Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE
Environmental Processes
Biodegradation of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Plastic under Anaerobic Sludge and Aerobic Seawater Conditions: Gas Evolution and Microbial Diversity Shunli Wang, Keri Lydon, Evan M. White, Joe B. Grubbs III, Erin Lipp, Jason Locklin, and Jenna Jambeck Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06688 • Publication Date (Web): 19 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
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.
Page 1 of 35
Environmental Science & Technology
1
Biodegradation of Poly(3-hydroxybutyrate-co-3-
2
hydroxyhexanoate) Plastic under Anaerobic Sludge
3
and Aerobic Seawater Conditions: Gas Evolution
4
and Microbial Diversity
5
Shunli Wang,*,†, § Keri A. Lydon,#,¶ Evan M. White,§ Joe B. Grubbs III, § Erin K. Lipp, # Jason
6
Locklin,†,‡,§ and Jenna R. Jambeck†, ‡, § †
7 8 9 10 11 12
‡
College of Engineering, University of Georgia, Athens, Georgia 30602, United States
Department of Chemistry, University of Georgia, Athens, Georgia 30602, United States §
¶
New Materials Institute, University of Georgia, Athens, Georgia 30602, United States
Present address: School of Freshwater Sciences, University of Wisconsin-Milwaukee,
Milwaukee, Wisconsin 53204, United States #
Department of Environmental Health Science, University of Georgia, Athens, Georgia
13
30602, United States
14
KEYWORDS: Poly(3HB-co-3HHx); PHA; Biodegradability; Anaerobic digestion; Seawater;
15
Ocean
ACS Paragon Plus Environment
1
Environmental Science & Technology
16
Page 2 of 35
ABSTRACT
17
Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (poly(3HB-co-3HHx)) thermoplastics are a
18
promising biodegradable alternative to traditional plastics for many consumer applications.
19
Biodegradation measured by gaseous carbon loss of several types of poly(3HB-co-3HHx) plastic
20
were investigated under anaerobic conditions and aerobic seawater environments. Under anaerobic
21
conditions, the biodegradation of a manufactured sheet of poly(3HB-co-3HHx) and cellulose
22
powder were not significantly different from one another over 85 days with 77.1±6.1% and
23
62.9±19.7% of the carbon converted to gas, respectively. However, the sheet of poly(3HB-co-
24
3HHx) had significantly higher methane yield (p≤0.05), 483.8±35.2 mL·g-1 volatile solid (VS),
25
compared to cellulose controls, 290.1±92.7 mL·g-1 VS, which is attributed to a greater total carbon
26
content. Under aerobic seawater conditions (148 – 195 days at room temperature), poly(3HB-co-
27
3HHx) sheets were statistically similar to cellulose for biodegradation as gaseous carbon loss (up
28
to 83% loss in about 6 months), although the degradation rate was lower than that for cellulose.
29
The microbial diversity was investigated in both experiments to explore the dominant bacteria
30
associated with biodegradation of poly(3HB-co-3HHx) plastic. For poly(3HB-co-3HHx)
31
treatments, Cloacamonales and Thermotogales were enriched under anaerobic sludge conditions,
32
while Clostridiales, Gemmatales, Phycisphaerales and Chlamydiales were the most enriched
33
under aerobic seawater conditions.
34
INTRODUCTION
35
Traditional thermoplastics are primarily derived from fossil fuels like petroleum and natural gas
36
with production increasing rapidly since 1950. An estimated 335 million metric tons of
37
anthropogenic plastics were produced in 2016.1 Cumulatively, 8.3 billion metric tons of plastic has
ACS Paragon Plus Environment
2
Page 3 of 35
Environmental Science & Technology
38
been produced globally since 1950 with 76% of total production (6.3 billion metric tons) ending
39
up as waste.2 Only 9% of global plastics were recycled, 12% incinerated and 79% ended up in
40
landfills or in our environment. Plastic has a high volume-to-mass ratio, and with large quantities
41
landfilled, these materials occupy significant portions of landfill capacities. In addition,
42
mismanaged plastic waste may easily enter aquatic systems, with an estimated 4.8 – 12.7 million
43
metric tons of plastic waste entering the ocean globally.3 Traditional plastic materials are not
44
known to be biodegradable in the environment. However, UV light and weathering processes
45
fragment plastics into microplastics, materials that have become ubiquitous in our terrestrial and
46
aquatic systems,4 and can cause a wide array of harm to wildlife and ecosystems, including
47
potential implications for human health.5
48
Plastics that are useful and biodegradable have been proposed as a part of the solution to global
49
plastic management, with the addition of contributing to a more circular economy. However,
50
biodegradable plastic is not without controversy. Current “compostable” plastics were previously
51
interpreted as biodegradable, but such materials are not biodegradable in normal soil environments
52
or in the ocean.4,6 Compostable plastic, like polylactic acid ( PLA) will be biodegraded in industrial
53
composting systems with temperatures above 50°C, but not at an appreciable rate in typical home
54
compost, where conditions are much more variable. In addition, recycling streams may be
55
contaminated by compostable polymers like PLA. To add confusion to the biodegradable term,
56
some non-biodegradable plastics have been modified with oxo-degradable chemistries so that the
57
plastic fragments faster when exposed to UV light, which only exacerbates the microplastic
58
problem. Therefore, companies and consumers are demanding packaging that doesn’t have the
59
unintended consequences of traditional plastics at end of life. Some large consumer brands have
60
committed to making all of their packaging either recyclable or compostable by 2025, and various
ACS Paragon Plus Environment
3
Environmental Science & Technology
Page 4 of 35
61
non-compostable, non-recyclable single use plastic has been banned in some countries (e.g.
62
France). In response to these issues, polyhydroxyalkanoate (PHAs) products are being developed
63
to replace conventional petrochemical plastics to meet biodegradable packaging requirements.
64
Polyhydroxyalkanoates (PHAs) are polyesters synthesized by bacteria under limited nutrient
65
conditions with an excess carbon source.7 They have similar properties to traditional plastics (i.e.
66
polyethylene and polypropylene) but are also biodegradable under several conditions.8–10 Under
67
anaerobic conditions, PHA is initially hydrolyzed to smaller oligomers and ultimately monomeric
68
units, such as 3-hydroxybutyrate, which then is degraded to acetate and hydrogen, which
69
ultimately produces CH4 and CO2.11 Anaerobic studies determined that 63.4% to 87.0% of carbon
70
in different types of PHA samples including PHB, and PHBV (poly(hydroxybutyrate-co-
71
hydroxyvalerate) with 13 or 20% hydroxyvalerate (HV) content) was converted to biogas under
72
anaerobic digestion at 35°C.11,12 However, CH4 (a potent greenhouse gas) production from the
73
biodegradation of PHA was not thoroughly explored in these studies, leaving some gaps in the
74
evaluation and environmental impact of PHA. Under aerobic conditions in seawater, PHA is
75
colonized and hydrolyzed by extracellular depolymerases that fragment the material, which are
76
then subsequently biodegraded into CO2.13 Biodegradation of different types of PHA, including
77
p(3HB), p(3HB-co-5mol% 3HV), p(3HB-co-5mol% 3HHx), etc., in seawater from various
78
locations has been tested only by measuring the weight loss of samples.13,14 The method of
79
measuring loss of mass identified the degradation of PHA; however, mass can be lost solely from
80
fragmentation, and not biodegradation. Therefore, it is critical to explore the complete
81
biodegradation and mineralization of PHA in seawater.
