CELLULOLYTIC BIODEGRADATION OF COTTON FIBERS FROM A DEEP-OCEAN ENVIRONMENT
RUNYING CHEN, & KATHRYN A. JAKES
1.1 THE SS CENTRAL AMERICA SHIPWRECK
The trunk of textiles that provided materials for this research project belongs to the shipwreck of the SS Central America, a passenger steamship that sank in the Atlantic Ocean in 1857 (Herdendorf et al. 1995). For 130 years, the location of the SS Central America remained unknown, but in 1987 the Columbus-America Discovery Group, Columbus, Ohio, successfully located the ship using modern deep-ocean exploration technologies. The organization then conducted a systematic investigation of the shipwreck, assisted by members of an affiliated Adjunct Science and Education Program who aided in the study of the deep-ocean environment of the site.
The shipwreck lies at a depth of 2,200 m in a mainly foraminiferal ooze comprised of shells of numerous small sea animals. The water at this depth is influenced by the North Atlantic deepwater flow. It is cold, oxygen-rich, and less saline than surface water (Herdendorf et al. 1995). Specific conditions of seawater appropriate to this depth include: pressure, 220 atmospheres; dissolved oxygen, 5.85–6.40 ml/l; temperature, 2.81–3.77oC; salinity, 34.9–35.06%; and pH, 7.99–8.14 (Herdendorf et al. 1995).
1.2 THE RETRIEVED TRUNK AND ITS TEXTILES
Of the several pieces of luggage located on the ocean floor near the shipwreck, only two were retrieved. The first, raised in 1990, belonged to a newly married couple named Easton and was the source of the fabrics used in this study. The trunk remained under water in transport to Ohio and was refrigerated until unpacking some four weeks later.
Each item in the trunk was unpacked, examined, flash-frozen, and slowly dried prior to initiation of the work reported herein (Jakes and Mitchell 1992). Items of clothing removed from the trunk smelled sulfurous, were covered by areas of black deposits, and displayed variable states of preservation.
1.3 MARINE BACTERIA
Both aerobic and anaerobic bacteria are active in the marine environment. As dissolved oxygen decreases in deeper water or in the sediment, anaerobic bacteria dominate. Sulfate-reducing bacteria grow in the absence of oxygen by using sulfate as the terminal electron acceptor and releasing hydrogen sulfide (H2S) (Florian 1987b; Vetter et al. 1989). Hydrogen sulfide reacts with organic materials in the sediment to produce thiol compounds and with certain metal ions to form the corresponding metal sulfide. One example is the black ferrous sulfide deposited on marine artifacts formed by the reaction of H2S with iron oxide. Sulfate-reducing bacteria also produce sulfuric acid, which promotes iron corrosion, e.g., corrosion of the metal components of the wrecked ship (Herdendorf et al. 1995).
1.4 STUDIES OF MARINE TEXTILE ARTIFACTS
The textiles from the trunks recovered from the SS Central America shipwreck site have been studied since 1990 by Jakes and others of the Department of Consumer and Textile Sciences at Ohio State University (Wang 1992; Foreman and Jakes 1993; Jakes and Wang 1993; Crawford 1994; Hannel 1994; Wang and Jakes 1994; Srinivasan and Jakes 1997; Chen 1998). Since the initial assessment and drying of the materials, fibers taken from different textile items have been studied by optical microscopy, scanning electron microscopy, x-ray microanalysis, x-ray diffraction, and infrared microspectroscopy. Flax, cotton, wool, and silk were identified through their typical morphological features and also by infrared spectroscopy. Changes in the morphology are noted as indicators of degradation. For example, disruptions of cotton fiber morphology, which are evidence of degradation, include bulges (i.e., localized swollen areas whether due to expansion of the secondary wall or of the lumen); cracks (i.e., fissures horizontal to the length of the fiber); and fibrillation (i.e., longitudinal splitting along the length of the fibrils (Hearle et al. 1989; Jakes and Wang 1993; Wang and Jakes 1994; Peacock 1996b). Dark deposits, found both on the fiber surfaces and inside some fibers' lumina, contained iron and sulfur predominantly (Jakes and Wang 1993). A geochemist examined samples of this black material, recovered from within the Easton trunk, and reported finding both pyrite and geothite. The sample was a “mixture of oxidized, slightly oxidized, and reduced iron” and the material indicated that it had been “sitting close to the redox boundary” (Robbins 1996). To study the effect of the marine environment on textiles, an experiment was initiated in which standard textiles were immersed on the deep-ocean floor near the shipwreck for later retrieval (Wang 1992; Wang and Jakes 1994). These textiles were placed in the debris field of the wreck within the distance that other items of luggage were strewn out from the location of the ship itself.
