Microbial Biofilm of Plastic in Tropical Marine Environment and their Potential for Bioremediation of Plastic Waste

Plastic debris has become a global problem due to its widespread distribution and accumulation in the marine environment. Indigenous bacteria in the marine environment are able to quickly contaminate plastic surface and assemble to form successional plastisphere-specific bacterial. The formation of microbial biofilms on the plastic surface can indirectly initiate the degradation of plastic polymers. The environmental conditions of the tropical region make the growth of microbial biofilms become faster. However, the study on the biodiversity of microorganisms in marine plastic debris is limited to the northern hemisphere, which includes subtropical and temperate regions. This review provides current studies of biodiversity and community structure of plastisphere in tropical environments, including bacteria and microalgae, and their potential to degrade the plastic polymer. A systematic literature search has been conducted using Scopus with different combinations of keywords. In addition, Google Scholar databases were also used to find more studies on some specific topics, including plastic degrading organisms. The climate-associated areas have been grouped according to the latitude of the study site into tropical, subtropical, and temperate latitudes. The microorganisms analyzed in this review are only bacteria, actinobacteria, and microalgae.


INTRODUCTION
In modern society, plastic debris, both macro and micro-plastic, is extensively spread and accumulated in the marine environment and has become a global concern (Barnes et al., 2009;Van Cauwenberghe et al., 2015). Cosmetic microbeads, synthetic fibers from clothes, and many other products are micro-form fragments that directly propagate microplastics in the environment (Hwang et al., 2020). In 2016, more than 300 million metric tons of plastics had been produced including waste generation, and it has significantly risen worldwide (Geyer et al., 2017). In the environment, plastic debris degrades down to microplastic which high potentially enters the food chain directly and contaminates it through the degradation of chemical compounds (F. Galgani et al., 2015;Walther et al., 2018). In Indonesia, approximately 0.52 kg of plastic waste was generated by each person per year, and more than 80% of that total failed to be managed. Plastic debris has an 11% proportion of the total, whilst the well-managed plastic waste corresponds to about 10% (Jambeck et al., 2015). Along 50 km of coastline with more than 180 million inhabitants, approximately 5.4 million metric tons of plastic debris was generated, of which unmanaged waste is about 60%; consequently, more than 1 million metric tons ultimately became marine debris (Syakti et al., 2017).
It was widely recognized that plastic debris poses a risk to marine life and human health (Iñiguez et al., 2017). Therefore, this issue has been included in national and international policies and legislation such as stated in SDGs 14 by United Nation-related life underwaters the aims to "by 2025, prevent and significantly reduce marine pollution of all kinds, particularly Microbial Biofilm of Plastic in Tropical Marine Environment and their Potential for Bioremediation of Plastic Waste from land-based activities, including marine debris and nutrient pollution" (Zafeiridou et al., 2018). In the marine environment, plastic will break down into smaller microplastic fragments (less than 5 mm in diameter) due to physical and chemical processes such as ultraviolet radiation, oxidation, and mechanical forces (Galgani et al., 2018). Microplastics are ubiquitous and ingested by an extensive range of organisms due to their sizes, possibly endangering the ecosystem and public health (Maes et al., 2017).
When plastic debris accumulates in the marine environment, it will serve as a new habitat for various microorganisms. It provides a durable substrate that can be colonized by microorganisms and supports the growth of microbial biofilms. The ecosystem that has evolved to live in plastic environments is known as the plastisphere. Plastisphere is formed by the association of microorganisms with organic and inorganic substances. Indigenous bacteria in the marine environment able to quickly contaminate microplastics and assemble successional plastisphere-specific bacteria (Galgani et al., 2018). The film layer formed on plastic debris can affect the surface of the material properties and influence the colonizing microorganisms, which begin when microbes first attach to the plastic debris substrate (Rummel et al., 2017). After forming the aforementioned, an adhesion of other microorganisms can be provided by first coating. Extracellular polymeric substances (EPS) produced by microorganisms will facilitate cell adhesion to surfaces, bind other organic compounds in the environment, facilitate the metabolism of microorganisms using their extracellular enzymes and provide physical and mechanical stability (Flemming & Wingender, 2010; L. Galgani et al., 2018). It forms a more complex biofilm (Lobelle & Cunliffe, 2011). In addition, microalgae such as Diatomae, Cyanobacteria, and Coccolithophores groups are the fastest and earliest microbial groups to attach and form colonies on plastic substrates (Casabianca et al., 2019;Eich et al., 2015).
The microorganisms colonizing plastics were co-influenced by multiple factors, such as environmental factors, including nutrient and exposure duration, polymer types, biogeographical, and seasonal variability exposure . Their research showed that geographical location has a more significant influence on the formation of microbial biofilms. Microbial biofilm formation is faster in tropical waters than in subtropical waters (Schlundt et al., 2020). It was known that the tropical region has the greatest level of biodiversity. Understanding the biodiversity of indigenous microorganisms attached to the plastic in the tropical region will certainly be a useful database for the development of several things, including the efforts to overcome the massive plastic pollution. However, the number of studies in tropical regions is lesser than in other regions. The study on the biodiversity of microorganisms in marine plastic debris is limited to the northern hemisphere, including subtropical and temperate areas (Roager & Sonnenschein, 2019). In this review, the current status about the studies of biodiversity and community structure of plastisphere in a tropical environment, including bacteria and microalgae, and their potential to degrade plastic polymer, was provided.

