一件出土饱水木漆器文物中可培养细菌的鉴定及对木材的腐蚀作用

【背景】饱水木质文物易受到微生物侵害,目前国外围绕饱水木质文物微生物病害已开展多方面研究,并取得阶段性进展,而国内在饱水木质文物微生物学技术方面的报道比较少。【目的】研究保藏水环境中出土饱水木漆器F446及水中细菌的种类,以及对木材的腐蚀作用。【方法】采用16S rRNA基因序列分析方法及生理生化试验,对饱水木漆器F446及水环境中细菌进行鉴定,并选取典型菌按5×10~8个/瓶菌量接种马尾松心材(悬于无菌自来水中),37°C培养120 d,测试木材的损失率。【结果】从F446文物和水样中分离的53株细菌中,21株被鉴定为芽孢杆菌属(Bacillus),为优势菌属,其中蜡样芽孢杆菌(B.cereus)19株,病研所芽孢杆菌(B.idriensis)和苏云金芽孢杆菌(B.thuringiensis)各1株;11株菌被鉴定为短杆菌属(Brevibacterium),此外还有4株短波单孢菌属(Brevundimonas),5株粪产碱杆菌(Alcaligenes faecalis),5株Altererythrobacter,2株水氏黄杆菌(Flavobacterium mizutaii);另外,还有解糖假苍白杆菌(Pseudochrobactrum saccharolyticum)、梭型芽孢杆菌(Lysinibacillus fusiformis)、Leucobacter aridicollis、Ochrobactrum pseudogrignonense、类芽孢杆菌属(Paenibacillus)菌株各1株。菌株A5、A6分别为类芽孢杆菌(Paenibacillus)和Altererythrobacter属中的疑似新种。从典型菌中选取15株菌回接木材进行腐蚀试验,结果显示,9株细菌与对照组比较存在极显著差异,说明这些菌对马尾松木材有一定的腐蚀作用,但是腐蚀率非常低,最高仅1.38%,表明这些细菌对试验木材马尾松腐蚀并不严重。【结论】F446木漆器文物样品中优势菌属为芽孢杆菌属(Bacillus),水样中优势菌属依次为短杆菌属(Brevibacterium)、短波单孢菌属(Brevundimonas)和Altererythrobacter。从F446木漆器文物和水样中分离出的细菌对木材的降解非常缓慢,短期内腐蚀作用有限。     

Depth of burial,an important factor in controlling bacterial decay of waterlogged archaeological poles

Wood discs from two waterlogged softwood poles were investigated for variations in bacterial decay. Exterior and interior degradation was observed using light, transmission and scanning electron microscopy. Maximum water content (U_max) was measured and extent of decay from the surface towards the wood interior was investigated. Despite a well preserved appearance, the majority of the tracheids were heavily degraded. Erosion bacteria were identified as the main degraders and a significant difference in degree of decay was found between the upper and lower parts of each pole. Small differences in oxygen concentration, derived from depth of burial, are suggested as the controlling factor. Active erosion bacterial decay was found in the inner parts of the 1200-year old wooden poles.     

Fungal colonization on excavated prehistoric wood: Implications for in-situ display

Excavations at the Neolithic settlement of Dispilio, Greece, have revealed significant amounts of waterlogged wood, some of which is being considered for in-situ display. This study investigated the presence of fungi and their biodeterioration patterns on the excavated material. Data will be used to assess the current threat from degradation and to aid in the design of appropriate control measures for any future display strategy. Fungi were isolated using different culture media and species were identified from slide cultures using light and fluorescence microscopy. Wood micro-morphology was examined using scanning electron and light microscopy. Seventeen fungal species were identified, all of which are typical terrestrial species, suggesting that they have developed in the exposed, post-excavation environment, rather than under waterlogged burial conditions. According to the literature many of the isolated species are potential wood deteriogens. In addition to fungi, abundant bacterial growth was observed in most samples. Microscopic examination of wood cells showed patterns of decay associated with soft-rot fungi, and tunnelling and erosion bacteria, suggesting past attack related to the waterlogged burial environment. The results obtained indicated fungal colonization of exposed timbers, presenting a potential threat to their long-term survival. Any in-situ display strategy, such as continuous or periodic water-spraying of the excavated wood, should include measures to monitor and control fungal growth.     

High‐Throughput DNA sequencing of ancient wood

Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high‐throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long‐term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human‐induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro‐evolutionary response of trees to climate change and human forest management.     

