Culture-independent Molecular Methods Research Articles

Bacterial viability in the built environment of the home.

The built environment (BE) consists of human-made structures and, much like living organisms, is colonized by bacteria that make up the BE microbiome. The BE microbiome can potentially affect human health because of the constant proximity of these bacteria to humans. This has led to increasing public concern of whether the bacteria in the BE are harmful. Previous studies have used approaches based on DNA sequencing to assess the composition of the BE microbiome. However, the extent to which the bacterial DNA in the BE represents viable bacterial cells that could infect human hosts remains unknown. To address this open question we used both culture-based and culture-independent molecular methods to profile bacterial viability of the microbiomes from several BE sites. As part of an undergraduate-led project, we found that the vast majority of the bacterial DNA from the BE is not associated with viable bacteria, suggesting that most bacteria in the BE are dead. To begin to understand the determinants of bacterial viability in the BE we used mock bacterial communities to investigate the effects of temperature, relative humidity, and human interaction on bacterial viability. We found that relative humidity, temperature, and surface material did not have statistically significant effects on BE microbiome viability, but environmental exposure decreased bacterial viability. These results update our conception of the BE microbiome and begin to define the factors that affect BE microbiome viability.

Decoding the microbial universe with metagenomics: a brief insight.

A major part of any biological system on earth involves microorganisms, of which the majority are yet to be cultured. The conventional methods of culturing microbes have given fruitful outcomes yet have limitations. The curiosity for better understanding has led to the development of culture-independent molecular methods that help push aside the roadblocks of earlier methods. Metagenomics unifies the scientific community in search of a better understanding of the functioning of the ecosystem and its component organisms. This approach has opened a new paradigm in advanced research. It has brought to light the vast diversity and novelty among microbial communities and their genomes. This review focuses on the development of this field over time, the techniques and analysis of data generated through sequencing platforms, and its prominent interpretation and representation.

Yeast diversity during the spontaneous fermentation of wine with only the microbiota on grapes cultivated in Japan

Making wine via spontaneous fermentation without sulfur dioxide and commercial yeast (spontaneous winemaking) is increasing in recent year, but there is scant research regarding microbial communities present in Japan during spontaneous winemaking using culture-independent molecular methods. We analyzed fungal communities and populations during laboratory-scale spontaneous winemaking using sterilized labware to avoid winery-resident microbes. In the spontaneous fermentation of four grape varieties (Pinot Noir, Riesling, Koshu, and Koshusanjaku) grown in the same Japanese vineyard, our analysis of yeast and other fungal species by next-generation sequencing based on the ITS1 region demonstrated that Saccharomyces cerevisiae was eventually dominant in seven of 12 fermentation batches (three replications for each grape variety), whereas non-Saccharomyces species (e.g., Schizosaccharomyces japonicus, Lachancea dasiensis, and Hanseniaspora valbyensis) became dominant in four batches at the end of fermentation. In another batch, lactic acid bacteria (LAB) became dominant and the fermentation remained incomplete. Diverse microbes were involved in the spontaneous fermentation (particularly in Koshusanjaku), indicating that residual sugar remained and lactic and acetic acid largely increased. Compared to the control wine made with SO2 and commercial yeast, the concentration of lactic acid was 47-fold higher in the must dominated by L.dasiensis, and the concentrations of acetic acid and lactic acid were 10-fold and 20-fold higher in the must dominated by LAB, respectively. Even when indigenous S.cerevisiae became dominant, the finished wines obtained high sensory-analysis scores for complexity but low scores for varietal typicality, indicating the risk of fermentation with unselected wild yeast on the grapes grown in Japan.

