Polymerase Chain Reaction And Denaturing Gradient Gel Electrophoresis Research Articles

Analysis on Microbial Flora Changes During Processing and Storage of Spiced Goose Based on Pcr-Dgge Combined with Conventional Microbial Culture Methods

During the processing and storage process of spiced goose, samples were collected from raw goose meat (R0), spiced goose meat cooled for 2 hours (2h) and spiced goose meat stored for 1day (1d), 3 days (3d) and 5 days (5d). Microbial flora changes in spiced goose during the processing and storage process were analyzed by conventional microbial culture methods, polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE). The results indicated that more abundant microbial diversity was determined in goose meat through PCR-DGGE technology than conventional microbial culture methods. According to the PCR-DGGE results, the dominant bacteria in R0 and 2h were Aeromonas and Acinetobacter. On 1d, the dominant bacteria also included Weissella and Enterobacter, and on 3d, Aeromonas was no longer dominant. The dominant bacteria became Weissella, Enterococcus and Staphylococcus on 5d.

Impacts of Mannan oligosaccharides (MOS) on growth performance andgastrointestinal health of Nile tilapia (Oreochromis niloticus)

Objective: This study was undertaken to evaluate the potential role of Mannan oligosaccharides (MOS) on growth performance, liver and intestine tissue morphology, and gut microbiota of Nile tilapia (Oreochromis niloticus).Design: Controlled study.Animals: Three groups of Nile tilapia.Procedures: Three diets were formulated to contain 0 % of MOS 500 (MOS) as control (control), 0.05% of MOS plus adding MOS into water as well at level of 12.5 mg/l (MOS (feed +water), and the third group MOS was added only to water (MOS water) at the same level, and fed to Nile tilapia for 6 weeks.Results: Simultaneous water and feed additives with (MOS feed+water) at (0.05% of feed + 12.5 mg/l water) caused a significant increase in growth parameters (initial and final weight, weight gain (WG), specific growth rate (SGR), feed conversion ratio (FCR) and condition factor (k), when compared to both MOS added only in tank water (MOS water) and control groups. In addition, MOS in both treatment types increased survival rates significantly compared to the control. Polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) analysis showed that a single species or species that contained chemically equivalent DNA dominated the intestinal tract of all Nile tilapia regardless of dietary treatment. While MOS had no adverse effect of intestinal histology, an increase in the villi length was recorded.Conclusion and clinical relevance: Collectively, our results indicate that MOS added to feed and water could be used to enhance the growth performances, increase the survivability and exert beneficial effects on the gut microbiota of Nile tilapia.

Impacts of Mannan oligosaccharides (MOS) on growth performance and gastrointestinal health of Nile tilapia (Oreochromis niloticus)

Objective: This study was undertaken to evaluate the potential role of Mannan oligosaccharides (MOS) on growth performance, liver and intestine tissue morphology, and gut microbiota of Nile tilapia (Oreochromis niloticus). Design: Controlled study. Animals: Three groups of Nile tilapia. Procedures: Three diets were formulated to contain 0 % of MOS 500 (MOS) as control (control), 0.05% of MOS plus adding MOS into water as well at level of 12.5 mg/l (MOS (feed +water), and the third group MOS was added only to water (MOS water) at the same level, and fed to Nile tilapia for 6 weeks. Results: Simultaneous water and feed additives with (MOS feed+water) at (0.05% of feed + 12.5 mg/l water) caused a significant increase in growth parameters (initial and final weight, weight gain (WG), specific growth rate (SGR), feed conversion ratio (FCR) and condition factor (k), when compared to both MOS added only in tank water (MOS water) and control groups. In addition, MOS in both treatment types increased survival rates significantly compared to the control. Polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) analysis showed that a single species or species that contained chemically equivalent DNA dominated the intestinal tract of all Nile tilapia regardless of dietary treatment. While MOS had no adverse effect of intestinal histology, an increase in the villi length was recorded. Conclusion and clinical relevance: Collectively, our results indicate that MOS added to feed and water could be used to enhance the growth performances, increase the survivability and exert beneficial effects on the gut microbiota of Nile tilapia.

