Investigating the Microbial Community Associated with Plastic Marine Debris
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Investigating the Microbial Community Associated with Plastic Marine Debris: An Experimental Colonization Study in the Coastal Waters of Woods Hole, MA, USA A Senior Thesis Presented to The Faculty of the Department of Organismal Biology and Ecology, The Colorado College By Keven Dooley Bachelor of Arts Degree in Biology 18th day of May, 2015 __________________________________________ Dr. Mark Wilson Primary Thesis Advisor _________________________________________ Dr. Marc Snyder Secondary Thesis Advisor Introduction The light weight, durability, and low cost of production of plastic have made it an everyday feature of our lives. In 2013, global plastic production increased by 3.9%, from 288 to 299 million metric tons (PlasticsEurope, 2014). This increase is the continuation of a trend that has been observed since plastic was first mass produced in the 1950’s. Between 1976 and 2013, global plastic production increased by a factor of six (PlasticsEurope, 2013). Accompanying this trend in production is a corresponding trend in plastic waste generation. A 2008 review of U.S. municipal solid waste reported a nine-fold increase in plastic waste generation between 1970 and 2008 (EPA, 2009). Although no reliable estimate of plastic input to the ocean has been established, the significant increase in global plastic production and plastic waste generation suggests the amount of plastic entering the ocean has been increasing over the past several decades. Floating plastic marine debris was first detected in the western North Atlantic Ocean in the early 1970’s (Carpenter and Smith, 1972; Carpenter et al., 1972; Colton and Knapp, 1974). These studies reported a wide distribution of plastic fragments and pellets throughout the western North Atlantic Ocean. However, only within the last decade have studies begun to shed light on the spatial distribution and abundance of plastic debris within the global ocean. These studies have documented high concentrations of microplastic debris within each of the gyres of the Pacific, Atlantic, and Indian Oceans (Law et al., 2010; Cozar et al., 2014; Law et al., 2014). Ocean gyres are large systems of convergence that occur within the northern and southern regions of the world’s oceans (Dore et al., 2008). The total amount of plastic debris in global, open-ocean surface waters was conservatively estimated to be between 7,000 and 35,000 tons (Cozar et al., 2014). The plastic resins observed in surface waters are predominantly 1 polyethylene, polypropylene, and polystyrene foam (Carpenter et al., 1972; Andrady, 2011; Oberbeckmann et al., 2014), all of which possess densities lower than that of seawater. These plastic resins are the most commonly used resins for the production of disposable packaging, one of the largest sources of plastic waste (PlasticsEurope, 2013). In addition to documenting distribution and abundance, several studies have investigated plastic accumulation trends. In the eastern North Atlantic Ocean, Thompson et al. (2004) reported an order of magnitude increase in surface plastic concentration between the 1960s- 1970s and the 1980s-1990s, and no significant increase in abundance between the 1980s and 1990s. These results are supported by another study which found no significant increase in surface plastic abundance in the western region of the North Atlantic subtropical gyre between 1986 and 2008 (Law et al., 2010). In the eastern North Pacific, Goldstein et al. (2012) reported a 2 orders of magnitude increase in plastic abundance between 1972-1987 and 1999-2010. However, another North Pacific study presenting a larger data set reported a more conservative 1 order of magnitude increase between 1972-1985 and 2002-2012 (Law et al., 2014). Ultimately, these studies suggest there has been less accumulation of plastic debris in the ocean surface than might be expected from the rapid increase in plastic production and waste generation since the 1970s. The discrepancy between plastic waste generation and ocean accumulation suggests mechanisms exist that remove and sequester a large quantity of plastic marine debris. These mechanisms are likely a complex assortment of biotic and abiotic processes including: photo- oxidative degradation and fragmentation, biofouling leading to sinking (Andrady, 2011), degradation by microorganisms (Zettler et al. 2013), and ingestion (Davison and Asch, 2011; Cozar et al., 2014). Many of the proposed processes can be mediated by interactions with marine 2 microorganisms. Apart from the roles microorganisms play in the eventual fate of plastic marine debris, interactions between microbial communities and plastic debris have the potential to cause significant effects on ocean ecosystems. The