Hydrolysis of Ionic Phthalic Acid Based Polyesters by Wastewater

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Hydrolysis of ionic phthalic acid based polyesters by wastewater microorganisms and their enzymes Karolina Haernvall, Sabine Zitzenbacher, Katrin Wallig, Motonori Yamamoto, Michael Bernhard Schick, Doris Ribitsch, and Georg M. Guebitz Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b00062 • Publication Date (Web): 27 Mar 2017 Downloaded from http://pubs.acs.org on March 29, 2017

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Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Environmental Science & Technology

% Biodegradation (CO2 evolution)

100

O

O

O

OH

O

O

O SO 3

-

5

Released TA

O

(mmol/mol polymer)

O HO

0

O HO O

O

O

O

OH

O

O

O SO 3

-

ACS Paragon Plus Environment

PpelaLip PpCutA

0 0

10 20 Time (days)

30


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1

Hydrolysis of ionic phthalic acid based polyesters by

2

wastewater microorganisms and their enzymes

3

Karolina Haernvall

4

Doris Ribitsch *, Georg M. Guebitz

5

a

6

Austria

7

b

BASF SE, Carl-Bosch-Strasse 38, 67056 Ludwigshafen am Rhein, Germany

8

c

BOKU - University of Natural Resources and Life Sciences, Institute for Environmental Biotechnology, Konrad

9

Lorenz Strasse 20, 3430 Tulln an der Donau, Austria

a

a, 1

, Sabine Zitzenbacher

a, 2

, Katrin Wallig

a, 3

b

b

, Motonori Yamamoto , Michael Bernhard Schick ,

a, c

ACIB - Austrian Centre of Industrial Biotechnology GmbH, Konrad Lorenz Strasse 20, 3430 Tulln an der Donau,

10

1

Present address: AstraZeneca AB (Dfind Science and Engineering AB), Astraallén, 152 57 Södertälje, Sweden

11

2

Present address: Richard Bittner AG, Ossiacherstrasse 7, 9560 Feldkirchen, Austria

12

3

Present address: Shire, Industriestrasse 67, 1221 Wien, Austria

13

* Corresponding author: Doris Ribitsch, [email protected], phone: (+43) 1 /47654-97475, fax: (+43) 1/47654-

14

97409

15

ABSTRACT

16

Water-soluble polyesters are used in a range of applications today and enter wastewater treatment plants after

17

product utilization. However, little is known about extracellular enzymes and aquatic microorganisms involved in

18

polyester biodegradation and mineralization. In this study, structurally different ionic phthalic acid based polyesters

19

(the number average molecular weights (Mn) 1,770 to 10,000 g/mol and semi crystalline with crystallinity below

20

1%) were synthesized in various combinations. Typical wastewater microorganisms like Pseudomonas sp. were

21

chosen for in-silico screening towards polyester hydrolyzing enzymes. Based on the in-silico search, a cutinase from

22

Pseudomonas pseudoalcaligenes (PpCutA) and a putative lipase from Pseudomonas pelagia (PpelaLip) were

23

identified. The enzymes PpCutA and PpelaLip were demonstrated to hydrolyze all structurally different polyesters. 1


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24

Activities on all the polyesters were also confirmed with the strains P. pseudoalcaligenes and P. pelagia. Parameters

25

identified to enhance hydrolysis included increased water solubility and polyester hydrophilicity as well as shorter

26

diol chain lengths. For example, polyesters containing 1,2-ethanediol were hydrolyzed faster than polyesters

27

containing 1,8-octanediol. Interestingly, the same trend was observed in biodegradation experiments. This

28

information is important to gain a better mechanistic understanding of biodegradation processes of polyesters in

29

WWTPs where the extracellular enzymatic hydrolysis seems to be the limiting step.

30

Keywords: biodegradation, sulfonated aromatic polyester, household product, cutinase, lipase, Pseudomonas sp..

31

1. INTRODUCTION

32

Ionic phthalic acid based polyesters are used in a wide range of applications today including the production of

33

plastics, water soluble lubricates, pharmaceuticals, antifreeze agents, cosmetics and surfactants. After utilization of

34

the product, a high proportion of polyesters enter wastewater treatment plants (WWTP) via sewage systems and

35

may pass the WWTP unaffected, ending up in aquatic environments . Hence, knowledge about biodegradation of

36

ionic polyesters in WWTPs is essential. Generally, biodegradation of polymers in WWTPs is a two-step process

37

where microbial extracellular enzymes initially hydrolyze polymers into smaller oligomers or monomers which can

38

then be taken up by microorganisms and further be degraded and mineralized. The crucial and rate-limiting step

39

for biodegradation of polymers is nevertheless the initial enzymatic hydrolysis.

40

communities and especially the microbial extracellular enzymes are playing a crucial role in biodegradation

41

processes of polymers in WWTPs. Enzymatic hydrolysis and microbial degradation of small phthalic acid esters in

42

WWTP has been studied since the 1930s and received increasing attention more recently due to advances in

43

analytical methods to identify traces in aquatic systems.

44

mechanism of phthalic acid esters in WWTPs is related to microbial activities while abiotic hydrolysis appears

45

negligible under most environmental conditions, confirming the importance of biological pathways.

46

Microorganisms identified as being important for phthalic acid ester degradation in WWTPs are, for instance,

47

Pseudomonas sp, Bacillus sp and Rodococcus sp.

48

stutzeri were isolated from activated sludge and proven to degrade phthalic acid esters.

1

3-6

1, 2

Therefore, microbial

It was demonstrated that the main degradation

4, 7

. The strains Pseudomonas fluorescence and Pseudomonas 8, 9

For enzymatic 2



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10,

49

hydrolysis of phthalic acid esters, extracellular hydrolases were shown to be essential, like cutinases (EC 3.1.1.74)

50

11

51

polyester materials, for example a lipase from Pseudomonas cepacia was shown to hydrolyze poly(butylene

52

succinate-co-terephthalate) (PBST)

53

poly(butylene succinate-co-butylene adipate) (PBSA) . One study focused on enzymatic hydrolysis and microbial

54

degradation of ionic phthalic acid esters and showed that nitroterephthalic acid esters were enzymatically

55

hydrolyzed by lipases from P. fluorescens and P. cepacia and degraded by several Pseudomonas strains, like

56

P. fluorescens, P. aeruginosa, P, chlororaphis, P. oleovorans and P. putida.

57

esters are oligomeric substances and therefore there is still a lack of knowledge related to the biodegradation of

58

polymeric substances as polyesters in WWTPs. Only few studies have focused on biodegradation of polyethylene

59

glycols in WWTP

60

understanding of biodegradation processes of phthalic acid based polyesters in WWTPs is crucial to gain knowledge

61

about the fate of these polyesters in the environment. Therefore, in this study structurally different ionic phthalic

62

acid based polyesters were synthesized to systematically investigate their enzymatic hydrolysis and microbial

63

hydrolysis. Aerobic biodegradation of the polyesters in simulated freshwater with WWTP sludge as inoculum was

64

also investigated.

