Plastic under Anaerobic Sludge and Aerobic Seawater Conditions

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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.

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Biodegradation of Poly(3-hydroxybutyrate-co-3-

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hydroxyhexanoate) Plastic under Anaerobic Sludge

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and Aerobic Seawater Conditions: Gas Evolution

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and Microbial Diversity

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Shunli Wang,*,†, § Keri A. Lydon,#,¶ Evan M. White,§ Joe B. Grubbs III, § Erin K. Lipp, # Jason

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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

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30602, United States

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KEYWORDS: Poly(3HB-co-3HHx); PHA; Biodegradability; Anaerobic digestion; Seawater;

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Ocean

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ABSTRACT

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Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (poly(3HB-co-3HHx)) thermoplastics are a

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promising biodegradable alternative to traditional plastics for many consumer applications.

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Biodegradation measured by gaseous carbon loss of several types of poly(3HB-co-3HHx) plastic

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were investigated under anaerobic conditions and aerobic seawater environments. Under anaerobic

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conditions, the biodegradation of a manufactured sheet of poly(3HB-co-3HHx) and cellulose

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powder were not significantly different from one another over 85 days with 77.1±6.1% and

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62.9±19.7% of the carbon converted to gas, respectively. However, the sheet of poly(3HB-co-

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3HHx) had significantly higher methane yield (p≤0.05), 483.8±35.2 mL·g-1 volatile solid (VS),

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compared to cellulose controls, 290.1±92.7 mL·g-1 VS, which is attributed to a greater total carbon

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content. Under aerobic seawater conditions (148 – 195 days at room temperature), poly(3HB-co-

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3HHx) sheets were statistically similar to cellulose for biodegradation as gaseous carbon loss (up

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to 83% loss in about 6 months), although the degradation rate was lower than that for cellulose.

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The microbial diversity was investigated in both experiments to explore the dominant bacteria

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associated with biodegradation of poly(3HB-co-3HHx) plastic. For poly(3HB-co-3HHx)

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treatments, Cloacamonales and Thermotogales were enriched under anaerobic sludge conditions,

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while Clostridiales, Gemmatales, Phycisphaerales and Chlamydiales were the most enriched

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under aerobic seawater conditions.

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INTRODUCTION

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Traditional thermoplastics are primarily derived from fossil fuels like petroleum and natural gas

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with production increasing rapidly since 1950. An estimated 335 million metric tons of

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anthropogenic plastics were produced in 2016.1 Cumulatively, 8.3 billion metric tons of plastic has

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been produced globally since 1950 with 76% of total production (6.3 billion metric tons) ending

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up as waste.2 Only 9% of global plastics were recycled, 12% incinerated and 79% ended up in

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landfills or in our environment. Plastic has a high volume-to-mass ratio, and with large quantities

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landfilled, these materials occupy significant portions of landfill capacities. In addition,

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mismanaged plastic waste may easily enter aquatic systems, with an estimated 4.8 – 12.7 million

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metric tons of plastic waste entering the ocean globally.3 Traditional plastic materials are not

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known to be biodegradable in the environment. However, UV light and weathering processes

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fragment plastics into microplastics, materials that have become ubiquitous in our terrestrial and

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aquatic systems,4 and can cause a wide array of harm to wildlife and ecosystems, including

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potential implications for human health.5

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Plastics that are useful and biodegradable have been proposed as a part of the solution to global

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plastic management, with the addition of contributing to a more circular economy. However,

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biodegradable plastic is not without controversy. Current “compostable” plastics were previously

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interpreted as biodegradable, but such materials are not biodegradable in normal soil environments

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or in the ocean.4,6 Compostable plastic, like polylactic acid ( PLA) will be biodegraded in industrial

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composting systems with temperatures above 50°C, but not at an appreciable rate in typical home

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compost, where conditions are much more variable. In addition, recycling streams may be

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contaminated by compostable polymers like PLA. To add confusion to the biodegradable term,

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some non-biodegradable plastics have been modified with oxo-degradable chemistries so that the

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plastic fragments faster when exposed to UV light, which only exacerbates the microplastic

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problem. Therefore, companies and consumers are demanding packaging that doesn’t have the

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unintended consequences of traditional plastics at end of life. Some large consumer brands have

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committed to making all of their packaging either recyclable or compostable by 2025, and various

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non-compostable, non-recyclable single use plastic has been banned in some countries (e.g.