Apart from those related to the SS Central America, only a limited number of marine textile studies have been reported in the literature. In general, these reports focus on a method of conservation, such as cleaning, drying, and storage;rarely is the degradation process explored. The conservation of wet organic artifacts, including textiles, was reviewed by Jenssen(1987). The fabric structure of the sails recovered from the Wasa and the treatment employed in their conservation was described (Bengtsson 1975a, 1975b). The textiles from the Mary Rose were examined by scanning electron microscopy (Ryder 1984). In the case of the Defence, linen, hemp, and silk fibers from the textiles were identified (Morris and Seifert 1978). Recognizing the need to study the degradation process of organic artifacts (other than wood) in the marine environment, Florian (1987a) outlined the chemical and physical structural changes that are possible.
1.5 CELLULOSE BIODEGRADATION
The decomposition of cellulose by bacteria and fungi occurs readily in damp, warm environments, resulting in material loss and deterioration of performance. A detailed summary of the bacteria and fungi associated with the biodegradation of cotton is reported in Siu (1951). Hearle et al. (1989) present micrographs displaying the morphological changes that result from degradation. Peacock (1996a, 1996b) describes cellulosic textiles in studies of biodegradation including burial experiments. Biodegradation of cellulose by marine fungi requires the presence of oxygen, so it is limited to oxygenated seawater and does not proceed within the anoxic sediment, while bacterial degradation of cellulose can proceed not only in seawater but also within the sediment to a depth of approximately 60 cm (Florian 1987a).
Micro-organisms are classified by the temperature range at which they reach optimum activity. Thermophilic bacteria and fungi grow fast in the temperature range 45–70�C, while mesophiles grow readily in the range of 25–45�C, and psychrophiles grow at temperatures around 10�C (Siu 1951). The bacteria described in the literature concerning cotton textile degradation nearly all fall into the mesophilic type, but bacterial cultures grown from water samples from the second trunk removed from the site of the SS Central America contained obligate psychrophiles that were short, gram negative rods (Herdendorf et al. 1995).
Most cellulose-decomposing bacteria are most active around pH 7.0–8.0, while fungi thrive in acidic conditions. Light also affects the activity of bacteria and fungi. Some grow more quickly in the dark. Nutrients required include carbon, nitrogen, phosphorus, magnesium, sulfur, and trace elements. Sulfur is often supplied in the form of sulfate; cellulolytic fungi require 0.1 g/l sulfate to attain optimum cellulose-decomposing activity (Siu 1951). Thus sulfate reduction and cellulose degradation may be accomplished by the same bacteria.
1.6 CHARACTERISTICS OF CELLULOSE FIBERS DAMAGED BY BACTERIA AND FUNGI
Microbial attack on cotton fibers begins with digestion of the outer cuticular layer (Siu 1951; Heyn 1954; Peacock 1996b). A consequence of this phenomenon can be observed microscopically by treating the damaged fibers with a strong swelling agent, such as cuprammonium hydroxide or a mixture of alkali and carbon disulfide. Swelling of the secondary wall of normal cotton, restricted by the primary wall, which does not swell, results in the formation of “balloons” (Heyn 1954; Merkel 1984). Ballooning occurs along normal cotton fibers but not on fibers damaged by bacteria and fungi. Further, the histology of bacterial and fungal attack on cellulose fibers differs. In general, bacterial attack on cotton fibers proceeds from the outer layer of the fiber without wall penetration, while fungal hyphae often penetrate into the fiber wall, resulting in the production of small fiber fragments. Crystalline areas of cellulosic fibers are less accessible to attack by cellulolytic enzymes released by bacteria and fungi (Kr�ssig 1993). A prior swelling treatment can increase the number of accessible regions within a fiber and thereby increase biodegradability.
1.7 OBJECTIVES AND RESEARCH QUESTIONS OF THIS STUDY
The items of clothing recovered from the Easton trunk were intact and contiguous garments. A man's waistcoat (inventory no. 29178), selected for this research, was constructed with three fabrics: a blend of silk and cotton for front portions, a dark brown–colored dyed cotton twill for the back, and undyed plain cotton fabric for the lining. The condition of the outer portions of the garment was generally good, although random areas were covered with black or brown stains and deposits. The lining was more obviously damaged, with torn fragmented areas covered with black deposits. The black material readily flaked from the surface of the lining onto anything it touched. When samples were taken from the waistcoat, the brown-colored waistcoat back fabric remained intact, while the undyed lining broke into fragments. The objective of this study was to investigate why and how the two cotton fabrics from the same object and same environment exhibited differences in degradation. The fabrics' degradation was evaluated in three ways: (1) the morphological differences among specimens of the dyed and undyed samples before and after swelling treatments; (2) the elemental composition differences of specimens from the two fiber samples and of the deposits on their surfaces; (3) the evidence for biological degradation of the specimens based on their reactivity. Evaluation of the degradation of the same materials through examination of the infrared spectra of single fibers is described in Chen and Jakes (2000).