65

2. MATERIALS AND METHODS

66

12

and lipases (EC 3.1.1.3) . Extracellular hydrolases from Pseudomonas sp. were previously shown to hydrolyze

13

and a lipase from Pseudomonas aeruginosa was

able to hydrolyze

14

2.1.

15

However, all investigated phthalic acid

16-19

, but there are no studies about phthalic acid based polyesters. Thus, a better mechanistic

CHEMICALS, REAGENTS AND MICROORGANISMS TM

67

Dimethyl sulfoxide and ethanol were purchased from Merck Millipore, Difco

68

extract and peptone from Fluka. All other chemicals and solvents were purchased from Sigma-Aldrich. Carrez

69

reagent I and Carrez reagent II were prepared by dissolving 5.325 g K4[Fe(CN)6]*3H2O and 14.400 g ZnSO4*7H2O in

70

50 ml milliQ water. Pseudomonas pseudoalcaligenes (DSM 50188) and Pseudomonas pelagia (DSM 25163) were

71

obtained from the German Collection of Microorganism and Cell Cultures (DSMZ, Braunschweig, Germany).

72

2.2.

Marine Broth 2216 from VWR, meat

POLYESTER SYNTHESIS AND CHARACTERIZATION 3


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73


2.2.1.

GENERAL PROCEDURE FOR POLYESTER SYNTHESIS

74

5-Sodiumsulfoisophthalic acid dimethyl ester, diol and tetrabutyl titanate were mixed and heated up to 180-200 °C

75

for 50 min. The catalyst tetrabutyl titanate was used in a concentration of 100 ppm based on polymer. Methanol

76

was distilled off during the reaction. Afterwards, dimethyl terephthalate was added to the mixture and stirred at

77

180-200 °C for 45 min. Again, methanol was distilled off before the temperature of the mixture was increased to

78

240 °C. In parallel, a vacuum was gradually applied during 30 min to approximately 1 mbar. The increase of viscosity

79

during the reaction was measured by the torque (HEIDOLPH RZR 2052 stirrer) of the stirrer.

80

2.2.2.

POLYESTER CHARACTERIZATION 1

81

The synthesized polyesters were characterized by proton nuclear magnetic resonance ( H-NMR), gel permeation

82

chromatography (GPC), differential scanning calorimetry (DSC), carbon content and water solubility.

83

The 400-MHz H-NMR spectra of the polyesters were recorded on a Bruker AV 400 spectrometer at 25 °C for 2 min

84

and 45 seconds and the samples were dissolved in dimethyl sulfoxide (DMSO). The achieved H NMR spectra were

85

used to determine the copolyester composition by comparing the relative peak intensities for terephthalic acid

86

(TA), 5-sulfoisophthalic acid (NaSIP) and diol/glycol. Their peak areas were considered to be equivalent to the

87

moieties quantities. The polyester composition was calculated as following

1

1

% =

⁄ ∗ 100 1 ⁄ + ⁄ and

% =

⁄ ∗ 100 2 ⁄ + ⁄

88

where ANaSIP is the sum of NaSIP proton integrals, ATA is the sum of TA proton integrals, HNaSIP is the sum of

89

hydrogens in NaSIP, HTA is the sum of hydrogens in TA.

90

GPC analysis was performed using a conventional GPC apparatus (Agilent 1100 series) equipped with columns PSS

91

GRAM (8x50 mm), PSS GRAM 30A (8x300 mm), PSS GRAM 1000A (8x300 mm) and PSS GRAM 1000A (8x300 mm) 4



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92

(PSS) and a RI detector. The columns were kept at a temperature of 85 °C. The injection volume was 100 µl of a

93

4 mg/ml solution and the samples were eluted with dimethylacetamide with 0.5% lithium bromide with a flow rate

94

of 1 ml/min. The calibration was carried out with PMMA standard from PSS.

95

The differential scanning calorimetry (DSC) were performed according to the standard DIN EN ISO 11357, and was

96

carried out over a temperature range of -50 °C to 200 °C at a heating and cooling rate of 20 K/min. The crystallinity

97

(Xc) of sample was calculated as following:

=

*

m is

∆! ∗ 100 3 ∗ ∆!

98

where ∆Hm is the melting enthalpy, and the ∆H

the melting enthalpy of 100% crystalline PET, which is equal to

99

130 J/g which is considered to be close enough to the melting enthalpy of the synthesized polyesters.

20

100

Carbon content of the polyesters was determined using a LECO RC 612 Multiphase Carbon/Hydrogen/Moisture

101

Determinator (LECO Corporation, MI, US). The polyesters were combusted by the following program to determine

102

the organic carbon content, starting temperature 105 °C, hold for 60 sec, and increase to 580 °C at a rate of 60 °C

103

per sec. Carbon is measured with IR as CO2. Carrier gas was Oxygen 4.0. Calculation was based on external

104

standard.

105

The water solubility of the polyesters was investigated in 100 mM potassium phosphate buffer pH 7.0. The

106

samples, with a final concentration of 10 mg/ml, were heated and shaken in a thermomixer (60 °C, 1400 rpm) for

107

30 min.

108

109

2.3.

DNA SEQUENCING, ALIGNMENTS AND DEPOSITION OF SEQUENCE

DATA

110

Nucleotide sequences of synthetic genes were confirmed by custom service by Agowa (Germany). DNA analysis

111

was performed with Vector NTI Suite 10 (Life Technologies, Germany). BLAST search was performed using the

112

ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics, and sequences

113

of related proteins were aligned using the Clustal W program (Swiss EMBnet node server). The codon optimized 5


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114

nucleotide sequences of PpelaLip and PpCutA was deposited in the GenBank database under accession numbers

115

KU695573 (PpelaLip) and KU695574 (PpCutA).