Use of electron microscopy for aiding our understanding of wood biodegradation

Abstract: The use of electron microscopy (EM) has proved to be an invaluable tool for studying structural aspects of lignocellulose degradation by fungi and bacteria and therefore improving our understanding of wood biodegradation. The present review details the application of conventional (SEM, TEM, STEM), analytical (EM X-ray microanalysis, (EDXA)), and immunogold cytochemical EM procedures in the field and gives specific examples of its use for each of the known important types of microbial wood decay including bacterial (tunnelling and erosion), soft rot (cavity and erosion), brown rot and white rot (simultaneous decay and preferential lignin degraders), and discusses important advances made by adopting the techniques. The more recent use of immunogold cytochemistry for studying microbe and fungal enzyme-wood cell wall interactions and its application for localization of specific wood-degrading (laccase, Mn(II) and lignin peroxidases, and cellulases) and H₂O₂ producing (pyranose oxidase) enzymes in situ during white rot decay are also reviewed, as is the application of EM for studying non-enzymatic wood decay. Methods for labelling and detecting wood components (lignin, hemi- and cellulose) in situ by EDXA and enzyme immunogold cytochemistry are also outlined. The use of EM in wood biodegradation research is rapidly expanding and is seen as an important compliment to biochemical and chemical approaches. The future should see even greater advances in our understanding of wood decay as more advanced and recently developed EM techniques are also exploited.     

Characterization of wood components of Portuguese medieval dugout canoes with high-resolution solid-state NMR

High-resolution nuclear magnetic resonance of carbon 13 was employed to characterize archaeological wood samples from Portuguese medieval dugout canoes. Structural and chemical modifications were assessed by comparing the structural features of archaeological wood samples with modern wood. The main results concern the strong decrease of sugar moiety with a complete disappearance of hemicelluloses. During ageing in water environment, the β-O-4 inter-unit linkages of lignins have not been degraded. The features of the ¹³C NMR spectra clearly reveal that the products of degradation of the carbohydrates occurring during depletion of sugars were not recombined inside the lignocellulosic matrix. Finally, this article gives a clear illustration that although the water contents of archaeological wood can certainly be used as a good criterion concerning their state of degradation, solid-state ¹³C NMR lead to unambiguously data as far the structural properties of archaeological waterlogged woods are concerned.     

DendroCT – Dendrochronology without damage

The paper describes an evaluation of the applicability of computer tomography in archaeological dendrochronology. Two different computer tomographs were tested, a Siemens Somatom Emotion single slice scanner developed for medical use, and a Nikon Metrology model XT H 225 LC, which is an industrial type scanner. Both scanners were tested against air-dried, archaeological oak wood, and more limited experiments were made with waterlogged archaeological oak wood and archaeological oak wood which had been treated with high-molecular polyethyleneglycol as a conservation treatment. After scanning the resulting imagery were measured and analysed for dendrochronology using off-the-shelf software for handling and measuring on the images and the specialist programme DENDRO for the dendrochronological analyses.The results showed that only the industrial scanner produced sufficiently clear imagery to allow for dendrochronological analyses. In the scans it was possible to separate tree-rings down to 0.2 mm width, and it was possible to identify the sapwood–heartwood border when sufficient sapwood rings were present. It was found, however, that a visual inspection of the object was required to distinguish between sapwood and decayed wood. Comparisons between direct measurements of tree-rings and measurements based on CT-imagery revealed no significant differences. The scanning and subsequent dating of more than 90 objects showed that dendrochronological dating based on CT-scanning has a success rate equal to conventional dating, albeit more time consuming.The attempts to scan waterlogged and PEG-impregnated archaeological oak wood were unsuccessful, due to a low degree of contrast between the water/PEG and the preserved wood. The experiments were too limited to exclude, however, that a successful protocol can be developed also for these types of materials.     

Structural and functional microbial diversity in deadwood respond to decomposition dynamics

We investigated the changes in microbial community diversities and functions in natural downed wood at different decay stages in a natural oak forest in the Italian Alps, through metagenomics analysis and in vitro analysis. Alfa diversity of bacterial communities was affected by the decay stage and log characteristics, while beta diversity was mainly driven by log diameter. Fungal and archaeal beta diversities were affected by the size of the sampled wood (log diameter), although, fungi were prominently driven by wood decay stage. The analysis of genes targeting cell wall degradation revealed higher abundances of cellulose and pectin-degrading enzymes in bacteria, while in fungi the enzymes targeting cellulose and hemicellulose were more abundant. The decay class affected the abundance of single enzymes, revealing a shift in complex hydrocarbons degradation pathways along the decay process. Moreover, we found that the genes related to Coenzyme M biosynthesis to be the most abundant especially at early stages of wood decomposition while the overall methanogenesis did not seem to be influenced by the decay stage. Intra- and inter-kingdom interactions between bacteria and fungi revealed complex pattern of community structure in response to decay stage possibly reflecting both direct and indirect interactions.     