82
Identification of the microbial diversity under aerobic and anaerobic biodegradation conditions
83
for PHA can provide a better understanding of potential metabolic pathways for biodegradation
ACS Paragon Plus Environment
4
Page 5 of 35
Environmental Science & Technology
84
and facilitate design of waste treatment systems. Previous studies have isolated single species
85
biodegrading PHA under various conditions, such as Desulfotomaculum sp. from the oil-water
86
mixture in a production well,15 Nocardiopsis aegyptia,16 and Marinobacter sp.17 from marine
87
seashore sediment and the deep-sea floor. Volova et al. also detected the PHA degrading bacteria
88
(i.e., genera Pseudomonas, Pseudoalteromonas, among others) on the biofilm formed on PHA
89
after 160 days of exposure to the seawater from Dam Bay (Vietnam).14 Nevertheless, there is
90
limited literature available that explores shifts in whole microbial communities during the
91
biodegradation of PHA. Therefore, further studies on complex microbial communities, which
92
contain species proven to biodegrade PHA in diverse environments are needed.
93
In order to explore alternative materials to traditional plastics in various environments, this paper
94
investigates the biodegradability of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (poly(3HB-
95
co-3HHx)), a type of PHA, in an anaerobic setting representing a proper management scenario
96
(e.g., anaerobic digestion or a landfill), and in an aerobic setting akin to a mismanagement scenario
97
(e.g., ending up in the ocean). Specifically, the objectives of this study were to (1) quantify the
98
gaseous carbon loss and the CH4 production potential from poly(3HB-co-3HHx) under anaerobic
99
conditions, (2) evaluate the mineralization and changing characteristics of poly(3HB-co-3HHx)
100
under aerobic conditions in seawater using a controlled lab condition, and (3) identify and
101
characterize the microbial diversity occurring in the biodegradation of poly(3HB-co-3HHx) under
102
both conditions.
103
MATERIALS AND METHODS
104
Feedstock
105
Two forms of poly(3HB-co-3HHx) samples, sheet (PHA-S) and flake (PHA-F) trademarked as
106
Nodax, were provided by DaniMer Scientific (Bainbridge, Georgia, USA). Canola oil was used
ACS Paragon Plus Environment
5
Environmental Science & Technology
Page 6 of 35
107
as the bacterial feedstock to produce the poly(3HB-co-3HHx) used in this study. PHA-S has 7.1%
108
C6 (3-hydroxyhexanoate) content and 92.9% C4 (3-hydroxybutyrate) content while PHA-F has
109
6.5% C6 (3-hydroxyhexanoate) content and 93.5% C4 (3-hydroxybutyrate) content (Figure S1 and
110
S2). The two poly(3HB-co-3HHx) samples have similar glass transition temperatures (Tg = -4.7
111
°C and -5.7 °C for PHA-S and PHA-F, respectively), similar cold crystallization peaks (Tc = 68.2
112
°C and 72.5 °C) and energies (47.9 and 47.1 J·g-1), and comparable melting point transitions (Tm
113
peaks from 112 – 147 °C) (Figure S3 and S4). The molecular weight of PHA-S and PHA-F are
114
446,203 and 50,672 g·mol-1, respectively, which were designed for high and low molecular weight
115
based on their target applications. The molecular weights were tuned for the appropriate melt
116
viscosities that allow processing into sheets and films (Table S1). The positive control was
117
cellulose powder, from Sigmacell (cellulose type 101), which was used to monitor microbial
118
activities. The negative control was polypropylene pellets (PP) from LyondellBasell (Pro-fax
119
8523). PHA-S was cut into a size of 5 mm x 5 mm x 1 mm, and PHA-F was cut into a size
120
approximating these dimensions due to its fragility. Cellulose was used as received in powder
121
form, and PP pellets (approximately 10 mm diameter) were cut in half so they were approximately
122
5 mm x 5 mm x 1 mm. Samples of PP, PHA-S and PHA-F were prepared with the similar size and
123
shape to minimize the error caused by different sample morphology. The total carbon content of
124
cellulose, PP, PHA-S and PHA-F was 41.8±0.3, 87.9±0.1, 58.4±0.1 and 58.1±0.1%, respectively
125
(Table S1). Other characteristics of polymer samples are also shown in Table S1 and Figure S5.
126
Anaerobic biodegradation of poly(3HB-co-3HHx)
127
Inoculum
128
Approximately 22 L of inoculum were collected from a full scale anaerobic digester in a local
129
wastewater treatment plant operated at 37°C and primarily fed with a co-thickened blend of
ACS Paragon Plus Environment
6
Page 7 of 35
Environmental Science & Technology
130
primary sludge, waste activated sludge (WAS), and some fats, oils, and grease and high strength
131
waste (FOG/HSW). The inoculum was placed in a water-sealed glass flask at 38°C for five days
132
to deplete un-degraded biological residues in the inoculum before use in the experiment.18
133
Digestion assay
134
500 mL batch anaerobic digesters with a working volume of 300 mL were used for this study.
135
Each digester contained 119 mL inoculum, 181 mL deionized (DI) water and 2 grams of sample
136
to adjust the substrate to inoculum ratio to 0.7 – 0.8 (volatile solid base) and then placed in an
137
incubator shaker set at 38°C and 100 RPM. Blank digesters included only inoculum and DI water.
138
Each of the treatments and blanks were performed in triplicate. The digesters were sealed using
139
butyl rubber stoppers and aluminum crimps, and the headspace was purged using N2 gas. The
140
digesters were incubated for 85 days to obtain a complete profile of gas production. The biogas
141
quantity and composition was measured 1 to 2 times a week using a Eudiometer water column
142
(Selutec, Germany) at room temperature (24°C) and a GC-TCD, respectively, depending on the
143
quantity of gas produced. Gas production was calculated at standard temperature and pressure
144
(STP). The pH of the digester was monitored (1 mL removed to test) and then adjusted with NaOH
145
to pH 7.0 – 7.5 if the digester became too acidic from forming fatty acids during degradation. The
146
methods calculating biogas production and carbon loss from the samples are presented in the SI.
147
In addition, carbon loss from the samples in anaerobic biodegradation were modeled using a first
148
order hydrolysis equation, as suggested by previous studies18,19 and cumulative CH4 curves were
149
modeled using a modified Gompertz equation during the incubation period as shown in a previous
150
study20, which are described in the SI.
151
Aerobic biodegradation of poly(3HB-co-3HHx) in seawater
152
Inoculum
ACS Paragon Plus Environment
7
Environmental Science & Technology
Page 8 of 35
153
Seawater was collected from the coast of Georgia, USA on March 30th, 2016 using a 20-L carboy
154
with small vent for air exchange (GPS coordinates: 31°, 57’, 32.8’’ N, 081°, 00’, 24.4’’ W). The
155
seawater temperature at the collection site was 19.4 °C. The seawater was transported and stored
156
at ambient temperature in the laboratory for seven days before beginning the experiments.
