Pure Cultures Of Microorganisms Research Articles

The bio-cathode of microbial fuel cells systems: Performance analysis under different microbial community conditions

This study aims to investigate the conversion of different microbial community compositions in the cathode chamber in a dual-chamber bioelectrochemical system. A denitrifying bacterial community, or sludge, was enriched for denitrification by microbial consortia and subsequently acclimatized within the cathodic chamber of microbial fuel cells, either within a mixed microbial community sludge microbial fuel cell (M-MFC) or a pure culture of microorganisms in a microbial fuel cell (P-MFC). The bioelectrochemical systems were treated with different nitrate concentrations in the cathodic chamber: the MFC with low concentration nitrate (MFC1, 25 mg/L nitrate; MFC2 50 mg/L nitrate), the MFC with medium concentration nitrate(MFC3, 75 mg/L nitrate; MFC4 100 mg/L nitrate), and MFC with high concentration nitrate (MFC5, 125 mg/L nitrate; MFC6 150 mg/L nitrate; MFC7 175 mg/L nitrate), and the initial COD in the anodic chamber was based on demand(C to N ratio is 5). M-MFC exhibited better-electrogenerated capability than P-MFC. However, P-MFC exhibited better nitrogen removal performance compared to M-MFC. The best performance was attained by both types of MFCs when the nitrate content was 100 mg/L. The maximum output voltages in M-MFC4 and P-MFC4 were 449 mV and 171 mV, respectively. The average conversion rate of M-MFC4 and P-MFC4 were 2.9 mg*L−1 *h−1 and 10.5 mg*L−1 *h−1, respectively. The analysis of the microbial community of two treated MFCs in the cathode chamber in the optimal state indicated that non-electrode denitrification predominantly occurs in P-MFC. The cathode chambers were mostly populated by denitrifying microorganisms. The dominant species in M-MFC were Azonexus, Zoogloea, Ignavibacterium, and Pirellula, and the dominant species in P-MFC were Comamonas, Aeromonas, and Acidovorax. The species was assessed by qPCR analysis, and the results validated the use of a bacterial consortium for domestication as a more suitable method to improve the stability and performance of the MFCs.

Conservation techniques for promising bacteria in the oil industry to preserve biological properties

Background: The conservation of valuable bacterial strains is crucial for various scientific, industrial, and environmental applications. Microorganisms with oil-emulsifying and oil-displacing properties are potentially significant for biotechnologies applied in the oil industry, particularly in such areas as bioremediation and tertiary enhanced oil recovery. To supply enterprises with pure cultures of microorganisms, they should be constantly maintained in the collection conditions in an active state while monitoring the preservation of their biotechnological properties. Therefore, keeping microorganism strains in working conditions and preserving their valuable properties are important for almost any work with microorganisms, ranging from primary research to their use in the production of various biopreparations.
 Aim: The article focuses on studying a method for preserving bacteria that are useful in the oil industry. This method involves modifying the technique of microencapsulating microorganism cells in alginate gel by adding glycerin, which is used as an agent with biostatic action.
 Materials and methods: The subject of research are eight hydrocarbon-oxidizing cultures of microorganisms that were sourced from the Department of Biotechnology of the al-Farabi Kazakh National University. Of these, four cultures were spore-bearing, while the other four were non-spore-bearing. The research employed microbiological methods of cultivation and storage of microorganisms in both solid and liquid media under aerobic conditions. In addition, Cooper's method was used to determine oil emulsification index and statistical methods for data analysis.
 Results: It has been found that adding glycerin (15% vol.) as a biostatic to the gel-forming matrix of sodium alginate can ensure long-term (up to 6 months) cell viability for the studied bacteria in the range of 88-96% while maintaining functionality of immobilized cells. The values of the bacteria’s oil emulsification remained at the pre-conservation levels, whereas traditional storage methods result in a lower number of viable cells after six months. It should be noted that after six months of being stored in encapsulated form with glycerin, the viability of non-sporе-forming Pseudomonas cultures is lower (88-91%) than spore-forming Bacillus (95-98%). This correlation is also is observed for traditional methods.
 Conclusion: The modern method of preserving bacteria allows for their long-term storage while maintaining functionality and viability.

