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Synthesis of short-chain-length and medium-chain-length polyhydroxyalkanoate blends from activated sludge by manipulating octanoic acid and nonanoic acid as carbon sources Zheng Chen, Chuanpan Zhang, Liang Shen, Heng Li, Yajuan Peng, Haitao Wang, Ning He, Qingbiao Li, and Yuanpeng Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04001 • Publication Date (Web): 28 Sep 2018 Downloaded from http://pubs.acs.org on September 29, 2018

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Journal of Agricultural and Food Chemistry

1

Synthesis

of

short-chain-length

and

medium-chain-length

2

polyhydroxyalkanoate blends from activated sludge by manipulating

3

octanoic acid and nonanoic acid as carbon sources

4

Zheng Chen1,2,3,4, Chuanpan Zhang1, Liang Shen1, Heng Li2, Yajuan Peng1, Haitao Wang1, Ning

5

He1, Qingbiao Li1,5, Yuanpeng Wang1,∗

6

1

7

Engineering, Xiamen University, Xiamen 361001, People’s Republic of China

8

2

9

Kah Kee College, Xiamen University, Zhangzhou 363105, People’s Republic of China

Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical

Department of Environmental Science, School of Environmental Science and Engineering, Tan

10

3

11

University, Wenzhou 325035, People’s Republic of China

12

4

13

of Fujian Normal University, Fuqing 350300, People’s Republic of China

14

5

15

Republic of China

Zhejiang Provincial Key Laboratory of Watershed Science and Health, Wenzhou Medical

Key Laboratory of Measurement and Control System for Coastal Environment, Fuqing Branch

College of Food and Biological Engineering, Jimei University, Xiamen 361021, People’s

16

∗

Corresponding author: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, No. 422, Southern Siming Road, Xiamen 361005, China. E-mail address: [email protected] (Y. Wang) 1 ACS Paragon Plus Environment


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Abstract

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The effects of octanoic acid/nonanoic acid and acclimation time on the synthesis of short-

19

chain-length and medium-chain-length PHA blends from activated sludge were investigated.

20

An increased concentration (847~1366 mg/L) of PHAs was resulted from 4-month acclimation

21

compared with the concentration derived from 2-month acclimation (450~1126 mg/L). The

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content of octanoic acid had a positive linear relationship with the content of even-numbered

23

carbon monomers among the PHAs. The blending products were identified mainly with scl-

24

PHAs during the 2-month acclimation period and were thereafter dominated by mcl-PHAs until

25

4 months of acclimation. Thermal properties analysis demonstrated that the products derived

26

from 4-month acclimation were a mixture of scl-PHAs and mcl-PHAs rather than a copolymer

27

of

28

Pseudofulvimonas, Paracoccus and Blastocatella were the dominant genera that might be

29

responsible for scl-PHAs production during the 2-month acclimation period, whereas

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Comamonas and Pseudomonas that were responsible for mcl-PHAs production then became

31

the dominant genera after 4-months acclimation.

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Key words: activated sludge; polyhydroxyalkanoates; substrate; acclimation time; microbial

33

community.

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Abbreviations: PHAs, polyhydroxyalkanoates; scl-PHAs, short-chain-length PHAs; mcl-

35

PHAs,

36

PHBHHx, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate); PHB, poly(3-hydroxybutyrate);

37

PHV, poly(3-hydroxyvalerate); 3HA, 3-hydroxyalkanoate; 3HB, 3-hydroxybutyrate; 3HV, 3-

scl-PHAs

and

mcl-PHAs.

medium-chain-length

High-throughput

PHAs;

PHBHV,

sequencing

results

indicated

that

poly(hydroxybutyrate-hydroxyvalerate);

2 ACS Paragon Plus Environment

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hydroxyvalerate; 3HHx, 3-hydroxyhexanoate; 3HHp, 3-hydroxyheptanoate; 3HO, 3-

39

hydroxyoctanoate; 3HN, 3-hydroxynonanoate; PCoA, Principal Coordinate Analysis.

