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Purpose Maritime cultural relics from the Nanhai No. 1 shipwreck were immersed in a buffer to maintain stability. To better monitor the changes in the composition of microorganisms in the buffer and, thus, prevent the damage to artifacts caused by harmful microorganisms. Methods In September and November 2019, we conducted high-throughput sequencing of water samples from four types of maritime cultural relics (ironware, porcelain, axe, and hull wood) to reveal the composition and changes in microbial communities. In addition, we isolated culturable microorganisms and conducted biocide sensitivity tests and lignin and cellulose degradation tests. Results Visible microbial colonization was observed in the water samples collected from the buffer solutions of iron- ware, porcelain, axe, and hull wood of the Nanhai No. 1 shipwreck; additionally, apparent differences in the composi- tion of microorganisms in the water samples collected from different cultural relics and different collection times of the same cultural relics were noted. Few species of bacteria and fungi from the microbial community observed in the maritime cultural relics were cultured, and it was noted that various biocides had certain inhibitory effects on them. Some dominant strains had lignin and cellulose degradation abilities and could only grow under specific environ- mental conditions. Conclusion We found apparent differences in the composition of microorganisms obtained from different cultural relics and different collection times of the same cultural relics. This study can provide data support for better pro - tection of maritime cultural relics obtained from the Nanhai No. 1 shipwreck and provide a theoretical basis for the biological protection of other maritime cultural relics. Keywords Nanhai No. 1 shipwreck, Maritime cultural relics, High-throughput sequencing, Microbial communities, Biodeterioration † 2 Jiao Pan and Yeqing Han equally contributed to this work. Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, Department of Microbiology, College of Life *Correspondence: Sciences, Nankai University, Tianjin 300071, China Naisheng Li National Centre for Archaeology, Beijing 100013, China email@example.com Institute for Cultural Heritage and History of Science & Technology, University of Science and Technology Beijing, Beijing 100083, China © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Pan et al. Annals of Microbiology (2023) 73:8 Page 2 of 12 bacteria (Gao et al. 2017). Metal cultural relics may also Background undergo biodeterioration and biological corrosion (Hu The Nanhai No. 1 is a Chinese wooden merchant ship, and Bi 2007; Song et al. 1992; Zhang and Hao 1998; Yu which sang into the South China Sea during the South- and Yang, 2018; Nugarli and Bartolini, 1997). Zarasvand ern Song Dynasty between 1127 and 1279 AD. It was analyzed typical corrosion parts with pitting and black salvaged as a whole in 2007, preserved in the Maritime corrosion products at the bottom of the hull and found a Silk Road Museum in Guangdong Province, and exca- large number of sulfate-reducing bacteria, most of which vated in 2013 (Cui 2019; Wu and Zhang 2008). To date, were typical corrosive bacteria belonging to Desulfovibrio more than 180,000 pieces of maritime cultural relics sp. (Zarasvand and Rai 2016). have been unearthed from the Nanhai No. 1 shipwreck, When biodeterioration of cultural relics occurs, meas- including porcelain, ironware, gold, and other precious ures must be taken to eliminate the damage caused by relics (Cui 2018). biodeterioration to the main body of cultural relics. The Owing to the change in environmental temperature most commonly used method is the use of safe and green and humidity after the unearthing of the cultural relics chemical biocides. For example, isothiazolinone bioc- from the seawater, they were inevitably prone to micro- ides, biotin, catechin, and nanoscale zinc oxide have been bial damage. Maritime cultural relics from the Nanhai widely used for the protection of cultural relics (Ji et al. No. 1 shipwreck can be divided into wooden hull and 2016; Jing et al. 2017; Ríos et al. 2012; Polo et al. 2010; Li ship carrying cultural relics. Under the synergistic action et al. 2010). At present, the biocide used in Nanhai No. 1 of salt and various microorganisms, wooden cultural rel- is isothiazolinone biocide Euxyl K100. Additionally, the ics had undergone serious physical and chemical damage detection of biodeterioration is indispensable. The vigor - and biodegradation (Hocker 2010; Fors et al. 2012; Fors ous development of next-generation sequencing technol- and Sandström 2006). The original wood components, ogy has led it to be widely used in various biological and including cellulose and lignin, were degraded and lost; medical detection fields (Goodwin et al. 2016; Shokralla the moisture content in wood cells had increased, and et al. 2012). The next-generation sequencing technology the supporting force between wood fibers had decreased, combined with traditional microbial isolation and iden- resulting in the decay of the structure of wooden cultural tification could solve these problems efficiently (Ward relics (Li et al. 1984; Bjordal et al. 