METHODS
The literature research was performed using Scopus with different combinations of the following keywords: plastic, macroplastic, microplastic, microorganism, bacteria, actinobacteria, plankton, plastisphere, biofilm, biofouling, degradation, marine, ocean, and coastal. In addition, the Google Scholar databases were also used to find more studies on some specific topics, including plasticdegrading organisms. The literature research was concluded on September 17th, 2021. The studies published after this date are not included in the reference list. Review articles were excluded and only research studies were included. Duplicate articles generated due to different keyword combinations were removed. The climate-associated areas were grouped according to the latitude of the study site into tropical (0-23.5°N or S), subtropical (23.5-40°N or S), and temperate latitudes (>40°N or S). The organisms analyzed in this review are only bacteria, actinobacteria and microalgae, whereas fungi are not included. Furthermore, the articles that contain the tropical region were analyzed.

PLASTISPHERE
Plastic will become a new habitat for microorganisms, known as plastisphere . The plastic in marine waters will be exposed to UV radiation, heat, oxidation, biodegradation, and mechanical disruption. This process causes the plastic to be cut into smaller sizes, either macro, micro, or even nanoplastic. Due to the air and ocean current conditions, plastics will be suitable substrates for the microbial community. The macroplastic waste in the waters is a durable substrate and provides an attachment site for various kinds of micro and macro-organisms in the seas. Indigenous bacteria in aquatic habitats will be able to attach and form a biofilm layer within about one week after plastic materials, such as PET (polyethylene terephthalate), PVC (polyvinyl chloride), and PS (polystyrene), submerged in water. In addition, the microalgae from the Diatom, Cyanobacteria, and Coccolithophores groups are among the fastest and earliest microbial groups to attach and form colonies on plastic substrates covered by microbial biofilms (Casabianca et al., 2019;Eich et al., 2015).
Biofilm microbial growth can cause damage to the plastic surface and initiate the polymer degradation process (Rummel et al., 2017). The plastic degradation process is a complex process involving a consortium of microorganisms in the biofilm.