116

2.4.

ENZYME EXPRESSION AND PURIFICATION

117

Cutinase 1 from Thermobifida cellulosilytica was expressed and purified as earlier described by Herrero Acero et al.

118

(2011).

119

2.4.1.

ENZYME EXPRESSION IN E.COLI

120

Genes coding for lipase from P. pelagia and cutinase A from P. pseudoalcaligenes were codon optimized for

121

expression in E. coli and synthesized without natural signal peptides by GeneArt® (Life Technologies). The synthetic

122

genes were cloned over NdeI and HindIII restriction sites into pET26b(+) (Novagen, Merck KGaA, Germany) and

123

transformed into E. coli BL21-Gold(DE3). Freshly transformed E. coli BL21-Gold(DE3) cells were used to inoculate

124

20 ml LB medium supplemented with 40 mg/ml kanamycin. Cultivation was performed overnight at 37 °C and

125

150 rpm. The overnight culture was used to inoculate a 500 ml shake flask containing 200 ml of the same medium

126

to an OD600 of 0.1. The culture was incubated at 37 °C and 150 rpm until an OD600 of 0.8 was reached. In case of

127

PpCutA, the culture was cooled down to 20 °C and induced by adding IPTG to reach a final concentration of

128

0.05 mM. After 50 h of expression the cells were harvested by centrifugation at 4 °C and stored at -20°C. PpelaLip

129

was expressed as inclusion bodies at 37 °C and 0.05 mM IPTG for 30 h. Like before, cells were harvested by

130

centrifugation at 4 °C and stored at -20 °C.

131

2.4.2.

PURIFICATION OF PPCUTA

132

Cell pellets from 400 ml cell culture were resuspended in 50 ml Ni-NTA Lysis Buffer (20 mM sodium phosphate

133

pH 7.4, 10 mM imidazole, 500 mM NaCl). Resuspended cells were sonicated with three-times 30 s pulses under ice

134

cooling (Vibra Cell, Sonics Materials, Meryin/Satigny, Switzerland). Lysates were then centrifuged (60 min, 4 °C,

135

18,000 rpm) and purified according to the manufacturer’s protocol (IBA GmbH, Goettingen, Germany). Finally, the

6



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136

buffer was exchanged to 100 mM Tris-HCl pH 7.0 using PD-10 desalting columns (GE Healthcare). Purified enzyme

137

was stored at -20 °C.

138

2.4.3.

REFOLDING AND PURIFICATION OF PPELALIP

139

Cell pellet obtained from 800 ml culture was resuspended in 50 ml of lysis buffer (50 mM Tris-HCl pH 8, 5 mM

140

EDTA, 10% saccharose). After adding of lysozyme to an end concentration of 0.8 mg/ml and incubation for 30 min

141

at room temperature, 50 ml Triton lysis buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5% Triton X-100) were added.

142

Lysis was performed by sonication for 5 x 20 sec at 4 °C. Inclusion bodies were separated by centrifugation (30 min

143

and 12,000 g) and the pellet was washed again with 100 ml Triton lysis buffer. After a further sonication for

144

5 x 10 sec at 4 °C and centrifugation (30 min and 12,000 g), pellet was resuspended in 100 ml resuspension buffer

145

(50 mM Tris-HCl pH 8, 5 mM EDTA), centrifuged (12,000 g, 4 °C, 30 min) and resuspended in 15 ml distilled water.

146

Then, 30 ml solubilization buffer consisting of 8 M urea, 50 mM sodium phosphate pH 7.0, 1 mM EDTA and 5 mM

147

DDT were slowly added and stirred for 1 h at 25 °C. After sonication (12,000 g, 4 °C, 30 min), 0.1 g cystine

148

solubilized in 0.4 ml of 5 M NaOH was added and stirred for 10 min at pH 9.5. The pH was adjusted to pH 8.0 using

149

concentrated H3PO4 and the solution was dropped within 3 h at 4 °C to 3000 ml renaturation buffer (50 mM sodium

150

phosphate pH 8.0, 1 mM EDTA, 0.02% NaN3, 5 mM cysteine hydrochloride monohydrate). Renaturation was

151

performed for 48 h at 4 °C without stirring. For purification, renaturation solution was adjusted to pH 7.4 using

152

concentrated H3PO4 and purified according to the manufacturer’s protocol (IBA GmbH, Goettingen, Germany).

153

Buffer was exchanged for 100 mM Tris-HCl pH 7.0 using PD-10 desalting columns (GE Healthcare). Purified enzyme

154

was stored at -20 °C.

155

2.5.

PROTEIN QUANTIFICATION AND ENZYME ACTIVITY

156

The Bradford based Bio-99 Rad Protein Assay (Bio-Rad Laboratories GmbH, Munich, Germany) with bovine serum

157

albumin as standard was used to determine protein concentrations of purified enzymes. The protein assay was

158

performed according to the manufacturers’ instruction.

159

7


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160

Enzyme activity was measured using a photometric esterase assay based on the soluble substrate p-nitrophenyl

161

acetate. As a first step, p-Nitrophenyl acetate (pNPA) was dissolved in DMSO and diluted in 50 mM TRIS-HCl pH 7.0

162

buffer with a final concentration of 3.636 mM pNPA and 10% DMSO in the final assay mixture. Activity

163

measurements were performed in 980 µl of the buffer solution and started by addition of 20 µl of enzyme solution.

164

In parallel, a blank reaction was prepared using 20 µl buffer instead of enzyme solution. The increased absorbance

165

was continuously measured for 5 minutes at 405 nm on a spectrophotometer (Cary WinUV Agilent) at 25 °C. The

166

measurement was performed in triplicates. Molar attenuation coefficient was measured to be e 9.031 l/mol at

167

405 nm. The hydrolysis of pNPA to p-nitrophenol leads to an absorbance increase at 405 nm indicating an esterase

168

activity. The activity of all tested enzymes was calculated in Units (U). One U is defined as the amount of enzyme

169

that is needed to catalyze the conversion of 1 μmol of substrate per minute under the given conditions.

170 171

The stability of the enzymes PpCutA and PpelaLip were investigated in 100 mM potassium phosphate buffer pH 7.0

172

at 28 °C during a time frame of seven days.