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France). In response to these issues, polyhydroxyalkanoate (PHAs) products are being developed

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to replace conventional petrochemical plastics to meet biodegradable packaging requirements.

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Polyhydroxyalkanoates (PHAs) are polyesters synthesized by bacteria under limited nutrient

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conditions with an excess carbon source.7 They have similar properties to traditional plastics (i.e.

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polyethylene and polypropylene) but are also biodegradable under several conditions.8–10 Under

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anaerobic conditions, PHA is initially hydrolyzed to smaller oligomers and ultimately monomeric

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units, such as 3-hydroxybutyrate, which then is degraded to acetate and hydrogen, which

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ultimately produces CH4 and CO2.11 Anaerobic studies determined that 63.4% to 87.0% of carbon

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in different types of PHA samples including PHB, and PHBV (poly(hydroxybutyrate-co-

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hydroxyvalerate) with 13 or 20% hydroxyvalerate (HV) content) was converted to biogas under

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anaerobic digestion at 35°C.11,12 However, CH4 (a potent greenhouse gas) production from the

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biodegradation of PHA was not thoroughly explored in these studies, leaving some gaps in the

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evaluation and environmental impact of PHA. Under aerobic conditions in seawater, PHA is

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colonized and hydrolyzed by extracellular depolymerases that fragment the material, which are

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then subsequently biodegraded into CO2.13 Biodegradation of different types of PHA, including

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p(3HB), p(3HB-co-5mol% 3HV), p(3HB-co-5mol% 3HHx), etc., in seawater from various

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locations has been tested only by measuring the weight loss of samples.13,14 The method of

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measuring loss of mass identified the degradation of PHA; however, mass can be lost solely from

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fragmentation, and not biodegradation. Therefore, it is critical to explore the complete

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biodegradation and mineralization of PHA in seawater.

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Identification of the microbial diversity under aerobic and anaerobic biodegradation conditions

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for PHA can provide a better understanding of potential metabolic pathways for biodegradation

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and facilitate design of waste treatment systems. Previous studies have isolated single species

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biodegrading PHA under various conditions, such as Desulfotomaculum sp. from the oil-water

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mixture in a production well,15 Nocardiopsis aegyptia,16 and Marinobacter sp.17 from marine

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seashore sediment and the deep-sea floor. Volova et al. also detected the PHA degrading bacteria

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(i.e., genera Pseudomonas, Pseudoalteromonas, among others) on the biofilm formed on PHA

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after 160 days of exposure to the seawater from Dam Bay (Vietnam).14 Nevertheless, there is

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limited literature available that explores shifts in whole microbial communities during the

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biodegradation of PHA. Therefore, further studies on complex microbial communities, which

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contain species proven to biodegrade PHA in diverse environments are needed.

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In order to explore alternative materials to traditional plastics in various environments, this paper

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investigates the biodegradability of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (poly(3HB-

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co-3HHx)), a type of PHA, in an anaerobic setting representing a proper management scenario

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(e.g., anaerobic digestion or a landfill), and in an aerobic setting akin to a mismanagement scenario

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(e.g., ending up in the ocean). Specifically, the objectives of this study were to (1) quantify the

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gaseous carbon loss and the CH4 production potential from poly(3HB-co-3HHx) under anaerobic

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conditions, (2) evaluate the mineralization and changing characteristics of poly(3HB-co-3HHx)

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under aerobic conditions in seawater using a controlled lab condition, and (3) identify and

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characterize the microbial diversity occurring in the biodegradation of poly(3HB-co-3HHx) under

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both conditions.