Radiosensitivity of fungi isolated from waterlogged archaeological wood

Waterlogged archaeological timbers of theMary Rose were shown to support a range of fungi, including marine soft rot fungi. These isolates, and other wood degrading fungi, were inactivated by gamma irradiation at doses of 3.1–15.0 kGy. No clear pattern of variation in radioresistance appeared between the Ascomycota, Basidiomycota and Deuteromycota. Terrestrial fungi were generally more resistant than marine fungi. Little variation in radioresistance was observed between vegetative hyphae and sporulating cultures/fruiting bodies. Sublethal doses resulted in reduced viability in some species. Gamma irradiation is suggested as a possible means of controlling fungal colonisation of waterlogged archaeological wood.     

Response of the microbial community to water table variation and nutrient addition and its implications for in situ preservation of organic archaeological remains in wetland soils

Wetland environments can preserve organic archaeological remains because of their anaerobic nature. The ongoing discovery of archaeological sites in wetlands is associated with a lack of funds for excavation and preservation. This situation has led to the consideration of preservation in situ the preferred option for dealing with the majority of waterlogged archaeological remains in England. To expand our understanding of the burial environment, we studied changes in environmental variables along with counts of total bacteria and microbial ¹⁴C-leucine assimilation down the soil profile at two wetlands in the North of England. Soil cores were sampled at five depth intervals between 10 and 100 cm. To test whether the addition of nutrients induces bacterial activity in the soil, inorganic phosphate and combined nitrogen were added to soil samples and the rate of ¹⁴C-leucine assimilation was recorded. Redox potential readings were positive above the water table and negative below. The total number of bacteria and the ¹⁴C-leucine assimilation rates differed among sites, but always decreased with increasing soil depth. Nutrient availability was limiting for the microbial communities in the upper soil horizons, but did not appear to limit those in the lower soil. These results allow a better understanding of the physico-chemical and microbiological conditions that potentially favour or inhibit the decomposition of organic archaeological remains at the studied wetlands.     

Trees Underground: New Insights into Trees and Woodmanship in South-east England c. AD 800–1300

Summary

Study of wood recovered from waterlogged archaeological sites in London reveals much about the trees that grew in the city's hinterland between about AD 800 and 1300. Tentative reconstructions of some specific types of trees and ‘treeland’ have been made by combining three-dimensional recording of the timbers and roundwood with examination of how the wood was used, tree-ring analysis, species identification and experimental work in ancient woodland. It has also been possible to gain some appreciation of general trends during the mediaeval period such as the intensification of woodland management and the disappearance of wildwood conditions.     

New dendroarchaeological evidence of water well constructions reveals advanced Early Neolithic craftsman skills

The first European settlements accompanied by crop and livestock farming occurred approximately 7500 years ago. In this agrarian society, wood was one of the most important raw materials, most notably for construction, but only a little is known about wood use and woodworking technology. Hence, archaeological wooden finds are of particular importance. Dendroarchaeological studies combine the analysis of external and internal characteristics of archaeological wood: traces of tools on the wooden surfaces and the shape of timbers provide information about woodworking techniques, the tree rings allow dendrochronological dating and provide a paleoecological archive. In 2015 and 2016, two water wells with wooden linings from the Early Neolithic Period were discovered in the Czech Republic close to the towns of Velim (Bohemia) and Uničov (Moravia). The timbers were excellently preserved under waterlogged conditions. Here, we present a dendroarchaeological study including tree-ring and woodworking analyses. Furthermore, we consider former forest species composition. Overall, 15 lumbers from both wells were successfully dated by dendrochronology. The oaks used for the wells from Velim and Uničov were felled in 5196/5195 BCE and 5093–5085 BCE, respectively. Additionally, the taxa of 1859 wooden fragments, such as charcoals, branches and chips, were wood anatomically identified. The well lining from Velim with a hollowed tree trunk is already known from other Central European locations. In contrast, the construction from Uničov is unique for this period. Until now, the advanced construction design formed by four corner posts with longitudinal grooves and inserted horizontal planks was only known several thousands years later. The Early Neolithic wells from Uničov and Velim are the oldest archaeological discoveries of wooden artefacts in the Czech Republic that have been dendrochronologically dated. The tree-ring width series extend the Czech oak tree-ring width chronology more than 300 years into the past.     

