The development of co-cultures of clostridial strains which combine different physiological traits represents a promising strategy to achieve the environmentally friendly production of biofuels and ...chemicals. For the optimization of such co-cultures it is essential to monitor their composition and stability throughout fermentation. FISH is a quick and sensitive method for the specific labeling and quantification of cells within microbial communities. This technique is neither limited by the anaerobic fermenter environment nor by the need of prior genetic modification of strains. In this study, two specific 23S rRNA oligonucleotide probes, ClosKluy and ClosCarb, were designed for the monitoring of C. kluyveri and C. carboxidivorans, respectively. After the optimization of hybridization conditions for both probes, which was achieved at 30% (v/v) formamide, a high specificity was observed with epifluorescence microscopy using cells from different pure reference strains. The discriminating properties of the ClosKluy and ClosCarb probes was verified with samples from heterotrophic co-cultures in anaerobic flasks as well as autotrophic stirred-tank bioreactor co-cultures of C. kluyveri and C. carboxidivorans. Besides being suited to monitor defined co-cultures of these two species, the new specific FISH oligonucleotide probes for C. kluyveri and C. carboxidivorans additionally have potential to be applied in environmental studies.
The interest and exploration of biodiversity in subsurface ecosystems have increased significantly during the last 2 decades. The aim of this study was to investigate the in vitro probiotic ...properties of spore-forming bacteria isolated from deep caves. Two hundred fifty spore-forming microbes were enriched from sediment samples from 10 different pristine caves in Algeria at different depths. Isolates showing nonpathogenic profiles were screened for their potential to produce digestive enzymes (gliadinase and beta-galactosidase) in solid and liquid media, respectively. Different probiotic potentialities were studied, including (i) growth at 37°C, (ii) survival in simulated gastric juice, (iii) survival in simulated intestinal fluid, and (iv) antibiotic sensitivity and cell surface properties. The results showed that out of 250 isolates, 13 isolates demonstrated nonpathogenic character, probiotic potentialities, and ability to hydrolyze gliadin and lactose in solution. These findings suggest that a selection of cave microbes might serve as a source of interesting candidates for probiotics. IMPORTANCE Previous microbial studies of subsurface ecosystems like caves focused mainly on the natural biodiversity in these systems. So far, only a few studies focused on the biotechnological potential of microbes in these systems, focusing in particular on their antibacterial potential, antibiotic production, and, to some extent, enzymatic potential. This study explores whether subsurface ecosystems can serve as an alternative source for microbes relevant to probiotics. The research focused on the ability of cave microbes to degrade two substrates (lactose and gliadin) that cause common digestive disorders. Since these enzymes may prove to be useful in food processing and in reducing the effect of lactose and gliadin digestion within intolerant patients, isolation of microbes such as in this study may expand the possibilities of developing alternative strategies to deal with these intolerances.
Edible-fungal-based solid-state fermentation holds promise for sustainable food and biofuel production. Understanding the role of microbial communities in fungal substrates is crucial. Birch-based ...substrates were treated with autoclaving (121 °C, at 2 bar) or hot air pasteurization (75–100 °C), followed by incubation with and without shiitake (Lentinula edodes) inoculum. Mycelial growth was monitored by CO2 release and microbial biomass by phosphate-lipid fatty acid (PLFA). DNA sequencing was used to analyze the microbial communities. Results showed successful colonization of shiitake on all substrates, regardless of pasteurization temperatures and coexisting microbes. Total microbial respiration (CO2) and PLFA biomass showed no significant differences between pasteurization regimes. However, significant microbial differences were found between shiitake-inoculated and non-inoculated treatments. DNA sequencing revealed the dominance of Phyllobacterium, Sphingomonas, and Pelomonas genera in all inoculated substrates, while non-inoculated substrates were abundant in Bacillus spp. and Paenibacillus spp. of the Firmicutes phylum. This study provides preliminary insights into the microbial community in birch-based shiitake substrates, facilitating further investigation of bacteria involved in shiitake mycelium growth promotion and biochemical conversion for biofuel production.
Microbial mats in sulfidic cave streams offer unique opportunities to study redox-based biogeochemical nutrient cycles. Previous work from Lower Kane Cave, Wyoming, USA, focused on the aerobic ...portion of microbial mats, dominated by putative chemolithoautotrophic, sulfur-oxidizing groups within the Epsilonproteobacteria and Gammaproteobacteria. To evaluate nutrient cycling and turnover within the whole mat system, a multidisciplinary strategy was used to characterize the anaerobic portion of the mats, including application of the full-cycle rRNA approach, the most probable number method, and geochemical and isotopic analyses. Seventeen major taxonomic bacterial groups and one archaeal group were retrieved from the anaerobic portions of the mats, dominated by Deltaproteobacteria and uncultured members of the Chloroflexi phylum. A nutrient spiraling model was applied to evaluate upstream to downstream changes in microbial diversity based on carbon and sulfur nutrient concentrations. Variability in dissolved sulfide concentrations was attributed to changes in the abundance of sulfide-oxidizing microbial groups and shifts in the occurrence and abundance of sulfate-reducing microbes. Gradients in carbon and sulfur isotopic composition indicated that released and recycled byproduct compounds from upstream microbial activities were incorporated by downstream communities. On the basis of the type of available chemical energy, the variability of nutrient species in a spiraling model may explain observed differences in microbial taxonomic affiliations and metabolic functions, thereby spatially linking microbial diversity to nutrient spiraling in the cave stream ecosystem.