Shaping the subway microbiome through probiotic-based sanitation during the COVID-19 emergency: a pre–post case–control study

BackgroundThe COVID-19 pandemic has highlighted the extent to which the public transportation environment, such as in subways, may be important for the transmission of potential pathogenic microbes among humans, with the possibility of rapidly impacting large numbers of people. For these reasons, sanitation procedures, including massive use of chemical disinfection, were mandatorily introduced during the emergency and remain in place. However, most chemical disinfectants have temporary action and a high environmental impact, potentially enhancing antimicrobial resistance (AMR) of the treated microbes. By contrast, a biological and eco-sustainable probiotic-based sanitation (PBS) procedure was recently shown to stably shape the microbiome of treated environments, providing effective and long-term control of pathogens and AMR spread in addition to activity against SARS-CoV-2, the causative agent of COVID-19. Our study aims to assess the applicability and impact of PBS compared with chemical disinfectants based on their effects on the surface microbiome of a subway environment.ResultsThe train microbiome was characterized by both culture-based and culture-independent molecular methods, including 16S rRNA NGS and real-time qPCR microarray, for profiling the train bacteriome and its resistome and to identify and quantify specific human pathogens. SARS-CoV-2 presence was also assessed in parallel using digital droplet PCR. The results showed a clear and significant decrease in bacterial and fungal pathogens (p < 0.001) as well as of SARS-CoV-2 presence (p < 0.01), in the PBS-treated train compared with the chemically disinfected control train. In addition, NGS profiling evidenced diverse clusters in the population of air vs. surface while demonstrating the specific action of PBS against pathogens rather than the entire train bacteriome.ConclusionsThe data presented here provide the first direct assessment of the impact of different sanitation procedures on the subway microbiome, allowing a better understanding of its composition and dynamics and showing that a biological sanitation approach may be highly effective in counteracting pathogens and AMR spread in our increasingly urbanized and interconnected environment.2f-kwnMvWGEoeLQ9ACdzrQVideo

Preventing Laboratory-Acquired Brucellosis in the Era of MALDI-TOF Technology and Molecular Tests: A Narrative Review

Brucellosis is one of the most common etiologies of laboratory-acquired infections worldwide, and handling of living brucellae should be performed in a Class II biological safety cabinet. The low infecting dose, multiple portals of entry to the body, the wide variety of potentially contaminated specimens, and the unspecific clinical manifestations of human infections facilitate the unintentional transmission of brucellae to laboratory personnel. Work accidents such as spillage of culture media cause only a small minority of exposures, whereas &gt;80% of events result from unfamiliarity with the phenotypic features of the genus, misidentification of isolates, and unsafe laboratory practices such as working on an open bench without protective goggles or gloves or the aerosolization of bacteria. The bacteriological diagnosis of brucellae by traditional methods is simple and straightforward but requires extensive manipulation of the isolates, and, nowadays, many laboratory technicians are not familiar with the genotypic features of the genus, resulting in inadvertent exposure and contagion. Detection of brucellar infections by culture-independent molecular methods is safe, but the identification of the organism using MALDI-TOF technology is not hazard-free, requiring an initial bacterial inactivation step to avoid transmission. Unfortunately, these novel and safer methods are costly and frequently unavailable in resource-limited endemic countries.

Recent advances in understanding the ecology of the filamentous bacteria responsible for activated sludge bulking.

Activated sludge bulking caused by filamentous bacteria is still a problem in wastewater treatment plants around the world. Bulking is a microbiological problem, and so its solution on species-specific basis is likely to be reached only after their ecology, physiology and metabolism is better understood. Culture-independent molecular methods have provided much useful information about this group of organisms, and in this review, the methods employed and the information they provide are critically assessed. Their application to understanding bulking caused by the most frequently seen filament in Japan, 'Ca. Kouleothrix', is used here as an example of how these techniques might be used to develop control strategies. Whole genome sequences are now available for some of filamentous bacteria responsible for bulking, and so it is possible to understand why these filaments might thrive in activated sludge plants, and provide clues as to how eventually they might be controlled specifically.