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Introduction

42

Polyhydroxyalkanoates (PHAs) are excellent candidate biomaterials to replace

43

conventional thermoplastics (polyethylene and polypropylene) because of their favorable

44

biodegradability and biocompatibility.1-3 Based on the number of carbon atoms in their

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repeating units, PHAs can be classified into medium-chain-length PHAs (mcl-PHAs), with 6

46

to 14 carbon atoms in monomeric constituents (e.g., 3HHx, 3HHp, 3HO and 3HN), and short-

47

chain-length-PHAs (scl-PHAs), with only 3 to 5 carbon atoms in one repeating 3-

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hydroxyalkanoate (3HA) unit (e.g., 3HB and 3HV).4, 5 Although scl-PHAs have preferable hard

49

crystalline mechanical properties, their range industrial applications are usually narrow due to

50

their brittle physical properties.6, 7 Compared to scl-PHAs, mcl-PHAs are better elastic materials

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and biological rubbers because they do not become brittle even far below the freezing point.8

52

However, the industrial application of mcl-PHAs is limited because their melting temperatures

53

are mostly close to room temperature. Therefore, the synthesis of blends might balance the

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negative and positive impacts on the practical application of scl-PHAs and mcl-PHAs.

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Considered an economical production strategy, using activated sludge for PHA production

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has become a more attractive alternative biosynthesis technology than the traditional synthesis

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route using pure microbial cultures because activated sludge can accumulate PHAs under

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unsteady conditions arising from an intermittent feeding regime and variation in the presence

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of electron acceptors.9, 10 In addition, this alternative approach has rapidly expanded due to the

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selection of microbes capable of producing PHA from activated sludge through supplementing

61

with alternate carbon substrates.11,

12

Inspired by this strategy, many recent studies have


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demonstrated that activated sludge can be used to achieve favorable productivity of PHB,

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PHBHV and PHBHHx.13,

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synthesized using feedstocks with different compositions.15 Thus, substrate characteristics and

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acclimated parameters in active sludge can be critical factors affecting microbial metabolism

66

and the compositions of intracellular storage products.

14

Moreover, blends of scl-PHAs and mcl-PHAs have been

67

In recent years, extensive studies on PHA production from activated sludge have been

68

preferentially carried out on the production of scl-PHAs or mcl-PHAs.16, 17 The monomeric

69

compositions and polymeric structures of PHAs can be adjusted by modifying the species and

70

proportions of added carbon sources or other nutrients.18 Relatively high productivity from

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activated sludge has been achieved for the production of scl-PHAs, including PHB, PHV and

72

PHBHV.11, 14 Nevertheless, these studies have mostly focused on the production of scl-PHAs.

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Previously, Bengtsson and Pisco et al.19, 20 demonstrated that using mixed cultures enriched in

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fermented sugar cane molasses, microorganisms produced PHAs containing significant

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amounts of the mcl-monomer 3HHx. Although this investigation probed the feasibility of using

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activated sludge for mcl-PHAs production, more information regarding the fermentation

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strategies of activated sludge regarding the efficient production of mcl-PHAs is still needed.

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Pseudomonas and Comamonas have been identified as mcl-PHA-producing bacterial

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genera.21, 22 Moreover, octanoic acid and nonanoic acid are commonly regarded as bioavailable

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fatty acids supporting not only the growth of mcl-PHA-producing microorganisms through

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assimilation but also stimulating the production of mcl-PHAs through microbial metabolism.23,

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24

Thus, adding external substrates like octanoic acid and nonanoic acid to activated sludge may



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not only increase the growth of mcl-PHA-producing microorganisms but also regulate the

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constituents of PHAs. However, it is still unknown how the monomer units of the produced

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PHA respond to the metabolism and growth of PHA-producing microorganisms in octanoic-

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acid- and/or nonanoic-acid-acclimated sludge. Therefore, using octanoic acid and/or nonanoic

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acid to acclimate activated sludge seems to be a promising strategy for shifting the compositions

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of polymers, which may influence the abundance of specific microbes and thereby regulate the

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final PHA constituents.

