youngand M Sullivan GR (1993)458 Dynamics of phenol degradation by Pseudomonas putida459 Biotechnol Bioeng 41572ndash579460 Anselmo AM Novais JM (1992) Degradation of phenol by461 immobilized mycelium of Fusarium floccieferum in con-462 tinuous culture Water Sci Technol 25161ndash168463 Bolanos ML Varesche MBA Zaiat M Foresti E (2001) Phenol464 degradation in horizontal-flow anaerobic immobilized465 biomass (HAIB) reactor under mesophilic conditions466 Water Sci Technol 44(4)167ndash174

467Chitra S Sekaran G Padmavathi S Gowri C (1995) Removal468of phenol from wastewater using a biocatalyst In Pro-469ceedings of the national symposium frontiers in applied470environmental microbiology pp 74ndash77471Chtourou M Ammar E Nasri M Medhioub K (2004) Isolation472of a yeast Trichosporon cutaneum able to use low473molecular weight phenolic compounds application to474olive mill waste water treatment J Chem Technol Bio-475technol 79869ndash878476Dimov K Samardjieva Pavlov P (1983) Laboratory practice477on technology of synthetic fibers Technica Sofia BG478Fiest CF Hegeman GD (1969) Phenol and benzoate metabo-479lism by Pseudomonas putida J Bacteriol 100869ndash877480Godjevargova T Aleksieva Z Ivanova D (2000) Cell immo-481bilization of Trichosporon cutaneum strain with phenol482degradation ability on new modified polymer carries483Process Biochem 35699ndash704484Godjevargova T Ivanova D Aleksieva Z Burdelova G (2006)485Biodegradation of phenol by immobilized Trichosporon

486cutaneum R57 on modified polymer membranes Process487Biochem 412342ndash2346488Gonzalez G Herrera MG Pena MM (2001) Biodegradation of489phenol in a continuous process comparative study of490stirred tank and fluidized-bed bioreactors Bioresour491Technol 76245ndash251492Hua L Sun ZH Leng Y (2005) Continuous biocatalytic reso-493lution of DL-pantolactone by cross-linked cells in494membrane bioreactor Process Biochem 401137ndash1142495Hudges EJL Bayly RC Skurray RA (1984) Evidence for496isofunctional enzymes in the degradation of phenol m-497and p-toluate and p-cresol at catechol meta-cleavage498pathways in Alcaligenes eutrophus J Bacteriol 15879ndash83499Jones KH Trudgill PW Hopper DJ (1995) Evidence of two500pathways for the metabolism of phenol by Aspergillus

501fumigatus Arch Microbiol 163176ndash181502Jose G Sanchez M Theodore T (2002) Membranes and mem-503brane reactors Wiley-VCH Veriag GmbH Weinheim504Karel S Libichi S Robertson C (1985) The immobilization of505whole cells engineering principles Chem Eng Sci506401321ndash1354

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

Fig 8 Concentration

profile of phenol in a

recirculating system with

immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

507 Kowalska M Bodzek M Bohdziewicz J (1998) Biodegradation508 of phenols and cyanides using membranes immobilized509 microorganisms Process Biochem 33189ndash197510 Loh K Chung T Ang W (2000) Immobilized-cell membrane511 bioreactor for high-strength phenol wastewater J Environ512 Eng ASCE 12675ndash79513 Marrot B Barrios-Martinez A Moulin P Roche N (2006)514 Biodegradation of high phenol concentration by activated515 sludge in an immersed membrane bioreactor Biochem516 Eng J 30174ndash183517 Mordocco A Kuek C Jenkins R (1999) Continuous degradation518 of phenol at low concentration using immobilized Pseu-

519 domonas putida Enzyme Microb Technol 25530ndash536520 Pazarlioglu NK Telefoncu A (2005) Biodegradation of phenol521 by Pseudomonas putida immobilized on activated pumice522 particles Process Biochem 401807ndash1814523 Santos VL Heilbuth NM Braga T Monteiro AS Linardi VR524 (2003) Phenol degradation by aGraphium sp FIB4 isolated525 from industrial effluents J Basic Microbiol 43238ndash248526 Shetty KV Kalifathulla I Srinikethan G (2007a) Performance527 of pulsed plate bioreactor for biodegradation of phenol528 J Hazard Mater 140346ndash352529 Shetty KV Ramanjaneyulu R Srinikethan G (2007b) Biolog-530 ical phenol removal using immobilized cells in a pulsed

531plate bioreactor effect of dilution rate and influent phenol532concentration J Hazard Mater 149452ndash459533Stephenson T Judd S Jefferson B Brindle K (2000) Mem-534brane bioreactors for wastewater treatment IWA535Publishing London UK pp 129ndash136536Sundar P Daniel D Krastanov A (2005) Biodegradation of537phenol by immobilized Aspergillus awamori cells Bulg J538Agric Sci 1161ndash71539Uzunova K Vassileva A Ivanova V Spasova D Tonkova A540(2002) Thermostable exo-inulinase production by semi-541continuous cultivation of membrane-immobilized Bacillus542sp 11 cells Process Biochem 37863ndash868543Wang Z Wan L Xu Z (2007) Surface engineerings of poly-544acrylonitrile-based asymmetric membranes towards545biomedical applications an overview J Membr Sci5463048ndash23547Yiang Y Wen J Li H Yang S Hu Z (2005) The biodegra-548dation of phenol at high initial concentration by the yeast549Candida tropicalis Biochem Eng J 24243ndash247550Yordanova G Ivanova D Godjevargova T Krastanov A (2006)551Biodegradation of phenol by immobilized Aspergillus

552awamori NRRL3112 cells Food science engineering and553technologies conference Scientific works Plovdiv554pp 260ndash265

555

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

327 results suggest better degradation times were

328 obtained with the immobilized system prepared in

329 the presence of phenol This can be explained with

330 the fact that the strain could have adapted to the

331 substrate during the immobilization process All

332 further experiments were carried out with the immo-

333 bilized system prepared in presence of phenol

334 The biodegradation ability of the immobilized

335 system onto polyacrylonitrile membrane towards

336 phenol in a concentration range from 02 to

337 08 g l-1 was studied As can be seen from Fig 4

338 the increase of phenol concentration resulted in

339increased biodegradation time For instance the

340biodegradation cycle at concentration of 04 g l-1

341was 66 h while at 07 g l-1 the cycle lasted for 135 h

342The results obtained with the selected immobilized

343system were compared to those with the free strain

344For this purpose the spores (08 9 107 ml-1)

