Search Results (101)

To determine the evolution of microbial community and microbial shift under anaerobic processes, this study investigates the use of denaturing gradient gel electrophoresis (DGGE). In the DGGE, short- and medium-sized DNA fragments are separated based on their melting characteristics, and this technique is used in this study to understand the dominant bacterial community in mesophilic and thermophilic anaerobic digestion processes. Dairy manure is known for emitting greenhouse gases (GHGs) such as methane, and GHG emissions from manure is a biological process that is largely dependent on the manure conditions, microbial community presence in manure, and their functions. Additional efforts are needed to understand the GHG emissions from manure and develop control strategies to minimize the biological GHG emissions from manure. To study the microbial shift during anaerobic processes responsible for GHG emission, we conducted a series of manure anaerobic digestion experiments, and these experiments were conducted in lab-scale reactors operated under various temperature conditions (28 °C, 36 °C, 44 °C, and 52 °C). We examined the third variable region (V3) of the 16S rRNA gene fingerprints of bacterial presence in anaerobic environment by PCR amplification and DGGE separation. Results showed that bacterial community was affected by the temperature conditions and anaerobic incubation time of manure. The microbial community structure of the original manure changed over time during anaerobic processes, and the community composition changed substantially with the temperature of the anaerobic process. At Day 0, the sequence similarity confirmed that most of the bacteria were similar (>95%) to Acinetobacter sp. (strain: ATCC 31012), a Gram-negative bacteria, regardless of temperature conditions. At day 7, the sequence similarity of DNA fragments of reactors (28 °C) was similar to Acinetobacter sp.; however, the DNA fragments of effluent of reactors at 44 °C and 52 °C were similar to Coprothermobacter proteolyticus (strain: DSM 5265) (similarity: 97%) and Tepidimicrobium ferriphilum (strain: DSM 16624) (similarity: 100%), respectively. At day 60, the analysis showed that DNA fragments of effluent of 28 °C reactor were similar to Galbibacter mesophilus (strain: NBRC 10162) (similarity: 87%), and DNA fragments of effluent of 36 °C reactors were similar to Syntrophomonas curvata (strain: GB8-1) (similarity: 91%). In reactors with a relatively higher temperature, the DNA fragments of effluent of 44 °C reactor were similar to Dielma fastidiosa (strain: JC13) (similarity: 86%), and the DNA fragments of effluent of 52 °C reactor were similar to Coprothermobacter proteolyticus (strain: DSM 5265) (similarity: 99%). To authors’ knowledge, this is one of the few studies where DGGE-based approach is utilized to study and compare microbial shifts under mesophilic and thermophilic anaerobic digestions of manure simultaneously. While there were challenges in identifying the bands during gradient gel electrophoresis, the joint use of DGGE and sequencing tool can be potentially useful for illustrating and comparing the change in microbial community structure under complex anaerobic processes and functionality of microbes for understanding the consequential GHG emissions from manure. Full article

Traditional dairy products, especially cheeses, represent part of the cultural food heritage of many countries. In addition, these cheeses constitute microbiological “reservoirs”, of which many have been lost due to the introduction of the pasteurization of milk in the dairy industry. Increased awareness of the importance of microorganisms that make up the biodiversity of traditional cheeses, as well as the development of molecular methods in recent decades, have enabled efforts to identify and preserve them. Traditional Livno cheese is a full-fat hard cheese, considered one of the most famous traditional cheeses of Bosnia and Herzegovina and is seasonally produced from a mixture of raw sheep’s milk supplemented with cow’s milk. Often, Livno cheese has variable quality, due to microbial contamination and poor milk quality. In this study, traditional Livno cheese was studied during the ripening of cheeses produced by different producers during two seasons. Culture-dependent analyses were made during ripening using microbiological plating on suitable media. Likewise, culture-independent methods Denaturing Gradient Gel Electrophoresis (DGGE) and Automated Ribosomal Intergenic Spacer Analysis (ARISA) were used to elucidate the cheese microbiota. Results of analysis showed Lactococcus spp., Streptococcus spp., Lactobacillus spp., Pediococcus spp. and Leuconostoc spp. to be dominant species in traditional Livno cheese. However, when comparing the use of culture-dependent and culture-independent methods in the evaluation of Livno cheese microbiota, Enterococcus was not detected by culture-independent DGGE methods. The microbial population of both the milk and the environment determines the fermentation processes during cheese production and ripening, and thereby defines the quality of this cheese. The numbers of bacteria in the cheese were shown to be dependent on the manufacturer, the degree of ripening and the production season. Full article

