Cyanobacteria and Algae in Clouds and Rain in the Area of puy de Dôme, Central France

ABSTRACT The atmosphere contains diverse living microbes, of which the heterotrophic community has been the best studied. Microbes with other trophic modes, such as photoautotrophy, have received much less attention. In this study, culture-independent and dependent methods were used to examine the presence and diversity of oxygenic photoautotrophic microbes in clouds and rain collected at or around puy de Dôme Mountain, central France. Cloud water was collected from the summit of puy de Dôme (1,465 m above sea level [a.s.l.]) for cultivation and metagenomic analysis. Cyanobacteria, diatoms, green algae, and other oxygenic photoautotrophs were found to be recurrent members of clouds, while green algae affiliated with the Chlorellaceae were successfully cultured from three different clouds. Additionally, rain samples were collected below the mountain from Opme meteorological station (680 m a.s.l.). The abundance of chlorophyll a-containing cells and the diversity of cyanobacteria and green algae in rain were assessed by flow cytometry and amplicon sequencing. The corresponding downward flux of chlorophyll a-containing organisms to the ground, entering surface ecosystems with rain, varied with time and was estimated to be between ∼1 and >300 cells cm−2 day−1 during the sampling period. Besides abundant pollen from Pinales and Rosales, cyanobacteria of the Chroococcidiopsidales and green algae of the Trebouxiales were dominant in rain samples. Certain members of these taxa are known to be ubiquitous and stress tolerant and could use the atmosphere for dispersal. Overall, our results indicate that the atmosphere carries diverse, viable oxygenic photoautotrophic microbes and acts as a dispersal vector for this microbial guild. IMPORTANCE Information regarding the diversity and abundance of oxygenic photoautotrophs in the atmosphere is limited. More information from diverse locations is needed. These airborne organisms could have important impacts upon atmospheric processes and on the ecosystems they enter after deposition. Oxygenic photoautotrophic microbes are integral to ecosystem functioning, and some have the potential to affect human health. A better understanding of the diversity and the movements of these aeolian dispersed organisms is needed to understand their ecology, as well as how they could affect ecosystems and human health.

Download Full-text

The Diversity of Root-Associated Endophytic Fungi from Four Epiphytic Orchids in China

Diversity ◽

10.3390/d13050197 ◽

2021 ◽

Vol 13 (5) ◽

pp. 197

Author(s):

Tao Wang ◽

Miao Chi ◽

Ling Guo ◽

Donghuan Liu ◽

Yu Yang ◽

...

Keyword(s):

Endophytic Fungi ◽

Southern China ◽

Culture Method ◽

Amplicon Sequencing ◽

Community Diversity ◽

Mycorrhizal Symbiosis ◽

Epiphytic Orchids ◽

Culture Independent ◽

Almost All

Root-associated endophytic fungi (RAF) are found asymptomatically in almost all plant groups. However, little is known about the compositions and potential functions of RAF communities associated with most Orchidaceae species. In this study, the diversity of RAF was examined in four wild epiphytic orchids, Acampe rigida, Doritis pulcherrima, Renanthera coccinea, and Robiquetia succisa, that occur in southern China. A culture-independent method involving Illumina amplicon sequencing, and an in vitro culture method, were used to identify culturable fungi. The RAF community diversity differed among the orchid roots, and some fungal taxa were clearly concentrated in a certain orchid species, with more OTUs being detected. By investigating mycorrhizal associations, the results showed that 28 (about 0.8%) of the 3527 operational taxonomic units (OTUs) could be assigned as OMF, while the OTUs of non-mycorrhizal fungal were about 99.2%. Among the OMFs, Ceratobasidiaceae OTUs were the most abundant with different richness, followed by Thelephoraceae. In addition, five Ceratobasidium sp. strains were isolated from D. pulcherrima, R. succisa, and R. coccinea roots with high separation rates. These culturable Ceratobasidium strains will provide materials for host orchid conservation and for studying the mechanisms underlying mycorrhizal symbiosis.