345were cultivated in Czapekdox medium containing

34605 g l-1 phenol (Fig 5) Four cycles were carried

347out in the same manner The amount of non-bound

348spores in the first cycle of cultivation is equivalent

349to the amount of immobilized spores on PAN

350membrane with surface 40 cm2 in the first cycle

0

50

100

150

200

250

1 2 3 4 5 6 7

Cycles

Tim

e

h

Fig 3 Cycles duration of

phenol biodegradation

(05 g l-1) by two

immobilized systems

Aspergillus awamori NRRL

3112modified PAN

membrane obtained by

immobilization in presence

of 02 g l-1 phenol ( ) and

without phenol (j)

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol co

ncen

trati

on

g

l-1

Fig 4 Phenol

biodegradation (from 02 to

08 g l-1) by immobilized

system Aspergillus

awamori NRRL 3112

modified PAN membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

351 (wet weightmdash632 g) As can be seen from Fig 5 the

352 phenol degradation time was considerably greater for

353 the four cycles in these experiments This time

354 extension was confirmed by the calculated rate of

355 biodegradation (Table 1) When comparing the rates

356 of phenol biodegradation by the free and the immo-

357 bilized cells the greater stability of the immobilized

358 systems over the free cells becomes more prominent

359 after multiple cultivation in media containing phenol

360 with concentration 05 g l-1 Obviously the free cells

361 were inhibited to a greater extent by phenol (biodeg-

362 radation rate at Cycle 4mdash116 mg h-1) in

363 comparison with the immobilized cells (biodegrada-

364 tion rates at Cycle 4 are 625 and 367 mg h-1

365respectively for immobilized system obtained in the

366presence of 02 g l-1 phenol and the system without

367phenol)

368In order to distinguish between phenol adsorption

369on the membrane surface and phenol degradation by

370microorganisms the pure carrier was also studied for

371phenol adsorption For this purpose 40 cm2 pure

372membrane was immersed in 05 g l-1 phenol solution

373for 6 h The results are presented in Fig 6 Since the

374phenol concentration didnrsquot change significantly

375the only possible reason for the decrease of the

376phenol content was the degradation activity of the

377immobilized cells This simplifies the kinetic studies

378when membrane is to be used as a support material

0

02

04

06

08

0 100 200 300 400 500

Time h

Ph

en

ol

bio

de

gra

da

tio

n

gl-1

Fig 5 Phenol

biodegradation with

concentration 05 g l-1 by

strain Aspergillus awamori

NRRL 3112

0

02

04

06

08

0 1 2 3 4 5 6 7

Time h

Ph

en

ol

bio

deg

rad

ati

on

g

l-1

Fig 6 Adsorption of

phenol by modified PAN

membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

379 for the immobilization of Asp awamori for the

380 purpose of phenol degradation

381 The amount of cells (wet weight) on the membrane

382 of 40 cm2 was measured after the sixth cycle of

383 phenol biodegradationmdash1104 g cm-2 The signifi-

384 cant increase of the biomass with the increased

385 number of cycles is obvious (after immobilizationmdash

386 0158 g cm-2) Thus the biomass accumulated on

387 the membrane was approximately ten times higher

388 after six cycles of phenol biodegradation The high

389 operational stability of investigated biocatalysts was

390 confirmed by the scanning electron micrographs of

391 the polyacrylonitrile membraneimmobilized Asper-

392 gillus awamori NRRL at the beginning of repeated

393 batch cultivation and after the 7th cycle After the 7th

394 cycle of cultivation the observations showed large

395 groups of cells (Fig 7a) The significant increase of

396 biomass and mycelia formation on the membrane

397 surface after the 7th cycle of cultivation was inev-

398 itably followed by deceleration of the phenol

399 degradation rate

400Experiments with membrane bioreactor

401The next step of the research work involved exper-

402iments which were carried out in a bioreactor with

403spirally wound membrane in a regime of recircula-

404tion The spores were immobilized onto the

405membrane directly in the system as described in

406lsquolsquoMaterials and methodsrsquorsquo section Phenol biodegra-

407dation was carried out under aerobic conditions The

408operation parameters of the bioreactor are presented

409in Table 2 Fresh nutrient medium containing

41005 g l-1 phenol were added after each cycle Phenol

411concentration was monitored for degradation At the

412end of a cycle the system was washed with sterile

413distilled water and charged with nutrient medium and

414phenol for the next cycle Thus 10 cycles of 05 g l-1

415phenol biodegradation were run consecutively to

416determine the degradation time for each cycle

417(Fig 8) No significant difference was observed

418between the degradation times of the cycles from

419the second to the seventh (about 50 h) and between

420the biodegradation rates (100 mg h-1) After the 7th

421cycle the biodegradation rate began to decrease

422gradually and at 10 cycles it was 40 mg h-1 These

423results confirmed the advantages of the recycled

424reactor with spirally wound membrane The design of

425the bioreactor appeared to be quite effective provid-

426ing large membrane surface to bind the strain cells

427Besides phenol would flow tangentially which pre-

428vents membrane from a decrease of the flow velocity

429Conclusion

430A novel immobilized systemmdashAspergillus awamori

431NRRL 3112polyacrylonitrile membranewas obtained

432for the purpose of phenol biodegradation It was

433established that the immobilized system prepared in

434the presence of 02 g l-1 phenol was more effective

Fig 7 Scanning electron micrographs of immobilized system

Aspergillus awamori NRRL 3112modified PAN membrane

after 7th cycle (a) and before 1st cycle (b)

Table 2 Operation conditions of bioreactor

Parameter Value

Rate flow 15 ml min-1

Volume 300 ml

Initial phenol concentration 05 g l-1

Membrane surface area 100 cm2

Immobilized spores 0174 g cm-2 (wet weight)

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

435 than the system prepared in the phenol absence It was

436 revealed that the immobilized systems were more

437 stable andmore effective when biodegrading 05 g l-1

438 phenol in comparison with the free cells A bioreactor

439 was constructed containing spirally wound membrane

440 with immobilized spores of Aspergillus awamori

441 NRRL 3112 in a regime of recirculation which was

442 successfully applied for phenol biodegradation The

443 obtained results confirmed the advantages of the

444 recycled reactor with spirally wound membrane Thus

445 the immobilized microbial technology is an extremely

446 versatile approach that can be used for degradation of

447 toxic pollutants from the industrial effluents

448 Acknowledgments The authors gratefully acknowledge to449 the Bulgarian Ministry for Education and to the National450 Science Fund for their financial support and encouragement of451 the scientific research work in state universities