BIODIVERSITY OF PLASTISPHERE IN TROPICAL ENVIRONMENT Bacteria
Bacteria are the initiators of microorganisms and the most dominant organism in the plastisphere. The search of a literature study using the Scopus machine showed that 69 articles focused on microorganisms' biodiversity in plastic surfaces for both macro and microplastics during the last ten years. It is known that the tropical region has the greatest level of biodiversity. However, the number of papers on microorganisms' biodiversity in plastic surfaces is still low. Among these articles, 43.5% concern the studies on the biodiversity of microorganisms that colonize on plastic in the temperate zone, followed by 42% percent in the subtropics and 14.5% in the tropical region. Although, the effect of latitude is still controversial in the diversity of microorganisms Proteobacteria, Bacteriodetes, and Firmicutes were the dominant phyla found on microplastic surfaces on sandy beaches along the coast of Singapore. However, there are very significant differences in the dominant genera. The environmental factor influences the differences of dominant genera in these microplastics. The virgin area was dominated by Brachymonas, Pseudomonas, and Sphingobium, whereas Arcobacter, Albimonas, and Bacteroides dominated the moderately populated area. In the areas with a high impact of anthropogenic activities, the dominant bacteria are Erythrobacter, Cohaesibacter, and Hyphomonas. Interestingly, the number of dominant bacteria in pristine and moderate areas was only about 5-6% for each genus. Still, the Erytrobacter genera appeared in very high numbers, reaching 21% of the inhabitants of the plastisphere. Erythrobacter is known as a hydrocarbon-degrading bacteria (Curren & Leong, 2019). Generally, plastics are derived from petroleum products and have a long carbon chain structure. Therefore, the attachment of Erythrobacter on the plastic surface is closely related to the structure of the plastic.
Bacterial biofilm formation consists of several stages, including attachment, initiated when bacteria extrude extracellular polymeric substances (EPS); early-stage biofilm which is indicated by microorganisms that are already irreversibly attached; and mature biofilm (Renner & Weibel, 2011). In contrast to the existing natural biofilms, microbial biodiversity at the same stage of biofilm formation can be identified and compared using in situ experiments. However, from several studies on biofilm formation using in situ experiment, it can be concluded that there are different community structures and formation rates between temperate and tropical zones. 2020 also mentioned that the biofilm of plastic in the tropical ocean has a high diverse bacterial community. Early-stage biofilm formation is not only dominated by Rhodobacteraceae but also other genera from Alpha and Gammaproteobacteria. The complexity of microbial community in tropical zone is followed by the attachment of organic material and EPS production that occur faster compared to other zones. In situ experiments using nine different plastic types were conducted in the Zhangziang mangrove area for three months. All taxa of bacteria in the mangrove rhizosphere were also found on plastic surfaces. However, plastic biofilms have higher diversity with more taxa of bacteria. The capability of the plastic to adsorb organic and inorganic compounds facilitates the metabolism of microorganisms in the plastisphere and creates similar conditions with sediment only 2-3 cm apart. (Xie et al., 2021) showed that biofilms succession in plastic surfaces is influenced by the surrounding environment and the chemotaxis ability of bacteria. Bacterial growth on the surface of polymers such as acrylonitrile butadiene styrene (ABS), polycarbonate, and polyethylene terephthalate is more influenced by environmental factors than the type of plastic. The first month in the initial phase of The growth of microorganisms in plastic polymers is able to initiate the degradation of plastic polymers, affecting the density, buoyancy, and sinking rate of plastics (Rummel et al., 2017). Tropical mangrove areas being polluted by plastic debris can be a habitat for plastic-degrading microorganisms. Incubation of plastic material in the mangrove area for three months contributed a pitting on the plastic surface, i.e., low-density polyethylene, polyamide 6, and polyvinyl chloride. These results indicate a plastic degradation process carried out by microorganisms. Hydrocarbon degrading bacteria are known to be the . This polymer is composed of aminocaproic acid, which has a carbonyl functional group. Erytrobacter is known to be a bacterium that can utilize the carbonyl group for its metabolism (Xie et al., 2021). Therefore, the attachment of these genera on the plastic surface is closely related to the plastic structure. In addition, hydrocarbon-degrading bacteria were also detected dominantly in the areas with the highest microplastic pollution along the Singapore coast (Curren & Leong, 2019). Generally, plastics are derived from petroleum products and have a long carbon chain structure. It increases the potential use of hydrocarbon-degrading bacteria to overcome the massive plastic pollution.