The next-generation tools for risk assessment and precision food safety in the One Health continuum

Abstract Sequencing technologies and bioinformatics are transforming microbiology and their applications, including aspects of food quality and precision food safety and description of microbial communities in the One Health context. The use of these protocols includes, among others, a deep understanding of the genomes of microorganisms in pure culture using whole genome sequencing and importantly, metagenomics has allowed the extensive comprehension of the microbiota and microbiome of samples. Microbial communities from different sources (clinical samples, environment, food-producing animals) have increasingly been studied to describe the genetic diversity, functionality, and succession of food-borne pathogens and for studying functional microorganisms used for producing novel foods and also using fermentation. This study presents the methods used for the description of microbial communities in a model fermented food, including culturing with a novel in situ culturing approach, full-length 16S rRNA gene sequencing, and shotgun metagenomic sequencing with computational enrichment. Several microorganisms were identified both in culture and as high-quality metagenome-assembled genomes and included prominent probiotic species of the genus Gluconobacter, Liquorilactobacillus, Lactiplantibacillus, Lentilactobacillus and Lacticaseibacillus that have potential health benefits and applications in food production and environment health enhancement. These tools can revolutionise risk assessment approaches, allowing for the pinpointing of contamination events, pathogens, antimicrobial resistance spread, and description of unknown beneficial microorganisms, as in this model study. The integration of these methods in the One Health continuum will allow the precise connection of samples from clinical settings, animals, plants, and their shared environment, achieving a holistic approach to food safety and public health. Key messages • technologies are changing food safety by enabling comprehensive analysis of microbiome. This can aid in identifying contamination events, pathogens, and AMR spread, and promote One Health integration. • Use of sequencing in foods, enables the identification of novel microbial species, providing valuable insights into the functional microorganisms for production and their potential health benefits.

Identification of a deep-branching thermophilic clade sheds light on early bacterial evolution

It has been proposed that early bacteria, or even the last universal common ancestor of all cells, were thermophilic. However, research on the origin and evolution of thermophily is hampered by the difficulties associated with the isolation of deep-branching thermophilic microorganisms in pure culture. Here, we isolate a deep-branching thermophilic bacterium from a deep-sea hydrothermal vent, using a two-step cultivation strategy (“Subtraction-Suboptimal”, StS) designed to isolate rare organisms. The bacterium, which we name Zhurongbacter thermophilus 3DAC, is a sulfur-reducing heterotroph that is phylogenetically related to Coprothermobacterota and other thermophilic bacterial groups, forming a clade that seems to represent a major, early-diverging bacterial lineage. The ancestor of this clade might be a thermophilic, strictly anaerobic, motile, hydrogen-dependent, and mixotrophic bacterium. Thus, our study provides insights into the early evolution of thermophilic bacteria.

Opportunistic microbiota of breast milk and antimicrobial activity of milk whey at different periods of lactation

Object of study. The evaluation of the interaction between breast milk opportunistic microorganisms abundance and the milk whey antimicrobial activity at different periods of lactation.
 Materials and methods. 100 samples of breast milk from healthy breastfeeding mothers were inoculated on solid selective media, and then pure cultures of microorganisms were identified by MALDI-TOF mass spectrometry. The antimicrobial activity of the whey against a model culture of Candida albicans was evaluated by spectrophotometry.
 Results. 270 isolates represented by 36 species of 13 genera of opportunistic bacteria were obtained. None of the 100 samples contained opportunistic fungi. Staphylococci (7 species) and streptococci (11 species) were predominant. The most common were staphylococci S. epidermidis (70.2%) and S. aureus (20.8%), and streptococci S. mitis (27.7%) and S. oralis (21.8%). The total contamination (median) of opportunistic bacteria in the colostrum was 79 103 CFU/ml, transitional milk 4 103 CFU/ml, mature milk 5 102 CFU/ml. The antimicrobial activity of colostrum whey was 87.489.4%; transitional milk 88.2%; mature milk 63.481.9%. The total contamination had a high inverse correlation with the lactation period (r = 0.806) and a high positive correlation with the antimicrobial activity of whey (r = 0.699). Meanwhile, a significant decrease in contamination was noted after 1 month from the beginning of lactation, while a significant decrease in antimicrobial activity was observed after 8 months.
 Conclusions. The decrease of the breast milk contamination by opportunistic bacteria during the lactation period was found to be primary compared to the decrease of the whey antimicrobial activity. Thus, changes in the whey antimicrobial protection factors occur in response to changes of the opportunistic microbiota abundance.