452 References

453 Abd-El-Haleem D Beshay U Abdelhamid AO Moawad H454 Zaki S (2003) Effects of mixed nitrogen sources on bio-455 degradation of phenol by immobilized Acinetobacter sp456 strain W-17 Afr J Biotechnol 2(1)8ndash12457 Allsop PJ Chisti Y Moi-Youngand M Sullivan GR (1993)458 Dynamics of phenol degradation by Pseudomonas putida459 Biotechnol Bioeng 41572ndash579460 Anselmo AM Novais JM (1992) Degradation of phenol by461 immobilized mycelium of Fusarium floccieferum in con-462 tinuous culture Water Sci Technol 25161ndash168463 Bolanos ML Varesche MBA Zaiat M Foresti E (2001) Phenol464 degradation in horizontal-flow anaerobic immobilized465 biomass (HAIB) reactor under mesophilic conditions466 Water Sci Technol 44(4)167ndash174

467Chitra S Sekaran G Padmavathi S Gowri C (1995) Removal468of phenol from wastewater using a biocatalyst In Pro-469ceedings of the national symposium frontiers in applied470environmental microbiology pp 74ndash77471Chtourou M Ammar E Nasri M Medhioub K (2004) Isolation472of a yeast Trichosporon cutaneum able to use low473molecular weight phenolic compounds application to474olive mill waste water treatment J Chem Technol Bio-475technol 79869ndash878476Dimov K Samardjieva Pavlov P (1983) Laboratory practice477on technology of synthetic fibers Technica Sofia BG478Fiest CF Hegeman GD (1969) Phenol and benzoate metabo-479lism by Pseudomonas putida J Bacteriol 100869ndash877480Godjevargova T Aleksieva Z Ivanova D (2000) Cell immo-481bilization of Trichosporon cutaneum strain with phenol482degradation ability on new modified polymer carries483Process Biochem 35699ndash704484Godjevargova T Ivanova D Aleksieva Z Burdelova G (2006)485Biodegradation of phenol by immobilized Trichosporon

486cutaneum R57 on modified polymer membranes Process487Biochem 412342ndash2346488Gonzalez G Herrera MG Pena MM (2001) Biodegradation of489phenol in a continuous process comparative study of490stirred tank and fluidized-bed bioreactors Bioresour491Technol 76245ndash251492Hua L Sun ZH Leng Y (2005) Continuous biocatalytic reso-493lution of DL-pantolactone by cross-linked cells in494membrane bioreactor Process Biochem 401137ndash1142495Hudges EJL Bayly RC Skurray RA (1984) Evidence for496isofunctional enzymes in the degradation of phenol m-497and p-toluate and p-cresol at catechol meta-cleavage498pathways in Alcaligenes eutrophus J Bacteriol 15879ndash83499Jones KH Trudgill PW Hopper DJ (1995) Evidence of two500pathways for the metabolism of phenol by Aspergillus

501fumigatus Arch Microbiol 163176ndash181502Jose G Sanchez M Theodore T (2002) Membranes and mem-503brane reactors Wiley-VCH Veriag GmbH Weinheim504Karel S Libichi S Robertson C (1985) The immobilization of505whole cells engineering principles Chem Eng Sci506401321ndash1354

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

Fig 8 Concentration

profile of phenol in a

recirculating system with

immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

507 Kowalska M Bodzek M Bohdziewicz J (1998) Biodegradation508 of phenols and cyanides using membranes immobilized509 microorganisms Process Biochem 33189ndash197510 Loh K Chung T Ang W (2000) Immobilized-cell membrane511 bioreactor for high-strength phenol wastewater J Environ512 Eng ASCE 12675ndash79513 Marrot B Barrios-Martinez A Moulin P Roche N (2006)514 Biodegradation of high phenol concentration by activated515 sludge in an immersed membrane bioreactor Biochem516 Eng J 30174ndash183517 Mordocco A Kuek C Jenkins R (1999) Continuous degradation518 of phenol at low concentration using immobilized Pseu-

519 domonas putida Enzyme Microb Technol 25530ndash536520 Pazarlioglu NK Telefoncu A (2005) Biodegradation of phenol521 by Pseudomonas putida immobilized on activated pumice522 particles Process Biochem 401807ndash1814523 Santos VL Heilbuth NM Braga T Monteiro AS Linardi VR524 (2003) Phenol degradation by aGraphium sp FIB4 isolated525 from industrial effluents J Basic Microbiol 43238ndash248526 Shetty KV Kalifathulla I Srinikethan G (2007a) Performance527 of pulsed plate bioreactor for biodegradation of phenol528 J Hazard Mater 140346ndash352529 Shetty KV Ramanjaneyulu R Srinikethan G (2007b) Biolog-530 ical phenol removal using immobilized cells in a pulsed

531plate bioreactor effect of dilution rate and influent phenol532concentration J Hazard Mater 149452ndash459533Stephenson T Judd S Jefferson B Brindle K (2000) Mem-534brane bioreactors for wastewater treatment IWA535Publishing London UK pp 129ndash136536Sundar P Daniel D Krastanov A (2005) Biodegradation of537phenol by immobilized Aspergillus awamori cells Bulg J538Agric Sci 1161ndash71539Uzunova K Vassileva A Ivanova V Spasova D Tonkova A540(2002) Thermostable exo-inulinase production by semi-541continuous cultivation of membrane-immobilized Bacillus542sp 11 cells Process Biochem 37863ndash868543Wang Z Wan L Xu Z (2007) Surface engineerings of poly-544acrylonitrile-based asymmetric membranes towards545biomedical applications an overview J Membr Sci5463048ndash23547Yiang Y Wen J Li H Yang S Hu Z (2005) The biodegra-548dation of phenol at high initial concentration by the yeast549Candida tropicalis Biochem Eng J 24243ndash247550Yordanova G Ivanova D Godjevargova T Krastanov A (2006)551Biodegradation of phenol by immobilized Aspergillus

552awamori NRRL3112 cells Food science engineering and553technologies conference Scientific works Plovdiv554pp 260ndash265