157
Digestion assay
158
500 mL batch digesters were used for each assay with a working volume of 200 mL. The
159
digesters were placed on a platform shaker set at 100 RPM, which simulated ocean currents and
160
movement. Seawater (200 mL) and each sample (0.1 g) were added to a digester, prepared in
161
triplicate. The seawater was supplemented with nutrients (0.5 g·L-1 of NH4Cl and 0.1 g·L-1 of
162
KH2PO4).21 Blank digesters were composed of seawater with nutrients only and contained no
163
polymer samples. The digester was sealed using butyl rubber stoppers and aluminum crimps. The
164
O2 level in each digester headspace was kept to a minimum of 12% by purging CO2-free air with
165
a pump pulling air through a scrubbing bottle filled with NaOH solution. The digesters were
166
incubated from 148 to 195 days depending on continued biological activity in the samples. The
167
composition of headspace gas in each digester was measured more frequently (two or three times
168
per week) for the first month and less frequently (once every two weeks) during the last month,
169
depending on the O2 concentration in the digester headspace. CO2 production was calculated at
170
STP conditions. At the end of the experiment, the portion of samples that remained, PP and PHA-
171
S, were prepared for analysis by gently rinsing with 10 mL DI water and then air-drying for 3 days.
172
The pH and concentration of dissolved carbon in each sample assay were measured after the end
173
of the degradation experiment.
174
The CO2 production was calculated by adding CO2 in the digester headspace (multiplying 339
175
mL headspace and CO2 concentration) and CO2 dissolved in the 200 mL seawater. The dissolved
ACS Paragon Plus Environment
8
Page 9 of 35
Environmental Science & Technology
176
CO2 was calculated according to Henry’s law where CO2 concentrations in the headspace and
177
Henry’s law constant for CO2 at 25oC (1630 atm*molwater/molgas) were used. The CO2 left in the
178
headspace and dissolved in the seawater after each CO2-free air purge was subtracted from the
179
total CO2 production. The average CO2 production from the blanks was subtracted from that of
180
each sample to discriminate the biodegradation associated with the samples. Finally, the mass of
181
carbon emitted (grams) as CO2 from the sample was calculated using the ideal gas law at 1 atm
182
and 23°C. The carbon loss from samples in aerobic biodegradation was also modeled using Eq.
183
S2. The analytical and statistical methods used in this study are described in the SI.
184
Microbial diversity analysis
185
Sample collection
186
Upon completion of the anaerobic biodegradation experiment (day 85), 40 mL sludge was
187
collected from each digester and centrifuged for 10 min. From each centrifuged sample, 5 mL of
188
supernatant was filtered through a 0.45 µm pore size membrane (HVLP 04700, Millipore), and the
189
membranes were stored immediately at -80°C for further microbial analysis. The raw seawater (50
190
mL) was collected at the beginning of the experiment (day 0), filtered and stored as describe above.
191
Upon completion of the aerobic seawater biodegradation experiments for cellulose and PHA-F
192
treatments (day 148), 50 mL seawater was collected from each digester, filtered, and stored as
193
described above. This procedure was repeated for the other seawater microcosm treatments (blank,
194
PP, and PHA-S) as those experiments were completed (day 195). PHA-F and cellulose treatments
195
were terminated by day 148 because the added materials were fully degraded.
196
Microbial community analysis
197
DNA was extracted from the filters using the PowerSoil DNA Isolation kit (MO BIO
198
Laboratories, Inc). Extracted DNA was quantified (NanoDrop 1000, Thermo Scientific,
ACS Paragon Plus Environment
9
Environmental Science & Technology
Page 10 of 35
199
Wilmington, DE) and diluted 1:10 before being subjected to PCR amplification of the 16S rRNA
200
gene (primers 515F/806R).22 Two rounds of amplification were used to amplify, then subsequently
201
tag, the V4 16S RNA gene region as previously described.23,24 Custom Illumina adaptors with
202
barcode sequences were added during a second round of amplification (Table S2). Amplicons were
203
purified using equal volume SPRI magnetic beads (Sera-Mag SpeedBeads, Thermo Scientific,
204
Freemont, CA)25 with 96 well magnetic plate (Promega MagnaBot II) and quantified with a Qubit
205
Fluorometer (Thermo Fisher Scientific, Grand Island, NY) before storage at -20°C. Samples were
206
pooled to 10 nM in 10 mM Tris-HCL (pH 8) before being sent to the Georgia Genomics Facility
207
(GGF) (Athens, GA) where they were tested for quality using a Fragment Analyzer (Advanced
208
Analytical Technologies). Pooled samples were sequenced by GGF using v2 chemistry on an
209
Illumina MiSeq PE250.
210
Quantitative Insights into Microbial Ecology (QIIME) version 1.9.126 was used to merge pair-
211
ends (fastq-join) of Illumina MiSeq reads. Chimeric sequences were removed with UCHIME27
212
referenced against the RDP Gold database. The resulting sequences were used to pick operational
213
taxonomic units (OTUs) with the QIIME pipeline28,26,29,30 using open reference and taxonomy
214
assigned with Greengenes database (version 13_8).31,32 OTUs were aligned with PyNast and
215
FastTree was used to generate a phylogenetic tree.33 QIIME was then used to filter unwanted
216
sequences including: unassigned, mitochondria, and singletons. Sequence reads were normalized
217
to the smallest number of reads per sample to generate data sets with equal abundance. Sequences
218
were deposited into NCBI Bioproject (PRJNA419037).
219
QIIME was used to calculate the weighted UniFrac distance matrix34 to determine beta
220
diversity parameters. Differences in microbial community composition between samples were
221
determined in QIIME with permutation-based multivariate analysis (PERMANOVA) of the
ACS Paragon Plus Environment
10
Page 11 of 35
Environmental Science & Technology
222
weighted UniFrac distance matrix using the adonis function in vegan with 999 permutations.35
223
Microbial taxa (order level) were chosen for comparison relative to no treatment control (NTC)
224
if their relative abundance was at least 1% within a single treatment.
225
RESULTS AND DISCUSSION
226
Anaerobic biodegradation
227
The anaerobic biodegradation of samples was evaluated by gaseous carbon loss in the form of
228
biogas emitted from samples in the anaerobic inoculum. After 40 – 60 days of incubation,
229
54.6±15.6, 77.1±6.1 and 62.9±19.7% of total carbon for PHA-F, PHA-S and cellulose were
230
biochemically converted to biogas, respectively, and were not significantly different (p>0.05). As
231
expected, carbon conversion to biogas for PP (0.3±0.1%) was negligible (Table 1). These results
232
coincide with the work of previous studies.11,12 There were low concentrations and not
233
significantly different dissolved organic carbon left in the digesters of blanks for each treatment
234
(Table S3). Due to the dark color of anaerobic sludge, the powder sample residue (PHA-F) shape
235
could not be easily identified. Figure S6 illustrates that the PHA-S samples were gradually
236
biodegraded from the edges to the center. The PP sample, as pellets, floated on the surface of
237
anaerobic sludge during the entire period of incubation. Other than the residues identified, some
238
poly(3HB-co-3HHx) samples could also be in the form of unidentified microplastics in the
239
anaerobic sludge, which should be verified in future studies.