Download Full-text

Culture-Independent and Culture-Dependent Characterization of the Black Soldier Fly Gut Microbiome Reveals a Large Proportion of Culturable Bacteria with Potential for Industrial Applications

Microorganisms ◽

10.3390/microorganisms9081642 ◽

2021 ◽

Vol 9 (8) ◽

pp. 1642

Author(s):

Dorothee Tegtmeier ◽

Sabine Hurka ◽

Sanja Mihajlovic ◽

Maren Bodenschatz ◽

Stephanie Schlimbach ◽

...

Keyword(s):

Gut Microbiome ◽

Amplicon Sequencing ◽

Industrial Applications ◽

Culture Collection ◽

Rrna Gene ◽

Organic Substrates ◽

Culturable Bacteria ◽

Black Soldier Fly ◽

Culture Independent ◽

Culture Dependent

Black soldier fly larvae (BSFL) are fast-growing, resilient insects that can break down a variety of organic substrates and convert them into valuable proteins and lipids for applications in the feed industry. Decomposition is mediated by an abundant and versatile gut microbiome, which has been studied for more than a decade. However, little is known about the phylogeny, properties and functions of bacterial isolates from the BSFL gut. We therefore characterized the BSFL gut microbiome in detail, evaluating bacterial diversity by culture-dependent methods and amplicon sequencing of the 16S rRNA gene. Redundant strains were identified by genomic fingerprinting and 105 non-redundant isolates were then tested for their ability to inhibit pathogens. We cultivated representatives of 26 genera, covering 47% of the families and 33% of the genera detected by amplicon sequencing. Among these isolates, we found several representatives of the most abundant genera: Morganella, Enterococcus, Proteus and Providencia. We also isolated diverse members of the less-abundant phylum Actinobacteria, and a novel genus of the order Clostridiales. We found that 15 of the isolates inhibited at least one of the tested pathogens, suggesting a role in helping to prevent colonization by pathogens in the gut. The resulting culture collection of unique BSFL gut bacteria provides a promising resource for multiple industrial applications.

Download Full-text

Dynamic Subspecies Population Structure of Vibrio Cholerae in Dhaka, Bangladesh

10.21203/rs.3.rs-364004/v1 ◽

2021 ◽

Author(s):

Mohammad Islam ◽

Tania Nasreen ◽

Kevin Y. H. Liang ◽

Fatema-Tuz Johura ◽

Paul C. Kirchberger ◽

...

Keyword(s):

Vibrio Cholerae ◽

Temporal Dynamics ◽

Sampling Period ◽

Amplicon Sequencing ◽

Rapid Expansion ◽

Capital City ◽

Dhaka City ◽

Physiochemical Parameters ◽

Subspecies Level ◽

Abundance And Distribution

Abstract Cholera has been endemic to the Ganges delta for centuries. Although the causative agent, Vibrio cholerae, is autochthonous to coastal and brackish water, cholera occurs continually in Dhaka, the inland capital city of Bangladesh which is surrounded by fresh water. Despite the persistence of this problem, little is known about the environmental abundance and distribution of lineages of V. cholerae, the most important being the pandemic generating lineage (PG) consisting mostly of serogroup O1 strains. To understand spatial and temporal dynamics of PG and other lineages belonging to the V. cholerae species in surface water in and around Dhaka city, we used qPCR and high throughput amplicon sequencing. Seven different freshwater sites across Dhaka were investigated for six consecutive months and physiochemical parameters were measured in situ. Total abundance of V. cholerae was found to be relatively stable throughout the six months sampling period, with 2×105 to 4×105 genome copies/L at six sites and around 5 ×105 genome copies/L at the site located in the most densely populated part of Dhaka city. PG O1 V. cholerae was present in high abundance during the entire sampling period and composed between 24-92% of the total V. cholerae population, only showing occasional but sudden reductions in abundance. In instances where PG O1 lost its dominance, other lineages underwent a rapid expansion while the size of the total V. cholerae population remained almost unchanged. Intraspecies richness of V. cholerae was positively correlated to salinity, conductivity and total dissolved solids (TDS), while it was negatively correlated to dissolved oxygen (DO) concentration in water. Interestingly, negative correlation was observed specifically between PG O1 and salinity, even though the changes in this variable were minor (0-0.8 ppt). Observations in this study suggest that at the subspecies level, population composition of naturally occurring V. cholerae can be influenced by fluctuations in environmental factors, which can lead to altered competition dynamics among the lineages.