1999; Capretti et al. et al. 1990; Dissanayake et al. 2018; Jayawardena et al. 2008), whereas the ironware was ionized in seawater, and 2018), aiming to better protect and study cultural relics. various valences of sulfides were formed under the action Herein, we collected water samples from four different of microorganisms, forming insoluble or slightly soluble types of maritime cultural relics from the Nanhai No. 1 deposits on the surface and inside the wooden cultural shipwreck in September and November 2019, includ- relics. The cultural relics in the Nanhai No. 1 shipwreck ing ironware, porcelain, axe, and hull wood (Fig. 1 and are mainly porcelain and few metal. Owing to seawater Table 1). The microbial community was comprehen - immersion and biological influence, hard and dense con - sively analyzed by optical microscopy, high-throughput cretions were formed on the surfaces of the porcelain and sequencing, and traditional microbial isolation and iden- metal cultural relics, which were difficult to remove. In tification methods. Biocide susceptibility tests and lignin addition, the soluble salts in porcelain and metal cultural and cellulose degradation tests of culturable microorgan- relics were dissolved and recrystallized according to the isms were performed. Our results provide a theoretical changes in environmental factors, resulting in various basis for the protection of maritime cultural relics in the cultural relic diseases. future. During the excavation and protection of maritime cul- tural relics, some biodeterioration problems may arise. Results Bacteria, fungi, and other microorganisms are a major Microbial diversity analysis by high‑throughput problem in the process of cultural heritage protection sequencing because of their potential for biodeterioration (Sterflinger To reveal the composition of the microbial community, and Piñar 2013). Different cultural relics may be prone to we performed high-throughput sequencing of the micro- different microbial colonizations. Wooden cultural relics organisms collected from the water samples of four types are rich in nutrients, hence promoting colonization and of cultural relics. Figure 2A and 2B show the distribution growth of microorganisms (Eleanor et al. 2008; Björdal of dominant bacteria in the 12 water samples. Figure 2A 2012). For example, fungal colonization was found on shows the distribution of the bacteria at the phylum a canoe (Zhang et al. 2019), waterlogged archeological level. Proteobacteria, at the phylum level, accounted for wood found in Sweden showed microbial decay (Bjor- the largest proportion of all samples; however, its abun- dal et al. 1999), and wooden relics from the Xiaobaijiao dance slightly varied, accounting for 47.03–97.38% of the No. 1 were damaged by erosion bacteria and tunneling P an et al. Annals of Microbiology (2023) 73:8 Page 3 of 12 Fig. 1 Sampling pictures of four kinds of maritime cultural relic immersion water samples. Sampled in September 2019 and November 2019, respectively. A Ironware. B Porcelain. C Axe. D Hull wood Table 1 Sampling information of four kinds of maritime cultural relic immersion water samples Cultural relics Sample name Sampling time Water pH Buffer temperature (℃) Ironware NH.I.1 and NH.I.2 2019.9 24.7 9.42 Deionized water, containing 5% sodium sesquicarbonate NH.I.3 2019.11 22.5 9.53 Deionized water, containing 5% sodium sesquicarbonate and 0.5% Isothiazolinone Euxyl K100 Porcelain NH.P.1 and NH.P.2 2019.9 20.1 7.82 Deionized water NH.P.3 2019.11 20.9 6.86 Deionized water Axe NH.A.1 and NH.A.2 2019.9 26.3 7.73 Deionized water NH.A.3 2019.11 21.3 6.65 Deionized water Hull wood NH.W1.1 and NH.W1.2 2019.9 24.5 6.83 Deionized water, containing 0.7‰ Isothiazolinone Euxyl K100 and 10 mmol/L EDTA-2Na NH.W1.3 2019.11 20.6 5.65 Deionized water, containing 0.5% Isothiazolinone Euxyl K100 and 10 mmol/L EDTA-2Na Pan et al. Annals of Microbiology (2023) 73:8 Page 4 of 12 Fig. 2 The relative abundance of microbial communities in four kinds of maritime cultural relic immersion water samples. The relative abundance is shown as a percentage. Phylum and genera are colored according to the legend on the right. A Relative abundance of bacteria in water samples at the phylum level. B Relative abundance of bacteria in water samples at the genera level. C Relative abundance of fungi in water samples at the phylum level. D Relative abundance of fungi in water samples at the genera level community. The samples also contained Actinobacteria significantly different from that collected in September, and Bacteroidetes. Figure 2B and Table S1 show the dis- and the most abundant bacteria were Sphingomonas, tribution of the bacteria at the genus level. The composi - accounting for 22.89%. Comparing the three ironware tion of bacteria, at the genus level, in each water sample water samples, we found a significant difference between was significantly different. The two parallel water sam - NH.I.1, NH.I.2, and NH.I.3 (fungi). Among them, the ples of the same cultural relic collected in September most abundant fungi were Fusarium, Diplocarpon, and were similar and were evidently different from the one Penicillium, accounting for 20.17%, 49.79%, and 31.67%, collected in November. Figure 2C and 2D show the dis- respectively. tribution