240
Figure 1a shows the evolution of gaseous carbon lost from samples under anaerobic
241
biodegradation conditions. The kinetic data in anaerobic biodegradation provide important
242
information for the design and operation of wastes treatment facilities. A first order decay model
243
was used to fit the experimental data for investigating the biodegradation kinetics (Eq.2).19 The
244
first order biodegradation rate constant (kh) of cellulose and poly(3HB-co-3HHx) samples showed
ACS Paragon Plus Environment
11
Environmental Science & Technology
Page 12 of 35
245
no significant difference (p>0.05), suggesting the microorganisms in each sample had similar rates
246
of biodegradation (Table 1), while the PP biodegradation rate constant was negligible. Few studies
247
have calculated the anaerobic biodegradation kinetics of biodegradable plastics. However,
248
biodegradable polymers such as PCL-starch blend and PBS had aerobic biodegradation rate
249
constants of 0.07 and 0.01 day-1, respectively.19 The kh rate constants determined in this study are
250
comparable at 0.02 day-1, which is interesting given that anaerobic degradation can sometimes be
251
a slower process. Here, the anaerobic biodegradation of poly(3HB-co-3HHx) occurs at a
252
comparable rate to other biodegradable plastics under aerobic conditions. No such results were
253
reported in the previous literature to the best of our knowledge. The relatively fast anaerobic rate
254
could be due to the fast acidogenesis and methanogenesis under anaerobic conditions. During
255
incubation, the pH of the assay was used to evaluate digester health and acid accumulation. The
256
pH of the cellulose sample sharply dropped to 6.0±0.1 corresponding to the higher CO2
257
concentration in the digester headspace (Figure 1c and S2), indicating relatively active hydrolysis
258
at the beginning of incubation. The pH of the PHA-S and PHA-F digesters decreased to 7.5±0.1
259
and 7.2±0.1 at day 9, respectively, showing the hydrolysis of PHA-S was slower than that of PHA-
260
F. At the end of incubation, the pH of the poly(3HB-co-3HHx) samples was consistently 7.0±0.0,
261
corresponding with comparable dissolved organic carbon values for poly(3HB-co-3HHx) samples,
262
which also indicated that no acids accumulated in the digesters.
263
Figure 1b shows the evolution of CH4 produced from the samples in the anaerobic environment.
264
The modified Gompertz model is widely used to fit the cumulative CH4 production data, shown in
265
Eq. 3. PHA-S and PHA-F had CH4 yields of 483.8±35.2 and 337.5±100.3 mL·g-1 VS, respectively,
266
showing no significant difference between them (p>0.05), likely due to their similar total carbon
267
content. Correspondingly, cellulose and PP yielded 290.1±92.7 and 6.1±1.0 mL·g-1 VS,
ACS Paragon Plus Environment
12
Page 13 of 35
Environmental Science & Technology
268
respectively, where the CH4 yield from PP was not significantly different than the blank (p>0.05).
269
Generally, cellulose yielded less CH4 than poly(3HB-co-3HHx), probably because of its lower
270
total carbon content (41.8 ± 0.3%). This result is comparable to that of a previous study, where
271
approximately 533 mL·g-1 VS yielded from PHA (in the film form) at a thermophilic temperature
272
of 50°C.36 In addition, CH4 yields of poly(3HB-co-3HHx) samples were also similar to cheese and
273
food wastes, 454 – 787 and 419 – 535 mL·g-1 VS, respectively. This suggests that poly(3HB-co-
274
3HHx) would work with co-digestion of common organic wastes in an anaerobic digester or a
275
landfill.37 The CH4 concentration in the biogas for cellulose and poly(3HB-co-3HHx) samples
276
stabilized at about 75% and 65% for 65 days (Figure S7), respectively, which is a relatively high
277
concentration of CH4 and may be used in landfill or digestion gas-to-energy recovery systems. The
278
Rm and λ of the Modified Gompertz equation for cellulose and poly(3HB-co-3HHx) samples were
279
not significantly different (p>0.05) (Table 1). Although CH4 yields were similar, the maximum
280
specific CH4 production rate (Rm) of poly(3HB-co-3HHx) materials, calculated as 12.3 to 13.0
281
mL·g-1 VS day-1, were lower than those of food wastes (40.9 – 55.5 mL·g-1 VS day-1). This was
282
due to the high microbial activities from the lower food wastes to inoculum ratio (1:2, VS/VS) in
283
the referenced study.37
284
Aerobic biodegradation in seawater
285
Figure 2 shows the CO2 production and gaseous carbon loss from the aerobic biodegradation of
286
samples in seawater. The cellulose and PHA-F experiments were operated for 148 days, and PP
287
and PHA-S were operated for 195 days. Cellulose and PHA-F had similar biodegradation rates but
288
PHA-F produced more CO2 due to the difference in total carbon content. PHA-S replicates had
289
high variability in biodegradation and CO2 production which ranged from 11.7% to 83.4% (Table
290
1). Cellulose and PHA-F were easily biodegraded due to their small particle sizes and rough
ACS Paragon Plus Environment
13
Environmental Science & Technology
Page 14 of 35
291
surface structures (Figure S6). PHA-F was biodegraded relatively quickly when compared to its
292
counterpart of PHA-S, which may be attributed to differences in processing conditions that define
293
the geometry as well as surface area, both of which impact rates of biodegradation. The variance
294
of the PHA-S samples is large (55.3±38.3%), and may have occurred due to a highly diverse
295
microbial colonization and activity on the surface of samples which could be impacted by seawater
296
agitation. This variance can also occur in the open environment, where the weather conditions of
297
ocean, microbial populations, and available nutrients (like phosphorus and nitrogen) vary naturally
298
and can limit microbial growth. PP had negligible biodegradation and CO2 production, which was
299
expected. While microorganisms colonize plastic in the natural environment, PP is not known to
300
be biodegraded due to the difficulty of enzymes to cleave purely aliphatic bonds lacking any
301
heteroatoms.38–40 The pH of seawater in all digesters dropped by the end of the experiment to a
302
range of 4.6±0.6 to 6.0±0.1 from an initial pH of 7.2±0.0, suggesting much of the dissolved organic
303
carbon existed in form of acids (Table S3). The dissolved inorganic carbon concentrations in the
304
seawater after experiments were very low, 0.5±0.2 to 1.6±0.9 mg·L-1, indicating that pH differences
305
negligibly impacted biodegradation calculations (Table S3).
306
The density of cellulose and poly(3HB-co-3HHx) is greater than that of seawater, so these
307
samples sunk to the bottom of the digester and were randomly moved around with the shaker table
308
movement of 100 rpm. Previous research by others shows that the ocean sediment has a consortium
309
of microorganisms that can accelerate the biodegradation of PHA.13 Similarly, in this study,
310
particles deposited on the bottom of the digester in the seawater are likely rich in microbial activity,
311
mimicking the natural environment. This allowed for easier attachment and colonization of
312
microbes to relatively smooth PHA-S pieces. Furthermore, once two or more pieces of the PHA-
ACS Paragon Plus Environment
14
Page 15 of 35
Environmental Science & Technology
313
S samples randomly came into physical contact, they often adhered to each other to form a base,
314
further facilitating more microbial growth (Figure S6).