Biodeterioration of Mayan archaeological sites in the Yucatan Peninsula, Mexico

Many of the monuments of the Mayan civilization are suffering deterioration caused by environmental factors (high temperatures and relative humidities), increasing contamination from natural and anthropogenic sources, and by the action of micro- and macro-biological communities. Archaeological sites and historical monuments in the Mayan area were constructed with different limestones which offer different resistances to degradation by the various types of contamination. Two different sampling sites were chosen at the archaeological site of Uxmal in the Yucatan Peninsula, Mexico. Heterotrophic bacteria, cyanobacteria and different fungi were isolated and classified taxonomically. The other archaeological site chosen for this study was the fortress of Tulum, located at the side of the Caribbean Sea and exposed to chloride of marine spray and sand erosion. In this case, heterotrophic aerobic and anaerobic bacteria, cyanobacteria and fungi were isolated from the four sampling areas selected. In both archaeological sites crust deposits were observed by using light microscopy, SEM and ESEM. Surface analyses were made by means of EDAX and electron microprobe. Possible mechanisms of stone decay, based on the type of microorganisms isolated, the physico-chemical characteristics of the constructional materials and environmental factors are discussed.     

Wood construction more strongly shapes deadwood microbial communities than spatial location over 5 years of decay

Diverse communities of fungi and bacteria in deadwood mediate wood decay. While rates of decomposition vary greatly among woody species and spatially distinct habitats, the relative importance of these factors in structuring microbial communities and whether these shift over time remains largely unknown. We characterized fungal and bacterial diversity within pieces of deadwood that experienced 6.3–98.8% mass loss while decaying in common garden ‘rotplots’ in a temperate oak-hickory forest in the Ozark Highlands, MO, USA. Communities were isolated from 21 woody species that had been decomposing for 1–5 years in spatially distinct habitats at the landscape scale (top and bottom of watersheds) and within stems (top and bottom of stems). Microbial community structure varied more strongly with wood traits than with spatial locations, mirroring the relative role of these factors on decay rates on the same pieces of wood even after 5 years. Co-occurring fungal and bacterial communities persistently influenced one another independently from their shared environmental conditions. However, the relative influence of wood construction versus spatial locations differed between fungi and bacteria, suggesting that life history characteristics of these clades structure diversity differently across space and time in decomposing wood.     

应用高通量技术分析F446饱水木漆器中微生物群落结构多样性

【背景】饱水保藏木漆器的环境中,微生物数量多、种类丰富,木漆器易受到微生物的腐蚀。【目的】研究木漆器上的微生物群落结构,分析饱水木漆器的微生物病害信息。【方法】采用Illumina MiSeq高通量测序技术对木漆器和保藏水样中的细菌进行群落结构分析。【结果】木质样品与水样品中的微生物群落多样性较丰富并在分布上存在一定差异。门水平上,木质样品共有7个优势菌门(相对丰度1%),分别为Proteobacteria(64.00%)、Acidobacteria(14.70%)和Actinobacteria(3.83%)等;水样共有6个优势菌门,分别为Proteobacteria(61.26%)、Acidobacteria(8.25%)和Planctomycetes(4.88%)等。属水平上木质样品共有8个优势菌属(相对丰度1%),分别为Phenylobacterium(16.24%)、Acidobacteria-Gp6(9.68%)和Rhodoplanes(6.45%)等;水样共有10个优势菌属,分别为Naxibacter(9.03%)、Acidobacteria-Gp6(3.84%)和Nevskia(3.27%)等。未分类菌在门和属上,木质样品分别占8.70%和50.68%;水样品分别占12.83%和59.35%。【结论】木漆器饱水保藏环境中木质样品与水样品的微生物群落结构均比较丰富,在门和属水平上水样品比木质样品复杂。另外,被测样品中可能含有大量潜在新菌。     

Sapwood and heartwood affect differentially bacterial and fungal community structure and successional dynamics during Quercus petraea decomposition

In forests, bacteria and fungi are key players in wood degradation. Still, studies focusing on bacterial and fungal successions during the decomposition process depending on the wood types (i.e. sapwood and heartwood) remain scarce. This study aimed to understand the effect of wood type on the dynamics of microbial ecological guilds in wood decomposition. Using Illumina metabarcoding, bacterial and fungal communities were monitored every 3 months for 3 years from Quercus petraea wood discs placed on forest soil. Wood density and microbial enzymes involved in biopolymer degradation were measured. We observed rapid changes in the bacterial and fungal communities and microbial ecological guilds associated with wood decomposition throughout the experiment. Bacterial and fungal succession dynamics were very contrasted between sapwood and heartwood. The initial microbial communities were quickly replaced by new bacterial and fungal assemblages in the sapwood. Conversely, some initial functional guilds (i.e. endophytes and yeasts) persisted all along the experiment in heartwood and finally became dominant, possibly limiting the development of saprotrophic fungi. Our data also suggested a significant role of bacteria in nitrogen cycle during wood decomposition.     