of the dominant fungi in the 12 water samples. Among the three porcelain water samples, the micro- High-throughput results for fungi in water samples NH. bial composition in the two parallel water samples W1.1 and NH. W1.2 were obtained from a previous study (NH.P.1 and NH.P.2) collected in September slightly dif- (Han et al. 2021). Figure 2C shows the distribution of the fered. Among them, the most abundant bacteria were fungi at the phylum level. Ascomycota was dominant at Panacagrimonas and Nevskia, accounting for 17.91% the phylum level in 10 samples; however, the abundance and 11.67%, respectively. One water sample (NH.P.3) col- slightly varied. In addition, Rozellomycota and Basidio- lected in November significantly differed from that col - mycota were dominant in NH.A.3 and NH.W1.3, respec- lected in September, and the most abundant bacteria tively. Figure 2D and Table S2 show the distribution of were Flavobacterium, accounting for 7.84%. The most fungi at the genus level. We found significant differences abundant fungi in NH.P.1, NH.P.2, and NH.P.3 were Cla- in the composition of fungi in different cultural relics and dosporium, Penicillium, and Trichoderma, accounting for different collection times of the same cultural relics. 12.99%, 61.36%, and 7.26%, respectively. Among the three ironware water samples, the micro- Among the three axe water samples, the micro- bial composition in the two parallel water samples bial composition in the two parallel water samples (NH.I.1 and NH.I.2) collected in September was relatively (NH.A.1 and NH.A.2) collected in September was rela- similar, and Hyphomicrobium was the most abundant tively similar, and the most abundant bacteria were bacteria, accounting for 25.90% and 25.51%, respectively. Pseudonocardia, accounting for 15.39% and 16.51%, One water sample (NH.I.3) collected in November was respectively. One water sample (NH.A.3) collected in P an et al. Annals of Microbiology (2023) 73:8 Page 5 of 12 Isolation and identification of culturable microorganisms November significantly differed from that collected in Maritime cultural relic immersion water samples were September, and the most abundant bacteria were Terri- evenly applied to lysogeny broth (LB) and potato dextrose monas, accounting for 24.84%. In contrast, the two par- agar (PDA) media after gradient dilution. After cultur- allel water samples (NH.A.1 and NH.A.2) collected in ing, purification, and identification, five culturable bacte - September were relatively similar, and the most abun- ria and fungi were isolated (Table 2). The single colony dant fungi were Penicillium, accounting for 76.22% and morphology and microstructure (mycelia and spores) of 45.11%, respectively. One water sample (NH.A.3) col- these fungi were observed and recorded using an optical lected in November significantly differed from that col - microscope (Fig. 3A–E). lected in September, and the most abundant fungi were Fusarium, accounting for 20.99%. The susceptibility test of culturable fungi and dominant Among the three hull wood samples, the bacterial bacteria to biocides composition in the two parallel water samples (NH. Five culturable fungi were obtained from maritime cul- W1.1 and NH.W1.2) collected in September slightly dif- tural relic immersion water samples. Among the five cul - fered. Among them, the most abundant bacteria were turable bacteria, the high-throughput results showed that Pseudomonas and Brucella, accounting for 13.57% and Pseudomonas sp. (NH.W1-B1) was the only dominant 16.41%, respectively. One water sample (NH.W1.3) col- bacteria. Therefore, we used the filter paper diffusion lected in November significantly differed from that col - method to conduct biocide susceptibility tests on five lected in September, and the most abundant bacteria culturable fungi and one dominant bacteria. The inhibi - were Sphingomonas, accounting for 79.97%. Among the tory effects of different biocides vary based on the strain three hull wood water samples, the fungal composition in of bacteria and fungi (Fig. 4). Different biocides had a the two parallel water samples (NH.W1.1 and NH.W1.2) certain inhibitory effect on the five fungi, among which collected in September was relatively similar, and the D7 was the best, and P91 and K100 were inferior. The most abundant fungi were Scedosporium, accounting for results showed that P91 and 20 N inhibited Pseudomonas 65.45% and 79.10%, respectively. One water sample (NH. sp. (NH.W1-B1), while K100 and D7 had no inhibitory W1.3) collected in November was significantly different effect. from that collected in September, and the most abundant fungi were Cutaneotrichosporon, accounting for 25.26%. Ligninolytic and cellulolytic enzymatic activity These results show that there were significant differ - of Pseudomonas sp. (NH.W1‑B1) ences in the composition of microorganisms in different We conducted lignin and cellulose degradation tests on cultural relics and different collection times of the same the dominant bacteria Pseudomonas sp. (NH.W1-B1). cultural relics. We found that in the sodium lignosulfonate agar Table 2 Molecular identification of culturable strains isolated from four kinds of water samples Bacteria Closet relative strain Phylum Similarity (%) Accession