315
In this experiment, the lower density PP pellets remained suspended on the seawater surface. For
316
the polymers that float (e.g., PP, PE and expanded polystyrene (EPS)), UV-induced oxidation and
317
other physical interferences break the plastics into microplastics whose diameters are generally
318
less than 1 – 5 mm, impacting wildlife and ecosystems.41,42 Conversely, poly(3HB-co-3HHx) will
319
sink to the bottom of an aquatic environment, where it will be exposed to microorganisms in the
320
sediment. While PHA should always be managed through waste management systems on land, if
321
this material ends up in the environment, this work shows that a solid form of poly(3HB-co-3HHx)
322
(PHA-S) biodegrades in a sample of seawater over 6.5 months (12% to 83%) (Table 1), which is
323
statistically similar to cellulose, although at a slower rate (see subsequent discussion on rate
324
constants). Similar to the anaerobic biodegradation, the aerobic biodegradation rate constants were
325
calculated by fitting the experimental data onto the first order decay model. Cellulose and PHA-F
326
had the same biodegradation rate constants (kh) of 0.019±0.003 and 0.019±0.000 day-1,
327
respectively, consistent with the reported value of 0.01 and 0.07 day-1 for the aerobic
328
biodegradation of PCL-starch blend and PBS in a respirometer operated at 25 °C.19 However, those
329
values were an order of magnitude higher than PHA-S, 0.004±0.003 day-1. The biodegradation
330
after the 148 – 195 day experiments for each sample illustrated this as well (89.6±5.9, 55.3±38.3
331
and 88.6±0.6% for cellulose, PHA-S, and PHA-F, respectively) (Table 1).
332
After 148 days of biodegradation, cellulose and PHA-F residues could not be visually identified
333
in the seawater, so these sample residues were unable to be collected for physical characterization;
334
however, the PP and PHA-S were collected and characterized. The PP weight loss was negligible
335
(-0.4±0.5%), corresponding to the negligible gaseous carbon loss of 0.0±0.1%, further illustrating
ACS Paragon Plus Environment
15
Environmental Science & Technology
Page 16 of 35
336
that the PP plastic was not biodegraded in the seawater during the experimental timeframe.43 For
337
PHA-S, the calculated CO2-C loss (55.3±38.3%) and measured weight loss are comparable
338
(51.9±29.2%), presented in Table 1 and Table S3, respectively. However, measuring weight loss
339
was challenging for partially degraded poly(3HB-co-3HHx) samples due to physisorbed materials,
340
such as microbial cells or salts, so the properties of residues were further characterized by SEM,
341
TGA, and optical microscopy.
342
SEM analysis was used to identify the microstructure of PP and PHA-S samples surface before
343
and after seawater treatment (Figure 3). For PP, the surface remained primarily intact and
344
unchanged. However, the PHA-S displays an eroded morphology. For the PHA-S sample with
345
only 11.7% CO2 carbon loss, a high diversity of bacteria and algae were found on the edges and
346
surface. The edge of the particle had extensive pitting, indicating high microbial activity on this
347
section. Some microorganisms appear to be diatoms of different species and sizes. These SEM
348
images suggest that PHA residues were partially enzymatically hydrolyzed and converted to
349
microbial biomass. This means the actual biodegradation rate of PHA materials like poly(3HB-co-
350
3HHx) could be higher than the value of CO2-C loss, since the mass of carbon converted to
351
microbial cells was not included in this study. In addition, the biodegradation rate of PHA in the
352
seawater using the weight loss method, reported by previous studies, could be underestimated since
353
PHA residues converted to microbial biomass were not considered.13,14
354
Thermogravimetric analysis (TGA) and differential thermal analysis (DTA) were used to
355
monitor the thermal decomposition profiles of samples before and after the experiment (Figure
356
S8). The profile of PP shows negligible difference after the experiment, with the maximum
357
decomposition temperature changing slightly from approximately 462°C to 454°C, suggesting
358
only slight physical aging of sample under light oxidization and microbial attack, which was
ACS Paragon Plus Environment
16
Page 17 of 35
Environmental Science & Technology
359
further corroborated by small cracks on the PP surface. However, for the PHA-S sample the onset
360
decomposition temperature increased and the TGA mass loss decreased, indicating a physical or
361
chemical change in the samples. The decomposition temperature change of the samples could be
362
attributed to the high molecular weight compounds produced by microbes or mineral compounds
363
(e.g. Mg(OH)2) adsorbed by the bacteria or algae on the samples. In the TGA test, one sample of
364
the PHA-S had more ash (37%), likely due to adsorbed minerals. Since seawater used in this study
365
contained 8,108 ppm of Na and 922.6 ppm of Mg and 274.6 ppm of Ca (Table S4), microorganisms
366
may have absorbed and retained salts during the biodegradation process. Furthermore, Figure 4 (f)
367
shows deposits of crystalline structures which are likely salt residues retained on the samples after
368
incubation.
369
Changes in molecular weight (Mw and Mn) and dispersity (Mw/Mn) of PHA-S were examined
370
before and after biodegradation. For example, in PHA-S-1 with 70.7% CO2 carbon loss, the
371
resulting Mw slightly decreased from 446,203 to 431,496 (g·mol-1) and the dispersity (Mw/Mn) was
372
not changed (Table S3), suggesting that the bulk interior of the recovered sample had been not
373
degraded. This result agrees with previous work which showed that Mw only slightly decreased
374
after biodegradation of PHA films in seawater.14
375
Microbial community composition
376
Phylogenetic similarities for the microbial communities showed significant differences in both
377
the anaerobic sludge (PERMANOVA, F4,9=10.47, p=0.001; Table S5) and aerobic seawater
378
experiments (PERMANOVA, F5,10=3.11, p=0.001; Table S6) with 82% and 61% of variation in
379
samples attributed to PHA-S and PHA-F treatments, respectively. In anaerobic sludge digesters,
380
bacterial orders Cloacamonales, Thermotogales, p_TA06 (no class, no order), and noTP_H7 were
381
enriched in PHA-S, PHA-F and cellulose treatments relative to NTC. Previous studies have shown
ACS Paragon Plus Environment
17
Environmental Science & Technology
Page 18 of 35
382
that the first two genera are able to anaerobically degrade oil, benzene, toluene, ethylbenzene and
383
xylene (BTEX), under sulfur and hydrocarbon rich environments.44,45 Xanthomonadales,
384
Sphingomonadales,
385
Pirellulales, Cytophagales, Gemmatales, Phycisphaerales, Chlorophyta and Chlamydiales,
386
totaling twelve bacterial orders, were enriched in PHA-S and PHA-F treatments relative to NTC
387
in aerobic seawater microcosms only (Figure 4). Seven of these bacterial orders were also enriched
388
in the cellulose treatment. Clostridiales, Gemmatales, Phycisphaerales and Chlamydiales were
389
most enriched in PHA-S and PHA-F treatments. These genera were also found to exist in the
390
environments of marine sediment to biodegrade total petroleum hydrocarbons (TPHs),46 wetlands
391
in China,47 wastewater treatment system to biodegrade tannery wastewater48 or biofilms in an old
392
drinking water network.49 This is the first study reporting these bacteria play important roles in the
393
biodegradation of poly(3HB-co-3HHx). It is possible that these bacteria have adaptions to utilize
394
poly(3HB-co-3HHx) as carbon and/or energy sources under anaerobic and aerobic conditions. It
395
should be noted that the microbial analysis was conducted for all samples on the liquid phase after
396
cumulative gas generation plateaued; therefore some microbial characterization from various
397
stages of degradation and from the solid surface were not characterized. Future work will include
398
expansion of samples collected for microbial analysis.
Chromatiales,
Clostridiales,
Rhodobacterales,
Planctomycetales,
399
For understanding the large variance of biodegradation of PHA-S under aerobic seawater
400
conditions, the individual bacteria group in each replicate digester was investigated (Table S7).