Soil phenolic acids mainly come from crop residue and root exudates, which are often reported as allelochemicals affecting crop growth and soil microbial community. Here, two phenolic acid compounds, p-hydroxyphenylacetic acid (HPA) and p-hydroxybenzoic acid (HBA) were amended to the soils and were incubated under room conditions, and the soil samples were collected from soybean and corn fields separately. The soil microbial community was determined by PCR-DGGE (polymerase chain reaction-denatured gradient gel electrophoresis) and clone methods. Microbial biomass carbon (MBC) was measured based on the fumigation–extraction method. The result revealed that HPA/HBA amendment significantly affected soil bacterial and fungal community structures. HPA/HBA enriched some distinct bacteria and fungi. The induced bacteria were mainly Gemmatimonas, Bacillus, and Acidobacteria, while the induced fungi were mainly Penicillium and Aspergillus. HPA amendment enhanced the more bacterial quantities of corn and soybean soils than HBA. The fungal diversity in corn and soybean soils treated with HBA was higher than that treated with HPA. It was speculated that the diversity of degrading HBA fungi was higher than that of degrading HPA. This study comprehensively analyzed the effects of phenolic acids on soil microbial community and increased the understanding of the monoculture barrier to agricultural production. Full article

Simultaneous bioproduction of hydrogen and ethanol from cheaper waste feedstock has the potential for the development of a more cost-effective biofuel generation process. Crude glycerol (CG), a by-product of the biodiesel industry, is a renewable resource, abundant, sold at low prices and available worldwide. However, the main CG limitations in fermentation processes are mainly related to the presence of impurities and the lack of nitrogen sources, both acting on microbial activity. In this study, a fermentation process with CG was improved using a highly specific microbial consortium called GlyCeroL (GCL). The process was developed in fed-batch fermentation mode using not diluted substrate and carried out under non-sterile conditions and at increasing amounts of the substrate (from 20 to 80 gL⁻¹ of glycerol). The results showed higher H₂ (from 6 to 8 LL⁻¹) and EtOH (from 13 to 20 gL⁻¹) production by increasing glycerol concentration from 20 to 40 gL⁻¹. On the other hand, a decrease in glycerol degradation efficiency (from 75 to 56%) was observed. Then, the nitrogen sparging strategy was applied. Using CG of 40 gL⁻¹, process improvement was achieved, leading to the increased production of hydrogen (10 LL⁻¹) but not that of ethanol (20 gL⁻¹). A further increase to 60 gL⁻¹ of glycerol produced a slight increment of EtOH (21 gL⁻¹) and H₂ (11 gL⁻¹) but a sharp decrease in glycerol degradation efficiency (41%). Acetate, as the main impurity of CG, was an additional carbon source for GCL microorganisms contributing to EtOH production and increasing that of lactic acid to restore the redox balance. The Denaturing Gradient Gel Electrophoresis (DGGE) fingerprint at the end of all fed-batch fermentations supported the robustness of GCL functional units and their adaptability to fermentation conditions. Full article

Genes of host plants and parasitic nematodes govern the plant–nematode interaction. The biological receptors and parasitism effectors are variable among plant species and nematode populations, respectively. In the present study, hatch testing and bioassays on cabbage, oilseed radish, and mustard were conducted to compare the biological characteristics among six populations of the beet cyst nematode Heterodera schachtii. Genetic patterns of the vap1 gene for the studied populations were distinct as shown by denaturing the gradient gel electrophoresis of PCR-amplified gene fragments. Concurrently, significant differences in the hatching rates, number of penetrated J2 in roots, and eggs/cyst ratios among the six nematode populations for the three cruciferous species were observed. In conclusion, analyzing the population genetic structure of H. schachtii plays a pivotal role in illustrating the variability in the plant–nematode interaction among its populations and plant species, which in its role leads to developing nematode management depending on plant resistance. Full article