number Source NH.P-B1 Staphylococcus epidermidis Firmicutes 100% MN511770.1 Porcelain water sample NH.P-B2 Burkholderia sp. Proteobacteria 99% MN263850.1 Porcelain water sample NH.P-B3 Bacillus subtilis Firmicutes 100% MG705979.1 Porcelain and axe water sample NH.W1-B1 Pseudomonas sp. Proteobacteria 100% JQ660555.1 Hull wood water sample NH.W1-B2 Ochrobactrum sp. Proteobacteria 100% KJ127515.1 Hull wood water sample Fungi Closet relative strain Phylum Similarity (%) Accession number Source NH.I-F1 Cladosporium sp. Deuteromycotina 98% MH325932.1 Ironware water sample NH.I-F2 Cladosporium sp. Deuteromycotina 98% MG746382.1 Ironware water sample NH.I-F3 Cladosporium sp. Deuteromycotina 99% HM535372.1 Ironware and axe water sample NH.P-F1 Penicillium sp. Ascomycota 98% KJ001170.1 Porcelain water sample NH.A-F1 Cladosporium sp. Deuteromycotina 99% MH884146.1 Axe water sample NH.W1-1 Penicillium sp. Ascomycota 100% GU212865.1 Hull wood water sample  NH.W1-2 Penicillium citrinum Ascomycota 100% MN398977.1 Hull wood water sample  NH.W1-3 Scedosporium apiospermum Ascomycota 100% FJ713053.1 Hull wood water sample  NH.W1-4 Fusarium solani Ascomycota 100% MN066126.1 Hull wood water sample  NH.W1-5 Cladosporium sp. Deuteromycotina 99% MN265985.1 Hull wood water sample  Pan et al. Annals of Microbiology (2023) 73:8 Page 6 of 12 Fig. 3 Single colony morphology and microstructure (mycelia and spores) of culturable fungi from water samples. The fungi were inoculated onto the PDA medium and incubated at 28° C for 3 days. The scale is 10 μm. A Cladosporium sp. (NH.I-F1). B Cladosporium sp. (NH.I-F2). C Cladosporium sp. (NH.I-F3). D Penicillium sp. (NH.P-F1). E Cladosporium sp. (NH.A-F1). Bar = 10 µm Fig. 4 The sensitivity of culturable fungi and dominant bacteria to biocides. The size of the inhibition zone indicates the inhibitory effect of biocides on culturable strains. The fungi were cultured at 28℃ for 3 days, and the bacteria were cultured at 37℃ for 1 day Fig. 5 A Pseudomonas sp. (NH.W1-B1) could grow on the sodium lignosulfonate agar medium. B Decolorizing circles of Pseudomonas sp. (NH. W1-B1) grown on the aniline blue agar medium for 5 days. C Decolorizing circles of Pseudomonas sp. (NH.W1-B1) grown on the remazol brilliant blue agar medium for 5 days. D Decolorizing circles of Pseudomonas sp. (NH.W1-B1) grown on the CMC-Na agar medium for 5 days medium with only sodium lignosulfonate as the carbon that Pseudomonas sp. (NH.W1-B1) can produce cellu- source, Pseudomonas sp. can be grown using sodium lose-degrading enzymes. Therefore, we concluded that lignosulfonate (Fig. 5A), whereas on an aniline blue Pseudomonas sp. (NH.W1-B1) had lignin and cellulose agar medium and a remazol brilliant blue agar medium, degradation abilities. decolorizing circles appeared around Pseudomonas sp. (NH.W1-B1) (Fig. 5B, C), indicating that it can produce Discussion lignin-degrading enzymes. In addition, in the CMC-Na Herein, we combined high-throughput sequencing tech- agar medium, after staining with Congo red, a visible nology with traditional microbial isolation and identifica - decolorizing circle (Fig. 5D) was observed, indicating tion methods to detect and explore the biodeterioration P an et al. Annals of Microbiology (2023) 73:8 Page 7 of 12 of four types of maritime cultural relic immersion water lower and Actinobacteria was significantly higher than samples from the Nanhai No. 1 shipwreck. Through com - that of other cultural relic water samples. At the bacterial prehensive detection of microorganisms, we evaluated genus level, the major bacterial species found in ironware the preservation status of maritime cultural relics and water samples were Hyphomicrobium, Pseudonocardia, provided data support for solving biological problems. Sphingomonas, and Pseudomonas and in axe water sam- First, we analyzed the water samples using an optical ples were Pseudonocardia, Nevskia, and Terrimonas. At microscope and observed several fungi-like microor- the genus level, three ironware and one axe water sam- ganisms, indicating evident microbial colonization. We ples were dominated by Ascomycota and one axe water then performed high-throughput sequencing and tradi- sample was dominated by Rozellomycota. At the fungal tional microbial isolation and identification. The results genus level, the major fungal species in ironware water showed that among the five strains of culturable bacteria, samples were Fusarium, Diplocarpon, Penicillium, and Pseudomonas sp. (NH.W1-B1) was the only dominant Acremonium and in the axe water samples were Penicil- bacteria in the hull wood water samples. Additionally, lium and Fusarium. Most of the dominant bacteria in the through lignin and cellulose degradation tests, we found ironware and axe water samples grew over a wide range. that Pseudomonas sp. (NH.W1-B1) had certain lignin Some bacteria can participate in the metabolism of sulfur and cellulose degradation abilities. Furthermore, the bio- and iron, such as Hyphomicrobium (Haaijer et al. 2008), cide sensitivity test showed that biocides P91 and 20 N Pseudonocardia (Pan et al. 2015), and Pseudomonas (Sul- had inhibitory effects on Pseudomonas sp. (NH.W1-B1). tan and Faisal 2016). Some dominant bacteria tend to Furthermore, all the five culturable fungi