International Biodeterioration & Biodegradation 59 (2007) 170–179 Microbial colonization of polymeric materials for space applications and mechanisms of biodeterioration: A review Ji-Dong Gu a,b, a Surface Biology and Environmental Microbiology Group, South China Sea Institute of Oceanography, Chinese Academy of Sciences, 164 Xingang Road West, Guangzhou 510301, PR China b Laboratory of Environmental Microbiology and Toxicology, Department of Ecology & Biodiversity, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, PR China Received 18 March 2005; received in revised form 28 November 2005; accepted 31 August 2006 Available online 24 October 2006 Abstract Biodeterioration of polymeric materials affects a wide range of industries. Formation of microbial biofilms on surfaces of materials being considered for use on the International Space Station was investigated. The materials included fiber-reinforced polymeric composites, adhesive sealant, polyimide insulation foam, Teflon cable insulation, and aliphatic polyurethane coatings. In simulation experiments, bacterial biofilms formed readily on the surfaces of the materials at a wide range of temperatures and relative humidity. The biofilm population was dominated by Pseudomonas aeruginosa, Ochrobactrum anthropi, Alcaligenes denitrificans, Xanthomonas maltophila, and Vibrio harveyi. Subsequently, degradation of polymeric materials was mostly a result of both fungal and bacterial colonization in sequence, and fungi may have advantages in the early phase of surface colonization over bacteria, especially on relatively resistant polymeric materials. These microorganisms are commonly detected on spacecraft on hardware and in the air. Furthermore, degradation of polymeric materials was documented with electrochemical impedance spectroscopy (EIS). The mechanisms of deterioration of polymeric materials were due to the availability of carbon source from the polymer, such as additives, plasticizers, and other impurities, in addition to the polymeric matrices. Microbial degradation of plasticizer phthalate esters is discussed for the microorganisms involved and the biochemical pathways of degradation. Current results suggest that candidate materials for use in space missions need to be carefully evaluated for their susceptibility to microbial biofilm formation and biodegradation. r 2006 Elsevier Ltd. All rights reserved. Keywords: Biofilms; Degradation; Plasmids; Polymeric materials; Resistance; Space station 1. Introduction Synthetic polymers can be potential substrates for heterotrophic microorganisms, depending on chemical structures, composition and bonds, and the dominant microflora. Biodegradability of polymers also depends on molecular weight, crystallinity, and the physical form of the relevant materials (Gu et al., 2000; Gu, 2004), while initial adhesion of bacteria on surfaces of materials may be mediated by surface physical characteristics, e.g., hydro- phobicity and smoothness. As a general rule, an increase in molecular weight of homopolymer may result in a decline of polymer degradation rate by both abiotic and biological processes. In contrast, monomers, dimers, and oligomers of the polymer repeating units can be much more easily degraded and mineralized quickly by the natural popula- tion of microflora. An increase in molecular weight of polymers tends to result in a decrease in solubility in water, making them unfavorable for microbial attack because bacteria, most of the time, require that the substrate be transported through the cellular membrane first, then degraded/metabolized by cellular enzymes, and assimilated by the cells in the synthesis of cellular components. ARTICLE IN PRESS www.elsevier.com/locate/ibiod 0964-8305/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2006.08.010 Corresponding address. Department of Ecology & Biodiversity, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, PR China. Tel.: +852 2299 0605; fax: +852 2517 6082. E-mail address: [email protected].
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International Biodeterioration & Biodegradation 59 (2007) 170–179
www.elsevier.com/locate/ibiod
Microbial colonization of polymeric materials for space applicationsand mechanisms of biodeterioration: A review
Ji-Dong Gua,b,�
aSurface Biology and Environmental Microbiology Group, South China Sea Institute of Oceanography, Chinese Academy of Sciences,
164 Xingang Road West, Guangzhou 510301, PR ChinabLaboratory of Environmental Microbiology and Toxicology, Department of Ecology & Biodiversity, The University of Hong Kong, Pokfulam Road,
Hong Kong SAR, PR China
Received 18 March 2005; received in revised form 28 November 2005; accepted 31 August 2006
Available online 24 October 2006
Abstract
Biodeterioration of polymeric materials affects a wide range of industries. Formation of microbial biofilms on surfaces of materials
being considered for use on the International Space Station was investigated. The materials included fiber-reinforced polymeric
composites, adhesive sealant, polyimide insulation foam, Teflon cable insulation, and aliphatic polyurethane coatings. In simulation
experiments, bacterial biofilms formed readily on the surfaces of the materials at a wide range of temperatures and relative humidity. The
biofilm population was dominated by Pseudomonas aeruginosa, Ochrobactrum anthropi, Alcaligenes denitrificans, Xanthomonas
maltophila, and Vibrio harveyi. Subsequently, degradation of polymeric materials was mostly a result of both fungal and bacterial
colonization in sequence, and fungi may have advantages in the early phase of surface colonization over bacteria, especially on relatively
resistant polymeric materials. These microorganisms are commonly detected on spacecraft on hardware and in the air. Furthermore,
degradation of polymeric materials was documented with electrochemical impedance spectroscopy (EIS). The mechanisms of
deterioration of polymeric materials were due to the availability of carbon source from the polymer, such as additives, plasticizers, and
other impurities, in addition to the polymeric matrices. Microbial degradation of plasticizer phthalate esters is discussed for the
microorganisms involved and the biochemical pathways of degradation. Current results suggest that candidate materials for use in space
missions need to be carefully evaluated for their susceptibility to microbial biofilm formation and biodegradation.
r 2006 Elsevier Ltd. All rights reserved.