Bioremediation of organic/heavy metal contaminants by mixed cultures of microorganisms: A review

Abstract Although microbial remediation has been widely used in the bioremediation of various contaminants, in practical applications of biological remediation, pure cultures of microorganisms are seriously limited by their adaptability, efficiency, and capacity to handle multiple contaminants. Mixed cultures of microorganisms involve the symbiosis of two or more microorganisms. Such cultures exhibit a collection of the characteristics of each microorganism species or strain, showing enormous potential in the bioremediation of organic or heavy metal pollutants. The present review focuses on the mixed cultures of microorganisms, demonstrating its importance and summarizing the advantages of mixed cultures of microorganisms in bioremediation. Furthermore, the internal and external relations of mixed culture microorganisms were analyzed with respect to their involvement in the removal process to elucidate the underlying mechanisms.

Rapid identification of new isolates of Acidipropionibacterium acidipropionici by fluorescence in situ hybridization (FISH)

Acidipropionibacterium acidipropionici is widely used for many applications, such as propionic acid production, cereal silage, and also as probiotic. Due to this plethora of applications, new isolates of A. acidipropionici with improved features are being searched for. These new isolates must be accurately identified, however, most approaches become expensive and time-consuming when the number of isolates is high. On the contrary, fluorescence in situ hybridization allows the affordable, reliable, and rapid identification of microorganisms in pure cultures and environmental and medical samples. Therefore, the aim of this work was to apply a fluorescent in situ hybridization probe for the reliable identification of new A. acidipropionici isolates. To this end, probe Pap446, specific for A. acidipropionici, was validated by hybridization assays with strains of this species from different origins, other species of the same genus or family, and unrelated genera. Eight isolates with propionibacterium characteristics were obtained from milk and feces of cows. Probe Pap446, hybridized only with isolates III and VI. The identity of these isolates was further confirmed by PCR using group and species-specific primers for propionibacteria and 16S rDNA sequencing.

Aspects of Mannheimia haemolytica cryopreservation

Mannheimia haemolytica causes severe pneumonia and septicemia in ruminant animals. The study aims to create an effective protective media for the long‐term storage of microbial cultures of M. haemolytica in a veterinary laboratory. The protective nutrient media were based on Hottinger's nutrient broth. Glycerol and ethylene glycol were used as cryoprotectants. Two protective media were obtained. Medium #1 consisted of nutrient broth (90%) and glycerin (10%), and medium #2 had nutrient broth (90%) and ethylene glycol (10%) in it. A homogeneous suspension 1.0 McFarland units turbidity was prepared from a pure culture of microorganisms (3.0 x 108 CFU/ml). The suspension was added to ampoules with an appropriate protective medium. The inoculated media were placed in a cryogenic storage dewar in liquid nitrogen at ‐196 ° C. The viability of M. haemolytica was determined by inoculation on blood agar and counting the number of living bacteria. The initial inoculum concentration of M. haemolytica was 3.0x107 CFU/ml. After 18‐20 hours of cultivation, it becomes 7.0x109 CFU/ml. The survival and safety of bacteria depend on the cryoprotectant. The results of the experiment showed that 10% glycerol provides 98.6% survival of M. haemolytica. The number of viable cells after cryopreservation was 6.9x109 CFU/ml. Ethylene glycol showed a 95.7% survival rate. The number of viable cells after cryopreservation was 6.7x109 CFU/ml. Without cryoprotectant, M. haemolytica showed a 40.0% survival rate with only 2.8x109 CFU/ml viable cells after defrosting. M. haemolytica causes turbidity and sediment formation at the bottom of the tube on Hottinger broth. After growing on semi‐liquid media (semi‐liquid agar with the addition of glucose or serum), bacteria show bipolar staining in smears. On Hottinger broth, with the addition of 10% defibrinated ram blood M. haemolytica formed small dewy colonies with an even edge without a hemolysis zone. The final species identification was performed by the proteomic method using MALDI‐TOF‐SM. All used media keep microorganisms' cultures in a stable, viable state. Thus, they can be successfully used for the storage of bacteria at low‐temperature conditions.