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In this work, the biosynthesis of blends of scl-PHAs and mcl-PHAs from activated sludge

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acclimated with various ratios of octanoic acid and nonanoic acid was investigated, focusing

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on the role of octanoic acid/nonanoic acid and acclimation time in the biosynthesis of products.

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Additionally, the relationships among the content of nonanoic and the mole fraction of even-

94

/odd-carbon-numbered PHA, and the shift of microbial communities were evaluated. The

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findings of this study will be valuable for regulating the biosynthesis of blends of scl-PHAs and

96

mcl-PHAs from activated sludge.

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Methods and materials

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Batch experiments

99

In this study, carbon sources and acclimation time were considered to be two important

100

factors influencing the synthesis of PHAs. To enrich PHA-accumulating organisms, activated

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sludge (sampled from the secondary sedimentation tank of a municipal wastewater treatment

102

plant in Xiamen, China) was acclimated in a 5.0-L sequencing batch reactor (SBR). The SBR

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was operated in sequential mode with a 24-h cycle composed of three phases, i.e., a 23-h aerobic


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phase, a 40-min settling phase and a 20-min withdrawing phase. At the beginning of the settling

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phase, 1 L of slurry mixture was collected for batch experiments. The composition of the

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medium was modified according to a recipe for PHA synthesis by both wild and recombinant

107

pure cultures. In our study, the culture medium was composed of (per liter) 0.456 g NH4Cl,

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0.0408 g KH2PO4, 0.108 g K2HPO4·3H2O, 0.6 g MgSO4·7H2O, 0.07 g CaCl2, and 2 mL trace

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element solution. The components of the trace element solution were (per liter) 1.5 g

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FeCl3·6H2O, 0.15 g H3BO4, 0.12 g CoCl2·6H2O, 0.12 g MnCl2·4H2O, 0.12 g ZnSO4·7H2O,

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0.06 g Na2MoO4·2H2O, 0.03 g CuSO4·5H2O, 0.03 g KI, and 0.01 g thiocarbamide (CN2H4S).

112

An air compressor with aeration rate of 2 L/min was applied to guarantee that approximately

113

6.4 mg/L dissolved oxygen (DO) was kept in the aeration phase. The reactors were stirred at a

114

speed of 120 rpm during the entire test period.

115

To study the influence of carbon source substrates, aerobic batch tests were conducted to

116

investigate the impacts of substrates on blend biosynthesis. In each batch test, the acclimated

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sludge that was collected from the withdraw phase was then fed with a defined concentration

118

of mixed-carbon substrate (that contained different test ratios of octanoic acid to nonanoic acid

119

in the chemical oxygen demand (COD) concentration) and transferred into a glass reactor (1 L)

120

for the accumulation of PHAs. The initial supplemented concentrations of COD in the mixed-

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carbon substrate were set as 0.9, 1.8 and 2.7 g/L. The tested ratios (based on the mass) of

122

octanoic acid to nonanoic acid in the COD concentration of each supplemented mixed-carbon

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substrate were 5:0, 4:1, 3:2, 2:3, 1:4 and 0:5. The tests were operated under continuous aerobic

124

conditions with an aeration rate of 2 L/min at 30 °C and pH 7.0. The stirring rate was maintained



125

at 400 ± 5 rpm using a mechanical agitator. Two months of acclimation was conducted to

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determine the biosynthesis of PHAs. During this acclimation period, approximately 1 L slurry

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mixture was discharged to monitor the COD parameters, the content of the mixed liquor

128

suspended solids (MLSS), pH and DO. The sampled sludge was concentrated by precipitating