This study investigated the effect of initially set anodic potentials (−0.3, −0.2, −0.1 and +0.1 V) on voltage production and microbial community in electroactive biofilm reactors (EBRs) treating synthetic and domestic wastewater (WW). In phase 1, EBRs were acclimated with different anodic potentials for synthetic and domestic WW. EBR (SE4) poised with +0.1 V showed the highest maximum power density (420 mW/m²) for synthetic WW, while EBR (DE3) poised with −0.1 V showed the highest maximum power density (235 mW/m²) for domestic WW. In phase 2, the EBRs were operated with a fixed external resistance (100 Ω for synthetic WW and 500 Ω for domestic WW) after the applied potentials were stopped. The EBRs showed slightly different voltage productions depending on the WW type and the initial anodic potential, but both EBRs applied with +0.1 V for synthetic (SE4) and domestic (DE4) WW showed the highest voltage production. Principal component analysis results based on denaturing gel gradient electrophoresis band profiles showed that the microbial community was completely different depending on the WW type. Nevertheless, it was found that the microbial community of EBRs applied with a negative potential (−0.3, −0.2, and −0.1 V) seemed to shift to those of EBRs applied with a positive potential (+0.1 V) regardless of WW type. Therefore, positive anodic potential is an important operating factor in electroactive biofilm development and voltage generation for rapid start-up. Full article

Hydrogen sulfide (H₂S) is a toxic and corrosive component that commonly occurs in biogas. In this study, H₂S removal from swine-waste biogas using sulfur-oxidizing Paracoccus versutus CM1 immobilized in porous glass (PG) and polyurethane foam (PUF) biofilters was investigated. Bacterial compositions in the biofilters were also determined using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The biofilters were first tested on a laboratory scale under three space velocities (SV): 20, 30, and 40 h⁻¹. Within 24 h, at an SV of 20 h⁻¹, PG and PUF biofilters immobilized with P. versutus CM1 removed 99.5% and 99.7% of H₂S, respectively, corresponding to the elimination capacities (EC) of 83.5 and 86.2 gm⁻³ h⁻¹. On a pilot scale, with the horizontal PG-P. versutus CM1 biofilter operated at an SV of 30 h⁻¹, a removal efficiency of 99.7% and a maximum EC of 113.7 gm⁻³ h⁻¹ were achieved. No reduction in methane content in the outlet biogas was observed under these conditions. The PCR-DGGE analysis revealed that Paracoccus, Acidithiobacillus, and Thiomonas were the predominant bacterial genera in the biofilters, which might play important roles in H₂S removal. This PG–P. versutus CM1 biofiltration system is highly efficient for H2S removal from swine-waste biogas. Full article

Chlorpyrifos (CP), a widely used pesticide, and its metabolite 3,5,6-trichloro-2-pyridinol (3,5,6-TCP), are xenobiotic compounds detected in many biomes, notably in marine sediments, all over the world. These compounds are posing a serious environmental and health problem given their toxicity to wildlife and possible exposure effects to human neurodevelopment. Microorganisms at CP-impacted environments could harbor metabolic capabilities that can be used as indicators of the biological effects of the contaminant and could encode selected functions reactive against contaminants. Those features could be used for microbial ecotoxicology applications by collectively using analytical, enzymatic, microbiological and toxicological techniques in order to assess the biological effects of pollutants and other environmental/climatic stressors in ecosystems. The objective of this study was to assess the variability in the metabolic responses of yeast isolates from CP-contaminated marine sediments as potential biological indicators for microbial ecotoxicology testing. Sediment samples from a South Caribbean tropical shore (Cartagena Bay, Colombia) were collected, and deoxyribonucleic acid (DNA) was recovered from lyophilized aliquots. The DGGE (Denaturing Gradient Gel Electrophoresis) technique targeting fungal Internal Transcribed Spacer (ITS) showed the great diversity of fungal types. Simultaneously, yeast strains were isolated from the freshly collected sediment samples. Physiological characterization including API 20C and antibiosis tests, growth patterns at salt concentrations (2/4/10/25%), temperatures (4/25/37/45 °C), esterase activity assay and resistance tests to CP/TCP toxicity resulted in 10 isolated yeast strains, identified as Candida spp. (6), Cryptococcus spp. (3). and Rhodotorula spp. (1), showing promising characteristics to be used as a test for yeast-based ecotoxicity indicators. The patterns of carbohydrate assimilation, low antibiosis, presence of esterases/lipases, growth in a wide range of temperatures and salt concentrations, and tolerance to minimal inhibitory concentrations of CP and TCP are factors useful for testing environmental samples. Full article