Study of the efficiency of the inhibitory effect of antimicrobial preparations in the production of sugar substances

This article presents the relevance of the issue of inhibition of microbiological processes at different stages of processing of sugar-containing raw materials, in particular ensuring the microbiological purity of juices and syrups in the production of sugar substances. Information on the composition of microbiota contaminating raw materials in the production of beet sugar, raw cane sugar, food syrups from sugar sorghum is analyzed.
 Experimental data on the evaluation of the effectiveness of a number of antimicrobial preparations in terms of growth retardation of pure cultures of microorganisms and the effect of disinfection during the introduction of preparations are presented.
 The greatest danger in production is caused by the development of spore-forming and mucus-forming bacteria, which led to the choice of research objects.
 Thus, the effect of antimicrobial preparations on microorganisms: mesophilic bacteria Bacillus subtilis, B. megatherium, thermophilic bacteria B. stearothermophilus, mucus-forming bacteria of the genus Leuconostos, was investigated. In addition, the effect of antimicrobials on mycelial fungi of the genus Aspergillus was studied.
 Based on the conducted experimental studies, the range of costs of the studied antimicrobial preparation for the suppression of contaminating microflora in the production of sugar and food syrups from vegetable raw materials was determined

Principles of the distribution of wheat with fusariois disease, its effect on yield, harm and disease measures in the case of Uzbekistan

This paper shows the role of Fusarium fungi in the farms of Kibray district of Tashkent province, which cause sudden yellowing of wheat fields before ripening, reducing the symptoms of the disease and the yield. The amount of grain in a diseased plant is reduced by 42% compared to the grain in a healthy plant, and the weight is reduced by 57.1%. Pure cultures of microorganisms were isolated from samples taken from diseased fields. The article is based on the results of scientific experiments and contains 2 tables and figures. The information presented in the article will serve as a scientific basis for further study of the prevalence and harm of wheat fusarium wilt.

Application of biochemical typing in veterinary medicine in bee enterobacterioses to determine Klebsiella Pneumoniae

The article presents laboratory diagnostics (in vitro), namely, identification of pure culture of pathogenic bacteria of Klebsiella Pneumoniae species in case of enterobacteriosis in bees in winter-spring and summer-autumn times. The purpose of the study was the biochemical typification of bacteria of the species Klebsiella Pneumoniae with humane medicine methods, that isolated in the case of dysbacteriosis of bees which have a characteristic symptomatic complex of a decrease in the strength of bee families, which leads to a decrease in the resistance of the bee family, their diarrhea, crawling, and then swarming or death of bees. Contamination of beehive frames and walls of beehive by fecation leads to sharp deterioration of the apiary's veterinary and sanitary condition and significant economic damage for beekeepers. Pure culture of pathogenic bacteria served as an object for experiment. The Family of the bacteria was established earlier – Enterobacteriaceae, and was confirmed by “Zhytomyr Regional State Laboratory of the State Service of Ukraine for Food Safety and Consumer Protection”. Laboratory diagnostics of dysbiosis in bees caused by enterobacteria was performed in such a sequence: 1. Sowing of pathological material taken from sick bees on selective nutrient media for enterobacteria and extraction of pure culture; 2. Microscopy of typical colonies; 3. Determination of bacteria genus; 4. Determination of bacteria motor activity: 5. Urease test; 6. Indole test; 7. Phenyalaalanine test; 8. Study of basic enzymatic properties of bacteria. In a series of laboratory biochemical studies of pure culture microorganisms isolated from mixed culture from diseased bees the isolated bacterial strain belongs to the Family Enterobacteriaceae, Genus Klebsiella, Species Klebsiella pneumoniae. The novelty of the application of the algorithm for determining the species of Klebsiella pneumoniae enterobacteriae allows to diagnose dysbacterioses in winter-spring and summer-autumn times clearly and economically. The isolated Klebsiella pneumoniae bacteria serve as experimental cultures for testing drugs of various directions and actions in laboratory conditions and are kept at the Department of Microbiology, Pharmacology and Epizootology, Faculty of Veterinary Medicine of Polissya National University (formerly Zhytomyr National Agroecological University). Further application of complex diagnostics of enterobacteriosis of bees, including methods of biochemical typification, will allow to expand etiological factors of bee family collapse.