accounted for a live in a polluted environment, such as Sphingomonas certain proportion of the water samples. In addition, cul- (Leys et al. 2004), Nevskia (Hao et al. 2009), and Terri- turable fungi were sensitive to isothiazolinone biocides. monas (Meng et al. 2020). The dominant fungal species Through the combination and comparison of the data in ironware and axe water samples have a certain heavy from high-throughput sequencing and traditional micro- metal (Rasool and Irum 2014a, b; Rasool and Irum 2014a, bial isolation and identification, we drew the following b) and high salt tolerance (Grishkan and Nevo 2003). conclusions: (1) there were significant differences in the Fusarium can metabolize sulfur and iron (Etemadzadeh composition of microorganisms in water samples from et al. 2016), and Penicillium often colonizes cultural relics different cultural relics; (2) there were significant differ - (Zhang et al. 2019). At present, research on the biodete- ences in the composition of microorganisms in water rioration of cultural relics is mostly focused on wooden samples from the same cultural relics at different col - cultural relics (Zhang et al. 2019; Liu et al. 2017), mural lection times; (3) there were few species of bacteria and cultural relics (Ma et al. 2020; Suphaphimol et al. 2022), fungi that could be cultured, and some dominant strains and stone cultural relics (Zhang et al. 2019), with few may only grow under specific environmental conditions. studies on metal cultural relics. However, we believe that There were certain differences in the biodeterioration in the process of excavation and protection of metal cul- of the different maritime cultural relics. In this study, tural relics, the problem of biodeterioration cannot be ironware and axes were metal cultural relics containing ignored. Additionally, microorganisms that can metabo- iron. Maritime metal cultural relics have concretions lize sulfur and iron may appear during the preservation that are difficult to remove and exhibit a high degree of process of some iron-containing cultural relics. corrosion (Wan 2019). When maritime metal cultural Among all maritime cultural relics unearthed from the relics are salvaged, they need to be rapidly cleaned, rust Nanhai No. 1 shipwreck, porcelain was the most abun- removed, immersed in a buffer, and moisturized. After dant. After porcelain is unearthed from the seawater, it stabilization, they were subjected to subsequent treat- needs to be immersed in deionized water for static desali- ments. The ironware used in this study was immersed nation after removing the surface concretion. Hence, the in deionized water containing sodium sesquicarbonate porcelain used in this study was immersed in deionized and biocide. The pH of the water samples in September water. The pH of the water samples in September and and November exceeded 9.0, which was alkalescent, and November was approximately neutral, and the aver- the average water temperature was 23.6℃. The axe relics age water temperature was 20.5℃. The high-throughput were immersed in deionized water. The pH of the water results showed that Proteobacteria was dominant at the samples in September and November was approximately phylum level. At the genus level, the major bacterial spe- neutral, and the average water temperature was 23.8℃. cies were Panacagrimonas, Nevskia, and Flavobacte- The high-throughput results showed that at the phylum rium. At the phylum level, Ascomycota was dominant. At level, although ironware and axe relics were dominated by the genus level, the major fungal species detected were Proteobacteria, similar to other cultural relic water sam- Cladosporium, Penicillium, and Trichoderma. Thus, the ples, the proportion of Proteobacteria was significantly dominant bacteria in porcelain water samples, such as Pan et al. Annals of Microbiology (2023) 73:8 Page 8 of 12 Panacagrimonas (Xue 2015), Nevskia (Hao et al. 2009), For the biological issues of cultural relics, we need to and Flavobacterium (high salt tolerance) (Yoon et al. regularly monitor biodeterioration and take targeted 2010) (Li et al. 2019), can be isolated from polluted envi- protection measures by observing and studying changes ronments. The dominant fungal species in porcelain in microbial composition. Additionally, deionized water water samples are often found to colonize the surface of should be used for immersion in cultural relics, the cultural relics (Zhang et al. 2019; Zhang et al. 2019) and ion concentration and conductivity of water should be have a high salt tolerance (Mbarki et al. 2017). Porcelain monitored regularly, and the deionized water should be itself is not an organic cultural relic and is not easy to replaced in time to prevent the accumulation of ions and corrode and oxidize. Overall, the preservation conditions nutrients. Finally, the use of biocides is important for are relatively stable. However, owing to its extraordinary controlling biodeterioration. However, the use of bioc- historical significance and value, its protection requires ides involves a series of problems, such as drug resistance attention. and health (Glécia et al. 2018; Kathiravan et al. 2012), so The Nanhai No. 1 shipwreck is an ancient wooden ship, biocides need to be carefully