This study aims to create a quantitative polymerase chain reaction (q-PCR) methodology for monitoring the hydrogen-producing mixed cultures enriched from elephant dung using alpha-cellulose as a carbon source through five generations of repetitive sub-culture. The enriched thermophilic mixed cultures from the fifth cultivation cycle gave the highest hydrogen yield of 170.3 mL H₂/g cellulose and were used to generate hydrogen from sawdust. Clostridium sp. and Thermoanaerobacterium sp. were the dominant bacteria in thermophilic mixed cultures with high hydrogen yield, according to polymerase chain reaction-denatured gradient gel electrophoresis (PCR-DGGE). q-PCR primers Chis150F and ClostIR, TherF and TherR, and BacdF and BacdR were developed to amplify the 16S rRNA genes of Clostridium sp., Thermoanaerobacterium sp., and Bacillus sp., respectively, for the quantification of hydrogen-producing bacteria in biohydrogen fermentation. Similar q-PCR analysis of Clostridium sp., Thermoanaerobacterium sp., and Bacillus sp. 16S rRNA gene amplification during hydrogen production from cellulose and sawdust revealed increasing gene copy number with time. The molecular approaches developed in this study can be used to monitor microbial communities in hydrogen fermentation processes efficiently. Full article

The aim of this study was to determine the activity and structure of microbial communities in soils contaminated with heavy metals (HMs). To achieve this goal, soil samples were taken from two contaminated sites (i.e., Piekary Śląskie and Bukowno) in Poland. A wide range of methods were applied, including: total and metal-tolerant culturable bacteria enumeration; microbial community structure analysis using the phospholipid fatty acid method (PLFA); denaturing gradient gel electrophoresis (PCR-DGGE); and metabolic activity using BIOLOG and EcoPlate^TM. Our studies showed that HMs negatively affected microbial community structure and activity in polluted soils. Apart from the contamination with HMs, other soil parameters like soil pH and water also impacted microbial community structure and growth. Metal-tolerant bacterial strains were isolated, identified and tested for presence of genes encoding HM tolerance using the polymerase chain reaction (PCR) methodology. Contamination with HMs in the tested areas was found to lead to development of metallotolerant bacteria with multiple tolerances toward Zn, Ni, Cd and Cu. Different genes (e.g., czcA, cadA and nccA) encoding HM efflux pumps were detected within isolated bacteria. Culturable bacteria isolated belonged to Proteobacteria, Actinobacteria and Bacteroidetes genera. Among non-culturable bacteria in soil samples, a significant fraction of the total bacteria and phyla, such as Gemmatimonadetes and Acidobacteria, were found to be present in all studied soils. In addition, bacteria of the Chloroflexi genus was present in soil samples from Piekary Śląskie, while bacteria of the Firmicutes genus were found in soil samples from Bukowno. Full article

The aim of this review was to investigate the effectiveness of the Human Oral Microbe Identification Microarray (HOMIM) in identifying and quantifying bacterial species of the oral microbiome in periodontal disease. The search for articles was conducted in CENTRAL, CINAHL, MEDLINE and EMBASE by two reviewers, and included articles published in English between January 1990 and December 2021. The selected articles were human observational studies in adults between 18 and 65 years, presenting specific predefined keywords. Articles were initially selected by title and abstract; articles that met the inclusion criteria were analyzed for methodological quality using a detailed checklist for quality assessment. Data were extracted and reported using the PRISMA tool. The study design, sample, follow-up period, collection and microbial analysis methods, statistical treatment, results and discussion were quality assessed and risk of bias was evaluated using the Cochrane Risk-of-Bias tool. A narrative synthesis approach was used to synthesize and interpret the extracted data. From the initial search, 2931 articles were retrieved; 51 of these were then selected after screening by title and abstract. Subsequently, 8 articles met the inclusion after full-text reading and were classed according to methodological quality as high (2), moderate (3) or low (3). Studies included in this review were of high and medium quality. Data from the Human Oral Microbe Identification Microarray (HOMIM) provide much more robust results, showing major shifts between periodontal health and periodontal disease. Compared to earlier techniques such as Denaturing Gradient Gel Electrophoresis (DGGE), HOMIM represents a more effective approach for quantification due to its high sensitivity; thus, it is able to identify a high prevalence of periodontal pathogens and novel species in low abundance. The literature provides moderate evidence that the Human Oral Microbe Identification Microarray (HOMIM) is more effective in identifying and quantifying bacterial species of the oral microbiome in periodontal disease, compared to earlier molecular and non-molecular methods such as Denaturing Gradient Gel Electrophoresis (DGGE) and a culture-based approach with phenotypic tests. Full article