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MATERIALS AND METHODS

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Feedstock

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Two forms of poly(3HB-co-3HHx) samples, sheet (PHA-S) and flake (PHA-F) trademarked as

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Nodax, were provided by DaniMer Scientific (Bainbridge, Georgia, USA). Canola oil was used

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as the bacterial feedstock to produce the poly(3HB-co-3HHx) used in this study. PHA-S has 7.1%

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C6 (3-hydroxyhexanoate) content and 92.9% C4 (3-hydroxybutyrate) content while PHA-F has

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6.5% C6 (3-hydroxyhexanoate) content and 93.5% C4 (3-hydroxybutyrate) content (Figure S1 and

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S2). The two poly(3HB-co-3HHx) samples have similar glass transition temperatures (Tg = -4.7

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°C and -5.7 °C for PHA-S and PHA-F, respectively), similar cold crystallization peaks (Tc = 68.2

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°C and 72.5 °C) and energies (47.9 and 47.1 J·g-1), and comparable melting point transitions (Tm

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peaks from 112 – 147 °C) (Figure S3 and S4). The molecular weight of PHA-S and PHA-F are

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446,203 and 50,672 g·mol-1, respectively, which were designed for high and low molecular weight

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based on their target applications. The molecular weights were tuned for the appropriate melt

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viscosities that allow processing into sheets and films (Table S1). The positive control was

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cellulose powder, from Sigmacell (cellulose type 101), which was used to monitor microbial

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activities. The negative control was polypropylene pellets (PP) from LyondellBasell (Pro-fax

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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

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approximating these dimensions due to its fragility. Cellulose was used as received in powder

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form, and PP pellets (approximately 10 mm diameter) were cut in half so they were approximately

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5 mm x 5 mm x 1 mm. Samples of PP, PHA-S and PHA-F were prepared with the similar size and

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shape to minimize the error caused by different sample morphology. The total carbon content of

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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

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(Table S1). Other characteristics of polymer samples are also shown in Table S1 and Figure S5.

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Anaerobic biodegradation of poly(3HB-co-3HHx)

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Inoculum

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Approximately 22 L of inoculum were collected from a full scale anaerobic digester in a local

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wastewater treatment plant operated at 37°C and primarily fed with a co-thickened blend of

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primary sludge, waste activated sludge (WAS), and some fats, oils, and grease and high strength

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waste (FOG/HSW). The inoculum was placed in a water-sealed glass flask at 38°C for five days

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to deplete un-degraded biological residues in the inoculum before use in the experiment.18

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Digestion assay

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500 mL batch anaerobic digesters with a working volume of 300 mL were used for this study.

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Each digester contained 119 mL inoculum, 181 mL deionized (DI) water and 2 grams of sample

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to adjust the substrate to inoculum ratio to 0.7 – 0.8 (volatile solid base) and then placed in an

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incubator shaker set at 38°C and 100 RPM. Blank digesters included only inoculum and DI water.

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Each of the treatments and blanks were performed in triplicate. The digesters were sealed using

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butyl rubber stoppers and aluminum crimps, and the headspace was purged using N2 gas. The

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digesters were incubated for 85 days to obtain a complete profile of gas production. The biogas

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quantity and composition was measured 1 to 2 times a week using a Eudiometer water column

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(Selutec, Germany) at room temperature (24°C) and a GC-TCD, respectively, depending on the

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quantity of gas produced. Gas production was calculated at standard temperature and pressure

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(STP). The pH of the digester was monitored (1 mL removed to test) and then adjusted with NaOH

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to pH 7.0 – 7.5 if the digester became too acidic from forming fatty acids during degradation. The

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methods calculating biogas production and carbon loss from the samples are presented in the SI.

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In addition, carbon loss from the samples in anaerobic biodegradation were modeled using a first

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order hydrolysis equation, as suggested by previous studies18,19 and cumulative CH4 curves were

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modeled using a modified Gompertz equation during the incubation period as shown in a previous

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study20, which are described in the SI.

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Aerobic biodegradation of poly(3HB-co-3HHx) in seawater

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Inoculum

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Seawater was collected from the coast of Georgia, USA on March 30th, 2016 using a 20-L carboy

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with small vent for air exchange (GPS coordinates: 31°, 57’, 32.8’’ N, 081°, 00’, 24.4’’ W). The

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seawater temperature at the collection site was 19.4 °C. The seawater was transported and stored

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at ambient temperature in the laboratory for seven days before beginning the experiments.