selected and used. and although tens of thousands of precious cultural rel- ics have been unearthed, its maritime wooden cultural Conclusions relics are considered the most important and have the In summary, by analyzing the composition of microbial utmost research and protection value. For some of the communities and detecting damages caused by biode- wooden cultural relics scattered from the shipwreck, the terioration in the four typical maritime cultural relics in concretion should first be cleaned and then immersed the Nanhai No. 1 shipwreck, we achieved a comprehen- in deionized water for moisturization, stabilization, and sive conclusion regarding the biological protection of the preliminary desalination treatment. The hull wood relic cultural relics in the Nanhai No. 1 shipwreck. We found in this study was immersed in deionized water containing significant differences in the composition of microorgan - EDTA-2Na and a biocide. The pH of the water samples isms in different cultural relics and different collection in September and November was weakly acidic, and the times of the same cultural relics. Employing and combin- average water temperature was 22.6℃. The high-through - ing high-throughput sequencing technology with tradi- put results showed that Proteobacteria was dominant at tional microbial isolation and identification methods is the phylum level. At the genus level, the major bacterial crucial to comprehensively detect biodeterioration. Our species were Pseudomonas, Brucella, and Sphingomonas. research provides data support for better protection of At the phylum level, two water samples were dominated maritime cultural relics in the Nanhai No. 1 shipwreck by Ascomycota and one water sample was dominated by and a theoretical basis for the biological protection of Basidiomycota. At the fungal genus level, the main fungal other maritime cultural relics. species detected were Scedosporium, Pseudallescheria, and Cutaneotrichosporon. Thus, the dominant bacteria in hull wood water samples can grow in acidic and high- Materials and methods salt environments (Dhail 2012; Li et al. 2015; Li et al. Sample collection 2018). Additionally, most of the dominant bacteria and The research objects were four types of maritime cul - fungi can be isolated from polluted environments (Leys tural relics: ironware, porcelain, axe, and hull wood, et al. 2004; Obayori et al. 2008; Cycon et al. 2016; Roug- which were immersed in a buffer. The Maritime Silk eron et al. 2015). They also have the ability to degrade Road Museum, Yangjiang City, Guangdong Province, the lignocellulose, such as Pseudomonas (Yang et al. 2018), archeological excavation site of the Nanhai No. 1 ship- Sphingomonas (Masai et al. 1999), Scedosporium (Han wreck, was the sample collection site. Before immer- et al. 2021), and Cutaneotrichosporon (Yaguchi et al. sion, the concretions on the surface of the cultural relics 2020). The lignin and cell degradation tests showed that were thoroughly cleaned. Samplings were performed in the major bacterial species Pseudomonas (NH.W1-B1) September and November 2019. In September, we col- had the ability to degrade lignin and cellulose. Therefore, lected two 50-mL parallel water samples from each of more attention should be paid to the Pseudomonas genus. the four types of cultural relic tanks, labeled NH.I.1 and As the Nanhai No. 1 is a large ancient maritime wooden NH.I.2 (from ironware), NH.P.1 and NH.P.2 (from porce- ship, the growth of microorganisms possibly had an irre- lain), NH.A.1 and NH.A.2 (from axe), and NH.W1.1 and versible impact on its wooden structure during the long- NH.W1.2 (from hull wood). In November, we collected term excavation process, which needs greater attention. one 50-mL water sample from each of the four types of Hence, the protection of maritime cultural relics should cultural relic tanks, labeled NH.I.3, NH.P.3, NH.A.3, and be multifaceted. Biological issues need to be highly val- NH.W1.3 (from ironware, porcelain, axe, and hull wood, ued in addition to conventional cultural relic protection. respectively). The sampling pictures and information of P an et al. Annals of Microbiology (2023) 73:8 Page 9 of 12 the immersed water samples of the four types of mari- and purification step for 1–3 times, we obtained pure time cultural relics are shown in Fig. 1 and Table 1. We cultures of bacteria and fungi and stored them on LB and used 50-mL aseptic centrifuge tubes to collect water PDA agar slant culture media at 4 °C. In order to iden- samples, followed by cryopreservation and transport to tify the species of bacteria, we first extracted the DNA the laboratory for subsequent experiments. from pure bacterial cultures (Abdellaoui et al. 2011) and then used 341F/907R primers to amplify the 16S rRNA Microscopic analysis region of bacteria (Yin et al. 2019). A band of approxi- We used an optical microscope (Nikon E200, Japan) to mately 600 bp in size was obtained. To identify the fungal observe the diluted water samples and recorded them species, we used the T5 Direct PCR Mix kit (TSINGKE, under a 400 × and 1000 × microscope. Additionally, the China) to amplify the fungal culture (Han et al. 2021). microstructures of mycelia and spores of culturable fungi The primers used were ITS1/ITS4 (White et al. 1990), were observed using an optical microscope and recorded and a band of approximately 600 bp in size was obtained. under a 400 × microscope. The PCR products of the above bacteria and fungi were sent to GENEWIZ (Beijing, China) for Sanger sequenc- ing, and the base sequences obtained were compared Total DNA extraction and high‑throughput sequencing with the NCBI databases to determine the bacterial and We extracted total DNA from the 12 water samples. fungal species. Raw sequencing data were downloaded We filtrated 50-mL water samples through a 0.22-µm from the NCBI’s Sequence Read Archive database (study size filter membrane and used the DNeasy PowerWater accession number: PRJNA708180). Kit (QIAGEN, Germany) to extract the total DNA from the filter membrane. Total DNA was sent to Novogene Susceptibility test of culturable microorganisms Genome Sequencing Company. The S5-16SV4 gene to biocides region was amplified with the primers F: GTG CCA Isothiazolinone has been used for cultural relic pro- GCMGCC GCG GTAA and R: GGA CTA CHVGGG tection to inhibit the growth of microorganisms. We TWT CTAAT for bacteria, and the S5-ITS1-5F region selected four types of isothiazolinone derivatives, namely was amplified with the primers F:GCG GTA ATT CCA ® ® Preventol D7 (Lanxess, Germany), Preventol BIT 20 N GCT CCA A and R:AAT CCR AGA ATT TCA CCT CT for (Lanxess, Germany), Preventol P91 (Lanxess, Germany), fungi. First, splice and quality control of the offline data and Euxyl K100 (Schulke, Germany) to test the sensi- (raw PE) using the Illumina Novaseq sequencing plat- tivity of the isolated culturable microorganisms to bio- form were performed to obtain clean tags, followed by cides; the filter paper diffusion method was used to test chimera filtering to obtain effective tags that can be used the sensitivity. First, fungal and bacterial cultures were for subsequent analysis. The effective tags of all samples inoculated evenly in PDA and LB media, respectively. were clustered by operational taxonomic units (OTUs) Five 3-layer aseptic filter papers with a diameter of 7 mm with 97% identity, and the sequences of OTUs were were placed on the media. Next, 15 μL of four different annotated. Based on the results of species annotation, we biocides (0.5%) was added to the filter paper, and distilled selected the top 10 species with the highest abundance in water was added to the middle filter paper as a negative each sample or group at each classification level (phylum, control. The PDA and LB media were cultured at 28℃ genus, and disciplines) and generated a cylindrical accu- for 3 days and 37℃ for 1 day, respectively. The inhibitory mulation diagram of the relative abundance of species. effects of different biocides on different culturable micro u Th s, the community composition of bacteria and fungi organisms were observed and compared. The larger the in the water samples was analyzed using high-throughput diameter of the inhibition zone, the higher is the sensitiv- sequencing. ity of culturable microorganisms to biocides (Zhang et al. 2019). The biocide susceptibility test was repeated thrice. Isolation and identification of culturable microorganisms To isolate culturable microorganisms from the water Ligninolytic and cellulolytic enzymatic activity of dominant samples, we prepared LB and PDA media to culture and bacteria isolate bacteria and fungi, respectively. First, the water −2 Different media were used to identify the cellulose and samples were diluted in a gradient, and then 10 and −3 lignin degradation ability of the dominant bacteria. We 10 dilutions were evenly applied to the surface of the prepared four types of media: sodium lignosulfonate agar LB and PDA media, respectively; these media were cul- medium, aniline blue agar medium, remazol brilliant tured at 37℃ and 28℃, respectively, for 1–5 days. We blue agar medium, and CMC-Na agar medium (Yin et al. then isolated and purified bacteria and fungi of different 2019). The ability to decompose cellulose and lignin was sizes, colors, and shapes. After repeating the isolation Pan et al. Annals of Microbiology (2023) 73:8 Page 10 of 12 Björdal CG (2012) Microbial degradation of waterlogged archaeological wood. evaluated by observing the size of the decolorizing circle J Cult Herit 13:S118–S122. https:// doi. org/ 10. 1016/j. culher. 2012. 02. 003 after culturing at 37℃ for 5 days and marking whether Capretti C, Macchioni N, Pizzo B, Galotta G, Giachi G, Giampaola D (2008) The the strain could grow (Yin et al. 2019). characterization of waterlogged archaeological wood: the three Roman ships found in Naples (Italy). Archaeometry 50(5):855–876. https:// doi. org/ 10. 1111/j. 1475- 4754. 2007. 00376.x Supplementary information Cui J (2018) The mystery of the 800-year shipwreck of Nanhai No.1. Travel The online version contains supplementary material available at https:// doi. World 004:55–59 org/ 10. 1186/ s13213- 022- 01705-4. Cui Y (2019) Brief summery on excavation of Nanhai No1 shipwreck. Study Nat Cultural Heritage 4(10):14–20 Cycon M, Mrozik A, Piotrowska-Seget Z (2016) Bioaugmentation as a strategy Additional file 1: Supplementary Table 1. Relative abundance of domi- for the remediation of pesticide-polluted soil: a review. Chemosphere nant bacteria among four kinds of maritime cultural relic immersion water 172(APR):52–71. https:// doi. org/ 10. 