The haloarchaeal diversity of four hypersaline alkaline lakes from the Wadi El-Natrun depression (Northern Egypt) was investigated using culture-independent polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) of 16S rRNA gene phylotypes, which was combined with remote sensing and geographic information system (GIS) data to highlight the distribution pattern of the microbial diversity in water and sediment samples. The majority of archaeal sequences identified in all four lakes belonged to the phyla Euryarchaeota and Crenarchaeota. Sediment samples from Beida Lake and water samples from El-Hamra Lake showed the highest levels of archaeal diversity. Sequence similarities ≥ 95% were found between six of the acquired clones and uncultured Halorhabdus, Euryarchaeota, and archaeon clones. In addition, two clones shared a high level of sequence similarity (97%) with unclassified archaea, while other nine clones exhibited 96% to 99% sequence similarity with uncultured archaeon clones, and only one clone showed 97% identity with an uncultured Crenarchaeota. Likewise, 7 DGGE bands presented a sequence similarity of 90 to 98% to Halogranum sp., Halalkalicoccus tibetensis, Halalkalicoccus jeotgali, uncultured Halorubrum, Halobacteriaceae sp., or uncultured haloarchaeon. In conclusion, while the variety of alkaliphilic haloarchaea in the examined soda lakes was restricted, the possibility of uncovering novel species for biotechnological applications from these extreme habitats remains promising. Full article

With the advance in gene technology, genetically modified (GM) crops have increased in recent years. GM crops offer us various benefits. However, there are potential risks of GM crops for the environment. In this study, the impacts of transgenic plants on soil microbial community structures were assessed. Two varieties of soybean (Glycine max L.) and hot pepper (Capsicum annuum L.), which introduced the herbicide-resistant gene, bar, were used in this study. The effects of GM crops on soil microbial community structures were investigated using a cultural method, the denaturing gradient gel electrophoresis (DGGE) procedure, and 16S rRNA gene sequencing on the Illumina platform. Additionally, the persistence of transgenes was monitored using a quantitative real-time PCR procedure. The cultural method, DGGE analysis, and the amplicon-based community profile indicated that soil microbial communities were not significantly different between GM and non-GM lines. The level of the bar gene in GM soybean plots greatly increased when the crops were actively growing, but thereafter gradually decreased to the initial level. Meanwhile, the level of the bar gene in GM hot pepper plots repeatedly increased and decreased according to the flowering stages. These results indicated that soil microbial community structures were not significantly affected. Full article

Recirculation technology has been emerging in the marine aquaculture industry. The microbiome developed in recirculation aquaculture systems (RASs) is an important factor for the optimal operation of these systems and fish welfare. In this study, the microbial community dynamics in the water column and the biofilms of a marine RAS with Mediterranean species of gilthead sea bream and sea bass were investigated, while physicochemical conditions were also monitored. Microbiological, culture, and non-culture analyses based on PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) fingerprints were performed on the water column and biofilm developed on stainless-steel surfaces. According to the obtained results, feed administration seemed to cause changes in pH and TAN, as well as drive changes in the bacterial abundance in the water column. Tested surfaces were colonized within 24 h and sessile cells were stabilized in terms of density within 6 days. DGGE fingerprints indicated the stability of the microbial community in water and a dynamic succession in the community of the biofilms. The fish pathogen Tenacibaculum discolor was found to colonize the biofilm and the water column. The main findings confirmed that RAS technology can be used as a control strategy for the stability of the water microbial community, that there is a dynamic succession of the dominant species in the biofilm communities, and that pathogenic bacteria can be dominant in the latter. Full article

The leafless orchids are rare epiphytic plants with extremely reduced leaves, and their aerial roots adopted for photosynthesis. The beneficial plant–microbial interactions contribute significantly to host nutrition, fitness, and growth. However, there are no data available on the bacterial associations, inhabiting leafless orchids. Here, we describe the diversity of cyanobacteria, which colonize the roots of greenhouse Microcoelia moreauae and Chiloschista parishii. The biodiversity and structure of the cyanobacterial community were analyzed using a complex approach, comprising traditional cultivable techniques, denaturing gradient gel electrophoresis (DGGE), and phylogenetic analysis, as well as the light and scanning electron microscopy (SEM). A wide diversity of associated bacteria colonize the root surface, forming massive biofilms on the aerial roots. The dominant populations of filamentous nitrogen-fixing cyanobacteria belonged to the orders Oscillatoriales, Synechococcales, and Nostocales. The composition of the cyanobacterial community varied, depending on the nitrogen supply. Two major groups prevailed under nitrogen-limiting conditions, belonging to Leptolyngbya sp. and Komarekiella sp. The latter was characterized by DGGE profiling and sequencing, as well as by its distinctive features of morphological plasticity. The leading role of these phototrophophic and diazotrophic cyanobacteria is discussed in terms of the epiphytic lifestyle of the leafless orchids. Full article