Download Full-text

The Interior Surfaces of Wooden Barrels Are an Additional Microbial Inoculation Source for Lambic Beer Production

Applied and Environmental Microbiology ◽

10.1128/aem.02226-18 ◽

2018 ◽

Vol 85 (1) ◽

Cited By ~ 5

Author(s):

J. De Roos ◽

D. Van der Veken ◽

L. De Vuyst

Keyword(s):

Microbial Community ◽

Amplicon Sequencing ◽

Microbial Inoculation ◽

Culture Independent ◽

Pressure Water ◽

Cleaning Procedures ◽

Environmental Air ◽

Beer Production ◽

Different Characteristics ◽

Culture Dependent

ABSTRACTTraditional lambic beer production takes place through wort inoculation with environmental air and fermentation and maturation in wooden barrels. These wooden casks or foeders are possible additional inoculation sources of microorganisms for lambic worts. To date, however, these lambic barrels have been examined only with culture-dependent techniques, thereby missing a portion of the microorganisms present. Moreover, the effects of the cleaning procedures (involving high-pressure water and/or fumigation) and the barrel type on the microbial community structures of the interior surfaces of wooden lambic barrels were unclear. The culture-dependent plating and culture-independent amplicon sequencing of swab samples obtained from the interior surfaces of different wooden casks and foeders used for traditional lambic beer production in Belgium revealed that the microbial compositions of these surfaces differed statistically throughout the barrel-cleaning procedures applied. At the end of the cleaning procedures, amplicon sequencing still detected fermentation- and maturation-related microorganisms, although only a few colonies were still detectable using culture-dependent methods. It is possible that some of the surviving microorganisms were missed due to the presence of many of these cells in a viable but not culturable state and/or engrained deeper in the wood. These surviving microorganisms could act as an additional inoculation source, besides brewery air and brewery equipment, thereby helping to establish a stable microbial community in the wort to diminish batch-to-batch variations in fermentation profiles. Furthermore, the microbial compositions of the interior barrel surfaces differed statistically based on the barrel type, possibly reflecting different characteristics of the lambic barrels in terms of age, wood thickness, and wood porosity.IMPORTANCEAlthough the coolship step is generally regarded as the main contributor to the spontaneous inoculation by environmental air of fresh worts for lambic beer production, it is known that microorganisms often associate with specific surfaces present in a brewery. However, knowledge about the association of microorganisms with the interior surfaces of wooden lambic barrels is limited. To clarify the role of casks and foeders as additional microbial inoculation sources, it was important to determine the influence of the barrel characteristics and the cleaning procedures on the microbial communities of the interior barrel surfaces. Moreover, this helped to elucidate the complex spontaneous lambic beer fermentation and maturation process. It will allow further optimization of the lambic beer production process, as well as the wooden-barrel-cleaning procedures applied.

Download Full-text

Lactococcus lactis Diversity Revealed by Targeted Amplicon Sequencing of purR Gene, Metabolic Comparisons and Antimicrobial Properties in an Undefined Mixed Starter Culture Used for Soft-Cheese Manufacture

Foods ◽

10.3390/foods9050622 ◽

2020 ◽

Vol 9 (5) ◽

pp. 622

Author(s):

Sabrina Saltaji ◽

Olivier Rué ◽

Valérie Sopena ◽

Sophie Sablé ◽

Fatoumata Tambadou ◽

...