555

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

351 (wet weightmdash632 g) As can be seen from Fig 5 the

352 phenol degradation time was considerably greater for

353 the four cycles in these experiments This time

354 extension was confirmed by the calculated rate of

355 biodegradation (Table 1) When comparing the rates

356 of phenol biodegradation by the free and the immo-

357 bilized cells the greater stability of the immobilized

358 systems over the free cells becomes more prominent

359 after multiple cultivation in media containing phenol

360 with concentration 05 g l-1 Obviously the free cells

361 were inhibited to a greater extent by phenol (biodeg-

362 radation rate at Cycle 4mdash116 mg h-1) in

363 comparison with the immobilized cells (biodegrada-

364 tion rates at Cycle 4 are 625 and 367 mg h-1

365respectively for immobilized system obtained in the

366presence of 02 g l-1 phenol and the system without

367phenol)

368In order to distinguish between phenol adsorption

369on the membrane surface and phenol degradation by

370microorganisms the pure carrier was also studied for

371phenol adsorption For this purpose 40 cm2 pure

372membrane was immersed in 05 g l-1 phenol solution

373for 6 h The results are presented in Fig 6 Since the

374phenol concentration didnrsquot change significantly

375the only possible reason for the decrease of the

376phenol content was the degradation activity of the

377immobilized cells This simplifies the kinetic studies

378when membrane is to be used as a support material

0

02

04

06

08

0 100 200 300 400 500

Time h

Ph

en

ol

bio

de

gra

da

tio

n

gl-1

Fig 5 Phenol

biodegradation with

concentration 05 g l-1 by

strain Aspergillus awamori

NRRL 3112

0

02

04

06

08

0 1 2 3 4 5 6 7

Time h

Ph

en

ol

bio

deg

rad

ati

on

g

l-1

Fig 6 Adsorption of

phenol by modified PAN

membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

379 for the immobilization of Asp awamori for the

380 purpose of phenol degradation

381 The amount of cells (wet weight) on the membrane

382 of 40 cm2 was measured after the sixth cycle of

383 phenol biodegradationmdash1104 g cm-2 The signifi-

384 cant increase of the biomass with the increased

385 number of cycles is obvious (after immobilizationmdash

386 0158 g cm-2) Thus the biomass accumulated on

387 the membrane was approximately ten times higher

388 after six cycles of phenol biodegradation The high

389 operational stability of investigated biocatalysts was

390 confirmed by the scanning electron micrographs of

391 the polyacrylonitrile membraneimmobilized Asper-

392 gillus awamori NRRL at the beginning of repeated

393 batch cultivation and after the 7th cycle After the 7th

394 cycle of cultivation the observations showed large

395 groups of cells (Fig 7a) The significant increase of

396 biomass and mycelia formation on the membrane

397 surface after the 7th cycle of cultivation was inev-

398 itably followed by deceleration of the phenol

399 degradation rate

400Experiments with membrane bioreactor

401The next step of the research work involved exper-

402iments which were carried out in a bioreactor with

403spirally wound membrane in a regime of recircula-

404tion The spores were immobilized onto the

405membrane directly in the system as described in

406lsquolsquoMaterials and methodsrsquorsquo section Phenol biodegra-

407dation was carried out under aerobic conditions The

408operation parameters of the bioreactor are presented

409in Table 2 Fresh nutrient medium containing

41005 g l-1 phenol were added after each cycle Phenol

411concentration was monitored for degradation At the

412end of a cycle the system was washed with sterile

413distilled water and charged with nutrient medium and

414phenol for the next cycle Thus 10 cycles of 05 g l-1

415phenol biodegradation were run consecutively to

416determine the degradation time for each cycle

417(Fig 8) No significant difference was observed

418between the degradation times of the cycles from

419the second to the seventh (about 50 h) and between

420the biodegradation rates (100 mg h-1) After the 7th

421cycle the biodegradation rate began to decrease

422gradually and at 10 cycles it was 40 mg h-1 These

423results confirmed the advantages of the recycled

424reactor with spirally wound membrane The design of

425the bioreactor appeared to be quite effective provid-

426ing large membrane surface to bind the strain cells

427Besides phenol would flow tangentially which pre-

428vents membrane from a decrease of the flow velocity

429Conclusion

430A novel immobilized systemmdashAspergillus awamori

431NRRL 3112polyacrylonitrile membranewas obtained

432for the purpose of phenol biodegradation It was

433established that the immobilized system prepared in

434the presence of 02 g l-1 phenol was more effective

Fig 7 Scanning electron micrographs of immobilized system

Aspergillus awamori NRRL 3112modified PAN membrane

after 7th cycle (a) and before 1st cycle (b)

Table 2 Operation conditions of bioreactor

Parameter Value

Rate flow 15 ml min-1

Volume 300 ml

Initial phenol concentration 05 g l-1

Membrane surface area 100 cm2

Immobilized spores 0174 g cm-2 (wet weight)

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

435 than the system prepared in the phenol absence It was

436 revealed that the immobilized systems were more

437 stable andmore effective when biodegrading 05 g l-1

438 phenol in comparison with the free cells A bioreactor

439 was constructed containing spirally wound membrane

440 with immobilized spores of Aspergillus awamori

441 NRRL 3112 in a regime of recirculation which was

442 successfully applied for phenol biodegradation The

443 obtained results confirmed the advantages of the

444 recycled reactor with spirally wound membrane Thus

445 the immobilized microbial technology is an extremely

446 versatile approach that can be used for degradation of

447 toxic pollutants from the industrial effluents

448 Acknowledgments The authors gratefully acknowledge to449 the Bulgarian Ministry for Education and to the National450 Science Fund for their financial support and encouragement of451 the scientific research work in state universities

452 References

453 Abd-El-Haleem D Beshay U Abdelhamid AO Moawad H454 Zaki S (2003) Effects of mixed nitrogen sources on bio-455 degradation of phenol by immobilized Acinetobacter sp456 strain W-17 Afr J Biotechnol 2(1)8ndash12457 Allsop PJ Chisti Y Moi-Youngand M Sullivan GR (1993)458 Dynamics of phenol degradation by Pseudomonas putida459 Biotechnol Bioeng 41572ndash579460 Anselmo AM Novais JM (1992) Degradation of phenol by461 immobilized mycelium of Fusarium floccieferum in con-462 tinuous culture Water Sci Technol 25161ndash168463 Bolanos ML Varesche MBA Zaiat M Foresti E (2001) Phenol464 degradation in horizontal-flow anaerobic immobilized465 biomass (HAIB) reactor under mesophilic conditions466 Water Sci Technol 44(4)167ndash174