401
Chlorophyta (41%), Phycisphaerales (17%) and Xanthomonadales (10%) were the dominant
402
bacterial orders in the first replicate, Sphingomonadales (23%), Clostridiales (16%) and
403
Chlorophyta (16%) were the dominant bacterial orders in the second replicate, and Chlorophyta
404
(41%), Phycisphaerales (15%) and Alphaproteobacteria (12%) were the dominant bacterial orders
ACS Paragon Plus Environment
18
Page 19 of 35
Environmental Science & Technology
405
in the third replicate. Corresponding to the increased biodegradation of three replicates (11.7 –
406
83.4%), Sphingomonadales, Clostridiales, Rhodobacterales, Gemmatales, Bacillales and
407
Solirubrobacterales had increased in relative abundances, which may suggest these bacterial
408
orders could improve PHA-S biodegradation. In addition, the dominant bacterial orders in
409
seawater at day 0 (before added in the digester) and day 198 (in blank digesters after experiments)
410
are also presented in Table S8 to indicate the microbial diversity shift before and after the
411
experiment.
412
ACS Paragon Plus Environment
19
Environmental Science & Technology
(a)
Page 20 of 35
(b)
413 (c)
414 415
Figure 1. Gaseous carbon loss (a), CH4 yield (b) and pH (c) in the digester from the anaerobic
416
biodegradation of samples. The pH of cellulose treatment was adjusted at day 9 due to the low pH
417
of 6.0±0.1. PHA-S and PHA-F indicate the sheet and flake forms of poly(3HB-co-3HHx) samples,
418
respectively. PP indicates polypropylene pellets.
419 420 421 422
ACS Paragon Plus Environment
20
Page 21 of 35
Environmental Science & Technology
(a)
423 (b)
424 425
Figure 2. CO2 production (a) and CO2 carbon loss from samples (b) in aerobic biodegradation in
426
seawater. The headspace of each digester was purged using CO2 free air at day 14, 26, 35, 44, 55,
427
85, 107, 127, and 163. PHA-S and PHA-F indicate the sheet and flake forms of poly(3HB-co-
428
3HHx) samples, respectively. PP indicates polypropylene pellets.
429 430 431
ACS Paragon Plus Environment
21
Environmental Science & Technology
Page 22 of 35
432 433
Figure 3. SEM micrographs of PP and PHA-S samples. (a) and (b) show the PP surface change
434
before and after the experiment. (c) shows the PHA-S surface before the experiment. (d) and (e)
435
show the PHA-S-3 surface of center and edge, respectively, after the experiment. (f) shows the
ACS Paragon Plus Environment
22
Page 23 of 35
Environmental Science & Technology
436
PHA-S-1 surface of center after the experiment. PHA-S and PHA-F indicate the sheet and flake
437
forms of poly(3HB-co-3HHx) samples, respectively. PP indicates polypropylene pellets.
438
ACS Paragon Plus Environment
23
Environmental Science & Technology
Page 24 of 35
439 440
Figure 4. Heat map showing fold-change of bacterial orders enriched and depleted relative to no
441
treatment control (NTC) for (A) anaerobic sludge and (B) aerobic seawater. PHA-S and PHA-F
442
indicate the sheet and flake forms of poly(3HB-co-3HHx) samples, respectively. PP indicates
443
polypropylene pellets.
444
ACS Paragon Plus Environment
24
Page 25 of 35
Environmental Science & Technology
Table 1. First order kinetic and modified Gompertz model parameters of CH4 production and gaseous carbon loss from samples. Parameters1
Cellulose
PP
PHA-S
PHA-F
Modified Gompertz equation Anaerobic biodegradation Pm (mL·g-1)
262.3b±84.0
7.9c±0.8
489.8a±49.9
336.9ab±102.4
Rm (mL·g-1 day-1)
14.5a±3.2
0.4b±0.1
12.3a±1.8
13.1a±3.5
λ (day)
6.1ab±1.5
10.9a±2.3
5.1b±2.3
1.8b±1.0
CH4 yield2 (mL·g-1)
266.9b±85.3
6.0c±1.0
483.8a±35.2
336.5ab±100.0
CH4 yield (mL·g-1 Volatile Solid)
290.1b±92.7
6.1c±1.0
483.8a±35.2
337.5ab±100.3
First order kinetics Anaerobic biodegradation kh (day-1)
0.03a±0.01
0.00
0.02a±0.00
0.02a±0.01
Biodegradation as Gaseous carbon loss (%) at 85 days
62.9a±19.7
0.3b±0.1
77.1a±6.1
54.6a±15.6
0.004b±0.003
0.019a±0.000
Aerobic biodegradation-seawater kh (day-1)
0.019a±0.003
NA3
Biodegradation as Gaseous carbon loss (%) at 148-195 days
85.4, 96.4, 87.0 (89.6a±5.9)
-0.1, 0.0, -0.1 70.7, 83.4, 11.7 (0.0b±0.1) (55.3a±38.3)
88.4, 89.4, 88.1 (88.6a±0.6)
1
The different letters (superscript a,b,c) indicate the significant difference (P≤0.05).
2
The values are on the wet weight basis.
3
NA is not available.
445
ACS Paragon Plus Environment
25
Environmental Science & Technology
Page 26 of 35
446
ASSOCIATED CONTENT
447
Supporting Information.
448
This Supporting Information is available free of charge via the Internet at http://pubs.acs.org.
449
Supplementary materials and methods. Characteristics of samples. Barcodes for forward and
450
reverse primers used for 16S rRNA gene sample tagging. Characteristics of anaerobic sludge or
451
seawater, and samples after the experiments. Properties of anaerobic sludge and natural seawater
452
used as the inoculum. PERMANOVA table for the analysis of the weighted UniFrac distance
453
matrix to test the main effects of treatment on anaerobic digester bacterial communities.
454
PERMANOVA table for the analysis of the weighted UniFrac distance matrix to test the main
455
effects of treatment on aerobic seawater bacterial communities. The dominant bacterial orders
456
(relative abundance > 1%) in three digester replicates of PHA-S under aerobic seawater conditions.
457
The dominant bacterial orders (relative abundance > 1%) at day zero and day 195 in three replicates
458
of blank digesters under aerobic seawater conditions. 1H NMR of poly(3HB-co-3HHx) Sheet
459
(PHA-S). 1H NMR of poly(3HB-co-3HHx) Flake (PHA-F). DSC of poly(3HB-co-3HHx) (PHA)
460
Sheet (PHA-S). DSC of poly(3HB-co-3HHx) (PHA) Flake (PHA-F). GPC overlay of poly(3HB-
461
co-3HHx) (PHA) samples before and after biodegradation. Visualization of samples before and
462
after the experiments. CH4 and CO2 concentrations in the digester in anaerobic biodegradation.
463
TGA and DTG of both PP and PHA-S before and after the seawater condition experiment.
464
AUTHOR INFORMATION
465
Corresponding Author
466
* Phone: 706-383-7014; Fax: 706-542-8806; Email:
[email protected] 467
ORCID
ACS Paragon Plus Environment
26
Page 27 of 35
Environmental Science & Technology
468
Shunli Wang: 0000-0002-1456-8727
469
Author Contributions
470
S.W. and J.R.J. designed the research. S.W. performed the experiments. K.A.L. and E.K.L.
471
designed and performed microbial analysis. E.M.W., J.B.G and J.L. characterized and analyzed
472
the poly(3HB-co-3HHx). S.W., K.A.L., E.M.W., J.B.G, E.K.L., J.L. and J.R.J. analyzed the data
473
and contributed the manuscript preparation. All authors have given approval to the final version
474
of the manuscript.
475
Notes
476
The authors declare no competing financial interest.