173

2.6.

POLYESTER HYDROLYSIS BY ENZYMES

174

Polyester powders (10 mg/ml) were incubated in 1 ml of 100 mM potassium phosphate buffer pH 7.0 and 1 µM

175

enzyme. Before the addition of enzyme, polyesters were solubilized by incubation in a thermomixer at 60 °C and

176

14000 rpm for 30 min. Samples were incubated at 28 °C on a rotary shaker at 150 rpm for 7 days. Experiments

177

were run in triplicates. In parallel, polyesters and enzyme were incubated in pure buffer as blank reactions. Before

178

HPLC analysis, enzymes were precipitated by addition of ice-cold methanol (1:1 vol/vol). Samples were acidified to

179

pH 4 and centrifuged (Hermle Z300K, MIDSCI, Missouri) for 15 minutes at 0 °C and 14000 rpm. Supernatant was

180

transferred to HPLC vials for further analysis.

181

2.7.

POLYESTER HYDROLYSIS BY MICROBIAL EXTRACELLULAR ENZYMES

182

Liquid cultures were grown in 100 ml Erlenmeyer flasks containing 25 ml of Media 1 (5.0 g/l peptone, 3.0 g/l meat

183

extract) for P. pseudoalcaligenes or Media 514 (Difco 2216, 5.00 g/l Bacto peptone, 1.00 g/l Bacto yeast extract,

8



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184

0.1 g/l Fe(III) citrate, 19.45 g/l NaCl, 5.90 g/l MgCl2 (anhydrous), 3.24 g/l Na2SO4, 1.8 g/l CaCl2, 0.55 g/l KCl, 0.16 g/l

185

NaHCO3, 0.08 g/l KBr, 34.00 mg/l SrCl2, 22.00 H3BO3, 4.00 mg/l Na-silicate, 2.40 mg/l NaF, 1.60 mg/l (NH4)NO3,

186

8.00 mg/l Na2HPO4) for P. pelagia according to the recommendations of DSMZ. Polyester powder was added to the

187

media to a final concentration of 1 mg/ml before autoclaving. Media were inoculated (1% vol/vol) using a 250 ml

188

pre-culture grown overnight in the same media. All cultures were incubated at 28 °C on a rotary shaker at 150 rpm

189

for 7 days. Experiments were run in duplicates. In parallel, cultures with 10 mg/ml of TA and 10 mg/ml NaSIP were

190

performed.

191

Cultures were centrifuged at 4000 rpm for 15 minutes (Centrifuge 5810, Eppendorf) and supernatants were used

192

for further HPLC analysis. Before HPLC analysis, interfering substrates were precipitated with a modified version of

193

Carrez precipitation method.

194

were added to the supernatant. The mixture was shaken and incubated for 1 min at 25 °C. Subsequently, 20 µl

195

Carrez reagent II was added to the mixture which was shaken and incubated for 5 min at 25 °C. The mixtures were

196

centrifuged for 30 min at 25 °C with 14000 rpm (Hermle Z300K, MIDSCI, Missouri, US). Finally, supernatants were

197

filtered (0.45 µm nylon filters) and analyzed via HPLC-UV.

198

2.8.

21

Samples were acidified to a pH 4 with HCl. Thereafter, 20 µl of Carrez reagent I

DETERMINATION OF HYDROLYSIS PRODUCTS

199

The prepared hydrolysis samples from enzyme hydrolysis and microbial degradation test were analyzed on a HPLC-

200

UV system consisting of a Dionex UltiMate 181 3000 Pump (Dionex Cooperation, Sunnyvale, USA), a Dionex ASI-100

201

automated sample injector, a Dionex UltiMate 3000 column compartment and a Dionex UVD 340 U photodiode

202

array detector. Hydrolysis products were separated by a reversed phase column, XTerra® RP18, 3.5 μm, 3.0x150

203

mm column (Waters Corporation, Milford, USA) at 40 °C and 0.4 ml/min flow rate using a nonlinear gradient where

204

eluent A consisted of water, eluent B of methanol and eluent C of 0.01 N H2SO4. Separation was achieved by

205

increasing eluent B from 15 to 40% from 13 to 30 min, followed by an increase to 90% during 5 min which was kept

206

for 10 min before re-established to initial conditions within 5 min and equilibrated for 20 min. Eluent C was kept at

207

10% during the whole run. The expected release products TA and NaSIP were detected via UV spectroscopy and

208

were qualified and quantified by external calibration curves. 9


Page 11 of 28

209

210


2.9.

AEROBIC BIODEGRADATION TEST IN FRESHWATER WITH WWTP

SLUDGE AS INOCULUM

211

The CO2 Evolution Test (modified Sturm Test) was used to evaluate aerobic biodegradation of the ionic phthalic

212

acid based polyesters according to the guidelines OECD 301B. The test determines the ultimate biodegradability of

213

organic compounds by aerobic microorganisms in water, using a static aqueous test system as described by

214

Strotmann et al. (2004). Biodegradation tests were carried out in mineral media inoculated with municipal

215

activated sludge from the WWTP in Tulln, Austria, which treats 100% municipal wastewater with an average daily

216

flow around 3200 m /day (personal communication). Activated sludge was collected from the aeration tank. The

217

sludge was centrifuged (2500 rpm, 10 min) and pellet dissolved in defined inorganic mineral medium (OECD 301B)

218

to a concentration of 5 g/l. Suspension and test vessels with inorganic mineral medium were aerated for 7 days at

219

22 °C to reduce the extent of background CO2 production before being applied to the test system. The sludge

220

suspension was added to the test vessels to a final concentration of 30 mg/l 24 h before starting the test.

221

Polyesters were solubilized in milliQ water at 60 °C for 30 min before they were added to the test vessels to a final

222

concentration of 0.1 g/l. Test vessels were aerated with CO2-free air at 22 °C for at least 28 days. Biologically

223

produced CO2 was captured in 200 ml 5 M sodium hydroxide solution and quantified by TOC IR in triplicates. Each

224

test series was done in three parallel test vessels. In addition to the polyesters, positive controls with aniline as

225

reference substance was performed as well as blank values from the sludge were recorded.