467Chitra S Sekaran G Padmavathi S Gowri C (1995) Removal468of phenol from wastewater using a biocatalyst In Pro-469ceedings of the national symposium frontiers in applied470environmental microbiology pp 74ndash77471Chtourou M Ammar E Nasri M Medhioub K (2004) Isolation472of a yeast Trichosporon cutaneum able to use low473molecular weight phenolic compounds application to474olive mill waste water treatment J Chem Technol Bio-475technol 79869ndash878476Dimov K Samardjieva Pavlov P (1983) Laboratory practice477on technology of synthetic fibers Technica Sofia BG478Fiest CF Hegeman GD (1969) Phenol and benzoate metabo-479lism by Pseudomonas putida J Bacteriol 100869ndash877480Godjevargova T Aleksieva Z Ivanova D (2000) Cell immo-481bilization of Trichosporon cutaneum strain with phenol482degradation ability on new modified polymer carries483Process Biochem 35699ndash704484Godjevargova T Ivanova D Aleksieva Z Burdelova G (2006)485Biodegradation of phenol by immobilized Trichosporon

486cutaneum R57 on modified polymer membranes Process487Biochem 412342ndash2346488Gonzalez G Herrera MG Pena MM (2001) Biodegradation of489phenol in a continuous process comparative study of490stirred tank and fluidized-bed bioreactors Bioresour491Technol 76245ndash251492Hua L Sun ZH Leng Y (2005) Continuous biocatalytic reso-493lution of DL-pantolactone by cross-linked cells in494membrane bioreactor Process Biochem 401137ndash1142495Hudges EJL Bayly RC Skurray RA (1984) Evidence for496isofunctional enzymes in the degradation of phenol m-497and p-toluate and p-cresol at catechol meta-cleavage498pathways in Alcaligenes eutrophus J Bacteriol 15879ndash83499Jones KH Trudgill PW Hopper DJ (1995) Evidence of two500pathways for the metabolism of phenol by Aspergillus

501fumigatus Arch Microbiol 163176ndash181502Jose G Sanchez M Theodore T (2002) Membranes and mem-503brane reactors Wiley-VCH Veriag GmbH Weinheim504Karel S Libichi S Robertson C (1985) The immobilization of505whole cells engineering principles Chem Eng Sci506401321ndash1354

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

Fig 8 Concentration

profile of phenol in a

recirculating system with

immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

507 Kowalska M Bodzek M Bohdziewicz J (1998) Biodegradation508 of phenols and cyanides using membranes immobilized509 microorganisms Process Biochem 33189ndash197510 Loh K Chung T Ang W (2000) Immobilized-cell membrane511 bioreactor for high-strength phenol wastewater J Environ512 Eng ASCE 12675ndash79513 Marrot B Barrios-Martinez A Moulin P Roche N (2006)514 Biodegradation of high phenol concentration by activated515 sludge in an immersed membrane bioreactor Biochem516 Eng J 30174ndash183517 Mordocco A Kuek C Jenkins R (1999) Continuous degradation518 of phenol at low concentration using immobilized Pseu-

519 domonas putida Enzyme Microb Technol 25530ndash536520 Pazarlioglu NK Telefoncu A (2005) Biodegradation of phenol521 by Pseudomonas putida immobilized on activated pumice522 particles Process Biochem 401807ndash1814523 Santos VL Heilbuth NM Braga T Monteiro AS Linardi VR524 (2003) Phenol degradation by aGraphium sp FIB4 isolated525 from industrial effluents J Basic Microbiol 43238ndash248526 Shetty KV Kalifathulla I Srinikethan G (2007a) Performance527 of pulsed plate bioreactor for biodegradation of phenol528 J Hazard Mater 140346ndash352529 Shetty KV Ramanjaneyulu R Srinikethan G (2007b) Biolog-530 ical phenol removal using immobilized cells in a pulsed

531plate bioreactor effect of dilution rate and influent phenol532concentration J Hazard Mater 149452ndash459533Stephenson T Judd S Jefferson B Brindle K (2000) Mem-534brane bioreactors for wastewater treatment IWA535Publishing London UK pp 129ndash136536Sundar P Daniel D Krastanov A (2005) Biodegradation of537phenol by immobilized Aspergillus awamori cells Bulg J538Agric Sci 1161ndash71539Uzunova K Vassileva A Ivanova V Spasova D Tonkova A540(2002) Thermostable exo-inulinase production by semi-541continuous cultivation of membrane-immobilized Bacillus542sp 11 cells Process Biochem 37863ndash868543Wang Z Wan L Xu Z (2007) Surface engineerings of poly-544acrylonitrile-based asymmetric membranes towards545biomedical applications an overview J Membr Sci5463048ndash23547Yiang Y Wen J Li H Yang S Hu Z (2005) The biodegra-548dation of phenol at high initial concentration by the yeast549Candida tropicalis Biochem Eng J 24243ndash247550Yordanova G Ivanova D Godjevargova T Krastanov A (2006)551Biodegradation of phenol by immobilized Aspergillus

552awamori NRRL3112 cells Food science engineering and553technologies conference Scientific works Plovdiv554pp 260ndash265