Keywords: Biofilms; Degradation; Plasmids; Polymeric materials; Resistance; Space station
1. Introduction
Synthetic polymers can be potential substrates forheterotrophic microorganisms, depending on chemicalstructures, composition and bonds, and the dominantmicroflora. Biodegradability of polymers also depends onmolecular weight, crystallinity, and the physical form ofthe relevant materials (Gu et al., 2000; Gu, 2004), whileinitial adhesion of bacteria on surfaces of materials may be
e front matter r 2006 Elsevier Ltd. All rights reserved.
iod.2006.08.010
ing address. Department of Ecology & Biodiversity, The
mediated by surface physical characteristics, e.g., hydro-phobicity and smoothness. As a general rule, an increase inmolecular weight of homopolymer may result in a declineof polymer degradation rate by both abiotic and biologicalprocesses. In contrast, monomers, dimers, and oligomers ofthe polymer repeating units can be much more easilydegraded and mineralized quickly by the natural popula-tion of microflora. An increase in molecular weight ofpolymers tends to result in a decrease in solubility in water,making them unfavorable for microbial attack becausebacteria, most of the time, require that the substrate betransported through the cellular membrane first, thendegraded/metabolized by cellular enzymes, and assimilatedby the cells in the synthesis of cellular components.
ARTICLE IN PRESSJ.-D. Gu / International Biodeterioration & Biodegradation 59 (2007) 170–179 171
However, it should be pointed out that both abiologicaland biological processes may take their part in the ultimatedegradation of polymers in natural and artificial environ-ments, including manned spacecraft.
Microbial contamination of space stations is a knownproblem, and investigation of this topic has been carriedout previously in the Soviet Union and the US (Gitel’zon etal., 1981; Pierson et al., 1994; Castro et al., 2004;Novikova, 2004; Ott et al., 2004). Because of the presenceand activity of human beings, microorganisms are com-monly found on surfaces of materials and hardware and inthe air of the spacecraft (Castro et al., 2004; Novikova,2004). Furthermore, the microflora on the surfaces ofspacecraft do not differ greatly from those of our ambientenvironments on Earth because the microorganismsoriginate from human sources and preflight contamination(Gu, 2003a; Ott et al., 2004).
After initial attachment of microorganisms to surfaces ofmaterials, their growth forms extensive biofilms, anddamage to the materials may be observed. An example ofbiofilms on surfaces of a candidate polymeric materialtested for use on space stations is shown in Fig. 1,illustrating the high heterogeneity of the biofilm in terms ofmicrobial composition and also the physical form of thebiofilm spatially. Biofilms are highly heterogeneous andporous, containing 495% of water in their natural state.Growth of microorganisms was observed on surfaces ofmaterials in the space stations, including rubber seals(Gitel’zon et al., 1981), viewing windows (Castro et al.,2004) and various other types of hardware on spacecraftand space stations (Castro et al., 2004). Because of theincreasing application of polymeric materials in space andaviation, deterioration and degradation of engineeringmaterials are important considerations when selectingappropriate materials for such a program (Gu and Gu,2005) and also for better understanding the extent ofmicrobiological attack and problems in space.
Fig. 1. A scanning electron micrograph showing a natural bacterial
biofilm developed on the surface of an artificial polymeric substratum after
dehydration and critical point drying.