Fingerprinting Saccharomyces cerevisiae Strains Using Next Generation Sequencing of PCR Amplicons Generated from Delta Elements

Identifying beer spoilage microbes is readily accomplished using PCR analyses targeting specific types of microbes, but the general classification of wild yeast versus brewing yeast using cultivation independent molecular methods has remained challenging. The approach presented in this study utilized genetic fingerprint matching to determine if an unknown yeast isolate matches the fingerprint of catalogued brewing yeast strains. Interdelta Next Generation Sequencing (NGS) fingerprints were produced using PCR amplification of delta elements, also known as long terminal repeat sequences of transposons of yeast. Fingerprints identifying different yeast strains were generated by processing reads produced by NGS of the interdelta PCR amplicons using open source software. The interdelta NGS fingerprint comprises DNA sequences that can be recorded, compared, and utilized as a fixed reference for yeast strain identification. Interdelta NGS fingerprints were shown to be reproducible and capable of distinguishing between strains of brewing yeast and wild yeast. Experimental yeast contamination demonstrated the utility of the approach for identifying in-house brewing yeast cross contamination versus foreign wild yeast contamination.

Diversity of fungal assemblages in rhizosphere and endosphere of blueberry (Vaccinium spp.) under field conditions revealed by culturing and culture-independent molecular methods.

Mycorrhizae are important to plants in improving nutrient absorption and stress resistance. To study mycorrhizal fungal diversity in blueberry, we combined culture method and culture-independent molecular method to analyze the root endosphere and rhizosphere fungi in three different cultivars. We obtained 212 isolates with a culture method and classified them into 40 types according to their morphological characteristics. Then, we amplified the internal transcribed spacer sequence and found rich species diversity. With high-throughput sequencing, 561 operational taxonomic units (OTUs) were annotated based on a 97% similarity level cutoff. The alpha diversity index revealed that the fungal abundance and diversity in the rhizosphere were higher than those in the endosphere. The dominant phyla were Ascomycota and Basidiomycota and the dominant genus was Oidiodendron. We also constructed the plant-fungus symbiotic system by inoculating in vitro stock shoots, which lays a theoretical foundation for further research to develop and utilize the dominant mycorrhizal fungi of blueberry.

Cultured Bacteria Provide Insight into the Functional Potential of the Coral-Associated Microbiome.

ABSTRACTImproving the availability of representative isolates from the coral microbiome is essential for investigating symbiotic mechanisms and applying beneficial microorganisms to improve coral health. However, few studies have explored the diversity of bacteria which can be isolated from a single species. Here, we isolated a total of 395 bacterial strains affiliated with 49 families across nine classes from the coral Pocillopora damicornis. Identification results showed that most of the strains represent potential novel bacterial species or genera. We also sequenced and assembled the genomes of 118 of these isolates, and then the putative functions of these isolates were identified based on genetic signatures derived from the genomes and this information was combined with isolate-specific phenotypic data. Genomic information derived from the isolates identified putative functions including nitrification and denitrification, dimethylsulfoniopropionate transformation, and supply of fixed carbon, amino acids, and B vitamins which may support their eukaryotic partners. Furthermore, the isolates contained genes associated with chemotaxis, biofilm formation, quorum sensing, membrane transport, signal transduction, and eukaryote-like repeat-containing and cell-cell attachment proteins, all of which potentially help the bacterium establish association with the coral host. Our work expands on the existing culture collection of coral-associated bacteria and provides important information on the metabolic potential of these isolates which can be used to refine understanding of the role of bacteria in coral health and are now available to be applied to novel strategies aimed at improving coral resilience through microbiome manipulation.IMPORTANCE Microbes underpin the health of corals which are the building blocks of diverse and productive reef ecosystems. Studying the culturable fraction of coral-associated bacteria has received less attention in recent times than using culture-independent molecular methods. However, the genomic and phenotypic characterization of isolated strains allows assessment of their functional role in underpinning coral health and identification of beneficial microbes for microbiome manipulation. Here, we isolated 395 bacterial strains from tissues of Pocillopora damicornis with many representing potentially novel taxa and therefore providing a significant contribution to coral microbiology through greatly enlarging the existing cultured coral-associated bacterial bank. Through analysis of the genomes obtained in this study for the coral-associated bacteria and coral host, we elucidate putative metabolic linkages and symbiotic establishment. The results of this study will help to elucidate the role of specific isolates in coral health and provide beneficial microbes for efforts aimed at improving coral health.