555

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

379 for the immobilization of Asp awamori for the

380 purpose of phenol degradation

381 The amount of cells (wet weight) on the membrane

382 of 40 cm2 was measured after the sixth cycle of

383 phenol biodegradationmdash1104 g cm-2 The signifi-

384 cant increase of the biomass with the increased

385 number of cycles is obvious (after immobilizationmdash

386 0158 g cm-2) Thus the biomass accumulated on

387 the membrane was approximately ten times higher

388 after six cycles of phenol biodegradation The high

389 operational stability of investigated biocatalysts was

390 confirmed by the scanning electron micrographs of

391 the polyacrylonitrile membraneimmobilized Asper-

392 gillus awamori NRRL at the beginning of repeated

393 batch cultivation and after the 7th cycle After the 7th

394 cycle of cultivation the observations showed large

395 groups of cells (Fig 7a) The significant increase of

396 biomass and mycelia formation on the membrane

397 surface after the 7th cycle of cultivation was inev-

398 itably followed by deceleration of the phenol

399 degradation rate

400Experiments with membrane bioreactor

401The next step of the research work involved exper-

402iments which were carried out in a bioreactor with

403spirally wound membrane in a regime of recircula-

404tion The spores were immobilized onto the

405membrane directly in the system as described in

406lsquolsquoMaterials and methodsrsquorsquo section Phenol biodegra-

407dation was carried out under aerobic conditions The

408operation parameters of the bioreactor are presented

409in Table 2 Fresh nutrient medium containing

41005 g l-1 phenol were added after each cycle Phenol

411concentration was monitored for degradation At the

412end of a cycle the system was washed with sterile

413distilled water and charged with nutrient medium and

414phenol for the next cycle Thus 10 cycles of 05 g l-1

415phenol biodegradation were run consecutively to

416determine the degradation time for each cycle

417(Fig 8) No significant difference was observed

418between the degradation times of the cycles from

419the second to the seventh (about 50 h) and between

420the biodegradation rates (100 mg h-1) After the 7th

421cycle the biodegradation rate began to decrease

422gradually and at 10 cycles it was 40 mg h-1 These

423results confirmed the advantages of the recycled

424reactor with spirally wound membrane The design of

425the bioreactor appeared to be quite effective provid-

426ing large membrane surface to bind the strain cells

427Besides phenol would flow tangentially which pre-

428vents membrane from a decrease of the flow velocity

429Conclusion

430A novel immobilized systemmdashAspergillus awamori

431NRRL 3112polyacrylonitrile membranewas obtained

432for the purpose of phenol biodegradation It was

433established that the immobilized system prepared in

434the presence of 02 g l-1 phenol was more effective

Fig 7 Scanning electron micrographs of immobilized system

Aspergillus awamori NRRL 3112modified PAN membrane

after 7th cycle (a) and before 1st cycle (b)

Table 2 Operation conditions of bioreactor

Parameter Value

Rate flow 15 ml min-1

Volume 300 ml

Initial phenol concentration 05 g l-1

Membrane surface area 100 cm2

Immobilized spores 0174 g cm-2 (wet weight)

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

435 than the system prepared in the phenol absence It was

436 revealed that the immobilized systems were more

437 stable andmore effective when biodegrading 05 g l-1

438 phenol in comparison with the free cells A bioreactor

439 was constructed containing spirally wound membrane

440 with immobilized spores of Aspergillus awamori

441 NRRL 3112 in a regime of recirculation which was

442 successfully applied for phenol biodegradation The

443 obtained results confirmed the advantages of the

444 recycled reactor with spirally wound membrane Thus

445 the immobilized microbial technology is an extremely

446 versatile approach that can be used for degradation of

447 toxic pollutants from the industrial effluents

448 Acknowledgments The authors gratefully acknowledge to449 the Bulgarian Ministry for Education and to the National450 Science Fund for their financial support and encouragement of451 the scientific research work in state universities

452 References

453 Abd-El-Haleem D Beshay U Abdelhamid AO Moawad H454 Zaki S (2003) Effects of mixed nitrogen sources on bio-455 degradation of phenol by immobilized Acinetobacter sp456 strain W-17 Afr J Biotechnol 2(1)8ndash12457 Allsop PJ Chisti Y Moi-Youngand M Sullivan GR (1993)458 Dynamics of phenol degradation by Pseudomonas putida459 Biotechnol Bioeng 41572ndash579460 Anselmo AM Novais JM (1992) Degradation of phenol by461 immobilized mycelium of Fusarium floccieferum in con-462 tinuous culture Water Sci Technol 25161ndash168463 Bolanos ML Varesche MBA Zaiat M Foresti E (2001) Phenol464 degradation in horizontal-flow anaerobic immobilized465 biomass (HAIB) reactor under mesophilic conditions466 Water Sci Technol 44(4)167ndash174

467Chitra S Sekaran G Padmavathi S Gowri C (1995) Removal468of phenol from wastewater using a biocatalyst In Pro-469ceedings of the national symposium frontiers in applied470environmental microbiology pp 74ndash77471Chtourou M Ammar E Nasri M Medhioub K (2004) Isolation472of a yeast Trichosporon cutaneum able to use low473molecular weight phenolic compounds application to474olive mill waste water treatment J Chem Technol Bio-475technol 79869ndash878476Dimov K Samardjieva Pavlov P (1983) Laboratory practice477on technology of synthetic fibers Technica Sofia BG478Fiest CF Hegeman GD (1969) Phenol and benzoate metabo-479lism by Pseudomonas putida J Bacteriol 100869ndash877480Godjevargova T Aleksieva Z Ivanova D (2000) Cell immo-481bilization of Trichosporon cutaneum strain with phenol482degradation ability on new modified polymer carries483Process Biochem 35699ndash704484Godjevargova T Ivanova D Aleksieva Z Burdelova G (2006)485Biodegradation of phenol by immobilized Trichosporon

486cutaneum R57 on modified polymer membranes Process487Biochem 412342ndash2346488Gonzalez G Herrera MG Pena MM (2001) Biodegradation of489phenol in a continuous process comparative study of490stirred tank and fluidized-bed bioreactors Bioresour491Technol 76245ndash251492Hua L Sun ZH Leng Y (2005) Continuous biocatalytic reso-493lution of DL-pantolactone by cross-linked cells in494membrane bioreactor Process Biochem 401137ndash1142495Hudges EJL Bayly RC Skurray RA (1984) Evidence for496isofunctional enzymes in the degradation of phenol m-497and p-toluate and p-cresol at catechol meta-cleavage498pathways in Alcaligenes eutrophus J Bacteriol 15879ndash83499Jones KH Trudgill PW Hopper DJ (1995) Evidence of two500pathways for the metabolism of phenol by Aspergillus

501fumigatus Arch Microbiol 163176ndash181502Jose G Sanchez M Theodore T (2002) Membranes and mem-503brane reactors Wiley-VCH Veriag GmbH Weinheim504Karel S Libichi S Robertson C (1985) The immobilization of505whole cells engineering principles Chem Eng Sci506401321ndash1354