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Digestion assay

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500 mL batch digesters were used for each assay with a working volume of 200 mL. The

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digesters were placed on a platform shaker set at 100 RPM, which simulated ocean currents and

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movement. Seawater (200 mL) and each sample (0.1 g) were added to a digester, prepared in

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triplicate. The seawater was supplemented with nutrients (0.5 g·L-1 of NH4Cl and 0.1 g·L-1 of

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KH2PO4).21 Blank digesters were composed of seawater with nutrients only and contained no

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polymer samples. The digester was sealed using butyl rubber stoppers and aluminum crimps. The

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O2 level in each digester headspace was kept to a minimum of 12% by purging CO2-free air with

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a pump pulling air through a scrubbing bottle filled with NaOH solution. The digesters were

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incubated from 148 to 195 days depending on continued biological activity in the samples. The

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composition of headspace gas in each digester was measured more frequently (two or three times

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per week) for the first month and less frequently (once every two weeks) during the last month,

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depending on the O2 concentration in the digester headspace. CO2 production was calculated at

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STP conditions. At the end of the experiment, the portion of samples that remained, PP and PHA-

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S, were prepared for analysis by gently rinsing with 10 mL DI water and then air-drying for 3 days.

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The pH and concentration of dissolved carbon in each sample assay were measured after the end

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of the degradation experiment.

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The CO2 production was calculated by adding CO2 in the digester headspace (multiplying 339

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mL headspace and CO2 concentration) and CO2 dissolved in the 200 mL seawater. The dissolved

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CO2 was calculated according to Henry’s law where CO2 concentrations in the headspace and

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Henry’s law constant for CO2 at 25oC (1630 atm*molwater/molgas) were used. The CO2 left in the

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headspace and dissolved in the seawater after each CO2-free air purge was subtracted from the

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total CO2 production. The average CO2 production from the blanks was subtracted from that of

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each sample to discriminate the biodegradation associated with the samples. Finally, the mass of

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carbon emitted (grams) as CO2 from the sample was calculated using the ideal gas law at 1 atm

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and 23°C. The carbon loss from samples in aerobic biodegradation was also modeled using Eq.

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S2. The analytical and statistical methods used in this study are described in the SI.

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Microbial diversity analysis

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Sample collection

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Upon completion of the anaerobic biodegradation experiment (day 85), 40 mL sludge was

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collected from each digester and centrifuged for 10 min. From each centrifuged sample, 5 mL of

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supernatant was filtered through a 0.45 µm pore size membrane (HVLP 04700, Millipore), and the

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membranes were stored immediately at -80°C for further microbial analysis. The raw seawater (50

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mL) was collected at the beginning of the experiment (day 0), filtered and stored as describe above.

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Upon completion of the aerobic seawater biodegradation experiments for cellulose and PHA-F

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treatments (day 148), 50 mL seawater was collected from each digester, filtered, and stored as

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described above. This procedure was repeated for the other seawater microcosm treatments (blank,

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PP, and PHA-S) as those experiments were completed (day 195). PHA-F and cellulose treatments

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were terminated by day 148 because the added materials were fully degraded.

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Microbial community analysis

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DNA was extracted from the filters using the PowerSoil DNA Isolation kit (MO BIO

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Laboratories, Inc). Extracted DNA was quantified (NanoDrop 1000, Thermo Scientific,

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Wilmington, DE) and diluted 1:10 before being subjected to PCR amplification of the 16S rRNA

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gene (primers 515F/806R).22 Two rounds of amplification were used to amplify, then subsequently

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tag, the V4 16S RNA gene region as previously described.23,24 Custom Illumina adaptors with

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barcode sequences were added during a second round of amplification (Table S2). Amplicons were

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purified using equal volume SPRI magnetic beads (Sera-Mag SpeedBeads, Thermo Scientific,

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Freemont, CA)25 with 96 well magnetic plate (Promega MagnaBot II) and quantified with a Qubit

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Fluorometer (Thermo Fisher Scientific, Grand Island, NY) before storage at -20°C. Samples were

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pooled to 10 nM in 10 mM Tris-HCL (pH 8) before being sent to the Georgia Genomics Facility

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(GGF) (Athens, GA) where they were tested for quality using a Fragment Analyzer (Advanced

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Analytical Technologies). Pooled samples were sequenced by GGF using v2 chemistry on an

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Illumina MiSeq PE250.