Evaluation of the variations in chemical and microbiological properties of the sourdoughs produced with selected lactic acid bacteria strains during fermentation.

This research aimed to analyze variations in chemical properties, microbiological characteristics and generated volatile organic compounds (VOCs) profile during sourdough fermentation. Sourdoughs were collected from different cities in Turkey at two different times and lactic acid bacteria (LAB) in the samples were identified with culture-independent and culture-dependent molecular methods. According to culture-dependent methodology, thirteen LAB species were identified. Lactobacillus spp. were identified as the major group according to MiSeq Illumina analysis. Technological potential of commonly isolated LAB species was evaluated. Due to high frequency of isolation, Fructilactobacillus sanfranciscensis and Lactiplantibacillus plantarum strains were better investigated for their technological traits useful in sourdough production. Experimental sourdoughs were produced with mono- and dual-culture of the selected strains and chemical properties and microbiological characteristics, as well as VOCs profile of the sourdoughs, were subjected to multivariate analysis which showed the relevance of added starter, in terms of acidification and VOCs profile.

The Effect of Radiation and Chemoradiation Therapy on the Head and Neck Mucosal Microbiome: A Review.

Radiation (RT) and chemoradiation therapy (CRT) play an essential role in head and neck cancer treatment. However, both cause numerous side effects in the oral cavity, paranasal sinuses, and pharynx, having deleterious consequences on patients’ quality of life. Concomitant with significant advances in radiation oncology, much attention has turned to understanding the role of the microbiome in the pathogenesis of treatment-induced tissue toxicity, to ultimately explore microbiome manipulation as a therapeutic intervention. This review sought to discuss current publications investigating the impact of RT and CRT-induced changes on the head and neck microbiome, using culture-independent molecular methods, and propose opportunities for future directions. Based on 13 studies derived from a MEDLINE, EMBASE, and Web of Science search on November 7, 2021, use of molecular methods has uncovered various phyla and genera in the head and neck microbiome, particularly the oral microbiome, not previously known using culture-based methods. However, limited research has investigated the impact of RT/CRT on subsites other than the oral cavity and none of the studies aimed to examine the relationship between the head and neck microbiome and treatment effectiveness. Findings from this review provide helpful insights on our current understanding of treatment-induced oral mucositis, dental plaque, and caries formation and highlight the need for future research to examine the effect of RT/CRT on the sinonasal and oropharyngeal microbiome. In addition, future research should use larger cohorts, examine the impact of the microbiome on treatment response, and study the effect of manipulating the microbiome to overcome therapy resistance.

Perspective on the clone library method for infectious diseases.

Recently, culture-independent molecular methods, such as DNA sequencing techniques targeting the 16S-ribosomal RNA (rRNA) gene and/or other housekeeping genes with Sanger method-based technologies, next generation sequencing (NGS), and metagenomic analysis, have been developed for detecting microorganisms in the human body; these can provide information on microbiomes of samples from individuals with or without infectious diseases. Determining the bacterial species is crucial in identifying causative bacteria of upper and lower respiratory tract infections, especially for Streptococcus species, but NGS analysis is often not precise enough to identify bacteria at the species level. This review briefly introduces previous observations of the microbiome of samples from various respiratory and other infections assessed using the clone library method with Sanger sequencing of the 16S-rRNA gene. On analysis of 16S-rRNA gene-sequence data of bronchoalveolar lavage fluid obtained from pneumonia lesions in patients with bacterial pneumonia and lung abscess, anaerobes are often detected in non-elderly patients with pneumonia, and the detection rate of Staphylococcus aureus in patients with hospital-acquired pneumonia is lower than that previously reported. Analysis of pleural effusion samples from patients with pleurisy indicated a more important role of anaerobes than previous believed. The other topics reviewed include microbiomes of nontuberculous mycobacteriosis and lower respiratory tract infections in children with permanent tracheostomy due to neuromuscular disorders, in nasal discharge, in bacterial vaginosis, in the intracystic fluid of postoperative maxillary cyst, and in bacterial conjunctivitis; urine microbiota in urethritis; fecal microbiota; and newly detected infectious organisms in the human respiratory tract.