2. Microbiology of an enclosed environment
Because of human inhabitation, the interior of spacestations can be colonized by microorganisms; there aresuitable environmental conditions for growth in terms ofhumidity and temperature. The source of the microorgan-isms is either human or related to the contamination of thesystem pre-flight. Analysis of the microflora in the airsystem of both Mir and the International Space Station(ISS) indicated that the two most important groupscommonly found are fungi and bacteria. In monitoringprograms on land, any surfaces with a microbial density4100 cfu/25 cm2 is required to be disinfected before launch(Pierson et al., 1994). While in space, when visible growthof microorganisms is observed, on-board disinfectionshould be carried out using H2O2. For example, the crewof the Russian Salyut 6 found a ‘white film’ on parts of theinterior surfaces, including rubber straps of the exercisemachine, and the organisms were identified mainly asAspergillus sp., Penecillium sp., and Fusarium sp. On Salyut7, visible microbial growth on the hull, joints, and cables inthe working module was also observed and similarmicroflora were identified (Pierson et al., 1994). Bacteriaincluded Achromobacter sp., Acinetobacter calcoaceticus,Bacillus idosus, Corneybacterium sp., Desulfovibrio desul-
aureus, Staphylococcus sp., and Streptococcus sp. (Gitel’zonet al., 1981; Pierson et al., 1994). A list of bacteria isolatedand identified from the Russian and US spacecraft andstation is presented in Table 1. Staphylococcus, Coryne-
bacterium, Micrococcus, and Acinetobacter were found on55.5%, 36.0%, 27.5%, and 24.3%, respectively, of thesurface samples taken from Mir surfaces (Novikova, 2004).Similarly, Staphylococcus, Bacillus, Corynebacterium,Micrococcus, and Serratia were detected in 53.2%,34.0%, 16.0%, 13.8%, and 9.6% of the air samples; andFlavobacterium, Xanthomonas, Pseudomonas, Corynebac-
terium, Bacillus, Methylobacterium, and Kluyvera were in25.0%, 21.0%, 20.8%, 16.7%, 12.5%, 12.5%, and 12.5%of the condensate samples (Novikova, 2004). The occur-rence of Corynebacterium sp. and Staphylococcus epidermi-
dis was 33.2% and 30.0%, respectively, on structuralmaterials of Mir (Novikova, 2004). The data were derivedfrom 1177 samples.All of these organisms were identified based on
morphological and biochemical tests at that time; nodefinitive molecular markers, e.g., 16S rRNA gene forbacteria, were utilized. It is apparent from the Russian andAmerican space programs that the source of microorgan-isms can be minimized, but not eliminated, due topayloads, water, human inhabitation and activities; con-tamination of the surface is almost unavoidable due tomany unexpected spills or leakage, e.g., food, feces, urine,or vomit. In Mir, bacterial contamination was maintainedbelow 500 cfu/m3 air in 95% of the air samples, while fungi
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Table 1
Bacteria isolated from space programs conducted on mir and the international space station by Russia and the US
Category Genus/Species names of microorganismsa References
Surfaces in Mir Acinetobacter, Aeromonas, Bacillus, Corynebacterium, Enterobacter, Micrococcus, Novikova (2004)
Water dispensers on ISS Brayrhizobium japonicum, Methylobacterium fujisawaense, Ralstonia eutropha, Castro et al. (2004)
Sphingomonas sp.
Cargo containers on ISS Acinetobacter radioresistens, Brevundimonas diminuta, Curtobacterium luteum, Castro et al. (2004)
Micrococcus luteus, Staphylococcus pasteuri
ISS preflight Bacillus flexus, Staphylococcus epidermidis, S. diminuta, S. pasteuri Castro et al. (2004)
ISS in-flight Acinetobacter radioresistens, Bacillus pumilus, Coryneybacterium afermentans, Castro et al. (2004)
Oerskovia xanthineolytica, Staphylococcus aureus
aMicroorganisms present at o1% of the 1177 Mir samples collected are not included.bISS, International Space Station.
OligomersDimers
Monomers
Polymer
Microbial BiomassCO2H2O
Depolymerases
Microbial BiomassCH4/H2SCO2H2O
AnaerobicAerobic
Fig. 2. A schematic diagram of the processes involved in degradation of
polymeric materials under natural conditions, including both aerobic and
J.-D. Gu / International Biodeterioration & Biodegradation 59 (2007) 170–179172
were more variable, between 2 and 1.0� 104 cfu/m3 air(Novikova, 2004). It was therefore argued that selection ofstructural materials for the spacecraft is a crucial step in theoverall program (Pierson et al., 1994). An effective strategyin dealing with this issue is to select materials with knownresistance to microbial attack and possibly to modify easilydegradable components. Commonly detected fungi in freecondensate during NASA, Mir 6 and 7 flights wereAcremonium sp., Candida guilliermondii, C. lipolytica,Cladosporium sp., Fusarium sp., Penicillium sp., Rhodotor-
ula glutinis, and R. rubra (Ott et al., 2004). Penicillium spp.,Aspergillus spp., and Cladosporium spp. were detected in76.8%, 39.4%, and 27.2% of surfaces on Mir, respectively,while each of these genera was found in 75.8%, 76.6%, and24.2%, respectively, of the air samples (Novikova, 2004).
anaerobic, and the relevant degradation products (redrawn from Gu,
2003).
3. Microbial colonization and deterioration of material
surfaces
Dominant groups of microorganisms in space are verysimilar to those commonly observed to be associated withthe deterioration of engineering materials on land (Gu,2004; Gu et al., 1998a; Mitchell et al., 1996; Ott et al., 2004;Novikova, 2004); the biochemical pathways associatedwith polymer degradation are often determined by theenvironmental conditions and the microorganisms in-volved. Considering the availability of O2 as an electronacceptor, aerobic and anaerobic conditions are the twobroad categories to be considered. When O2 is available,e.g., with thin microbial biofilms or oxygenated conditions,aerobic microorganisms are mostly responsible for destruc-tion of complex materials. In contrast, under strictlyanaerobic conditions, e.g., in a thick biofilm or nichewhere oxygen is limited due to diffusion transport,
anaerobic microorganisms, including fermentative andsulfate-reducing bacteria, may become active and areresponsible for polymer deterioration (Gu et al., 2000;Gu, 2003b). Because of the difference in environmentalconditions and hence the presence of active microorgan-isms, the possible degradation processes will lead todifferent end-products, even though the biochemicalprocesses of hydrolysis are identical (Fig. 2). Theseenvironmental conditions are widely found in naturalenvironments and can also be simulated in the laboratorywith appropriate chemical media and inocula from therelevant environments (Gu, 2003a; Gu et al., 1992a, b,1993a, b, c, 1994; Gu and Gu, 2005). In addition, bothaerobic and strictly anaerobic microorganisms can coexistin natural environments in close association; both memberscan benefit from such associations, being spatially sepa-rated but biochemically connected.