Correlation between ammonia-oxidizing microorganisms and environmental factors during cattle manure composting

Cattle manure composting was performed in an aerated vessel. Community structure and diversity of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) were investigated using polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) techniques targeting the ammonia monooxygenase alpha subunit (amoA) gene and the correlation between AOB and AOA communities and environmental factors was explored. Thirteen (13) AOB sequences were obtained, which were closely related to Nitrosomonas spp., Nitrosomonas eutropha, and Nitrosospira spp. and uncultured bacteria, among which Nitrosomonas spp. were predominant. Excessively high temperature and high ammonium concentration were not favorable for AOB growth. Five AOA sequences, belonging to CandidatusNitrososphaera gargensis and to an uncultured archaeon, were obtained. During composting, community diversity of AOB and AOA fluctuated, with AOA showing a higher Shannon–Wiener index. The AOB community changed more dramatically in the mesophilic stage and the early thermophilic stage, whereas the most obvious AOA community succession occurred in the late thermophilic stage, the cooling stage and the maturity stage. Water content, total nitrogen (TN) and ammonium concentration were more relevant to the AOB community structure, while higher correlations were observed between ammonia, nitrate and TN and the AOA community. AOB community diversity was negatively correlated with pH (r = −0.938, p < 0.01) and water content (r = −0.765, p < 0.05), while positively correlated with TN (r = 0.894, p < 0.01). AOA community diversity was negatively correlated with ammonium concentration (r = −0.901, p < 0.01). Ammonium concentration played an important role in the succession of AOB and AOA communities during composting.

Adsorption of Cu2+ and mechanism by natural biofilm.

The biofilm culturing device fixed on the slides was vertically placed in the commonly called small Li Lake of Jiangnan University. The adsorption experiment of Cu2+ was carried out by mature biofilm. Besides, scanning electron microscope (SEM), polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy-energy spectrum (SEM-EDX) were used to analysis the effect of Cu2+ on the morphological structure of biofilm. The result indicated that when the initial concentration of Cu2+ was 5 mg·L-1, the absorption capacity of Cu2+ by unit mass biofilm is the maximum. More extracellular polymeric substances (EPS) were released by biofilm due to the stimulation of Cu2+. EPS was beneficial to the adsorption of Cu2+ by biofilm. After the adsorption of Cu2+, the bacterial diversity index decreased, while there were no significant differences in microbial communities on biofilm. Moreover, the main groups combining Cu2+ were the hydroxyl groups and amide groups in S-EPS and B-EPS. Ion exchange is a mechanism of the adsorption of Cu2+ by EPS.

The Effect of Different Medium on Enantioselectivity in Degradation of Bifenthrin.

Bifenthrin (BF) is a chiral pesticide with two enantiomers. In this work, its stereoselective degradation was investigated in plants and domesticated active sludge. Considering that the degradation to pesticides of plants was effected by external conditions, hence, nanometer materials (gothite) were added into soil to explore the effect of sorption on enantioselectivity in degradation of BF. The microbial community that was responsible for BF-biodegrading in active sludge was studied by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE). In plant researches, chiral difference was discovered in the degradation of pesticides that onions resulted in. The existence of gothite in soil not only reduced the amount of BF that plants uptake from the environment, but it also enlarged the chiral difference during the process, indicating that the absorption of gothite to pesticides had enantioselectivity, which may be generated by that adsorption site where BF combined with gothite located in the chiral center of the pesticide. In studies of sludge, BF can be effectively degraded and decline of both isomers followed first-order kinetics. However, there was no obvious stereoselective degradation in domesticated sludge. The analysis of DGGE revealed that active sludge maintained the rich microbial community in the whole process (H > 3). The H index increased in the early domestication, which may because BF had no direct toxicity to the bacteria. The bacteria were able to degrade the small dose of the pesticide and absorbed it as nutrition. The sequence results demonstrated that the variety of bacteria grew, instead of Acidobacteria, Betaproteobacteria, Sphingobacteriia and Alphaproteobacteria, Flavobacteria also appearing in sludge after domestication.