widespread distribution of plastic debris represents an opportunity for colonizing microorganisms to have significant effects across wide areas. One such effect is the transport of non-indigenous or harmful species. The common occurrence and extensive transport of floating plastic debris gives plastic significant potential to act as a dispersal vector (Barnes, 2002; Barnes and Milner, 2004). Studies have documented the introduction and dispersal of non-indigenous bryozoan species via floating plastic debris (Gregory, 1978; Winston, 1982). Additional studies have revealed the potential for plastic to act as a vector for the dispersal of harmful species such as those that contribute to harmful algal blooms (Maso et al., 2003) and potential pathogens such as members of the bacterial genus Vibrio (Zettler et al., 2013). Apart from the introduction and dispersal of plastic colonizers, the colonization of this novel and abundant substrate may have significant effects on open ocean ecosystems. Goldstein et al. (2012) revealed that the accumulation of plastic debris in the Northern Pacific subtropical gyre (NPSG) since the early 1970s has significantly enhanced oviposition success in the pelagic insect Halobates sericeus by increasing hard-substrate abundance. This increase in oviposition, and thus egg abundance, may cause a shift in energy transfer between pelagic and substrate associated communities as the biomass of a small number of Halobates eggs is equivalent to 9.2- 27.6% of daytime zooplankton biomass in the surface waters of the NPSG (Goldstein et al., 2012). The rapid introduction and expansion of this novel substrate, a previously limited feature of pelagic ecosystems, may cause substantial shifts in the structure of oceanic ecosystems. 3 Experimental colonization studies have been employed to investigate the colonization of plastic and structure of the colonizing community. Many studies investigating the process and mechanisms of colonization have employed glass as a substrate. The surface condition of glass and plastic resins are notably different: glass is a hydrophilic, biologically inert material while plastic resins are hydrophobic and bioactive. When introduced to a marine environment, the surfaces immediately experience biochemical conditioning through the adsorption of macromolecules like polysaccharides and glycoproteins (Wahl, 1989; Van Loosdrecht et al., 1990). Previous studies have reported initial surface conditions (e.g. hydrophobicity, charge, functional groups) influence the composition of the adsorbed chemical layer, which then plays an important role in mediating colonization (Marszalek et al., 1979; Wahl, 1989). This suggests substrates of differing surface characteristics may develop different colonizing communities. In a recent colonization study, Oberbeckmann et al. (2014) observed significant differences in microbial community structure between glass and polyethylene terephthalate (PET) deployed in the North Sea. Understanding how microbes interact with different colonization substrates is an important part of plastic debris research. If communities observed on plastic marine debris (PMD) are substantially different from those observed on other colonization substrates, extensive colonization of plastic debris may significantly shift the structure of substrate-associated microbial communities present in pelagic systems. This may alter existing interactions and result in novel interactions between substrate-associated and pelagic communities. The plastic itself may additionally serve as a novel source of energy within ocean ecosystems as plastic debris has been observed to host a suite of potential hydrocarbon- degrading organisms. Most studies investigating the composition of plastic colonizing marine 4 communities have observed the presence of hydrocarbon degrading taxa (Zettler et al., 2013; Harrison et al., 2014; Oberbeckmann et al., 2014; Reisser et al., 2014). However, few studies report explicit evidence of biodegradation. Zettler et al. (2013) and Reisser et al. (2014) both observed unknown organisms referred to as “pit formers” on plastic surfaces using scanning electron microscopy. These organisms were found interacting with pits in the plastic surface, proposed to have been formed through biodegradation. A study investigating the communities present on plastic samples collected from (as well as incubated within) the North Sea employed Fourier transform infrared (FTIR) spectroscopy to analyze the molecular structure of the plastic surface for evidence of biodegradation (Oberbeckmann et al., 2014). This study reported no chemical evidence of biodegradation on collected or incubated plastic samples; however, it did detect the presence of potential hydrocarbon-degrading organisms on incubated samples. The resins that characterize plastic