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

Fig 8 Concentration

profile of phenol in a

recirculating system with

immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

507 Kowalska M Bodzek M Bohdziewicz J (1998) Biodegradation508 of phenols and cyanides using membranes immobilized509 microorganisms Process Biochem 33189ndash197510 Loh K Chung T Ang W (2000) Immobilized-cell membrane511 bioreactor for high-strength phenol wastewater J Environ512 Eng ASCE 12675ndash79513 Marrot B Barrios-Martinez A Moulin P Roche N (2006)514 Biodegradation of high phenol concentration by activated515 sludge in an immersed membrane bioreactor Biochem516 Eng J 30174ndash183517 Mordocco A Kuek C Jenkins R (1999) Continuous degradation518 of phenol at low concentration using immobilized Pseu-

519 domonas putida Enzyme Microb Technol 25530ndash536520 Pazarlioglu NK Telefoncu A (2005) Biodegradation of phenol521 by Pseudomonas putida immobilized on activated pumice522 particles Process Biochem 401807ndash1814523 Santos VL Heilbuth NM Braga T Monteiro AS Linardi VR524 (2003) Phenol degradation by aGraphium sp FIB4 isolated525 from industrial effluents J Basic Microbiol 43238ndash248526 Shetty KV Kalifathulla I Srinikethan G (2007a) Performance527 of pulsed plate bioreactor for biodegradation of phenol528 J Hazard Mater 140346ndash352529 Shetty KV Ramanjaneyulu R Srinikethan G (2007b) Biolog-530 ical phenol removal using immobilized cells in a pulsed

531plate bioreactor effect of dilution rate and influent phenol532concentration J Hazard Mater 149452ndash459533Stephenson T Judd S Jefferson B Brindle K (2000) Mem-534brane bioreactors for wastewater treatment IWA535Publishing London UK pp 129ndash136536Sundar P Daniel D Krastanov A (2005) Biodegradation of537phenol by immobilized Aspergillus awamori cells Bulg J538Agric Sci 1161ndash71539Uzunova K Vassileva A Ivanova V Spasova D Tonkova A540(2002) Thermostable exo-inulinase production by semi-541continuous cultivation of membrane-immobilized Bacillus542sp 11 cells Process Biochem 37863ndash868543Wang Z Wan L Xu Z (2007) Surface engineerings of poly-544acrylonitrile-based asymmetric membranes towards545biomedical applications an overview J Membr Sci5463048ndash23547Yiang Y Wen J Li H Yang S Hu Z (2005) The biodegra-548dation of phenol at high initial concentration by the yeast549Candida tropicalis Biochem Eng J 24243ndash247550Yordanova G Ivanova D Godjevargova T Krastanov A (2006)551Biodegradation of phenol by immobilized Aspergillus

552awamori NRRL3112 cells Food science engineering and553technologies conference Scientific works Plovdiv554pp 260ndash265

555

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

435 than the system prepared in the phenol absence It was

436 revealed that the immobilized systems were more

437 stable andmore effective when biodegrading 05 g l-1

438 phenol in comparison with the free cells A bioreactor

439 was constructed containing spirally wound membrane

440 with immobilized spores of Aspergillus awamori

441 NRRL 3112 in a regime of recirculation which was

442 successfully applied for phenol biodegradation The

443 obtained results confirmed the advantages of the

444 recycled reactor with spirally wound membrane Thus

445 the immobilized microbial technology is an extremely

446 versatile approach that can be used for degradation of

447 toxic pollutants from the industrial effluents

448 Acknowledgments The authors gratefully acknowledge to449 the Bulgarian Ministry for Education and to the National450 Science Fund for their financial support and encouragement of451 the scientific research work in state universities

452 References

453 Abd-El-Haleem D Beshay U Abdelhamid AO Moawad H454 Zaki S (2003) Effects of mixed nitrogen sources on bio-455 degradation of phenol by immobilized Acinetobacter sp456 strain W-17 Afr J Biotechnol 2(1)8ndash12457 Allsop PJ Chisti Y Moi-Youngand M Sullivan GR (1993)458 Dynamics of phenol degradation by Pseudomonas putida459 Biotechnol Bioeng 41572ndash579460 Anselmo AM Novais JM (1992) Degradation of phenol by461 immobilized mycelium of Fusarium floccieferum in con-462 tinuous culture Water Sci Technol 25161ndash168463 Bolanos ML Varesche MBA Zaiat M Foresti E (2001) Phenol464 degradation in horizontal-flow anaerobic immobilized465 biomass (HAIB) reactor under mesophilic conditions466 Water Sci Technol 44(4)167ndash174

467Chitra S Sekaran G Padmavathi S Gowri C (1995) Removal468of phenol from wastewater using a biocatalyst In Pro-469ceedings of the national symposium frontiers in applied470environmental microbiology pp 74ndash77471Chtourou M Ammar E Nasri M Medhioub K (2004) Isolation472of a yeast Trichosporon cutaneum able to use low473molecular weight phenolic compounds application to474olive mill waste water treatment J Chem Technol Bio-475technol 79869ndash878476Dimov K Samardjieva Pavlov P (1983) Laboratory practice477on technology of synthetic fibers Technica Sofia BG478Fiest CF Hegeman GD (1969) Phenol and benzoate metabo-479lism by Pseudomonas putida J Bacteriol 100869ndash877480Godjevargova T Aleksieva Z Ivanova D (2000) Cell immo-481bilization of Trichosporon cutaneum strain with phenol482degradation ability on new modified polymer carries483Process Biochem 35699ndash704484Godjevargova T Ivanova D Aleksieva Z Burdelova G (2006)485Biodegradation of phenol by immobilized Trichosporon

486cutaneum R57 on modified polymer membranes Process487Biochem 412342ndash2346488Gonzalez G Herrera MG Pena MM (2001) Biodegradation of489phenol in a continuous process comparative study of490stirred tank and fluidized-bed bioreactors Bioresour491Technol 76245ndash251492Hua L Sun ZH Leng Y (2005) Continuous biocatalytic reso-493lution of DL-pantolactone by cross-linked cells in494membrane bioreactor Process Biochem 401137ndash1142495Hudges EJL Bayly RC Skurray RA (1984) Evidence for496isofunctional enzymes in the degradation of phenol m-497and p-toluate and p-cresol at catechol meta-cleavage498pathways in Alcaligenes eutrophus J Bacteriol 15879ndash83499Jones KH Trudgill PW Hopper DJ (1995) Evidence of two500pathways for the metabolism of phenol by Aspergillus