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Quantitative Insights into Microbial Ecology (QIIME) version 1.9.126 was used to merge pair-

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ends (fastq-join) of Illumina MiSeq reads. Chimeric sequences were removed with UCHIME27

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referenced against the RDP Gold database. The resulting sequences were used to pick operational

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taxonomic units (OTUs) with the QIIME pipeline28,26,29,30 using open reference and taxonomy

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assigned with Greengenes database (version 13_8).31,32 OTUs were aligned with PyNast and

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FastTree was used to generate a phylogenetic tree.33 QIIME was then used to filter unwanted

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sequences including: unassigned, mitochondria, and singletons. Sequence reads were normalized

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to the smallest number of reads per sample to generate data sets with equal abundance. Sequences

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were deposited into NCBI Bioproject (PRJNA419037).

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QIIME was used to calculate the weighted UniFrac distance matrix34 to determine beta

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diversity parameters. Differences in microbial community composition between samples were

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determined in QIIME with permutation-based multivariate analysis (PERMANOVA) of the

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weighted UniFrac distance matrix using the adonis function in vegan with 999 permutations.35

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Microbial taxa (order level) were chosen for comparison relative to no treatment control (NTC)

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if their relative abundance was at least 1% within a single treatment.

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RESULTS AND DISCUSSION

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Anaerobic biodegradation

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The anaerobic biodegradation of samples was evaluated by gaseous carbon loss in the form of

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biogas emitted from samples in the anaerobic inoculum. After 40 – 60 days of incubation,

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54.6±15.6, 77.1±6.1 and 62.9±19.7% of total carbon for PHA-F, PHA-S and cellulose were

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biochemically converted to biogas, respectively, and were not significantly different (p>0.05). As

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expected, carbon conversion to biogas for PP (0.3±0.1%) was negligible (Table 1). These results

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coincide with the work of previous studies.11,12 There were low concentrations and not

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significantly different dissolved organic carbon left in the digesters of blanks for each treatment

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(Table S3). Due to the dark color of anaerobic sludge, the powder sample residue (PHA-F) shape

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could not be easily identified. Figure S6 illustrates that the PHA-S samples were gradually

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biodegraded from the edges to the center. The PP sample, as pellets, floated on the surface of

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anaerobic sludge during the entire period of incubation. Other than the residues identified, some

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poly(3HB-co-3HHx) samples could also be in the form of unidentified microplastics in the

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anaerobic sludge, which should be verified in future studies.

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Figure 1a shows the evolution of gaseous carbon lost from samples under anaerobic

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biodegradation conditions. The kinetic data in anaerobic biodegradation provide important

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information for the design and operation of wastes treatment facilities. A first order decay model

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was used to fit the experimental data for investigating the biodegradation kinetics (Eq.2).19 The

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first order biodegradation rate constant (kh) of cellulose and poly(3HB-co-3HHx) samples showed

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no significant difference (p>0.05), suggesting the microorganisms in each sample had similar rates

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of biodegradation (Table 1), while the PP biodegradation rate constant was negligible. Few studies

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have calculated the anaerobic biodegradation kinetics of biodegradable plastics. However,

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biodegradable polymers such as PCL-starch blend and PBS had aerobic biodegradation rate

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constants of 0.07 and 0.01 day-1, respectively.19 The kh rate constants determined in this study are

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comparable at 0.02 day-1, which is interesting given that anaerobic degradation can sometimes be

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a slower process. Here, the anaerobic biodegradation of poly(3HB-co-3HHx) occurs at a

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comparable rate to other biodegradable plastics under aerobic conditions. No such results were

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reported in the previous literature to the best of our knowledge. The relatively fast anaerobic rate

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could be due to the fast acidogenesis and methanogenesis under anaerobic conditions. During

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incubation, the pH of the assay was used to evaluate digester health and acid accumulation. The

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pH of the cellulose sample sharply dropped to 6.0±0.1 corresponding to the higher CO2

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concentration in the digester headspace (Figure 1c and S2), indicating relatively active hydrolysis

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at the beginning of incubation. The pH of the PHA-S and PHA-F digesters decreased to 7.5±0.1

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and 7.2±0.1 at day 9, respectively, showing the hydrolysis of PHA-S was slower than that of PHA-

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F. At the end of incubation, the pH of the poly(3HB-co-3HHx) samples was consistently 7.0±0.0,

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corresponding with comparable dissolved organic carbon values for poly(3HB-co-3HHx) samples,

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which also indicated that no acids accumulated in the digesters.

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Figure 1b shows the evolution of CH4 produced from the samples in the anaerobic environment.