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Table 2
Polymeric materials tested for their susceptibility to degradation and
Both bacteria and fungi have been observed to colonizeon a very wide range of engineering and high-strengthpolymeric materials indiscriminately (Gu, 2003b; Gu et al.,1996a, b, 1998a, b, 2000; Mitchell et al., 1996) (Table 2).High-strength engineering materials are structurally im-portant for the spacecraft, while electronic insulationpolymers are essential in the operation systems command-ing the flight of the vehicle; polymers selected and used inelectronic industries are chemically synthesized withexceptionally high strength and resistance against degrada-tion, both chemical and biological processes. For example,the thermosetting polyimides are a major class of high-performance polymer used in electronic insulation andfiber-reinforced composites (Brown, 1982). Wide accep-tance of polyimides in the electronics industry (Lai, 1989;Verbicky, 1988) has drawn serious attention to the stabilityof these materials over long periods of use. The NationalResearch Council (NRC (National Research Council),1987) pointed out the need to document the potentialdeterioration of these polymers in the electronics industriesbecause data acquisition, information processing, andcommunication are critically dependent on their perfor-mance. The inter-layering of polyimides and electronics inintegrated circuits prompted several studies on the inter-
actions between materials and microorganisms (Gu, 2003a;Gu et al., 1998b, 2000; Mitton et al., 1996, 1998). Suchinformation is discussed in detail below.
3.1. Electronic insulation materials
Polyimides are widely used in load-bearing applications,e.g., struts, chassis, and brackets in automotive and aircraftstructures, due to their flexibility and compressive strength,as well as their chemical resistance to oils, grease, and fats,microwave transparency, and thermal resistance. Theirdielectrical properties are ideally suited for use in electro-nics, especially as high-temperature insulation materialsand passivation layers in the fabrication of integratedcircuits and flexible circuitry. The high flame resistance ofthis class of polymers provides a halogen-free flame-retardant material for aircraft interiors, furnishings, andwire insulation, and they are also commonly chosen forspace applications. Other possible uses include fibers forprotective clothing, advanced composite structures, adhe-sives, insulation tapes, foam, and optics operating at hightemperatures (Verbicky, 1988).Electronic packaging polyimides are particularly useful
in the aviation and space industries because of theiroutstanding performance and engineering properties, asindicated above. It is only recently that the biodeteriora-tion problem of these polymers has been investigated, usingpyromellitic dianhydride and 4,40-diaminodiphenyl etherwith molecular weight of 2.5� 105 (Mitton et al., 1996; Guet al., 1998b; Mitton et al., 1998). Using fungi isolated frompreviously deteriorated circuit parts, both polyimide filmsand sputter-coated silicon wafers were tested for degrada-tion, after inoculation with the fungal culture, usingelectrochemical impedance spectroscopy (EIS) (Mittonet al., 1996; Gu et al., 1998b; Mitton et al., 1998). Bothforms of polyimides were found to be susceptible todeterioration by a mixed culture of fungi (Gu et al., 1996b;Mitton et al., 1998). The fungi formed a biofilm on thesurface of the material and its degradation was confirmedby monitoring the dielectrical properties of the polymerfilm using EIS (Gu et al., 1996b). The dielectric propertiesof polyimides could be altered drastically following growthof a microbial biofilm (Mitton et al., 1996; Gu et al., 1996b;Mitton et al., 1998). At the end of the incubationexperiments, physically damaged but intact polyimide filmshowed a severe decrease of capacitance and increase ofconductance, further supporting the suggestion thatdegradation of polymer dielectric properties were solelydue to damage of polymer by fungal growth. It should bepointed out that this form of deterioration may be slowunder ambient conditions due to limited availability oforganic carbon and other nutrients for the colonizingmicroorganisms. However, the deterioration processes canbe accelerated in humid conditions or in enclosed environ-ments, e.g., submarines, space vehicles, and aircraft,because the humidity of these environments promotes thedevelopment of bacterial biofilms on surfaces, and different
ARTICLE IN PRESSJ.-D. Gu / International Biodeterioration & Biodegradation 59 (2007) 170–179174
microorganisms may form a succession, according to theconditions. Under such conditions, very small amounts ofimpurities or polymerized by-products will be able tosupport the growth of fungi preceding bacteria, as observedin our simulation study (Gu et al., 1996a). Changes ofmaterial properties, if not prevented, will affect commu-nications and control systems, resulting in catastrophicconsequences.