1016/j. chemo sphere. 2016. 12. 129 samples at the genus level. Supplementary Table 2. Relative abundance Da-Wa S, Li-Zhi M, Lu P, Qing-Lin M (2009) Coatings materials used for conser- of dominant fungi among four kinds of maritime cultural relic immersion vation of iron relics. Paint Coatings Ind 39(1):17–19 water samples at the genus level de los Ríos A, PérezOrtega S, Wierzchos J, Ascaso C (2012) Differential effects of biocide treatments on saxicolous communities: case study of the Segovia cathedral cloister (Spain). Int Biodeterior Biodegradation 67:64. https:// Acknowledgements doi. org/ 10. 1016/j. ibiod. 2011. 10. 010 We gratefully acknowledge the assistance of Prof. Hong Guo from the Institute Dhail S (2012) Isolation of potent biosurfactant producing bacteria from oil for Cultural Heritage and History of Science & Technology, University of Sci- spilled marine water and marine sediments. Afr J Biotech 11(103):16751– ence and Technology Beijing. 16757. https:// doi. org/ 10. 5897/ AJB12. 1510 Dissanayake AJ, Purahong W, Wubet T, et al (2018) Direct comparison of Authors’ contributions culture-dependent and culture-independent molecular approaches Jiao Pan: conceptualization, methodology, and writing—reviewing and reveal the diversity of fungal endophytic communities in stems of grape- editing. vine ( Vitis vinifera). Fungal Divers 90:85–107. https:// doi. org/ 10. 1007/ Yeqing Han: data curation and writing—original draft preparation. s13225- 018- 0399-3 Cen Wang, Yu Wang, Kaixuan Ma, and Xinduo Huang: software, data curation, Eleanor TL, Mitchella JI, Hotchkissa S, Eatona RA (2008) Bacterial diversity and validation. associated with archaeological waterlogged wood: ribosomal RNA Zhiguo Zhang, Jing Du, and Yue Chen: resources. clone libraries and denaturing gradient gel electrophoresis (DGGE). Int Naisheng Li: designed the whole project. Biodeterior Biodeg-Radation 61(1):106–116. https:// doi. org/ 10. 1016/j. The authors read and approved the final manuscript. ibiod. 2007. 07. 007 Etemadzadeh SS, Emtiazi G, Etemadifar Z (2016) Heterotrophic bioleaching of Funding sulfur, iron, and silicon impurities from coal by Fusarium oxysporum FE This work was supported by the Natural Science Foundation of Tianjin #1 and Exophiala spinifera FM with growing and resting cells. Curr Microbiol under Grant 19JCZDJC33700 and the National Key R&D Program of China #2 72(6):707–715. https:// doi. org/ 10. 1007/ s00284- 016- 1008-x under Grant 2020YFC1521805. Fors Y, Sandström M (2006) Sulfur and iron in shipwrecks cause conservation concerns. Chem Soc Rev 35(5):399–415. https:// doi. org/ 10. 1039/ b5070 Availability of data and materials 10b The raw sequencing data could be downloaded at the NCBI Sequence Read Fors Y, Jalilehvand F, Risberg ED, Björdal C, Phillips E, Sandström M (2012) Sulfur Archive (SRA) with the study accession number PRJNA708180. and iron analyses of marine archaeological wood in shipwrecks from Table S1: Relative abundance of dominant bacteria among four kinds of the Baltic Sea and Scandinavian waters. J Archaeol Sci 39(7):2521–2532. maritime cultural relic immersion water samples at the genus level. Table S2: https:// doi. org/ 10. 1016/j. jas. 2012. 03. 006 Relative abundance of dominant fungi among four kinds of maritime cultural Gao Meng-Ge, Zhang Qin-Fen, Jin Tao, Luo P, Li Q, Xu RL (2017) Observation relic immersion water samples at the genus level. and damage assessment of microbial diseases in some wooden cultural relics from the ancient marine shipwreck, Ningbo Xiaobaijiao No.1. Sci Declarations Conserv Archaeol 29(06):102–111 Glécia L, Sousa B, Guedes A, Barreto C, Brasil L (2018) Biocides used as additives Ethics approval and consent to participate to biodiesels and their risks to the environment and public health: a Not applicable. review. Molecules 23(10):2698. https:// doi. org/ 10. 3390/ molec ules2 31026 Consent for publication Goodwin S, Mcpherson JD, Mccombie WR (2016) Coming of age: ten years of Not applicable. next-generation sequencing technologies. Nat Rev Genet 17(6):333–351. https:// doi. org/ 10. 1038/ nrg. 2016. 49 Competing interests Grishkan I, Nevo E, Wasser SP (2003) Soil micromycete diversity in the hypersa- The authors declare that they have no competing interests. line dead sea coastal area. Israel. Mycological Progress 2(1):19–28. https:// doi. org/ 10. 1007/ s11557- 006- 0040-9 Haaijer S, Harhangi HR, Meijerink BB, Strous M, Pol A, Smolders AJ (2008) Received: 25 May 2022 Accepted: 28 November 2022 Bacteria associated with iron seeps in a sulfur-rich, neutral pH, freshwater ecosystem. Isme J 2:1231–1242. https:// doi. org/ 10. 1038/ ismej. 2008. 75 Han Y, Huang X, Wang Y, Du J, Ma K, Chen Y (2021) Fungal community and biodeterioration analysis of hull wood and its storage environment of the Nanhai No. 1 Shipwreck. Front Microbiol 11:609475.https:// doi. org/ 10. 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Annals of Microbiology – Springer Journals
Published: Feb 3, 2023
Keywords: Nanhai No. 1 shipwreck; Maritime cultural relics; High-throughput sequencing; Microbial communities; Biodeterioration
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