477
ACKNOWLEDGMENT
478
The authors are gratefully to acknowledge the support from an unrestricted gift from DaniMer
479
Scientific.
480
REFERENCES
481 482 483 484
1. PlasticsEurope. Plastics-the Facts 2017, An analysis of European plastics production, demand and waste data. 2017. 2. Geyer, R.; Jambeck, J. R.; Law, K. L. Production, use, and fate of all plastics ever made. Sci. Adv. 2017, 3 (7), e1700782.
485
3. Jambeck, J.R.; Geyer, R.; Wilcox, C.; Siegler, T.R.; Perryman, M.; Andrady, A.;
486
Narayan, R.; Law, K.L. Plastic waste inputs from land into the ocean. Science 2015,
487
347(6223), 768–771.
ACS Paragon Plus Environment
27
Environmental Science & Technology
Page 28 of 35
488
4. GESAMP. Sources, fate and effects of microplastics in the marine environment: part two
489
of a global assessment (Kershaw, P.J., and Rochman, C.M., eds). (IMO/FAO/UNESCO-
490
IOC/UNIDO/WMO/IAEA/UN/UNEP/UNDP Joint Group of Experts on the Scientific
491
Aspects of Marine Environmental Protection). 2016. Rep.Stud. GESAMP No. 93, 220 p.
492
5. Worm, B.; Lotze, H.K.; Jubinville, I.; Wilcox, C.; Jambeck, J. Plastic as a persistent
493 494 495 496 497 498 499 500 501 502
marine pollutant. Annu. Rev. Environ. Resour. 2017, 42, 1–26. 6. Lambert, S.; Wagner, M. Environmental performance of bio-based and biodegradable plastics: the road ahead. Chem. Soc. Rev. 2017, 46, 6855–6871. 7. Bhatt, R.; Shah, D.; Patel, K.C.; Trivedi, U. PHA-rubber blends: synthesis, characterization and biodegradation. Bioresour. Technol.2008, 99, 4615–4620. 8. Keshavarz, T.; Roy, I. Polyhydroxyalkanoates: bioplastics with a green agenda. Curr. Opin. Microbiol. 2010, 13, 321–326. 9. Reddy, C.S.K.; Ghai, R.; Rashmi, Kalia, V.C. Polyhydroxyalkanoates: an overview. Bioresour. Technol. 2013, 87, 137–146. 10. Thellen, C.; Coyne, M.; Froio, D.; Auerbach, M.; Wirsen, C.; Ratto, J.A. A processing,
503
characterization
504
polyhydroxyalkanoate (PHA) films. J. Polym. Environ. 2008, 16(1), 1–11.
505 506
and
marine
biodegradation
study
of
melt-extruded
11. Budwill, K.; Fedorak, P.M.; Page, W.J. Methanogenic degradation of poly(3hydroxyalkanoates). Appl. Environ. Microbiol.1992, 58(4), 1398–1401.
ACS Paragon Plus Environment
28
Page 29 of 35
Environmental Science & Technology
507
12. Gutierrez-Wing, M.T.; Stevens, B.E.; Theegala, C.S.; Negulescu, I.I.; Rusch, K.A.
508
Anaerobic biodegradation of polyhydroxybutyrate in municipal sewage sludge. J.
509
Environ. Eng. 2010, 136(7), 709–718.
510
13. Sridewi, N.; Bhubalan, K.; Sudesh, K. Degradation of commercially important
511
polyhydroxyalkanoates in tropical mangrove ecosystem. Polym. Degrad. Stab. 2006, 91,
512
2931–2940.
513
14. Volova, T.G.; Boyandin, A.N.; Vasil’ev, A.D.; Karpov, V.A.; Kozhevnikov, I.V.;
514
Prudnikova, S.V.; Rudnev, V.P.; Xuan, B.B.; Dung, V.V.; Gitel’zon, I.I. Biodegradation
515
of polyhydroxyalkanoates (PHAs) in the South China Sea and identification of PHA
516
degrading bacteria. Microbiol. 2011, 80(2), 252–260.
517 518 519
15. Çetin, D. Anaerobic biodegradation of poly-3-hydroxybutyrate (PHB) by sulfate reducing bacterium desulfotomaculum sp. Soil Sediment. Contam. 2009, 18:345–353. 16. Ghanem, N.B.; Mabrouk, M.E.S.; Sabry, S.A.; Ei-Badan, D.E.S. Degradation of
520
polyesters by a novel marine Nocardiopsis aegyptia sp. nov.: Application of Plackett-
521
Burman experimental design for the improvement of PHB depolymerase activity. J.
522
Gen. Appl. Microbiol. 2005, 51, 151–158.
523
17. Kasuya, K.; Mitomo, H. Identification of a marine benthic P(3HB)-degrading
524
bacterium isolate and characterization of its P(3HB) depolymerase. Biomacromolecules
525
2000, 1, 194-201.
526
18. Angelidaki, I.; Alves, M.; Bolzonella, D.; Borzacconi, L.; Campos, J. L.; Guwy, A. J.;
527
Kalyuzhnyi, S.; Jenicek, P.; van Lier, J. B. Defining the biomethane potential (BMP) of
ACS Paragon Plus Environment
29
Environmental Science & Technology
Page 30 of 35
528
solid organic wastes and energy crops: a proposed protocol for batch assays. Water Sci.
529
Technol. 2009, 59(5), 927–934.
530
19. Cho, H.S.; Moon, H.S.; Kim, M.; Nam, K.; Kim, J.Y. Biodegradability and
531
biodegradation rate of poly(caprolactone)-starch blend and poly(butylene succinate)
532
biodegradable polymer under aerobic and anaerobic environment. Waste Manage. 2011,
533
31, 475–480.
534
20. Yoon, Y.M.; Kim, S.H.; Oh, S.Y.; Kim, C.H. Potential of anaerobic digestion for material
535
recovery and energy production in waste biomass from a poultry slaughterhouse. Waste
536
Manage.2014, 34, 204–209.
537
21. ASTM-D6691. Standard Test Method for Determining Aerobic Biodegradation of
538
Plastic Materials in the Marine Environmental by a Defined Microbial Consortium or
539
Natural Sea Water Inoculum. 2009.
540
22. Caporaso, J. G.; Lauber, C. L.; Walters, W. A.; Berg-Lyons, D.; Lozupone, C. A.;
541
Turnbaugh, P. J.; Fierer, N.; Knight, R. Global patterns of 16S rRNA diversity at a
542
depth of millions of sequences per sample. Proc. Natl. Acad. Sci. U. S. A. 2011, 108
543
(S1), 4516−4522.
544
23. Lydon, K.A.; Glinski, D.A.; Westrich, J.R.; Henderson, W.M.; Lipp, E.K. Effects of
545
triclosan on bacterial community composition and Vibrio populations in natural
546
seawater microcosms. Elem. Sci. Anth. 2017, 5, 22.
547
24. Tinker, K.A.; Ottesen, E.A. The core gut microbiome of the American cockroach,
548
Periplaneta americana, is stable and resilient to dietary shifts. Appl. Environ. Microbiol.
549
2016, 82, 344–346.