Polyimide deterioration occurs through biofilm forma-tion and subsequent dielectrical changes in the polymer.Using EIS, a very sensitive technique for monitoring thedielectric constant of polymers (Mansfeld, 1995), fungalgrowth on polyimides was shown to yield distinctive EISspectra, indicative of increased conductivity and failingresistivity of the colonied polyimides over a year ofmonitoring (Gu et al., 1996b, 1998b). Two steps wereinvolved during degradation: An initial decline in coatingresistance was related to the partial ingress of water andionic species into the polymer matrices. This was followedby further deterioration of the polymer by fungal activity,resulting in a large decrease in resistance. Microorganismsinvolved were identified as Aspergillus versicolor, Clados-
porium cladosporioides, and Chaetomium sp. (Gu et al.,1996b, 1998b); these are commonly found in our atmo-sphere. The data suggest that polyimides are susceptible tomicrobial deterioration and also confirm the versatility ofEIS as a method for detecting biosusceptibility of polymersat an early stage. EIS is a very sensitive electrochemicaltechnique widely used by corrosion engineers to assesscorrosion at the oxidized surface layer. The new applica-tion in biodeterioration detection offers another applica-tion for this technique.
3.2. Structural and engineering composites
Fiber-reinforced polymeric composite materials(FRPCMs) are new materials widely used in aerospaceand aviation because of their easy molding and lightweight. The increasing usage of FRPCMs as structuralcomponents in aerospace applications has generated anurgent need to evaluate the biodegradability of this class ofmaterials. FRPCMs are also susceptible to attack bymicroorganisms, but the mechanisms involved are morecomplicated than with the previously discussed polyimides,due to the multi-components within any FRPCM (Guet al., 1996a, 1997). Impurities and additives, which canpromote microbial growth as potential sources of carbonand energy for environmental microorganisms, are widelyincorporated into the materials during manufacturingprocesses. In addition, no attention is given to minimizingthe contamination of microorganisms during fabrication ofeach component or each directional layer of fibers in resinssuch as ply. There is no environmental control duringstorage of these plys before overlaying them to the designedorientation of FRPCMs.
Natural populations of microorganisms are capable ofgrowth on surfaces of FRCPM coupons at both relatively
high (65–70%) and lower humidity (55–65%) (Gu et al.,1996a, 1997, 1998a; Mitchell et al., 1996). The accumula-tion of bacteria on surfaces of composites develops into athick biofilm layer and decreases the sensitivity ofmicroorganisms to further environmental changes. TheFRCPMs can provide enough organic carbon fordetectable growth of microorganisms to be observedusing microbiological techniques (Gu et al., 1996a, 1998a;Mitchell et al., 1996). More changes within materialmatrices can be detected by using highly sensitivetechniques such as EIS. Monitoring using EIS showedthat the resistance of FRPCMs, as reflected by capaci-tance, declined significantly after the initial three monthsof a one-year monitoring program (Gu et al., 1997). Cleardifferences resulting from biofilm development weredetected on FRCPMs used in aerospace applications(Gu et al., 1998a; Mitchell et al., 1996). Further studyindicated that many fungi are capable of utilizingchemicals, e.g., plasticizers, surface treatment chemicals,and impurities, introduced during composite manufac-ture (Gu et al., 1998a, 2000). Fungi are the mostaggressive microorganisms attacking FRCPMs overlonger periods, while bacteria cause limited damage.These observations clearly establish that materials canprovide nutrients for the growth of environmentalmicroorganisms and, because of this, systematic selectionof candidate materials, including fiber type, surfacecoating chemicals, and resins, is crucial in the long-termsuccess of space and aviation.Currently, a critical question remains about the effect of
FRPCM deterioration of the mechanical properties of thecomposite materials though separation—technically calleddelimination by engineers—of the fibers from resins; thishas been observed in laboratory experiments. Thorp et al.(1994) attempted to determine mechanical changes incomposite coupons after exposure to a mixed fungalculture in a simulation study. Mechanical tests showedthat no significant changes could be detected after 120 daysof exposure. It was suggested that methodologies were notsufficiently sensitive to detect surface changes duringincubation and the whole coupon-based technique cur-rently used would need to be modified for highersensitivity. Wagner et al. (1996) suggested an acoustictechnique as a means of detecting changes in the physicalproperties of the FRPCMs.A mixed culture of microorganisms, including a sulfate-
reducing bacterium, was used to show FRPCM deteriora-tion (Wagner et al., 1996). In contrast, we used a fungalconsortium, originally isolated from a degraded polymericcomposite, on a range of materials, including fluorinatedpolyimide/glass fibers, bismaleimide/graphite fibers, poly(ether–ether–ketone) (PEEK)/graphite fibers, and epoxy/graphite fibers (Gu et al., 1998a). This consortium ofmicroorganisms consisted of Aspergillus versicolor, Clados-
porium cladosporioides, and a Chaetomium sp. Whenanalyzing bacteria and fungi capable of growing on thegraphite and glass fibers of FRPCMs, only fungi were
ARTICLE IN PRESSJ.-D. Gu / International Biodeterioration & Biodegradation 59 (2007) 170–179 175
shown to cause deterioration detectable over more than350 days (Gu et al., 1997).
Physical and mechanical tests were not sufficientlysensitive to detect any significant physical changes in thematerials (Gu et al., 1997; Thorp et al., 1994). However, theresins were actively degraded, indicating that the materialswere at risk of failure. It is clear that both fiber surfacetreatment and resin processing supply enough carbon formicrobial growth (Gu et al., 1998a). It became clear thatFRPCMs are not immune to adhesion of environmentalmicroflora, and attack of materials may be observed (Guet al., 1998a; Mitchell et al., 1996). Unfortunately, themechanical evidence has not been established quantita-tively.