Keyword(s):

Microbial Diversity ◽

Starter Culture ◽

Antimicrobial Properties ◽

Amplicon Sequencing ◽

Antimicrobial Activities ◽

Operational Taxonomic Units ◽

Soft Cheese ◽

Culture Independent ◽

Identification Of Species ◽

Species Specific

The undefined mixed starter culture (UMSC) is used in the manufacture of cheeses. Deciphering UMSC microbial diversity is important to optimize industrial processes. The UMSC was studied using culture-dependent and culture-independent based methods. MALDI-TOF MS enabled identification of species primarily from the Lactococcus genus. Comparisons of carbohydrate metabolism profiles allowed to discriminate five phenotypes of Lactococcus (n = 26/1616). The 16S sequences analysis (V1–V3, V3–V4 regions) clustered the UMSC microbial diversity into two Lactococcus operational taxonomic units (OTUs). These clustering results were improved with the DADA2 algorithm on the housekeeping purR sequences. Five L. lactis variants were detected among the UMSC. The whole-genome sequencing of six isolates allowed for the identification of the lactis subspecies using Illumina® (n = 5) and Pacbio® (n = 1) technologies. Kegg analysis confirmed the L. lactis species-specific niche adaptations and highlighted a progressive gene pseudogenization. Then, agar spot tests and agar well diffusion assays were used to assess UMSC antimicrobial activities. Of note, isolate supernatants (n = 34/1616) were shown to inhibit the growth of Salmonella ser. Typhimurium CIP 104115, Lactobacillus sakei CIP 104494, Staphylococcus aureus DSMZ 13661, Enterococcus faecalis CIP103015 and Listeria innocua CIP 80.11. Collectively, these results provide insightful information about UMSC L. lactis diversity and revealed a potential application as a bio-protective starter culture.

Download Full-text

Algae colonisation of brick pavement at the University of Venda: A potential slippery hazard

Jàmbá Journal of Disaster Risk Studies ◽

10.4102/jamba.v11i2.689 ◽

2019 ◽

Vol 11 (2) ◽

Cited By ~ 1

Author(s):

Thabelo R. Munyai ◽

Thandi Sonqishe ◽

Jabulani R. Gumbo

Keyword(s):

Green Algae ◽

Inductively Coupled Plasma ◽

Limpopo Province ◽

Inductively Coupled ◽

High Solar Radiation ◽

Icp Ms ◽

Metal Contents ◽

Plasma Mass Spectrometry ◽

Cyanobacteria And Algae ◽

The University

A brick pavement, tramped by humans, is exposed to atmospheric elements, thus allowing cyanobacteria and algae to colonise. In this article, we report on the factors that contribute to the slipperiness of a brick pavement at the University of Venda in the Limpopo province of the South Africa. Samples were collected from brick surfaces either colonised by green algae (treated) or not (control). The samples were acid-digested and analysed for metals by Inductively Coupled Plasma Mass Spectrometry (ICP MS) in parts per billion (ppb). The treated bricks, with green algae, had average high metal contents (ppb): Al 9456.02, Ti 731.23, V 46.44, Cr 78.85, Mn 862.93, Fe 16295.18, Co 23.57, Ni 59.36, Cu 66.31, Zn 160.57, As 7.92, Se 10.45, Mo 6.74, Cd 5.19, Sn 4.65, Sb 2.31 and Pb 19.51. In contrast, control bricks had a low average of metal content (ppb) as follows: Al 2.99, Ti 0.28, V 4.04, Cr 1.42, Mn 4.29, Fe 20.89, Co 0.36, Ni 2.74, Cu 5.64, Zn 4.21, As 0.56, Se <3.00, Mo 0.88, Cd 0.01, Sn 1.05, Sb 0.04 and Pb 0.04. Other factors that promote algae colonisation include high solar radiation, neutral pH, nutrients, low electrical conductivity and total dissolved solids. The algae colonisation of brick pavement results in an unaesthetic sighting and a slippery surface that is hazardous to humans.