Effects Oxytetracycline on Bacterial Diversity in Livestock Wastewater

Abstract To determine the effect of oxytetracycline on the anaerobic biological treatment of livestock wastewater, we studied the removal efficiency of chemical oxygen demand (determined by K2Cr2O7, CODCr), methane production, and bacterial diversity under the stress of different concentrations of oxytetracycline. Results indicated that the removal of COD and the yield of methane were inhibited by oxytetracycline. With an increase in oxytetracycline concentration, methane production decreased from 392 to 207 mL (the control group was 448 mL) and COD removal efficiency fell from 92% to 84% (the control group was 94%). Results of PCR-DGGE (polymerase chain reaction and denaturing gradient gel electrophoresis) indicated that, with an increase in oxytetracycline concentration, the richness values of the bacterial community fell from 0.86 to 0.45 (the control group was 1). In addition, oxytetracycline reduced COD removal efficiency, methane production, and bacterial diversity in livestock wastewater. Results p...

Intestinal Lactobacillus community structure and its correlation with diet of Southern Chinese elderly subjects.

This study aimed to investigate the relationship between the intestinal Lactobacillus species and diet of elderly subjects in a longevity area in Southern China. Healthy elderly subjects ranging from 80 to 99 years old were respectively selected from the regions of Bama and Nanning, Guangxi, China. The nested polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) technology was used to analyze the intestinal Lactobacillus community structure. Results showed that Weissella confusa, L. mucosae, L. crispatus, L. salivarius, and L. delbrueckii were the representative Lactobacillus of elderly subjects. Among them, L. crispatus and L. delbrueckii were the dominant Lactobacillus of all species. In comparison to Nanning elderly subjects, the detection frequencies of W. confusa and L. salivarius were significantly increased in Bama elderly subjects (P < 0.01), whereas L. mucosae was significantly decreased (P < 0.01). Interestingly, it was also found that there were 4 kinds of representative Lactobacillus, which were significantly correlated with dietary fiber. W. confusa (P < 0.01) and L. salivarius (P < 0.05) were significantly positively correlated with the intake of dietary fiber, while L. mucosae (P < 0.01) and L. crispatus (P < 0.05) were significantly negatively correlated with the intake of dietary fiber, respectively. Results confirmed that different diets had obvious effects on the intestinal Lactobacillus community structure of elderly subjects in Southern China, which may provide a certain theoretical basis for the elderly's healthy food strategic design and probiotics product development.

Effect of dietary prebiotic inulin on growth, body composition and gut microbiota of Asian seabass (Lates calcarifer)

A feeding trial was conducted to study the effect of inulin on growth, body composition and gut microbiota of Lates calcarifer fingerlings (average weight: 7.14±0.05g). Inulin was supplemented at five different concentrations 0, 5, 10, 15 and 20gkg−1 in the diet (400gkg1 protein and 90gkg−1 lipid) of L. calcarifer. The results of the 60 days feeding trial revealed that dietary inulin supplementation had a significant effect on specific growth rate (SGR) (linear, P=0.029; quadratic, P=0.022) and feed conversion ratio (FCR) (linear, P=0.033; quadratic, P=0.003) between control and treatments. However, the final body weight (FBW) (quadratic, P=0.0138) and weight gain (WG) (quadratic, P=0.0150) followed a quadratic pattern. Supplementation of inulin did not affect whole body moisture and lipid. However, crude protein increased both linearly (linear, P=0.001) and quadratically (quadratic, P=0.001) in fish fed with 20gkg−1 inulin supplemented diets and a quadratic pattern in ash (P<0.05) was recorded. Similarly hepatosomatic index (HSI) significantly increased (linear, P=0.008; quadratic, P=0.048) in fish fed 15gkg−1 inulin. Polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) analysis of gut samples from inulin supplemented diet revealed the change in the gut microbial community of Asian seabass. It could therefore be inferred that inulin supplementation is beneficial in the diet of Asian seabass and supplementation at 15gkg −1 level is optimal for enhancing growth.