3.3. Polymer plasticizers and additives
Polymeric materials commonly contain a wide array ofchemicals to improve their processibility and productquality; these chemicals include phthalate esters, namelydimethyl phthalate esters (ortho-dimethyl phthalate ester,dimethyl isophthalate ester, and dimethyl terephthalateester), di-n-butyl phthalate ester, and dibutylbenzyl phtha-late ester (Gu, 2003b; Gu et al., 2004). These chemicals arenot covalently bound to the polymer resins and aretherefore susceptible to release into the environment. Thisclass of chemicals has been investigated for their biode-gradability by pure cultures of bacteria isolated fromactivated sludge (Wang et al., 2003a, b; Fan et al., 2004;Wang, 2004), mangrove sediments (Gu et al., 2005; Liet al., 2005a, b; Xu et al., 2005a, b; Li and Gu, 2006), anddeep-ocean sediment (Wang, 2005; Wang and Gu, 2006)(Table 3). These microorganisms are well characterized bymorphological, biochemical, and 16S rRNA gene analyses.The biochemical degradation mechanisms of plasticizerscommonly used in polymer formulation and processing arediscussed below.
Table 3
Degradability of plasticizers by bacteria isolated from various environments
Substrate Microorganism(s) involved Source of Ino
Dimethyl phthalate Comamonas acidovorans fy-
1,Xanthomonas maltophila, and
Sphingomonas paucimobilis
Activated slu
Dimethyl phthalate Rhodococcus rubber Sa Mangrove se
Dimethyl isophthalate Klebsiella oxytoca Sc, and
Methylobacterium mesophilicum Sr
Mangrove se
Dimethyl isophthalate Rhodococcus rubber Sa Mangrove se
Dimethyl terephthalate Rhodococcus rubber Sa Mangrove se
Dimethyl terephthalate Pasteurella multocida Sa Mangrove se
Di-n-butyl phthalate Pseudomonas fluorescens B-1 Mangrove se
Butylbenzyl phthalate Pseudomonas fluorescens B-1 Mangrove se
3.3.1. Isomers of dimethyl phthalate ester
Degradation of phthalate esters has been reported forselective isomers, but the best-characterized biochemicalpathway is that of phthalic acid. Information on degrada-tion of phthalate esters is comparatively very limited in theliterature (see review by Gu, 2003b). Our earlier researchhas been focused on the degradation of dimethyl phthalateester (DMP) and its isomers, and the biochemical pathwaysinvolved by pure cultures of bacteria (Gu et al., 2004). Oneimportant fact derived from the research was thatbiochemical cooperation by different bacteria was neededfor the utilization of ortho-dimethyl phthalate ester (Fig. 3)and dimethyl terephthalate ester (Fig. 4) (Gu et al., 2004;Li et al., 2005a, b; Wang, 2005). Sphingomonas paucimobilis
has been demonstrated as a very important participant inthe bacterial consortium degrading phthalic acid (Wanget al., 2003a; Fan et al., 2004; Wang et al., 2004). Thisstrain lacks the ability to hydrolyze ester bonds, requiredfor transforming DMP to monomethyl phthalate (MMP)(Fig. 3), but can degrade down-stream degradationintermediates. Mineralization of ortho-dimethyl phthalateester was carried out by a biochemical cooperation betweenArthrobacter sp., which can transform DMP to MMP, andSphingomonas yanoikuyae DOS01, which is distinct fromthe S. paucimobilis in the previous study, consumes thesubstrates DMP and MMP. However, S. yanoikuyae
DOS01 was incapable of degrading PA. In addition,Arthrobacter keyseri degraded several ortho-dimethylphthalate esters, including dimethyl phthalate, diethylphthalate, and di-n-butyl phthalate.
Variovorax paradoxus T4, isolated from deep-oceansediment of the South China Sea, was capable of utilizingdimethyl terephthalate ester as sole carbon and energysource (Fig. 4); the intermediates monomethyl terephtha-late (MMT) and terephthalic acid (TA) were also degraded.However, Sphingomoas sp. DOS01, also involved in thecomplete degradation of dimethyl terephthalate ester, wasonly capable of transforming dimethyl terephthalate esterto MMT without further degradation (Fig. 4) (Wang, 2005;
culum Degradation intermediates References
dge monomethyl phthalate, phthalic
acid
Wang et al. (2003a, b)
diment monomethylphthalate, phthalic
acid
Li et al. (2005a, b)
diment monoisophthalate,phthalic acid Gu et al. (2004)
diment monoisophthalate,phthalic acid Li et al. (2005a)
diment, monoterephthalate,phthalic acid Li et al. (2005a)
diment monoterephthalate,phthalaic acid Li et al. (2005b)
diment monobutyl phthalate,phthalic acid Xu et al. (2005a, b)
diment monobutyl phthalate, Xu et al.
Monobenzyl phthalate phthalic
acid
(unpublished data)
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OCH3
O
O
OCH3
OH
O
O
OCH3
OH
O
O
OH
OH
O
O
OHFurther Degradation
Further Degradation
Arthrobacter sp.