129

it for 24 h at 4 °C and then pretreated with sodium hydroxide according to the protocol described

130

by Vlyssides et al.25

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In addition, we also conducted comparative experiments to determine the changes in the

132

concentration and composition of PHAs resulting from acclimations for 2 months and 4 months

133

to further explore the influence of acclimation time. In this experiment, the same mixed-carbon

134

substrates were equally supplemented into the reactors. The mixed-carbon substrates were in

135

turn amended with 1.8 g/L of the initial COD that also fed with the same corresponding ratios

136

(octanoic acid to nonanoic acid on a mass basis) of 5:0, 4:1, 3:2, 2:3, 1:4 and 0:5. The other

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operation parameters were the same as those for our investigation of carbon source substrate

138

influence described above.

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Isolation of PHAs

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One liter of slurry was collected from the reactors and centrifuged (MIKRO 220R, Hettich,

141

Germany) at 12000 rpm for 10 min to remove the supernatant. Intracellular PHAs were

142

extracted using a dispersion system of sodium hypochlorite solution (100 mL, 13% v/v) and

143

chloroform (100 mL) mixture in 500-mL three-necked round-bottomed flasks. The mixture was

144

magnetically stirred for 1 h at 60 °C and then cooled to room temperature, and the upper phase

145

was discarded. The residual mixture was then centrifuged at 5000 rpm for 10 min to collect the


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lower organic phase. The isolated lower organic phase was further concentrated to

147

approximately 20 mL using a rotary evaporator and precipitated with cold methanol (5 times

148

the volume of the concentrate). Finally, the precipitated PHAs were separated from the liquid

149

mixture by centrifugation (5000 rpm for 10 min) and then dissolved in chloroform and vacuum

150

dried for 12 h at 60 °C.

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Analytical methods

152

The PHA monomer content in the sludge was determined using a methanolysis procedure

153

described by Albuquerque et al.26 The content of cell dry mass (CDM) was calculated according

154

to the weight of lyophilized cells per volume. PHBV, PHBHHx, 3HHp, 3HO, 3HN (purchased

155

from Chengdu Kamel Pharmaceutical Co., Ltd, China) were selected as the reference materials

156

for the measurement of PHAs. A centrifuged sample from the batch test was lyophilized,

157

digested, methylated, extracted and then analyzed by a gas chromatograph (7890A, Agilent

158

Technologies Co., Ltd., USA) equipped with a flame ionization detector (FID) and a 30 m×0.32

159

mm capillary column (HP-5, Agilent Technologies Co., Ltd., USA). The injection port and

160

detector temperatures were set as 200 and 220 °C, respectively, with N2 at 43 mL/min, H2 at 50

161

mL/min and air at 100 mL/min as carrier gases. The GC oven temperature was programmed as

162

follows: initial temperature of 80 °C for 1.5 min, first ramp (30 °C/min) up to 140 °C, which

163

was held for 2 min, then a second ramp (40 °C/min) up to 220 °C with an additional hold time

164

of 9 min. The sample injection volume was 1.0 µL with a split ratio of 50:1 (vent: column).

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To demonstrate the extracted products were blended mixtures of scl-PHA and mcl-PHA

166

rather than the copolymers of scl-PHA and mcl-PHA, the samples were run in a differential



167

scanning calorimeter (DSC, Netzsch-200F3, Germany). Samples were first heated from -80 °C

168

to 180 °C at a heating rate of 20 K/min (first heating scan). The melt samples were then rapidly

169

quenched to -80 °C at a melting rate of 20 K/min. Then, they were heated again to 180 °C at a

170

heating rate of 20 K/min (second heating scan). The glass transition temperature (Tg) was taken

171

as the midpoint of the heat capacity change. The melting temperature (Tm) and the heat of fusion

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were determined from the DSC endotherm. Meanwhile, thermogravimetric analysis (TGA) for

173

the isolated samples was carried out using a TA Instruments Q500 instrument (TA Instruments,

174

USA). Approximately 3~10 mg sample was loaded in an aluminum pan and heated from room

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temperature to 600 °C at a heating rate of 10 °C/min under N2.