Effect of karst rocky desertification on soil fungal communities in Southwest China.

Karst mountainous ecosystems are associated with karst rocky desertification (KRD), which can greatly impact soil structure and function. Despite the importance of soil microbes as a major factor maintaining ecosystem stability, we know little about the effect on soil fungal communities of KRD in karst regions. We investigated this relationship across a gradient of KRD soils from Guizhou, China by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE). Fungal diversity indices (Shannon-Wiener, richness, and evenness) significantly differed (P < 0.05) based on KRD severity, being lowest in moderately affected areas. Cluster analysis showed that the five sites examined clustered into two main groups according to KRD grade (high and low). Moreover, a homology search using sequences recovered from PCR-DGGE bands showed that the dominant fungi in each community varied remarkably, and included Aspergillus, Aphanoascus, Blastomyces, Fusarium, Glomus, Geomyces, Gibberella, Mortierella, Tetracladium, and Tumularia species, and an unclassified group. In conclusion, these findings demonstrate that KRD has a significant impact on soil fungal communities.

Bacaba beverage produced by Umutina Brazilian Amerindians: Microbiological and chemical characterization.

Bacaba chicha is a beverage prepared by the indigenous Umutina people from the bacaba fruit (Oenocarpus bacaba), a purple berry that is rich in fat and carbohydrates, as well as a source of phenolic compounds. In this study, samples of bacaba chicha beverage were collected, and the microbial community was assessed using culture-dependent and -independent techniques. The nutritional composition and metabolite profiles were analyzed, and species belonging to lactic acid bacteria (LAB) and yeasts were detected. The LAB group detected by culture-dependent analysis included Enterococcus hormaechei and Leuconostoc lactis. Polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) detected additional Propionibacterium avidum, Acetobacter spp., and uncultured bacteria. Pichia caribbica and Pichia guilliermondii were detected in a culture-dependent method, and Pichia caribbica was confirmed by PCR-DGGE analysis. The pH value of the beverage was 6.2. The nutritional composition was as follows: 16.47 ± 0.73 g 100 mL-1 dry matter, 2.2 ± 0.0 g 100 mL-1 fat, 3.36 ± 0.44 g 100 mL-1 protein, and 10.87 ± 0.26 g 100 mL-1 carbohydrate. The metabolites detected were 2.69 g L-1 succinic acid, 0.9 g L-1 acetic acid, 0.49 g L-1 citric acid, 0.52 g L-1 ethanol, and 0.4 g L-1 glycerol. This is the first study to identify microbial diversity in bacaba chicha spontaneous fermentation. This study is also the starting step in the immaterial record of this Brazilian indigenous beverage prepared from bacaba fruit.

Impact of continuous Salvia miltiorrhiza cropping on rhizosphere actinomycetes and fungi communities

Salvia miltiorrhiza Bunge (SMB) is an important herb that has been used in traditional Chinese medicine for centuries. Continuous SMB cropping can result in significant losses of yield and quality of the plant. The purpose of this study was to examine the variations in community structure of soil actinomycetes and fungi under continuous cropping of SMB in Sichuan, China. Four soil samples were analyzed by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE). The microbial diversity indices (Shannon-Wiener index, richness, and evenness) of the soil actinomycetes decreased along with continuous SMB cropping and rebound at the 3rd year, while that of soil fungi declined. Cluster analysis showed that the similarity between uncultivated soils and cropping soils declined with subsequent cropping periods. Homology search of sequences recovered from the DGGE bands showed that the actinomycete community in the studied soils was dominated by Actinobacteria, while dominant species of the fungal community varied remarkably, including Pleospora, Psathyrella, Pseudozyma, Rhizoctonia, Trichophaea and unclassified groups. Overall, these findings demonstrate that continuous SMB cropping has a significant impact on soil actinomycete and fungal communities.