Study of corrosion-active microorganisms of the coastal zone of the Baltic and Barents seas

The composition of microbial communities in coastal zones in the area of Kislogubskaya tidal power plant (the Barents Sea) and near Saint Petersburg Flood Prevention Facility Complex in the Gulf of Finland (Baltic Sea) was studied. The enrichment and pure cultures of microorganisms of various groups (sulfate-reducing, sulfur-oxidizing, nitrifying, ammonifying and iron-oxidizing bacteria) have been isolated, their cultural and physiological-biochemical properties, as well as their ability to induce corrosion of different materials used in the construction of hydraulic structures, have been studied.

Isothermal microcalorimetry for thermal viable count of microorganisms in pure cultures and stabilized formulations

BackgroundQuantification of viable microorganisms is an important step in microbiological research as well as in microbial product formulation to develop biological control products or probiotics. Often, the efficiency of the resulting product is dependent on the microbial cell density and their viability, which may decrease over time. Commonly, the number of viable cells is determined by serial dilution and plating techniques or flow cytometry. In 2017, we developed a mathematical model for isothermal microcalorimetry (IMC) data analysis and showed that the new method allows for a more rapid quantification of viable fresh and freeze-dried anaerobic Lactobacillus reuteri cells than traditional viable count methods.ResultsThis study developed the new method further by applying it to well-known aerophilic plant-beneficial microbial species (Pseudomonas brassicacearum, Bacillus amyloliquefaciens subsp. plantarum and Clonostachys rosea) used in biological control products. We utilized IMC to quantify viable cells in microbial pure cultures as well as when coated onto wheat seeds. The results from this study confirmed that thermal viable count methods are more rapid and sensitive than traditional viable count techniques. Most interestingly, a thermal viable count method was able to quantify microbes coated on seeds despite the presence of the natural microbiota of the seeds. Our results also showed that, in contrast to plating techniques for which clustered cells skew the results, IMC does not require single cells for accurate viable counts.ConclusionsThermal viable count methods are novel methods for the rapid quantification of divergent bacterial and fungal species and enhance the speed, sensitivity, and accuracy of routine viable counts of pure cultures and controlled microbiomes such as plant seed coatings.

Enhanced biodegradation of atrazine by Arthrobacter sp. DNS10 during co-culture with a phosphorus solubilizing bacteria: Enterobacter sp. P1

The interaction between pure culture microorganisms has been evaluated allowing for the enhanced biodegradation of various kinds of pollutants. Arthrobacter sp. DNS10 previously enriched in an atrazine-containing soil was capable of utilizing atrazine as the sole nitrogen source for growth, and Enterobacter sp. P1 is a phosphorus-solubilizing bacterium that releases various kinds of organic acids but lacks the ability to degrade atrazine. Whether strain P1 could enhance atrazine biodegradation by the degrader strain DNS10 was investigated in this experiment. Gas chromatography and high-performance liquid chromatography results showed that co-culture of both strains degraded 99.18 ± 1.00% of the atrazine (initial concentration was 100 mg L−1), while the single strain DNS10 only degraded 38.57 ± 7.39% after a 48 h culture, and the resulting concentration of the atrazine final metabolite cyanuric acid were 63.91 ± 3.34 mg L−1 and 26.60 ± 3.87 mg L−1, respectively. In addition, the expression of the atrazine degradation-related genes trzN, atzB and atzC in co-culture treatments was 6.61, 1.81 and 3.09 times that of the single strain DNS10 culture treatment. A substrates utilization test showed that the atrazine-degrading metabolites ethylamine and isopropylamine could serve as the nitrogen source to support strain P1 growth, although strain P1 cannot degrade atrazine or utilize atrazine for growth. Furthermore, the pH of the medium was significantly decreased when strain P1 utilized ethylamine and isopropylamine as the nitrogen source for growth. The results suggest that nondegrader strain P1 could promote the atrazine biodegradation when co-cultured with strain DNS10. This phenomenon is due to metabolite exchange between the two strains. Culturing these two strains together is a new biostimulation strategy to enhance the biodegradation of atrazine by culturing these two strains together.