226

3. RESULTS & DISCUSSION

227

The aim of this study was to mechanistically investigate enzymatic and microbial hydrolysis of ionic polyesters by

228

wastewater microorganisms and their enzymes. Therefore, the main focus was to elucidate influence of structural

229

differences on enzymatic and microbial hydrolysis. Moreover, aerobic biodegradation processes of the polyesters

230

based on WWTP sludge were investigated. To systematically investigated parameters expected to influence

231

hydrolysis rate were a set of structurally different ionic phthalic acid based polyesters synthesized. (Figure 1) The

3

10



Page 12 of 28

232

investigated parameters were (1) water solubility, (2) chain length of diol/glycol, (3) structure of diol (cyclic and

233

branched), and (4) hydrophilicity/hydrophobicity.

234

3.1.

SYNTHESIS AND CHARACTERIZATION OF POLYESTERS

235

Three polyesters were synthesized with different ratios of NaSIP (introduction charge) and TA, 10:90, 20:80 and

236

30:70, in order investigate the effect of water solubility. All other polyesters contained NaSIP:TA with a ratio of

237

30:70 while the chain length of the diol was altered. Moreover, polyesters with a cyclic and/or a branched diol were

238

synthesized to study possible steric effects. Finally, four polyesters were made with glycols of different chain

239

lengths to see whether the hydrophilicity/hydrophobicity of the diol would have an impact on the degradation.

240

(Figure 1, Table S1) The polyesters were successfully synthesized and their structure confirmed with H-NMR

241

measurements. (Figure S1) Polyester compositions, calculated by equation (1) and (2), are nearly equal to feed

242

compositions. (Table S1) A perfectly random sequence distribution of the polyesters is expected due to the two-

243

step polyester synthesis performed. The polyesters were also synthesized with an excess of diols or glycols to

244

ensure hydroxyl end groups enhancing hydrolytic stability.

245

determined by gel permeation chromatography (GPC) of the polyesters were in the range from 1,770 to

246

10,000 g/mol with polydispersity from 1.7 to 3.0. (Figure 1) All polyesters were found to be semi crystalline, with a

247

crystallinity below 1% except for polyesters containing 1,8-octanediol (C8) and 1,12-dodecanediol (C12) that

248

displayed a crystallinity of 4% or 12% calculated by equation (3). The glass transition temperature of the polyesters

249

was determined by DSC to range from 92 to -3 °C, continuously decreasing with increasing diol and glycol length.

250

(Figure 1) The carbon content for the polyesters C3(1,2) and C12 were determined to be 51.7 ± 0.90% carbon

251

content and 64.4± 0.61% carbon content (n=3), respectively. In addition, the solubility of the polyesters was

252

investigated, and the highest solubility was found for polyesters containing short diols and glycols. (Table S2)

1

22

The number average molecular weights (Mn)

253

11


Page 13 of 28


254 255

Figure 1. Properties of synthesized structurally varying polyester as models for polyesters used in household

256

products. Molecular weight distribution Mn (number average molecular weight) of polyesters obtained by GPC

257

(bars) and polydispersity (numbers). Dependence of glass transition temperatures ( Tg2, cooling) on the polyester

258

composition obtained by DSC (♦). All polyesters were found to be semi crystalline, with a crystallinity below 1%

259

except for polyesters containing 1,8-octanediol (C8) and 1,12-dodecanediol (C12) that displayed a crystallinity of

260

4% or 12%. Three groups of polyesters were compared. Left (purple): polyesters containing varying ratios of

261

terephthalic acid (TA), and 5-sulfoisophthalic acid (NaSIP) as given in brackets. Middle (blue): polyesters containing

262

TA and NaSIP in a ratio of 70:30 and diols with different chain length: C3(1,2): 1,2-Propanediol, C2: 1,2-Ethanediol,

263

C3: 1,3-Propanediol, C5: 1,5-Pentanediol, C6: 1,6-Hexanediol, C8: 1,8-Octanediol, C12: 1,12-Dodecanediol, Cyclic:

264

Cyclohexanedimethanol. Right (green): polyesters containing TA and NaSIP in a ratio of 70:30 and glycols with

265

different chain length: EG1: Ethylene glycol, EG2: Diethylene glycol, EG3: Triethylene glycol, EG4: Tetraethylene

266

glycol. C2(30:70), C2 and EG1 is identical.

267

3.2.

POLYESTER HYDROLYZING ENZYMES

12



Page 14 of 28

23

268

Sequences of polyester-degrading enzymes from microbial producers typically found in compost

were used to

269

identify potential polyester-degrading enzymes from typical wastewater microorganisms based on sequence

270

similarities. For the in-silico search, suitable enzyme sources in form of wastewater microorganisms and suitable

271

enzyme classes had to be identified. Pseudomonas sp. are frequently found in wastewater and sewage sludge

272

and have previously been isolated from activated sludge based on their degradation capacity of phthalic acid esters

273

8

274

polyester materials like PBST

275

cellulosilytica (Thc_Cut1) was selected for prescreening of the ionic phthalic acid polyesters since Thc_Cut1 was

276

previously reported to hydrolyze a variety of polyester materials like poly(ethylene furanoate) (PEF)

277

polyethylene terephthalate (PET)

278

experiments indicated that Thc_Cut1 was able to degrade ionic polyesters (Table S3) and therefore the sequence of

279

Thc_Cut1 was used to identify similar enzymes in wastewater microorganisms by in-silico search. The screening of

280

protein databases for extracellular hydrolases from Pseudomonas sp. resulted in identification of two hydrolytic

281

enzymes, namely a lipase from P. pelagia (PpelaLip)

282

revealing 12% and 65% similarity respectively to Thc_Cut1.

283

Genes coding for the identified enzymes were codon optimized and successfully expressed in E.coli without their

284

natural signal peptide. PpCutA was expressed in soluble form and purified from cleared lysate. However, PpelaLip

285

was produced as insoluble inclusion bodies as expected for lipases from Pseudomonas and had to be refolded to

286

obtain active enzyme. (Figure 2)

4, 7

. In addition, extracellular hydrolases from Pseudomonas sp. have previously been shown to hydrolyze aromatic 13

and PBSA

14

. Cutinase 1 from the typical compost microorganism Thermobifida

25

, and poly(butylene adipate-co-terephthalate) (PBAT)

27

24

,

26

. Indeed, preliminary

and a cutinase from P. pseudoalcaligenes (PpCutA)

28

13


Page 15 of 28


287 288

Figure 2. SDS-PAGE analysis (NuPage® 4-12% Bis-Tris) of the refolded and purified lipase PpelaLip from P. Pelagia.