Download Full-text

Bacteria from the Genus Arcobacter Are Abundant in Effluent from Wastewater Treatment Plants

Applied and Environmental Microbiology ◽

10.1128/aem.03044-19 ◽

2020 ◽

Vol 86 (9) ◽

Cited By ~ 5

Author(s):

Jannie Munk Kristensen ◽

Marta Nierychlo ◽

Mads Albertsen ◽

Per Halkjær Nielsen

Keyword(s):

Wastewater Treatment ◽

Pathogenic Bacteria ◽

Municipal Wastewater ◽

Wastewater Treatment Plants ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Content Type ◽

Effluent Quality ◽

Culture Independent ◽

Influent Wastewater

ABSTRACT Pathogenic bacteria in wastewater are generally considered to be efficiently removed in biological wastewater treatment plants. This understanding is almost solely based on culture-based control measures, and here we show, by applying culture-independent methods, that the removal of species in the genus Arcobacter was less effective than for many other abundant genera in the influent wastewater. Arcobacter was one of the most abundant genera in influent wastewater at 14 municipal wastewater treatment plants and was also abundant in the “clean” effluent from all the plants, reaching up to 30% of all bacteria as analyzed by 16S rRNA gene amplicon sequencing. Metagenomic analyses, culturing, genome sequencing of Arcobacter isolates, and visualization by fluorescent in situ hybridization (FISH) confirmed the presence of the human-pathogenic Arcobacter cryaerophilus and A. butzleri in both influent and effluent. The main reason for the high relative abundance in the effluent was probably that Arcobacter cells, compared to those of other abundant genera in the influent, did not flocculate and attach well to the activated sludge flocs, leaving a relatively large fraction dispersed in the water phase. The study shows there is an urgent need for new standardized culture-independent measurements of pathogens in effluent wastewaters, e.g., amplicon sequencing, and an investigation of the problem on a global scale to quantify the risk for humans and livestock. IMPORTANCE The genus Arcobacter was unexpectedly abundant in the effluent from 14 Danish wastewater treatment plants treating municipal wastewater, and the species included the human-pathogenic A. cryaerophilus and A. butzleri. Recent studies have shown that Arcobacter is common in wastewater worldwide, so the study indicates that discharge of members of the genus Arcobacter may be a global problem, and further studies are needed to quantify the risk and potentially minimize the discharge. The study also shows that culture-based analyses are insufficient for proper effluent quality control, and new standardized culture-independent measurements of effluent quality encompassing most pathogens should be considered.

Download Full-text

Guanicid and PHMG Toxicity Tests on Aquatic Organisms

Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis ◽

10.11118/actaun201664010129 ◽

2016 ◽

Vol 64 (1) ◽

pp. 129-134

Author(s):

Eva Poštulková ◽

Radovan Kopp

Keyword(s):

Acute Toxicity ◽

Green Algae ◽

Danio Rerio ◽

Guanidine Hydrochloride ◽

Aquatic Organisms ◽

Toxicity Tests ◽

Freshwater Algae ◽

Lc50 Value ◽

Desmodesmus Communis ◽

Cyanobacteria And Algae

The emergence and development of new algicidal products is caused by the ever increasing popularity of garden ponds as well as the use of these products in the fisheries sector, especially for disposal of cyanobacteria and algae. Most frequent means of combating cyanobacteria and algae are applications of algicidal substances. Newly developed algaecides include Guanicid and polyhexamethylene guanidine hydrochloride (PHMG). The aim of the study was to identify toxic effects of Guanicid and PHMG on zebrafish (Danio rerio) and green algae (Desmodesmus communis). We determined the acute toxicity in fish according to ČSN EN ISO 7346-1, and conducted the freshwater algae growth inhibition test according to ČSN ISO 8692 methodology. For inhibition tests with green algae we chose Guanicid and PHMG concentrations of 0.001, 0.005, and 0.010 ml/L. For fish short-term acute toxicity tests we chose Guanicid concentrations of 0.010, 0.050, 0.150, 0.200, 0.250, and 0.300 ml/L and PHMG concentrations of 0.010, 0.025, 0.050, 0.075, 0.100, and 0.125 ml/L. In case of zebrafish (Danio rerio), the LC50 value for Guanicid is 0.086 ml/L, while the LC50 value for PHMG is 0.043 ml/L. Effects of Guanicid on inhibition of green algae (Desmodesmus communis) appear highly significant (p < 0.010) at a concentration of 0.010 ml/L. For PHMG, these effects are highly significant (p < 0.001) at concentrations of 0.005 and 0.010 ml/L in 48 hours.