Evaluating gelling-agent mixtures as potential substitutes for bacteriological agar: an approach by mixture design

Gelling agents are components to solidify media cultures facilitating the isolation of microorganisms in pure cultures. Agar is the most used gelling agent in microbiological field because of properties like gelling temperature and a desirable texture (high firmness and low adhesiveness). A current problem with agar manufacture is that overexploitation of the algae Gelidium spp is affecting the species conservation and has increased agar’s market price by up to 300%. The aim of this research was to propose substitutes of bacteriological agar in terms of texture by using some reported gelling agents used by food and agroindustry. To determine the effects of the components on texture, were proposed a mixture-design screening to establish an optimal mixture that replace agar. The optimal result, according to the desirability function, were composed of arabic gum (2%), gelan gum (0.5%) and carrageenan (0.5%). This mixture exceeds the firmness of the reference matrix (agar at 2%) by 15% and is potentially 25% more cheaper and without significative statistical difference in growth CFU when compared to agar based media

Традиции и перспективы использования чистых культур микроорганизмов в отечественных технологиях ржаного хлеба на заквасках

Традиции и перспективы использования чистых культур микроорганизмов в отечественных технологиях ржаного хлеба на заквасках

Идентификация микроорганизмов с помощью инфракрасных Фурье-спектров

The need for novel techniques of rapid identification of pathogenic microorganisms arises from the massive spread of drug-resistant nosocomial strains and the emergence of centers for biohazard control. Fourier-transform infrared spectroscopy is a promising alternative to mass spectrometry as it is cost-effective, fast and suitable for field use. The aim of this work was to propose an algorithm for the identification of microorganisms in pure cultures based on the analysis of their Fourier transform infrared spectra. The algorithm is based on the automated principal component analysis of infrared spectra. Unlike its analogues described in the literature, the algorithm is capable of identifying bacteria regardless of the culture medium or growth phase. The training sample included the most prevalent causative agents of infections and sepsis in humans: Staphylococcus aureus (n = 67), Enterococcus faecalis (n = 10), Enterococcus faecium (n = 10), Klebsiella pneumoniae (n = 10), Escherichia coli (n = 10), Serratia marcescens (n = 10), Enterobacter cloacae (n = 10), Acinetobacter baumannii (n = 10), Pseudomonas aeruginosa (n = 10), and Candida albicans (n = 10). The model we built successfully passed a series of blind tests involving clinical isolates of 10 methicillin-resistant (MRSA) and 10 methicillin-sensitive (MSSA) Staphylococcus aureus strains as well as pair mixes of these cultures with clinical isolates of Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae.

ЗНАЧЕНИЕ ПРИСТЕНОЧНОЙ МИКРОБИОТЫ ТОЛСТОЙ КИШКИ В ПОДБОРЕ РАЦИОНАЛЬНОЙ АНТИБАКТЕРИАЛЬНОЙ ТЕРАПИИ У ПАЦИЕНТОВ С ЯЗВЕННЫМ КОЛИТОМ