Characterisation of Environmental Biofilms Colonising Wall Paintings of the Fornelle Cave in the Archaeological Site of Cales.

Caves present unique habitats for the development of microbial communities due to their peculiar environmental conditions. In caves decorated with frescoes, the characterization of microbial biofilm is important to better preserve and safeguard such artworks. This study aims to investigate the microbial communities present in the Fornelle Cave (Calvi Risorta, Caserta, Italy) and their correlation with environmental parameters. The cave walls and the wall paintings have been altered by environmental conditions and microbial activity. We first used light microscopy and scanning electron microscopy (SEM) and X-ray diffraction to characterise the biofilm structure and the mineral composition of substrata, respectively. Then, using both culture-dependent (Sanger sequencing) and culture-independent (automated ribosomal intergenic spacer analysis, ARISA) molecular methods, we demonstrated that the taxonomic composition of biofilms was different across the three substrata analysed and, in some cases, positively correlated with some environmental parameters. We identified 47 taxa in the biofilm samples, specifically 8 bacterial, 18 cyanobacterial, 14 algal and 7 fungal taxa. Fungi showed the highest number of ARISA types on the tuff rock, while autotrophic organisms (cyanobacteria and algae) on the frescoes exposed to light. This study confirms that caves constitute a biodiversity-rich environment for microbial taxa and that, in the presence of wall paintings, taxonomic characterization is particularly important for conservation and restoration purposes.

Factors related to the composition and diversity of wound microbiota investigated using culture-independent molecular methods: a scoping review.

All open wounds are often colonized by commensal microbes as a loss of skin can provide a ready portal of entry for microorganisms. Although the wound microbiota is known to be associated with wound infection and with delayed healing, the factors related to the formations of wound microbiota contributing to such poor clinical outcomes are not clear and have not led to effective infection prevention interventions. This review aimed to scope the factors related to the composition and diversity of wound microbiota that have been investigated using culture-independent molecular methods. Original articles on wound microbiota published from January 1986 to February 2020 were included in this review. Thirty-one articles met the inclusion criteria and were grouped according to wound types: chronic, acute, and animal model wounds. The factors identified were categorized according to patient characteristics, wound characteristics, treatment, and sampling. Although some studies reported the effect size of the factors, the values were small. No studies elucidated the mechanism of wound microbiota formation. The results of this scoping review highlight that the factors associated with the diversity of wound microbiota are poorly understood and that further studies are needed.

Rapid macrolide and amikacin resistance testing for Mycobacterium abscessus in people with cystic fibrosis.

Introduction. Mycobacterium abscessus complex (MABSC) is an environmental organism and opportunistic pathogen. MABSC pulmonary infections in people with cystic fibrosis are of growing clinical concern. Resistance data guide the use of macrolides and amikacin in MABSC pulmonary disease treatment. MABSC can acquire resistance against macrolides or amikacin via 23S or 16S rRNA gene mutations, respectively.Gap Statement. Current culture-based methods for MABSC detection and antibiotic resistance characterization are typically prolonged, limiting their utility to directly inform treatment or clinical trials. Culture-independent molecular methods may help address this limitation.Aim. To develop real-time PCR assays for characterization of key 23S or 16S rRNA gene mutations associated with constitutive resistance in MABSC.Methodology. We designed two real-time PCR assays to detect the key 23S and 16S rRNA gene mutations. The highly conserved nature of rRNA genes was a major design challenge. To reduce potential cross-reactivity, primers included non-template bases and targeted single-nucleotide polymorphisms unique to MABSC. We applied these assays, as well as a previously developed real-time PCR assay for MABSC detection, to 968 respiratory samples from people with cystic fibrosis. The results from the molecular methods were compared to those for gold standard culture methods and 23S and 16S rRNA gene sequencing.Results.The real-time PCR MABSC detection assay provided a sensitivity of 83.8 % and a specificity of 97.8 % compared to culture. The results from the real-time PCR resistance detection assays were mostly concordant (>77.4 %) with cultured isolate sequencing. The real-time PCR resistance detection assays identified several samples harbouring both resistant and susceptible MABSC, while culture-dependent methods only identified susceptible MABSC in these samples.Conclusion. Using the molecular methods described here, results for health care providers or researchers could be available days or weeks earlier than is currently possible via culture-based antibiotic susceptibility testing.