289

Lane 1: Fermentas® Prestained Protein Ladder, Lane 2 and 3: PpelaLip after purification by 6xHis Tag.

290

On the short chain ester 4-nitrophenyl butyrate PpCutA and PpelaLip had activities of 1.5 ± 0.2 U/mg and

291

0.85 ± 0.04 U/mg, respectively. At 28 °C, both enzymes retained about 40% of the hydrolytic activity after seven

292

days of storage. (Figure S2)

293

As a next step, activities of PpCutA and PpelaLip towards the synthesized ionic phthalic acid based polyester were

294

investigated. The optimal growth conditions for these microorganisms are about 28 °C and pH 7, which are

295

therefore expected to be suitable conditions for the enzymes. PpCutA and PpelaLip showed hydrolytic activity

296

against all polyesters. (Figure S3) In general, the hydrolytic pattern was similar for PpCutA and PpelaLip with the

297

highest activity towards polyesters containing glycols. PpCutA showed an overall higher hydrolytic activity

298

compared to PpelaLip. TA was the predominantly released monomer for both enzymes and all polyesters.

299

Since the enzymes were proven to successfully hydrolyze all synthesized polyesters, a more detailed analysis was

300

performed to identify parameters affecting hydrolysis. The following parameters were investigated: (1) water

301

solubility, (2) diol chain length, (3) diol structure (cyclic and branched), and (4) hydrophilicity/hydrophobicity.

302

First the effect of water solubility of polyesters on hydrolysis was investigated. Three polyesters with different

303

ratios of NaSIP and TA, C2(10:90), C2(20:80) and C2(30:70), were synthesized in order to achieve increasing water 14



Page 16 of 28

304

solubility. PpCutA and PpelaLip showed increasing amount of released TA and NaSIP with increasing water

305

solubility, even if PpCutA displayed an overall higher activity (Figure 3, Figure S4).

306

Theoretically, it could be assumed that the monomer content would reflect the amount of release products.

307

However, the released amount of TA increased with increasing NaSIP content which most likely was the result of

308

increasing water solubility. A two to threefold increased hydrolysis was achieved by simply increasing the water

309

solubility of the polyester. This indicates that water solubility has a great impact on enzymatic polyesters

310

hydrolysis. A similar tendency has previously been shown for phthalic acid esters.

311

affect enzymatic hydrolysis as Mn and Tg are not expected to play an essential role in the hydrolysis results since the

312

Mn of the polyesters are comparable and they all have similar Tg above incubation temperature. (Figure 1)

29

Other parameters known to

313 314

Figure 3. Released terephthalic acid from ionic phthalic acid based polyesters, with altered water solubility due to

315

different ratios of 5-sulfoisophthalic acid (NaSIP) and terephthalic acid (TA), after incubation with 1 μM of the

316

enzymes PpelaLip (black) and PpCutA (white) for 7 days at 28 °C. The dashed line shows mol% of NaSIP in the

317

polyester. C2(NaSIP:TA) 1,2-Ethanediol and the mol% ratio of NaSIP to TA. Each bar represents the average of three

318

independent samples; error bars indicate the standard deviation.

319

320

Furthermore, the impact of diol length on hydrolysis was investigated. For this purpose, polyesters with diol moiety

321

ranging from three to twelve carbon atoms (C3, C5, C6, C8 and C12) with similar molecular weights (4,80015


Page 17 of 28


322

5,850 g/mol, and water solubility were compared. (Figure 1, Table S2) PpelaLip and PpCutA show similar hydrolysis

323

patterns for these polyesters. (Figure 4) However, PpCutA displayed an overall higher activity and a higher

324

specificity compared to PpelaLip. Both enzymes displayed the highest activity for C5 with a decreasing activity with

325

increasing diol chain length. This fits well to previously published data where phthalate acid esters with shorter

326

hydrocarbon chains were more readily biodegradable compared to those with longer hydrocarbon chains.

327

explanation for the low degradation of C3 could be the high Tg (72 °C) compared to the other polyesters (34- -3 °C)

328

and incubation temperature (28 °C). The high Tg would also result in a less flexible polymer chain which in general

329

hampers enzymatic hydrolysis. It can also not be excluded that the reduced hydrolysis of C8 and C12 is connected

330

to the increased crystallinity, 4% or 12%, respectively, compared to the other polyesters with crystallinity below 1%

331

since increased crystallinity is likely to decrease hydrolysis.

29, 30

An

31, 32

332 333

Figure 4. Released terephthalic acid (TA) from polyesters with different diol chain length containing 5-

334

sulfoisophthalic acid (NaSIP) and TA in a ratio of 30:70. The polyesters were incubated with 1 μM of the enzymes

335

PpelaLip (black) and PpCutA (white) for 7 days at 28 °C. C3: 1,3-Propanediol, C5: 1,5-Pentanediol, C6: 1,6-

336

Hexanediol, C8: 1,8-Octanediol, C12: 1,12-Dodecanediol. Each bar represents the average of three independent

337

samples; error bars indicate the standard deviation.

338

16



Page 18 of 28

339

As a next step, the hydrolysis of cyclic diol based polyesters was investigated. PpCutA and PpelaLip were both able

340

to hydrolyze the cyclic polyester with a higher release of TA detected for PpCutA than for PpelaLip

341

(0.50 ± 0.04 mmol TA/mol polyester and 0.33 ± 0.02 mmol TA/mol polyester, respectively, n=3). However, the

342

hydrolysis for the cyclic diol was lower compared to all other polyesters except of C12. This can be attributed to the

343

polyester being less flexible, caused by the rigid diol, and having low water solubility. (Table S2) Decreased

344

biodegradation rate have also previously been demonstrated for polyesters with increasing cyclohexanedimethanol

345

content.