Biological resistance of chemically modified aspen composites

Aspen wood (Populus tremula L.) was chemically modified by a two-step procedure consisting of esterification with maleic anhydride (MA) and subsequent oligoesterification with MA and glycidyl methacrylate (GMA) or allyl glycidyl ether (AGE). This chemical modification procedure was carried out on solid wood, veneers and sawdust. The modified wood showed thermoplastic properties and could be thermally formed by hot-pressing. As a result, solid wood and the veneer samples had smooth, glossy surfaces, while a plastic-like material was produced on thermally forming the modified sawdust. The biological resistance of chemically modified and thermally formed samples was assessed by determination of weight loss following exposure to a decay fungus in a laboratory test and by a weathering test. Modified samples exposed to the white rot Coriolus versicolor for 12 weeks in the laboratory were more resistant to decay, with weight losses significantly lower than for the corresponding control samples. Solvents and thermal treatments employed in the chemical modification process had no significant effect on decay resistance of Aspen veneers. However, hot-pressing significantly improved decay resistance in unmodified wood samples by limiting hyphal colonisation of the wood structure. A microscopic comparison of chemically modified and unmodified wood samples was conducted to examine extent of fungal colonisation and decay. Chemical modification was also shown to enhance the weathering resistance of aspen wood to discoloration and surface erosion by UV and rainwater and to stain from mould fungi.     

Nitrogen-fixing bacteria in Douglas-fir residue decayed byFomitopsis pinicola

Summary Colonizing populations of nitrogen-fixing bacteria were measured in various decay stages of Douglas-fir logs infested withFomitopsis pinicola. Numbers of nitrogen-fixers and N-fixation rates in the wood increased as decay progressed. These increase in bacterial populations and N-fixing activity appeared related to increases in wood moisture content. Bacteria resemblingClostridium pasteurianum, Clostridium butyricum, andKlebsiella/Enterobacter spp. were isolated from the various wood decay stages.     

Non-destructive tree-ring measurements using a clinical 3T-MRI for archaeology

For dendrochronology, both high spatial resolution and a wide imaging area are required. However, there has been no study to satisfy these two requirement for a tree-ring measurement method using magnetic resonance imaging (MRI). In this study, we developed a ring-width measurement method (ultra-high-resolution T2 weighted imaging: uHR-T2WI) using clinical MRI that can scan images at high resolution and over a wide sample area for waterlogged wood, by repeating small fields of view (FOV).Incidentally, until now, the method using X-ray computed tomography (X-CT) has been developed for non-destructive ring-width measurement for analysis of wooden cultural heritage and excavated wooden artifacts, and is mainly successful for dried samples with a low moisture content. However, the contrast of X-CT images tends to reduce as the water content increases, and it has been reported that the attempts to visualize waterlogged and PEG-impregnated archaeological oak wood were unsuccessful owing to a low degree of contrast between the water/PEG and the preserved wood (Bill et al., 2012).Actually, the archaeologically excavated wood generally has very high moisture content (400%–800%), thereby reducing the suitability of X-CT for tree-ring analysis.On the other hand, MRI equipment images the density and state of hydrogen nuclei, and so the expected quality of MRI improves as the moisture content of the wood increases.With these factors, to use both modality complementary will be very useful for dendroarchaeology. However, direct comparison of the suitable range of each modality has not been reported. Therefore, we examined the difference between X-CT and MRI related to moisture content of wood, and examined the range of adaptation for each modality as a function of moisture content.By using the proposed method, we then obtained a tree-ring width series and evaluated the usefulness of the proposed method. The uHR-T2WI method was able to obtain tree-ring width series comparable to those of an optical scanner from images acquired at a spatial resolution of 0.05 mm in high moisture content wood. The results show that wood with a high moisture content (e.g., waterlogged wood measured immediately after excavation, or high-water-content lacquered wooden cultural property) can now be successfully analyzed as non-destructive methods with MRI.In conclusion, the complementarity of both MRI and X-CT will be very useful as non-destructive inspection methods for dendroarchaeology. Although further research should consider more wood species, our study suggests that the proposed method will significantly advance the field of dendrochronology, particularly when related to dendroarchaeology.