S. paucimobilis Arthrobacter sp.or
S. paucimobilis
Arthrobacter sp.or
S. paucimobilis
Fig. 3. Proposed biochemical pathways for degradation of ortho-dimethyl phthalate ester by Arthrobacter species and Sphingomonas paucimobilis isolated
from deep-ocean sediment of the South China Sea (Wang, 2005).
dimethyl terephthalate(DMT)
monomethyl terephthalate(MMT)
OCH3
3HCO
O
O
OH
3HCO
O
O
OH
HO
O
O
CO2 + H2O
dimethyl terephthalate(DMT)
monomethyl terephthalate(MMT)
terephthalate acid(TA)
OCH3
3HCO
O
O
OH
3HCO
O
O
OH
HO
O
O
(A)
(B)
terephthalate acid(TA)
Fig. 4. Proposed biochemical pathways for degradation of dimethyl terephthalate ester by Variovorax paradoxus T4 (A) and Sphingomonas yanoikuyae
DOS01 (B) isolated from deep-ocean sediment of the South China Sea (Wang, 2005).
J.-D. Gu / International Biodeterioration & Biodegradation 59 (2007) 170–179176
Wang and Gu, 2006), indicating the complexity ofbiochemical cooperation between different microorganismsin the natural environment.
3.3.2. Di-n-butyl phthalate ester and dibutylbenzyl phthalate
ester
Degradation of di-n-butyl phthalate ester was accom-plished by Pseudomonas fluorescence B-1 isolated frommangrove sediment (Xu et al., 2005a, b). Monobutylphthalate was the major intermediate produced duringthe initial phase of degradation, and a small amount ofphthalic acid was also produced. Detection of bothintermediates in this study suggests that similar biochem-ical mechanisms to the isomers of dimethyl phthalate estersare involved in degradation of di-n-butyl phthalate.A major difference was also apparent in that P. fluorescens
alone was capable of completely degradating di-n-phtha-late ester. Similarly, dibutylbenzylphthalate ester was firsttransformed to monobutylphthalate and monoben-zylphthalate and then phthalic acid by the sameP. fluorescens B-1 (Xu et al., 2005b). Results of this partof the study indicated that enzymes involved in the initialcleavage of the ester bonds were not selective, which is verydifferent from the similar enzymes catalyzing transforma-
tion of isomers of dimethyl phthalate ester. Because of thedifferences in selectivity of the hydrolytic esterases fordifferent substrates, further proteomics work will beneeded to elucidate the specific nature of the esterasesinvolved in degradation of this collection of environmentalpollutants.
4. Susceptibility testing methodologies
Currently available methods are not applicable forassessing deterioration of high-strength and engineeringmaterials due to their slow degradation rate. At the sametime, traditional methods rely heavily on observation andrating for characterization of the extent of degradation.Most of the time, the qualitative results reflect thedevelopment of biofilms on surfaces of materials (reviewby Gu, 2003a) and no scientifically sound inference ofmaterial integrity or damage can be made. Quantitativeand reliable methods are needed for screening and accuratedetermination of biodeterioration, especially early detec-tion methods for engineering applications (Gu and Gu,2005). A series of testing methods has been developed fortesting the biodeterioration and biodegradation of organicmaterials, particularly polymers of various chemical
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compositions and a range degradability (Gross et al., 1995;Gu, 2003a). Cellulose acetate with degree of substitution(d.s.) values from 0.8–2.5 was used for the validation of thetest methods conducted under thermophilic compostingand landfill simulations (Gu et al., 1992a, b, 1993a, b, c,1994). These methods can also accommodate polymers invarious physical forms, e.g., thick films and powders.
The highly sensitive and quantitative method of EIS hasbeen introduced in evaluation of polymer integrity ofpolyimide thin films, fiber-reinforced polymeric compo-sites, and aliphatic polyurethane top-coatings (Gu et al.,1996b, 1998b; Gu, 2004). With proper controls, withoutinoculation of microorganisms, and inoculated ones, theeffects of microorganisms and their growth on polymerdeterioration can be evaluated. The mechanisms may alsobe delineated by further study, after gaining informationabout the early changes. With the latest advances, newtechniques can be adopted in tests specific to the materialsand their use of environments, so that data generated ondifferent materials will provide a quantitative basis for thedescription of the biological deterioration potential.
5. Protective measures against microorganisms
Microbiological contamination of space travel andenclosed systems is a challenge for the successful applica-tion of any long-term program because damage ofmaterials by microorganisms will increase over time. Atthe same time, health-related problems may also appeardue to the increasing load of microorganisms in theenvironments. Control of microbial growth and propaga-tion on material surfaces can be achieved by using theproper criteria in selecting materials that are not easilysusceptible to microbial growth or utilization (Gu, 2004).Physical and chemical manipulations of the materials andthe artificial environments can also delay the initialestablishment of microorganisms on surfaces and thesubsequent development of biofilms; environmental con-trol by lowering humidity can slow the growth ofmicroorganisms (Gu et al., 1998a). Obviously, because ofthe presence of astronauts, there is a limit to the extent towhich the humidity can be manipulated. Experience fromRussian space programs on materials formulation byincorporation of biocides and physical cleaning clearlyshows both the practicality of application and also theproblems involved (Pierson et al., 1994). Similarly,temperature can be controlled to reduce the growth ofmicroorganisms (Gu et al., 1998a; Mitchell et al., 1996), toprevent potential contamination and to extend the lifetimeof the hardware. It should also be pointed out that verylittle is known about slow-growing bacteria on the surfaceof polymers (Gu and Pan, 2006). This group of bacteria hasbeen largely ignored, but their ability to utilize polymericmaterials may be higher than that of fast-growing bacteria.Such information will be very useful in the fundamentalunderstanding of material damage and half-life prediction.