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In addition, the structures of PHA monomers were identified by a 13C-nuclear magnetic

177

resonance (NMR) spectrometer (Bruker AV-400 NMR, Switzerland) at 200 MHz using the

178

chemical shift of tetramethylsilane as an internal standard. Extracted PHA samples (20 mg)

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were dissolved in 0.6 mL CHCl3. Then, the solution was filtered and transferred into NMR tube

180

for testing. The NMR spectra were fitted by Topspin 3.2 (Bruker, Switzerland). To further

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confirm the compositions of the blended products, a gas chromatograph coupled with mass

182

spectrometry (GC-MS, QP2010, Shimadzu, Japan) based on electron impact (EI) was used to

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determine the compositions of intracellular storage products. The gas chromatograph was

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equipped with a capillary column (30 m×0.25 mm). The column temperature was programmed

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to remain at 80 °C for 2 min and then to increase at a rate of 10 °C/min to 220 °C, and continue

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to increase at a rate of 60 °C/min to 290 °C, which was held for 10 min. The injection

187

temperature was set to 280 °C.


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188 189 190


Calculations The mass content of specific 3HA in the sludge is defined in Equation 1. C3HA =

mass of the specific 3HA in the dry sludge CDM of the dry sludge

(1)

191

The volumetric yield of PHAs in the reactor is expressed as Equation 2. The volumetric

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yields of PHAs derived from the substrates that contained 0.9, 1.8 and 2.7 g/L of COD are

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denoted as Y0.9, Y1.8 and Y2.7.

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YPHA (g/g COD)=

amount of produced PHAs amount of consumed organic substrates

(2)

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The mole fraction of specific 3HA among PHAs (denoted as 3HA mol%) is expressed in

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Equation 3, where m and M are the mass (g) and the molar mass (g/mol) of the specific 3HA.

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The molar masses of 3HB, 3HV, 3HHX, 3HHp, 3HO and 3HN were 86, 100, 114, 128, 142 and

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156 g/mol, respectively.

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F3HA (mol%)=

200

mspecific 3HA /Mspecific 3HA

(3)

∑all mspecific 3HA /Mspecific 3HA

DNA extraction, PCR amplification and sequencing

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Microbial DNA was extracted from 0.5 g sludge samples that accumulated the highest

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concentration of PHAs (stored at -20 °C) using a Fast DNA Spin Kit for soil (MP Biomedical)

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according to manufacturer’s protocols. The V3-V4 region of the bacteria 16S ribosomal RNA

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gene was amplified by PCR (95 °C for 3 min, followed by 27 cycles of 95 °C for 30 s, 55 °C

205

for 30 s, and 72 °C for 45 s, with a final extension at 72 °C for 10 min) using primers 338F

206

ACTCCTACGGGAGGCAGCAG and 806R GGACTACHVGGGTWTCTAAT, where the

207

barcode was an eight-base sequence unique to each sample. PCR was performed in triplicate

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20 μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each



209

primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA.

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Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel

211

Extraction Kit (Axygen Biosciences, Union City, CA, U.S.) according to the manufacturer’s

212

instructions and quantified using QuantiFluor™ -ST (Promega, U.S.). Purified amplicons were

213

pooled in equimolar amounts and paired-end sequenced (2 × 250) on an Illumina MiSeq

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platform according to the standard protocols. The raw reads were deposited into the NCBI

215

Sequence Read Archive (SRA) database.

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Raw fastq files were demultiplexed and quality-filtered using QIIME (version 1.17) with

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the following criteria: (i) 300 bp reads were truncated at any site receiving an average quality

218

score

Synthesis of Short-Chain-Length and Medium ... - ACS Publications

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