Benthic microalgae and invertebrates
Plastic debris provides a new surface for the growth of biofilm producers, such as bacteria and microalgae, which supported the growth of benthic invertebrates and later on formed a community consisting of both phototrophic and heterotrophic organisms (Eich et al., 2015).  Table 2.
On the other hand, benthic invertebrates, such as species from the taxa group of bryozoans, lepadomorph barnacles (Lepas), marine insect (Halobates), Asellote isopods, and marine worms were reported on drifted plastic debris sampled around the coastal waters of Australia (Reisser et al., 2014). A study on the rafting invertebrates on the marine plastic debris by Goldstein et al. (2014)

POTENTIAL CANDIDATE FOR BIOREMEDIATION
Substrate availability, surface characteristics, substrate form, and polymer molecular weight all contribute to the complex biodegradation process. Chemical, physical, or biological degradation can occur naturally. Microorganisms will act as effective bioremediation agents, contribute to the biological degradation process (Ammala et al., 2011;Harrison et al., 2018). There have been several studies on the use of microorganisms as bioremediation agents for plastic degradation. In bioremediation, microorganisms such as bacteria, actinomycetes, microalgae, and fungi, play a significant role. Plastic polymer, specifically petroleum-based plastic, show great promise for decomposition into monomers using bioremediation (Montazer et al., 2020). Microorganisms can biodegrade plastics and alter their physical qualities. In order to lessen plastic pollution in the environment, genetic engineering of microorganisms can enhance the enzymes produced by microorganisms to increase their ability to break down plastic waste into carbon dioxide.

Bacteria
Plastic waste is generally found in the areas where the population is quite dense, such as in coastal areas. Beaches have a high population density; thus, the abundance of plastic waste will also be high because it is influenced by the anthropogenic pressure. Proteobacteria and Bacteroides are two types of bacteria found in bacterial communities, and their distinctions vary by location. In previous studies, the characteristics of the bacterial community on the surface of marine microplastics revealed the same types  Oceanospirillales, and Burkholderiales are some of the most common orders of bacteria in the plastisphere. Primary colonizers and secondary consumers are thought to account for the majority of biofilm in inhabitants; nevertheless, species within this varied community could play a key role in the plastic degrading pathway. Many studies have been conducted in order to identify the greatest bacterial candidates for degrading plastics. However, the research on marine bacteria that can break down microplastics effectively is still limited, particularly in the tropics. In this review, the Publish or Perish program is used to conduct a review of the studies on the bacteria that can degrade plastics, particularly in the tropics; from the data obtained, only a few studies have been conducted, as shown in Table 3. Several research results were discovered on the marine bacteria originating from the tropics in degrading microplastics, including Alicagens faecalis (Nag et al., 2021),   Cho, et al. (2021). This indicates that the bacteria belonging to the Pseudomonas sp. genus are present and Bacillus sp. are the best microorganisms for degrading microplastics, particularly in the tropics.