The purpose of the study: optimization of diagnosis and treatment of patients with ulcerative colitis by selecting rational antibacterial therapy based on the results of microbiological examination of the microflora of the wall of the colon. Materials and methods: 35 patients with ulcerative colitis aged from 28 to 61 years who were on outpatient and inpatient treatment at the departments of coloproctology and gastroenterology of the SamSMU Clinic were examined. Collection of biopsy material of ulcerative defects of the mucous membrane of the large intestine was performed during the fibrocolonoscopy, and a pure culture of microorganisms was isolated from biopsy material using classical methods. The isolated cultures were identified using MALDI-TOF mass spectrometry. All the isolated cultures were sensitive to antibiotics by a disco-diffusion method. Results: In assessing the quantitative composition of the isolated microflora in patients with mild disease, the microflora in the wall of the colon was absent, or low titers of the sown strains (10²-10³ CFU per biopsy) were determined. In patients with an average severity and severe course of the disease in 50.0% and 53.8% of cases, respectively, a pronounced microbial contamination of the submucosal layer (the number of microorganisms 105-106 CFU per biopsy), a wide variety of species. When analyzing sensitivity to antibiotics of isolated strains, it was found that 45% of them have resistance to 1-2 groups of drugs, 33% have resistance to 3 or more groups. Only 22% of the strains were sensitive to all tested drugs. Conclusion: The eradication of a resistant flora is complex and, from our point of view, pathogenetically substantiated and requires the appointment of a combination therapy after microbiological examination of the biopsy specimen.

Assessing the metabolism of sedimentary microbial communities using the hydrogen isotopic composition of fatty acids

The hydrogen isotopic composition of fatty acids (FAs) has previously been shown to reflect the metabolism of microorganisms in pure culture, but has rarely been tested in the environment. Here, we report the abundance and hydrogen isotopic composition of polar lipid derived FAs extracted from surface sediments of the saline Lake Grevelingen (The Netherlands), at two different stations and during two seasons with oxic bottom water conditions during spring and hypoxic to anoxic conditions during late summer. These data are compared with the bacterial diversity revealed by 16S rRNA gene amplicon sequencing. All measured FAs were depleted in deuterium relative to the bottom water by 103‰ to 267‰. FAs associated with heterotrophic bacteria (i-15:0 and ai-15:0) showed the smallest fractionation (−103‰ to −185‰) while those derived from pelagic photoautotrophic phytoplankton (20:5) showed the largest fractionation (−230‰ to −267‰). Overall, the hydrogen isotope fractionation reflected in the majority of the more commonly occurring FAs (14:0, 16:0, 16:1ω7) is relatively large (−172‰ to −217‰). Together with the high relative abundance of the 20:5 FA, this suggests a substantial contribution from dead pelagic biomass settling from the water column to the sedimentary polar lipid derived FA pool and not from the in situ microbial communities. Therefore, the majority of the isotope signal in the fatty acids from surface sediments might not represent the general metabolism of the active sedimentary communities. Therefore, the input of pelagic biomass into sedimentary environments may bias the information contained in the hydrogen isotopic composition of FAs.

Metaproteomics method to determine carbon sources and assimilation pathways of species in microbial communities

Measurements of stable carbon isotope ratios (δ13C) are widely used in biology to address questions regarding food sources and metabolic pathways used by organisms. The analysis of these so-called stable isotope fingerprints (SIFs) for microbes involved in biogeochemical cycling and microbiota of plants and animals has led to major discoveries in environmental microbiology. Currently, obtaining SIFs for microbial communities is challenging as the available methods either only provide low taxonomic resolution, such as the use of lipid biomarkers, or are limited in throughput, such as nanoscale secondary ion MS imaging of single cells. Here we present "direct protein-SIF" and the Calis-p software package (https://sourceforge.net/projects/calis-p/), which enable high-throughput measurements of accurate δ13C values for individual species within a microbial community. We benchmark the method using 20 pure culture microorganisms and show that the method reproducibly provides SIF values consistent with gold-standard bulk measurements performed with an isotope ratio mass spectrometer. Using mock community samples, we demonstrate that SIF values can also be obtained for individual species within a microbial community. Finally, a case study of an obligate bacteria-animal symbiosis shows that direct protein-SIF confirms previous physiological hypotheses and can provide unexpected insights into the symbionts' metabolism. This confirms the usefulness of this approach to accurately determine δ13C values for different species in microbial community samples.