Download Full-text

Assessing anaerobic gut fungal (Neocalliamstigomycota) diversity using PacBio D1/D2 LSU rRNA amplicon sequencing and multi-year isolation

10.1101/2020.03.24.005967 ◽

2020 ◽

Author(s):

Radwa A. Hanafy ◽

Britny Johnson ◽

Noha H. Youssef ◽

Mostafa S. Elshahed

Keyword(s):

Ribosomal Rna ◽

Ribosomal Subunit ◽

Amplicon Sequencing ◽

Sequencing Technology ◽

Its1 Region ◽

5.8S Rrna ◽

Wide Range ◽

Culture Independent ◽

Long Read ◽

Pacbio Smrt Sequencing

AbstractThe anaerobic gut fungi (AGF, Neocallimastigomycota) reside in the alimentary tracts of herbivores where they play a central role in the breakdown of ingested plant material. Accurate assessment of AGF diversity has been hampered by inherent deficiencies of the internal transcribed spacer1 (ITS1) region as a phylogenetic marker. Here, we report on the development and implementation of the D1/D2 region of the large ribosomal subunit (D1/D2 LSU) as a novel marker for assessing AGF diversity in culture-independent surveys. Sequencing a 1.4-1.5 Kbp amplicon encompassing the ITS1-5.8S rRNA-ITS2-D1/D2 LSU region in the ribosomal RNA locus from fungal strains and environmental samples generated a reference D1/D2 LSU database for all cultured AGF genera, as well as the majority of candidate genera encountered in prior ITS1-based diversity surveys. Subsequently, a D1/D2 LSU-based diversity survey using long read PacBio SMRT sequencing technology was conducted on fecal samples from 21 wild and domesticated herbivores. Twenty-eight genera and candidate genera were identified in the 17.7 K sequences obtained, including multiple novel lineages that were predominantly, but not exclusively, identified in wild herbivores. Association between certain AGF genera and animal lifestyles, or animal host family was observed. Finally, to address the current paucity of AGF isolates, concurrent isolation efforts utilizing multiple approaches to maximize recovery yielded 216 isolates belonging to twelve different genera, several of which have no prior cultured-representatives. Our results establish the utility of D1/D2 LSU and PacBio sequencing for AGF diversity surveys, and the culturability of a wide range of AGF taxa, and demonstrate that wild herbivores represent a yet-untapped reservoir of AGF diversity.

Download Full-text

Rbec: a tool for analysis of amplicon sequencing data from synthetic microbial communities

ISME Communications ◽

10.1038/s43705-021-00077-1 ◽

2021 ◽

Vol 1 (1) ◽

Author(s):

Pengfan Zhang ◽

Stjin Spaepen ◽

Yang Bai ◽

Stephane Hacquard ◽

Ruben Garrido-Oter

Keyword(s):

Microbial Communities ◽

Amplicon Sequencing ◽

Fungal Communities ◽

Polymorphic Variation ◽

Sequencing Data ◽

Sequencing Errors ◽

Extensive Evaluation ◽

Culture Independent ◽

Reference Sequences ◽

Synthetic Microbial Communities

AbstractSynthetic microbial communities (SynComs) constitute an emerging and powerful tool in biological, biomedical, and biotechnological research. Despite recent advances in algorithms for the analysis of culture-independent amplicon sequencing data from microbial communities, there is a lack of tools specifically designed for analyzing SynCom data, where reference sequences for each strain are available. Here we present Rbec, a tool designed for the analysis of SynCom data that accurately corrects PCR and sequencing errors in amplicon sequences and identifies intra-strain polymorphic variation. Extensive evaluation using mock bacterial and fungal communities show that our tool outperforms current methods for samples of varying complexity, diversity, and sequencing depth. Furthermore, Rbec also allows accurate detection of contaminants in SynCom experiments.

Download Full-text