Actinobacteria
Among plastic polymer biodegraders, the most active taxa are fungi and actinobacteria (Tosin et al., 2012). Actinobacteria, often known as actinomycetes, are Gram-positive bacteria that have a high GC content. These bacteria can be found in a variety of ecological habitats, including soils, freshwater, and marine environments (Valan et al., 2012). Actinobacteria, one of many microorganisms, has been found attached to the marine environment plastic waste surface but seldom reported from tropical areas. On the basis of the literature study conducted, from 61 articles, sorted by 12 articles, only one article reported the presence of plastics-degrader actinobacteria in tropical environments. In contrast, most were reported from temperate regions.
Most actinobacteria are known as bioactive compounds producers, such as anticancer, antimicrobial, antifungal, and others, but recently those microorganisms have also been found to degrade complex polymers (Goodfellow, 1983;Sharma et al., 2014). Besides being known as a producer of many secondary metabolites, the marine actinobacteria are also reported for the degradation of oil spills, chlorobenzenes, aliphatic and polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), phenols, n-alkanes, heavy metal compounds, and plastic materials (Rathore et al., 2021). The actinobacteria are also reported to biodegrade various pollutants, such as pesticides, plastics, rubber, heavy metal, and other organic matters (Diez, 2010;Tseng et al., 2007). Actinobacteria are known to decompose a large number of biomolecules (lignin, cellulose, and hemicellulose) and to metabolize recalcitrant polymers such as long-chain n-alkanes (Lo Piccolo et al., 2011), xenobiotics (Borozan, 2013), pesticides (Alvarez et al., 2017), and rubber (Shivlata & Satyanarayana, 2015).
The use of actinobacteria in plastics waste bioremediation has raised the hopes that the plastic problem will be solved in the future (Oliveira et al., 2020). The improvement was such that the Rhodococcus ruber of marine origin is reported to degrade plastic waste significantly. This organism can degrade 8% of dry weight of plastic waste within 30 days under in vitro conditions (Andrady, 2011). The use of Rhodococcus ruber strain C208, the initial signs of degradation appeared after only 16 days. R. ruber degraded 7.5 percent of the initial weight of the microplastic in only two months and could use PE as the sole carbon source (Sivan et al., 2006). Amycolatopsis orientalis expressed an extracellular PLAdegrading enzyme with high degrading activity, which resulted in the total degradation of the PLA powder within 8 hours. Additionally, the Kibdelosporangium aridum strain showed a high capacity for biodegradation, degrading almost 97 percent of the initial polymer. This strain formed several small pits across the PLA surface (Jarerat et al., 2003).
Some of the actinobacteria mentioned above are marine-derived actinomycetes. These revealed high polymer degradation, as well as polymer structural and chemical changes. Therefore, the marine actinomycete counterparts appear to be quite effective in using polymers as a sole carbon source, especially for PLA degradation (Oliveira et al., 2020). Most of the papers reported actinobacteria from the temperate zone, but only some studies informed plastics-degrader actinobacteria from a tropical area. Auta et al. (2018) investigated the growth response and mechanism of degradation of polypropylene (PP) by Bacillus sp. and Rhodococcus sp. isolated from mangrove sediments in Peninsular Malaysia. These bacteria and actinobacteria strains were able to thrive on PP microplastic, as indicated by the polymer mass reduction. After 40 days of incubation, Rhodococcus sp. lost 6.4 percent of its weight while Bacillus sp. lost 4.0 percent of its weight.
Harshvardhan & Jha, (2013) reported sixty marine bacteria isolated from pelagic waters of the Arabian Sea coast, India, were screened for their ability to degrade low-density polyethylene; among them, Kocuria palustris M16, Bacillus pumilus M27 and Bacillus subtilis H1584 were positive and able to grow in a medium containing polythene as the sole carbon source.

Benthic microalgae
Marine microalgal taxa, particularly diatoms and cyanobacteria, are one of the first organisms that colonize a new substrate such as plastic debris. Those microalgae help the formation of biofilm by secreting polysaccharide substances that form Transparent Exopolymer Particles (TEPs) under turbulent conditions which help stabilize the biofilm and allow the diatoms or cyanobacteria cells to stick to the surface of the plastic debris (Casabianca et al., 2020; Ryabushko et al., 2021). However, whether the microalgae contribute directly towards the degradation of the plastic debris itself or not is still debatable. The microalgae were thought to contribute indirectly by promoting the growth of plastic degrading bacteria. The biofilm that formed with the help of microalgae and their metabolic waste could provide an additional substrate and nutrients for the bacteria that are capable of degrading some components of plastic, particularly the hydrocarbon compounds

CONCLUSIONS
The number of studies on microbial biodiversity of plastisphere in the tropical marine region is the least compared to the other regions. However, several studies have shown higher diversity and faster biofilm formation in tropical areas. The potential of microorganisms on plastic surfaces has not been studied further, although several studies have shown indications of the potential microorganisms as plastic degrading agents. The discovery of plastic degrading microorganisms in the tropical areas is also still very rare. Future research needs to focus on using the omics approach to understand gene or enzyme secretion by microorganisms in a biofilm of plastic to find their function. In addition, the isolation of microorganisms and analysis of their potential as plastic degrading from tropical marine environments is an essential part of further research.