Extended-culture and culture-independent molecular analysis of the airway microbiota in cystic fibrosis following CFTR modulation with ivacaftor

BackgroundTreatment with Ivacaftor provides a significant clinical benefit in people with cystic fibrosis (PWCF) with the class III G551D-CFTR mutation. This study determined the effect of CFTR modulation with ivacaftor on the lung microbiota in PWCF. MethodsUsing both extended-culture and culture-independent molecular methods, we analysed the lower airway microbiota of 14 PWCF, prior to commencing ivacaftor treatment and at the last available visit within the following year. We determined total bacterial and Pseudomonas aeruginosa densities by both culture and qPCR, assessed ecological parameters and community structure and compared these with biomarkers of inflammation and clinical outcomes. ResultsSignificant improvement in FEV1, BMI, sweat chloride and levels of circulating inflammatory biomarkers were observed POST-ivacaftor treatment. Extended-culture demonstrated a higher density of strict anaerobic bacteria (p = 0.024), richness (p = 1.59*10−4) and diversity (p = 0.003) POST-treatment. No significant difference in fold change was observed by qPCR for either total bacterial 16S rRNA copy number or P. aeruginosa density for oprL copy number with treatment. Culture-independent (MiSeq) analysis revealed a significant increase in richness (p = 0.03) and a trend towards increased diversity (p = 0.07). Moreover, improvement in lung function, richness and diversity displayed an inverse correlation with the main markers of inflammation (p < 0.05). ConclusionsFollowing treatment with ivacaftor, significant improvements in clinical parameters were seen. Despite modest changes in overall microbial community composition, there was a shift towards a bacterial ecology associated with less severe CF lung disease. Furthermore, a significant correlation was observed between richness and diversity and levels of circulating inflammatory markers.

Fermentation of African nightshade leaves with lactic acid bacterial starter cultures

The interest in the consumption of African indigenous leafy vegetables increased in African countries, e.g. Kenya, within the last years. One example of African indigenous leafy vegetables is African nightshade (Solanum scabrum) which is nutritious, rich in proteins and micronutrients and therefore could contribute to a healthy diet. African nightshade has several agricultural advantages. However, the most important disadvantage is the fast perishability which leads to enormous post-harvest losses. In this study, we investigated the fermentation of African nightshade as a post-harvest processing method to reduce post-harvest losses. The two lactic acid bacterial starter strains Lactiplantibacillus plantarum BFE 5092 and Limosilactobacillus fermentum BFE 6620 were used to inoculate fermentations of African nightshade leaves with initial counts of 106–107 cfu/ml. Uninoculated controls were conducted for each fermentation trial. Fermentations were performed both in Kenya and in Germany. The success of the inoculated starter cultures was proven by the measurement of pH values and determination of lactic acid concentration. Lactobacilli strains dominated the microbiota of the starter inoculated samples in contrast to the non-inoculated controls. This was supported by classical culture-dependent plating on different microbiological media as well as by the culture-independent molecular biological methods denaturing gradient gel electrophoresis and 16S rRNA gene high-throughput amplicon sequencing. We could demonstrate that the use of the selected starter cultures for fermentation of African nightshade leaves led to controlled and reliable fermentations with quick acidification. Thus, controlled fermentation with appropriate starter cultures is a promising method for post-harvest treatment of African nightshade leaves.