501fumigatus Arch Microbiol 163176ndash181502Jose G Sanchez M Theodore T (2002) Membranes and mem-503brane reactors Wiley-VCH Veriag GmbH Weinheim504Karel S Libichi S Robertson C (1985) The immobilization of505whole cells engineering principles Chem Eng Sci506401321ndash1354

0

02

04

06

08

1

0 200 400 600 800

Time h

Ph

en

ol

co

nc

en

tra

tio

n

gl-1

Fig 8 Concentration

profile of phenol in a

recirculating system with

immobilized system

Aspergillus awamori NRRL

3112modified PAN

membrane

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

507 Kowalska M Bodzek M Bohdziewicz J (1998) Biodegradation508 of phenols and cyanides using membranes immobilized509 microorganisms Process Biochem 33189ndash197510 Loh K Chung T Ang W (2000) Immobilized-cell membrane511 bioreactor for high-strength phenol wastewater J Environ512 Eng ASCE 12675ndash79513 Marrot B Barrios-Martinez A Moulin P Roche N (2006)514 Biodegradation of high phenol concentration by activated515 sludge in an immersed membrane bioreactor Biochem516 Eng J 30174ndash183517 Mordocco A Kuek C Jenkins R (1999) Continuous degradation518 of phenol at low concentration using immobilized Pseu-

519 domonas putida Enzyme Microb Technol 25530ndash536520 Pazarlioglu NK Telefoncu A (2005) Biodegradation of phenol521 by Pseudomonas putida immobilized on activated pumice522 particles Process Biochem 401807ndash1814523 Santos VL Heilbuth NM Braga T Monteiro AS Linardi VR524 (2003) Phenol degradation by aGraphium sp FIB4 isolated525 from industrial effluents J Basic Microbiol 43238ndash248526 Shetty KV Kalifathulla I Srinikethan G (2007a) Performance527 of pulsed plate bioreactor for biodegradation of phenol528 J Hazard Mater 140346ndash352529 Shetty KV Ramanjaneyulu R Srinikethan G (2007b) Biolog-530 ical phenol removal using immobilized cells in a pulsed

531plate bioreactor effect of dilution rate and influent phenol532concentration J Hazard Mater 149452ndash459533Stephenson T Judd S Jefferson B Brindle K (2000) Mem-534brane bioreactors for wastewater treatment IWA535Publishing London UK pp 129ndash136536Sundar P Daniel D Krastanov A (2005) Biodegradation of537phenol by immobilized Aspergillus awamori cells Bulg J538Agric Sci 1161ndash71539Uzunova K Vassileva A Ivanova V Spasova D Tonkova A540(2002) Thermostable exo-inulinase production by semi-541continuous cultivation of membrane-immobilized Bacillus542sp 11 cells Process Biochem 37863ndash868543Wang Z Wan L Xu Z (2007) Surface engineerings of poly-544acrylonitrile-based asymmetric membranes towards545biomedical applications an overview J Membr Sci5463048ndash23547Yiang Y Wen J Li H Yang S Hu Z (2005) The biodegra-548dation of phenol at high initial concentration by the yeast549Candida tropicalis Biochem Eng J 24243ndash247550Yordanova G Ivanova D Godjevargova T Krastanov A (2006)551Biodegradation of phenol by immobilized Aspergillus

552awamori NRRL3112 cells Food science engineering and553technologies conference Scientific works Plovdiv554pp 260ndash265

555

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of

UNCORRECTEDPR

OOF

UNCORRECTEDPR

OOF

507 Kowalska M Bodzek M Bohdziewicz J (1998) Biodegradation508 of phenols and cyanides using membranes immobilized509 microorganisms Process Biochem 33189ndash197510 Loh K Chung T Ang W (2000) Immobilized-cell membrane511 bioreactor for high-strength phenol wastewater J Environ512 Eng ASCE 12675ndash79513 Marrot B Barrios-Martinez A Moulin P Roche N (2006)514 Biodegradation of high phenol concentration by activated515 sludge in an immersed membrane bioreactor Biochem516 Eng J 30174ndash183517 Mordocco A Kuek C Jenkins R (1999) Continuous degradation518 of phenol at low concentration using immobilized Pseu-

519 domonas putida Enzyme Microb Technol 25530ndash536520 Pazarlioglu NK Telefoncu A (2005) Biodegradation of phenol521 by Pseudomonas putida immobilized on activated pumice522 particles Process Biochem 401807ndash1814523 Santos VL Heilbuth NM Braga T Monteiro AS Linardi VR524 (2003) Phenol degradation by aGraphium sp FIB4 isolated525 from industrial effluents J Basic Microbiol 43238ndash248526 Shetty KV Kalifathulla I Srinikethan G (2007a) Performance527 of pulsed plate bioreactor for biodegradation of phenol528 J Hazard Mater 140346ndash352529 Shetty KV Ramanjaneyulu R Srinikethan G (2007b) Biolog-530 ical phenol removal using immobilized cells in a pulsed

531plate bioreactor effect of dilution rate and influent phenol532concentration J Hazard Mater 149452ndash459533Stephenson T Judd S Jefferson B Brindle K (2000) Mem-534brane bioreactors for wastewater treatment IWA535Publishing London UK pp 129ndash136536Sundar P Daniel D Krastanov A (2005) Biodegradation of537phenol by immobilized Aspergillus awamori cells Bulg J538Agric Sci 1161ndash71539Uzunova K Vassileva A Ivanova V Spasova D Tonkova A540(2002) Thermostable exo-inulinase production by semi-541continuous cultivation of membrane-immobilized Bacillus542sp 11 cells Process Biochem 37863ndash868543Wang Z Wan L Xu Z (2007) Surface engineerings of poly-544acrylonitrile-based asymmetric membranes towards545biomedical applications an overview J Membr Sci5463048ndash23547Yiang Y Wen J Li H Yang S Hu Z (2005) The biodegra-548dation of phenol at high initial concentration by the yeast549Candida tropicalis Biochem Eng J 24243ndash247550Yordanova G Ivanova D Godjevargova T Krastanov A (2006)551Biodegradation of phenol by immobilized Aspergillus

552awamori NRRL3112 cells Food science engineering and553technologies conference Scientific works Plovdiv554pp 260ndash265

555

Biodegradation

123

Journal Medium 10532 Dispatch 26-3-2009 Pages 10

Article No 9259 h LE h TYPESET

MS Code BIOD813 h CP h DISK4 4

Au

tho

r P

ro

of