346

In order to investigate the impact of branched diol on hydrolysis, the polyesters containing 1,2-propanediol

347

(C3(1,2)) and 1,2-ethanediol (C2) were compared. PpCutA and PpelaLip were both able to hydrolyze the branched

348

diol based polyester with a higher release of TA detected for PpCutA than for PpelaLip (4.10 ± 0.02 mmol TA/mol

349

polyester and 2.12 ± 0.04 mmol TA/mol polyester, respectively, n=3). However, both PpCutA and PpelaLip

350

displayed a higher activity towards the polyester containing the linear diol, a six or fivefold increased amount of

351

released TA was observed. This was even though the molecular weight of the branched diol (1770 g/mol) was lower

352

when compared to the linear diol (6080 g/mol). Ejlertsson et al. (1997) investigated the influence of side chain

353

structure on degradation of phthalic acid esters and found that butyl 2-ethylhexyl phthalate (BEHP) with one

354

branched side chain was readily degradable while bis(2-ethylhexyl) phthalate (DEHP) with two branched side chains

355

was not degraded under methanogenic conditions.

356

Finally, the impact of the hydrophilicity on enzymatic hydrolysis was investigated. Four polyesters were synthesized

357

with glycols having different degree of ethoxy-units (n=1-4), EG1, EG2, EG3 and EG4. PpCutA and PpelaLip showed a

358

15-20 fold increased hydrolysis towards glycol based polyesters compared to diol based polyesters with

359

comparable chain length. This result indicates a higher hydrolysis rate for hydrophilic polyesters compared to

360

hydrophobic polyesters for both enzymes. It has previously been proven that introducing ethoxy units into

361

polyester chains radically increased biodegradation rate. Introduction of ethoxy-units has also been suggested as a

362

parameter for tuning biodegradation rate of polyesters.

363

influenced by the increased water solubility, since the glycol-containing polyesters displayed a significantly higher

364

water solubility compared to the diol-based polyesters. (Table S2) Nevertheless, the hydrolysis pattern was

33, 34

29

35

However, it cannot be excluded that the results might be

17


Page 19 of 28


365

different for PpCutA and PpelaLip. (Figure 5) PpCutA again displayed an overall higher activity and also a higher

366

specificity for EG2 and EG3 while PpelaLip instead had a constant increase of released TA with increasing glycol

367

chain length. The relatively high amount of release TA for EG2 for both enzymes can be explained by the calculated

368

polyester composition ( Table S1), which reveals a higher degree of TA in EG2 compared to feed rate while the

369

other polyesters displays a better correlation between feed rate and polyester composition.

370 371

Figure 5. Terephthalic acid (TA) released from polyesters containing glycols with 1 μM PpelaLip (black) and PpCutA

372

(white) after incubation for 7 days at 28 °C. EG1: Ethylene glycol, EG2: Diethylene glycol, EG3: Triethylene glycol,

373

EG4: Tetraethylene glycol. Each bar represents the average of three independent samples; error bars indicate the

374

standard deviation.

375

3.3.

POLYESTER HYDROLYSIS WITH MICROORGANISMS

376

The identified wastewater microorganisms P. pelagia and P. pseudoalcaligenes were incubated with the

377

synthesized ionic polyesters and the release of hydrolysis products was monitored. Indeed, both P. pelagia and

378

P. pseudoalcaligenes showed hydrolytic activities towards all the investigated polyesters indicated by the release of

379

terephthalic acid. For both P. pelagia and P. pseudoalcaligenes the highest hydrolytic activities were detected

380

towards glycol-containing polyesters. (Figure S5) Terephthalic acid was the major hydrolysis product for both

381

microorganisms in all cases. NaSIP could not be detected for P. pseudoalcaligenes due to matrix interference.

18



Page 20 of 28

382

The microorganisms also showed increasing hydrolytic activities with increasing water solubility just like the

383

enzymes PpCutA and PpelaLip. (Figure 6, top) However, the enzymes PpCutA and PpelaLip were less affected by the

384

decreased water solubility compared to the microorganisms, emphasizing the importance of polyester water

385

solubility for enhanced biodegradation. When comparing the impact of diol length on hydrolysis, both

386

microorganisms and enzymes showed decreasing activity with increasing diol chain length, which correlates well

387

with earlier published data. (Figure 6, middle)

388

towards the polyesters containing 1,5-pentanediol while P. pseudoalcaligenes had the highest activity on polyesters

389

containing 1,3-propanediol.

390

P. pelagia and P. pseudoalcaligenes hydrolytic activities were also higher and less affected by exchanging the linear

391

diol for a cyclic or branched diol compared to the hydrolytic activities of the enzymes PpCutA and PpelaLip.

392

P. pelagia and P. pseudoalcaligenes released comparable amounts of TA for the polyester with cyclic diol

393

(48.4 ± 3.5 mmol TA/mol polyester and 64.5 ± 14.3 mmol TA/mol polyester, respectively n=2), even if, in this case,

394

only the polyester with 1,12-dodecanediol showed a lower hydrolysis as compared to hydrolysis with enzymes. The

395

microorganisms also showed similar hydrolytic activities towards polyester-containing branched diol, C3(1,2)

396

(808.4 ± 64.2 mmol TA/mol polyester and 767.2 ± 64.4 mmol TA/mol polyester, n=2). The activities for the

397

microorganisms were only reduced twofold while the reduction was five and twentyfold for PpelaLip and PpCutA,

398

respectively, by exchanging the linear diol to the branched diol.

399

An interesting observation is that the hydrolytic activity for P. pelagia was not affected by increasing the glycol

400

chain length while P. pseudoalcaligenes in contrast showed a steady increased hydrolytic activity with increasing

401

glycol chain length. (Figure 6, bottom)

402

Overall, the same trend regarding hydrolytic activity of the strucurally different polyesters could be seen for the

403

two microorganisms, P. pelagia and P. pseudoalcaligenes, as for their enzymes, PpelaLip and PpCutA. Again, a great

404

impact of water solubility, chain length and hydrophilicity on the hydrolytic activities was seen.