ACS Paragon Plus Environment
30
Page 31 of 35
550 551
Environmental Science & Technology
25. Rohland, N.; Reich, D. Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res. 2012, 22, 939–946.
552
26. Caporaso, J. G.; Kuczynski, J.; Stombaugh, J.; Bittinger, K.; Bushman, F. D.; Costello,
553
E. K.; Fierer, N.; Pena, A. G.; Goodrich, J. K.; Gordon, J. I.; Huttley, G. A.; Kelley, S.
554
T.; Knights, D.; Koenig, J. E.; Ley, R. E.; Lozupone, C. A.; McDonald, D.; Muegge, B.
555
D.; Pirrung, M.; Reeder, J.; Sevinsky, J. R.; Turnbaugh, P. J.; Walters, W. A.;
556
Widmann, J.; Yatsunenko, T.; Zaneveld, J.; Knight, R. QIIME allows analysis of high-
557
throughput community sequencing data. Nat. Methods 2010, 7 (5), 335−336.
558 559 560
27. Edgar, R. C.; Haas, B. J.; Clemente, J. C.; Quince, C.; Knight, R. UCHIME Improves Sensitivity and Speed of Chimera Detection. Bioinformatics 2011, 27 (16), 2194−2200. 28. Wang, Q.; Garrity, G. M.; Tiedje, J. M.; Cole, J. R. Naive Bayesian classifier for rapid
561
assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ.
562
Microbiol. 2007, 73, 5261−5267.
563
29. Caporaso, J. G.; Bittinger, K.; Bushman, F. D.; DeSantis, T. Z.; Andersen, G. L.;
564
Knight, R. PyNAST: a flexible tool for aligning sequences to a template alignment.
565
Bioinformatics 2010, 26 (2), 266−267.
566 567 568
30. Edgar, R. C. Search and Clustering Orders of Magnitude Faster than BLAST. Bioinformatics 2010, 26 (19), 2460−2461. 31. DeSantis, T. Z.; Hugenholtz, P.; Larsen, N.; Rojas, M.; Brodie, E. L.; Keller, K.;
569
Huber, T.; Dalevi, D.; Hu, P.; Andersen, G. L. Greengenes, a chimera-checked 16S
570
rRNA gene database and workbench compatible with ARB. Appl. Environ. Microbiol.
571
2006, 72 (7), 5069−5072.
ACS Paragon Plus Environment
31
Environmental Science & Technology
572
Page 32 of 35
32. McDonald, D.; Price, M. N.; Goodrich, J.; Nawrocki, E. P.; DeSantis, T. Z.; Probst, A.;
573
Andersen, G. L.; Knight, R.; Hugenholtz, P. An improved Greengenes taxonomy with
574
explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J.
575
2012, 6, 610−618.
576 577 578 579 580
33. Price, M. N.; Dehal, P. S.; Arkin, A. P. FastTree 2- Approximately MaximumLikelihood Trees for Large Alignments. PLoS One 2010, 5(3), e9490. 34. Lozupone, C.; Knight, R. UniFrac: a New Phylogenetic Method for Comparing Microbial Communities. Appl. Environ. Microbiol. 2005, 71 (12), 8228−8235. 35. Oksanen, J.; Blanchet, F.G.; Friendly, M.; Kindt, R.; Legendre, P.; McGlinn, D.;
581
Minchin P.R.; O’hara, R.B.; Simpson, G.L.; Solymos, P.; Henry, M.; Stevens, H.;
582
Szoecs, E.; Wagner, H. ‘vegan’: Community Ecology Package. R package version,
583
2016, 2.4-0..
584
36. El-Mashad, H.M.; Zhang, R.; Greene, J.P. Anaerobic biodegradability of selected
585
biodegradable plastics and biobased products. J. Enivron. Sci. Eng. A 2012, 1(A), 108–
586
114.
587
37. Browne, J.D.; Allen, E.; Murphy, J.D. Evaluation of the biomethane potential from
588
multiple waste streams for a proposed community scale anaerobic digester. Environ.
589
Technol.2013, 34(13-14), 2027–2038.
590
38. Arutchelvi, J.; Sudhakar, M.; Arkatkar, A.; Doble, M.; Bhaduri, S.; Uppara, P.V.
591
Biodegradation of polyethylene and polypropylene. Indian J. Biotechnol. 2008, 7, 9–22.
592
39. Ghosh, S.K.; Pal, S.; Ray, S. Study of microbes having potentiality for biodegradation of
593
plastics. Environ. Sci. Pollut. Res. 2013, 20, 4339–4355.
ACS Paragon Plus Environment
32
Page 33 of 35
Environmental Science & Technology
594
40. Zettler, E.R.; Mincer, T.J.; Amaral-Zettler, L.A. Life in the “Plastisphere”: microbial
595
communities on plastic marine debris. Environ. Sci. Technol. 2013, 47, 7137–7146.
596
41. United Nations Environment Programme (UNEP). Biodegradable plastics and marine
597
litter. Misconceptions, concerns and impacts on marine environments. United Nations
598
Environment Programme (UNEP) 2015, Nairobi.
599
42. Browne, M.A.; Dissanayake, A.; Galloway, T.S.; Lowe, D.M.; Thompson, R.C. Ingested
600
microscopic plastic translocates to the circulatory system of the mussel, Mytilus edulis
601
(L.). Environ. Sci. Technol. 2008, 42, 5026–5031.
602 603 604
43. Tokiwa, Y.; Calabia, B.P.; Ugwu, C.U.; Aiba, S. Biodegradation of plastics. Int. J. Mol. Sci. 2009, 10, 3722–3742. 44. Shelton, J.L.; Akob, D.M.; Mclntosh, J.C.; Fierer, N.; Spear, J.R.; Warwick, P.D.;
605
McCray, J.E. Environmental drivers of differences in microbial community structure in
606
crude oil reservoirs across a methanogenic gradient. Front. Microbiol. 2016, 7, 1535.
607
45. Berlendis, S.; Lascourreges, J.; Schraauwers, B.; Sivadon, P.; Magot, M. Anaerobic
608
biodegradation of BTEX by original bacterial communities from an underground gas
609
storage aquifer. Environ. Sci. Technol. 2010, 44, 3621–3628.
610
46. Zhang, Z.; Lo, I.M.C. Biostimulation of petroleum-hydrocarbon-contaminated marine
611
sediment with co-substrate: involved metabolic process and microbial community.
612
Appl. Microbiol Biotechnol. 2015, 99, 5683–5696.
613
47. Cao, Q.; Wang, H.; Chen, X.; Wang, R.; Liu, J. Composition and distribution of
614
microbial communities in natural river wetlands and corresponding constructed
615
wetlands. Ecol. Eng. 2017, 98, 40–48.
ACS Paragon Plus Environment
33
Environmental Science & Technology
616
Page 34 of 35
48. Kim, I.; Ekpeghere, K.; Ha, S.; Kim, S.; Kim, B.; Song, B.; Chun, J.; Chang, J.; Kim,
617
H.; Koh, S. An eco-friendly treatment of tannery wastewater using bioaugmentation
618
with a novel microbial consortium. J. Environ. Sci. Heal. A 2013, 48, 1732–1739.
619
49. Henne, K.; Kahlisch, L.; Brettar, I.; Höfle, M.G. Analysis of structure and composition
620
of bacterial core communities in mature drinking water biofilms and bulk water of a
621
citywide network in Germany. Appl. Environ. Microbiol. 2012, 78(10), 3530–3538.
622
ACS Paragon Plus Environment
34
Page 35 of 35
623
Environmental Science & Technology
Abstract Art
CH4-C
CO2-C
Dissolved-C
CO2-C Residue Residue
624
ACS Paragon Plus Environment
35