Molecular Techniques to Study Microbial Wastewater Communities

wastewater treatment (WT) is of major importance on modern cities, removing wastewater pollutants resultant from anthropogenic activities. The unique abilities of microbes to degrade organic matter, remove nutrients and transform toxic compounds into harmless products make them essential players in waste treatment. The microbial diversity determines the metabolic pathways that may occur in WT and quality of treated wastewater. Therefore, understanding WT microbial community structure, distribution, and metabolic functioning is essential for development and optimization of efficient microbial engineering systems. Since cultivation methods can only detect a small fraction of the microbial diversity, the use of culture-independent molecular methods has circumvented this issue, allowing unprecedented access to genes and genomes used for microbial composition and function evaluation. Traditional approaches like RAPD, DGGE, ARDRA, RISA, SSCP, T-RFLP, and FISH and modern approaches like microarray, qPCR, and metagenomics are essential techniques for identifying and depicting the total microbial community structure and their interaction with environmental and biotic factors. Thus, this review describes traditional and state of the art molecular techniques which provide insights into phylogenetic and functional activities of microbial assemblages in a WT system.

Irradiation with medical diode laser as a new method of spot-elimination of microorganisms to preserve historical cellulosic objects and human health

The aim of the study was to optimise operating parameters of a diode laser (810 nm), that could be used to effectively disinfect very small, microbiologically contaminated spots on historical cellulosic objects from the Auschwitz-Birkenau State Museum, and thus limit unnecessary treatment of whole objects. The scope included: assessment of microbiological contamination of the historical materials using culture-dependent (MALDI-TOF MS, 16S rRNA sequencing and macro- and microscopic observations) and culture-independent (NGS sequencing) molecular methods; optimisation of laser irradiation based on the assessment of biocidal effectiveness against 14 microbial strains and determination of changes in colour, and evaluation of the impact of optimal laser variant on the optical (colour measurement spectrophotometry), morphological (SEM), structural (FTIR) and chemical (XPS) properties of the model and historical materials. The small spots were colonised with microorganisms harmful to the objects and posing a threat to human health. The best variant of laser parameters was: 0.3 W of power, continuous wave mode, 2 min of exposure and 2 repetitions, which resulted in 92.17–100.00% reduction in the number of bacteria and complete elimination of 5 out of 6 strains of filamentous fungi, and at the same time it did not have an adverse effect on the colour, morphology, structure and degree of oxidation of the tested materials. A slight increase in the surface polarity was not related to cellulose, but protective substances.

Biotic and Abiotic Imprints on Mg-Rich Stromatolites: Lessons from Lake Salda, SW Turkey

Modern hydrated Mg rich stromatolites are actively growing along the shallow shorelines of Lake Salda (SW Turkey). An integrated approach involving isotopic, mineralogical, microscopic, and organic/geochemical techniques along with culture-independent molecular methods were applied to various lake samples to assess the role of microbial processes on stromatolite formation. This study further explores the biosignature preservation potential of fossil stromatolites by comparing with textures, lipid profiles and isotopic composition of the modern stromatolites. Similar lipid profile and δ13C isotope values in active and fossil stromatolites argue that CO2 cycling delicately balanced between photosynthetic and heterotrophic (aerobic) activity as in the active ones may have regulated stromatolite formation in the lake. A decrease in the exopolymeric substances (EPS) profile of the mat and concurrent hydromagnesite precipitation imply a critical role for EPS in the formation of stromatolite. Consistently, a discrete, discontinuous lamination and clotted micropeloidal textures with cyanobacterial remnants in the fossil stromatolites likely refer to partial degradation of EPS, creating local nucleation sites and allowing precipitation of hydrated Mg minerals and provide a link to the active microbial mat in the modern stromatolites. Our results for the first time provide strong evidence for close coupling of cyanobacterial photosynthesis and aerobic heterotrophic respiration on hydromagnesite textures involved in the stromatolite formation of Lake Salda. The creation of photosynthesis induced high-pH conditions combined with a change in the amount and properties of the EPS and the repetition of these processes over time seems to be a possible pathway for stromatolite growth in the lake. Understanding these microbial symbioses and their mineralized records may provide new insights on the formation mechanism of Mg-rich carbonates not only for terrestrial geological records but also for planetary bodies like Mars, where hydrated Mg-carbonate deposits have been identified in possible paleolake deposits at Jezero crater, the landing site of the NASA Mars 2020 rover.