29, 30

P. pelagia, PpelaLip and PpCutA showed the highest activity

405

19


Page 21 of 28


406 20



Page 22 of 28

407

Figure 6. Comparison of hydrolysis of structurally different polyesters by P. pelagia and P. pseudoalcaligenes and

408

enzymes originating from these organisms PpelaLip and PpCutA, respectively. Released terephthalic acid (TA) from

409

hydrolysis of polyesters with 1 μM PpelaLip (black box), PpCutA (white box), P. pelagia (dark grey stack) and

410

P. pseudoalcaligenes (light grey stack) after 7 days of incubation at 28 °C. For the comparison of degradation

411

capacity of the microorganisms and the associated enzymes the release amount of TA for the ethylene glycol

412

containing polyester, C2(30:70), C2 and EG1, were set to 100% and the other polyesters were calculated

413

accordingly. TA: terephthalic acid, NaSIP: 5-sulfoisophthalic acid, C2(NaSIP:TA) Ethanediol and mol% ratio of NaSIP

414

and terephthalic acid, C3: 1,3-Propanediol, C5: 1,5-Pentanediol, C6: 1,6-Hexanediol, C8: 1,8-Octanediol, C12: 1,12-

415

Dodecanediol, EG1: Ethylene glycol, EG2: Diethylene glycol, EG3: Triethylene glycol, EG4: Tetraethylene glycol. Each

416

bar represents the average of three independent samples; error bars indicate the standard deviation.

417

The aerobic biodegradation of the polyesters C3(1,2) and C12 was also tested in freshwater with WWTP sludge as

418

inoculum according to OECD 301b, to evaluate the biodegradation and mineralization of the ionic phthalic acid

419

based polyesters. The biodegradation test is carried out by a known volume of mineral media inoculated with

420

WWTP sludge and a known concentration of the test substance. The mixture is held in the dark at 28 °C and it is

421

aerated with carbon dioxide-free air at a controlled rate. Degradation is followed over 28 days by determining the

422

carbon dioxide produced. The carbon dioxide is trapped in sodium hydroxide and quantified by TOC IR. The amount

423

of carbon dioxide produced from the test substance (corrected for that derived from the blank inoculum) is

424

expressed as a percentage of ThCO2. For a chemical to be classified as readily biodegradable it has to produce at

425

least 60% of (the theoretical CO2) ThCO2 within 28 days. Moreover, the pass values have to be reached in a 10-d

426

window starting counting from the day when 10% ThCO2 has been produced according to the guidelines OECD

427

301B. C3(1,2) had reached 10% of ThCO2 production after approximately 3 days and 50% of ThCO2 production

428

10 days later (Figure 7) which was less than the 60% required to be classified as readily biodegradable. However,

429

C3(1,2) still showed a relatively good biodegradation without any lag phase and a degradation up to 30% after only

430

10 days and 90% after 28 days. C12 however, showed a slightly slower biodegradation rate but was still degraded

431

up to 56% after 28 days and no lag phase was detected in this case either. This can be due to the microorganisms in

432

the WWTP sludge were probably adapted to similar polyesters. Polymer C12 reached 10% of ThCO2 production

433

after approximately 4 days and 30% is reached 10 days later. Based on these results C12 cannot be classified as 21


Page 23 of 28


434

readily biodegradable. The test was terminated after 28 days even if the polymers had not reached a plateau at this

435

time since 28 days is the normal test frame and the requirements for readily biodegradable chemicals following the

436

guidelines OECD 301B. However, the overall trend corresponds well to the results from the enzymatic hydrolysis

437

with PpCutA and PpelaLip, where C2(1,2) showed a higher hydrolysis rate compared to C12. The results are also in

438

agreement with previously reported biodegradation test of polyethylene glycols with different molecular weights in

439

freshwater media under aerobic conditions using microorganisms obtained from WWTP sludge.

16

440 441

Figure 7. Aerobic biodegradation of ionic phthalic acid based polyesters in freshwater using WWTP sludge as

442

inoculum expressed as CO2 evolution in percentage. The polyesters consisted of terephthalic acid (TA), 5-

443

sulfoisophthalic acid (NaSIP) and 1,2-propanediol (C2(1,2)) (white box) or 1,12-dodecandiol (C12) (black box) and

444

were used with a final concentration of 0.1 g/l. Aniline was used as reference with the same concentration.

445

Enzymatic hydrolysis of the same polyesters expressed as released amount of terephthalic acid (TA) after

446

incubation for 7 days at 28 °C with 1 µM enzyme.

447

In conclusion, we successfully identified, based on in-silico search, a cutinase from Pseudomonas pseudoalcaligenes

448

(PpCutA) and a putative lipase from Pseudomonas pelagia (PpelaLip) as effective polyester degraders. PpCutA and

449

PpelaLip were proven to hydrolyze structurally different ionic phthalic acid based polyesters. Similarly,

450

P. pseudoalcaligenes and P. pelagia were proved to hydrolyze these polyesters. The systematic study on enzymatic

451

and microbial hydrolysis of structurally different polyesters identified increasing water solubility and increasing

452

hydrophilicity to significantly enhance enzymatic hydrolysis rates. We also showed that increased diol chain lengths 22



Page 24 of 28

453

had a negative effect on hydrolysis rates as well as cyclic and branched diols when compared to linear diols.

454

Moreover it was demonstrated that ionic phthalic acid based polyesters were biodegraded and mineralized in

455

simulated freshwater with WWTP sludge as inoculum. This information is important both related to the fate of

456

ionic phthalic acid based polyesters in aquatic environments as well as to gain a better mechanistic understanding

457

of biodegradation processes of ionic phthalic acid based polyesters in WWTP. The improved knowledge along with

458

the identified polyester hydrolyzing enzymes and microorganisms are essential for developing enhanced microbial

459

and enzymatically based processes for WWTP.

460

ACKNOWLEDGEMENTS

461

We thank Marion Sumetzberger-Hasinger for help with TOC measurements. This work was supported by the

462

Federal Ministry of Science, Research and Economy (BMWFW), the Federal Ministry of Traffic, Innovation and

463

Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol, the Government of

464

Lower Austria and Business Agency Vienna through the COMET-Funding Program managed by the Austrian

465

Research Promotion Agency FFG.

466

SUPPORTING INFORMATION

467

1

468

cutinase 1 from T. cellulosilytica (Thc_Cut1), SDS-PAGE analysis (NuPage® 4-12% Bis-Tris) of the refolded and

469

purified lipase PpelaLip from P. Pelagia, stability of lipase PpelaLip from P. pelagia and cutinase PpCutA from

470

P. pseudoalcaligenes, polyester degradation with PpelaLip and PpCutA and polyester degradation with

471

P. pseudoalcaligenes and P. pelagia.

H NMR spectrum, polyester composition, solubility of the synthesized polyesters, polyester degradation with

472

473

23


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474

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Hydrolysis of Ionic Phthalic Acid Based Polyesters by Wastewater

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