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The modified Gompertz model is widely used to fit the cumulative CH4 production data, shown in

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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,

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showing no significant difference between them (p>0.05), likely due to their similar total carbon

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content. Correspondingly, cellulose and PP yielded 290.1±92.7 and 6.1±1.0 mL·g-1 VS,

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respectively, where the CH4 yield from PP was not significantly different than the blank (p>0.05).

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Generally, cellulose yielded less CH4 than poly(3HB-co-3HHx), probably because of its lower

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total carbon content (41.8 ± 0.3%). This result is comparable to that of a previous study, where

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approximately 533 mL·g-1 VS yielded from PHA (in the film form) at a thermophilic temperature

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of 50°C.36 In addition, CH4 yields of poly(3HB-co-3HHx) samples were also similar to cheese and

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food wastes, 454 – 787 and 419 – 535 mL·g-1 VS, respectively. This suggests that poly(3HB-co-

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3HHx) would work with co-digestion of common organic wastes in an anaerobic digester or a

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landfill.37 The CH4 concentration in the biogas for cellulose and poly(3HB-co-3HHx) samples

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stabilized at about 75% and 65% for 65 days (Figure S7), respectively, which is a relatively high

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concentration of CH4 and may be used in landfill or digestion gas-to-energy recovery systems. The

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Rm and λ of the Modified Gompertz equation for cellulose and poly(3HB-co-3HHx) samples were

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not significantly different (p>0.05) (Table 1). Although CH4 yields were similar, the maximum

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specific CH4 production rate (Rm) of poly(3HB-co-3HHx) materials, calculated as 12.3 to 13.0

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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

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due to the high microbial activities from the lower food wastes to inoculum ratio (1:2, VS/VS) in

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the referenced study.37

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Aerobic biodegradation in seawater

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Figure 2 shows the CO2 production and gaseous carbon loss from the aerobic biodegradation of

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samples in seawater. The cellulose and PHA-F experiments were operated for 148 days, and PP

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and PHA-S were operated for 195 days. Cellulose and PHA-F had similar biodegradation rates but

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PHA-F produced more CO2 due to the difference in total carbon content. PHA-S replicates had

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high variability in biodegradation and CO2 production which ranged from 11.7% to 83.4% (Table

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1). Cellulose and PHA-F were easily biodegraded due to their small particle sizes and rough

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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,

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particles deposited on the bottom of the digester in the seawater are likely rich in microbial activity,

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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-

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S samples randomly came into physical contact, they often adhered to each other to form a base,

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further facilitating more microbial growth (Figure S6).

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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

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sink to the bottom of an aquatic environment, where it will be exposed to microorganisms in the

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sediment. While PHA should always be managed through waste management systems on land, if

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this material ends up in the environment, this work shows that a solid form of poly(3HB-co-3HHx)

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(PHA-S) biodegrades in a sample of seawater over 6.5 months (12% to 83%) (Table 1), which is

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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

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after the 148 – 195 day experiments for each sample illustrated this as well (89.6±5.9, 55.3±38.3

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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;

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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

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that the PP plastic was not biodegraded in the seawater during the experimental timeframe.43 For

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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

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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

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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,

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bacterial orders Cloacamonales, Thermotogales, p_TA06 (no class, no order), and noTP_H7 were

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enriched in PHA-S, PHA-F and cellulose treatments relative to NTC. Previous studies have shown

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that the first two genera are able to anaerobically degrade oil, benzene, toluene, ethylbenzene and

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xylene (BTEX), under sulfur and hydrocarbon rich environments.44,45 Xanthomonadales,

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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

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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

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(41%), Phycisphaerales (15%) and Alphaproteobacteria (12%) were the dominant bacterial orders

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in the third replicate. Corresponding to the increased biodegradation of three replicates (11.7 –

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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.

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(b)

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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

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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

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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

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PHA-S-1 surface of center after the experiment. PHA-S and PHA-F indicate the sheet and flake

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forms of poly(3HB-co-3HHx) samples, respectively. PP indicates polypropylene pellets.

438

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Figure 4. Heat map showing fold-change of bacterial orders enriched and depleted relative to no

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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.

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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

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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

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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

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Page 35 of 35

623

Environmental Science & Technology

Abstract Art

CH4-C

CO2-C

Dissolved-C

CO2-C Residue Residue

624

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