Biocides are commonly applied in polymeric materials.Silver ion has been used as a microbial inhibitor againstmicroorganisms in water purification systems of Russianspacecraft, while iodine was chosen on the ISS by the US(Pierson et al., 1994). These chemicals have been shown tobe of variable efficacy at the initial stage in killingplanktonic bacterial cells. After adhesion to surfaces,biofilm bacteria show great resistance to a whole range ofbiocides (Mitchell et al., 1996; Gu, 2003a, b). Organicbiocides, used to prevent bacterial growth in industrialsystems, may selectively enrich the resistant population ofmicroorganisms on long-term of exposure (Gu et al.,1998a). Microorganisms are known to uptake genes fromthe environment through horizontal gene transfer. Noeffective solution to these problems is currently availableand alternative biocides have been screened from naturalproducts. Resistant microorganisms have often beenisolated because of the high mobility of resistant genes inthe environment; microorganisms are capable of incorpor-ating such genes into the cell to increase their survival. Thehigh frequency and diversity of plasmids found inenvironmental bacteria are an example illustrating theplasticity of bacteria (Wang et al., 2006; Zhang et al.,2006). These bacteria show a wide range of resistance toantibiotics and chemicals, including Hg2+. It should alsobe pointed out that very little information is availableabout the functional genes in the plasmids isolated fromenvironmental bacteria, e.g., Vibrio spp. (Wang et al., 2006;Zhang et al., 2006). In addition to their environmentalunacceptability, biocides also serve as a selection pressurefor development of a resistant population through muta-tion or up-taking resistance genetic elements from theenvironment. Current research by biologists and materialsscientists is focused on the prevention of adhesion ofmicroorganisms to surfaces, using surface treatments andmodifications. A less opportunistic pathogenic strain ofPseudomonas aeruginosa was detected on surfaces incor-porating Ag+ or Ag+ plus lectin, because both biocidaland initial adhesion inhibition were activated in suchtreatments (Gu et al., 2001).Since bacteria are capable of forming biofilms on
surfaces of materials, future tests should focus on thequantification of biofilm rather than descriptively showingbiofilms on surfaces using scanning electron microscopy. Inassaying the efficacy of biocides against bacteria, testsshould be conducted based on simulated biofilm conditionsrather than the traditional liquid culture efficacy tests (Guet al., 1998a, 2000). Because bacteria are more resistant toantibiotics and biocides after biofilms are formed anddeveloped, the planktonic cells of liquid cultures are nottruly representative of their actual condition on thesurfaces of materials (Gu, 2003a). And as mentionedabove, horizontal gene transfer allows bacteria to gainresistance to higher concentrations of biocides, metals, andantibiotics (Zhang et al., 2006).Prevention of biofilm formation and biodeterioration on
materials used in space should emphasize surface engineering
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of materials, so that initial attachment and susceptibility tomicroorganisms can be minimized. Biofilm microorganismscan be reduced greatly on surfaces. Early detection ofmaterial integrity is an important component in diagnosisand prevention of deterioration (Mansfeld, 1995). Newdetection technologies, including optical fiber, DNA probesand microarrays (Gu, 2003a), will find valuable applicationsin this field, and advance our understanding of themechanisms involved in bacterial adhesion and its preventionin the near future.
In conclusion, microbial contamination and colonizationof surfaces are problems facing the long-term applicationof space programs, particularly in terms of polymericmaterials used. Since both bacteria and fungi are commonmicroflora in spacecraft, the population size should bemaintained at a lower level to provide microbial safety tothe materials and also to the astronauts. Almost allmaterials are susceptible to colonization by environmentalmicroorganisms, including both fungi and bacteria; candi-date materials for space applications should be screenedusing quantitative methods for their susceptibility andshould be tested for the possible extent of deterioration anddegradation by microorganisms. Further coupling ofinformation derived from applied microbiology andmaterials science will allow the purpose-oriented designof materials for such specialized applications and extensionof service life.
Acknowledgements
Preparation of this manuscript was supported in part byresearch grants of the Chinese Academy of Sciences andthe University of Hong Kong.
References
Brown, G.A., 1982. Implications of electronic and ionic conductivities of
polyimide films in integrated circuit fabrication. In: Feit, E.D.,
Wilkins, C.W. (Eds.), Polymer Materials for Electronic Applications,
ACS Symposium Series, Vol. 184. American Chemical Society,
Washington, DC, pp. 151–169.
Castro, V.A., Thrasher, A.N., Healy, M., Ott, C.M., Pierson, D.L., 2004.
Microbial characterization during the early habitation of the Interna-
tional Space Station. Microbial Ecology 47, 119–126.