Biological wastewater treatment has been applied for more than a century to ameliorate anthropogenic damage to the environment. But only during the last decade the use of molecular tools allowed to ...accurately determine the composition, and dynamics of activated sludge and biofilm microbial communities. Novel, in many cases yet not cultured bacteria were identified to be responsible for filamentous bulking and foaming as well as phosphorus and nitrogen removal in these systems. Now, methods are developed to infer the in situ physiology of these bacteria. Here we provide an overview of what is currently known about the identity and physiology of some of the microbial key players in activated sludge and biofilm systems.
Summary
Lower Kane Cave, Wyoming (USA), has hydrogen sulfide‐bearing springs that discharge into the cave passage. The springs and cave stream harbour white filamentous microbial mats dominated by ...Epsilonproteobacteria. Recently, novel 16S rRNA gene sequences from the phylum Acidobacteria, subgroup 7, were found in these cave mats. Although Acidobacteria are ubiquitously distributed in many terrestrial and marine habitats, little is known about their ecophysiology. To investigate this group in Lower Kane Cave in more detail, a full‐cycle rRNA approach was applied based on 16S and 23S rRNA gene clone libraries and the application of novel probes for fluorescence in situ hybridization. The 16S and 23S rRNA gene clone libraries yielded seven and six novel acidobacterial operational taxonomic units (OTUs) respectively. The majority of the OTUs were affiliated with subgroups 7 and 8. One OTU was affiliated with subgroup 6, and one OTU could not be assigned to any of the present acidobacterial subgroups. Fluorescence in situ hybridization distinguished two morphologically distinct, rod‐shaped cells of the acidobacterial subgroups 7 and 8. Although the ecophysiology of Acidobacteria from Lower Kane Cave will not be fully resolved until cultures are obtained, acidobacterial cells were always associated with the potentially chemolithoautotrophic epsilon‐ or gammaproteobacterial filaments, suggesting perhaps a lifestyle based on heterotrophy or chemoorganotrophy.
Although there are several studies describing bacteria associated with marine fish, the bacterial composition associated with seahorses has not been extensively investigated since these studies have ...been restricted to the identification of bacterial pathogens. In this study, the phylogenetic affiliation of seahorse-associated bacteria was assessed by 16S rRNA gene sequencing of cloned DNA fragments. Fluorescence
in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rRNA analysis. Both methods revealed that
Vibrionaceae was the dominant population in
Artemia sp. (live prey) and intestinal content of the seahorses, while
Rhodobacteraceae was dominant in water samples from the aquaculture system and cutaneous mucus of the seahorses. To our knowledge, this is the first time that bacterial communities associated with healthy seahorses in captivity have been described.
Fluorescence
in situ hybridization (FISH) using fluorochrome-labeled DNA oligonucleotide probes has been successfully applied for
in situ detection of anaerobic ammonium oxidizing (anammox) bacteria. ...However, application of the standard FISH protocols to visualize anammox bacteria in biofilms from a laboratory-scale wastewater reactor produced only weak signals. Increased signal intensity was achieved either by modifying the standard FISH protocol, using peptide nucleic acid probes (PNA FISH), or applying horse radish peroxidase- (HRP-) labeled probes and subsequent catalyzed reporter deposition (CARD-FISH). A comparative analysis using anammox biofilm samples and suspended anammox biomass from different laboratory wastewater bioreactors revealed that the modified standard FISH protocol and the PNA FISH probes produced equally strong fluorescence signals on suspended biomass, but only weak signals were obtained with the biofilm samples. The probe signal intensities in the biofilm samples could be enhanced by enzymatic pre-treatment of fixed cells, and by increasing the hybridization time of the PNA FISH protocol. CARD-FISH always produced up to four-fold stronger fluorescent signals but unspecific fluorescence signals, likely caused by endogenous peroxidases as reported in several previous studies, compromised the results. Interference of the development of fluorescence intensity with endogenous peroxidases was also observed in cells of aerobic ammonium oxidizers like
Nitrosomonas europea, and sulfate-reducers like
Desulfobacter postgatei. Interestingly, no interference was observed with other peroxidase-positive microorganisms, suggesting that CARD-FISH is not only compromised by the mere presence of peroxidases. Pre-treatment of cells to inactivate peroxidase with HCl or autoclavation/pasteurization failed to inactive peroxidases, but H
2O
2 significantly reduced endogenous peroxidase activity. However, for optimal inactivation, different H
2O
2 concentrations and incubation time may be needed, depending on nature of sample and should therefore always be individually determined for each study.
PDF