알칼리도 제어에 의한 SBR 반응조에서의 부분아질산화

Abstract : In this study, major parameter of partial nitritation was investigated for the stable operation. In order to establish partial nitritation system, prevailing parameters such as temperature, BA (bicarbonate alkalinity) and pH were evaluated. As a result, it is inferred that appropriate bicarbonate alkalinity ratio (mg NaHCO 3 ･L -1 /mg Inf. NH 4+ -N･L -1 ) drives stable 50% partial nitritation at 32℃ and ambient temperature, respectively. Alkalinity ratio was proposed as new strategy for 50% partial nitritation without pH control in both temperature regimes. Because of the results, it was added amound of BA required only for 50% nitritation to inhibit nitratation. The effluent NO 2- -N/NH 4+ -N ratio reached almost 100% when initial bicarbonate alkalinity ratios (mg NaHCO 3 ･L -1 /mg Inf. NH 4+ -N･L -1 ) were 6.8 (R1) and 6.7 (R2), respectively. Polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) results demonstrated that AOB was the dominant nitrifying bacteria and NOB was negligible after adopting process control.

红壤区桉树人工林炼山后土壤肥力变化及其生态评价

Controlled burning is a traditional forest-clearing method used in Southern China. We analyzed the variability of soil fertility between controlled burning and non-burnt areas in a Eucalyptus robusta plantation forest of the red soil region, located in Hengxian County in Guangxi Zhuang Autonomous Region,China. Traditional incubation and culture-independent methods,such as the dilution-plate method,the polymerase chain reaction,and denaturing gradient gel electrophoresis( PCR-DGGE) were used. Possible effects on the soil ecological system were also evaluated. Soil organic matter( SOM), total and available nitrogen,phosphorus and potassium contents in the 0—3 cm soil layer only one week after burning were all higher than those in a non-burnt Eucalyptus forest. The number of soil culturable microbes,which includes bacteria, fungi and actinomycetes,and the biomass C and N in the 0—3 cm layer were also significantly higher than those in the nonburnt Eucalyptus forest. Indexes of chemical and biological properties in the middle layer( 3—25cm) of the burnt soils also showed the same trends observed in the surface soil layer. However,irregular changes were found in these chemical and biological indexes in soil layers below 25cm. This indicates that the chemical and biological properties of soils,including soil organic matter( SOM),total and available nitrogen,phosphorus and potassium,as well as the number of soil culturable microbes,and the biomass C and N in layer below 25cm were not significantly impacted by controlled burning. Furthermore,four months after controlled burning,except for total and available phosphorus contents,nutrients such as total and available nitrogen,potassium and SOM contents did not show the same trends with burning as were found after one week,and were all lower than those in the non-burnt forest soil. Also,the number of soil culturable microbes,and the biomass C and N in each layer of the burnt soils were also lower than those in the non-burnt soil. Thus,controlled burning in the Eucalyptus plantations in the red soil region of southern China has only short-term beneficial effects on soil fertility, and is not helpful for improving long-term soil fertility of Eucalyptus plantations. Indexes of soil bacterial diversity( Shannon-Wiener,H),Richness and Evenness( E H) in each layer after burning showed irregular changes. However,in the surface soil layers( 0—3cm) of the burnt treatments these indexes were all lower than those in the non-burnt treatment and hardly recovered to the same level as in the non-burnt treatment 4 months after burning. These results suggest that the method of controlled burning in southern China might not only result in a decline in soil fertility with time,but also lead to a decrease in the indexes of soil bacterial diversity,Richness and Evenness,in surface soils in these two periods after burning. Thus,based on the above results,controlled burning is not helpful for improving the soil fertility and maintaining the development of a sustainable surface soil ecosystem in Eucalyptus plantations in the red soil region of southern China.

Effect of Bacillus cereus Br on bacterial community and gossypol content during fermentation in cottonseed meal

To define new methods for the detoxification of free gossypol (FG) within cotton seed meal (CSM), FG during CSM fermentation with Bacillus. cereus Br were measured. Four conditions were studied: CSM inoculated with Br (Br + CSM), CSM without any inocula (CSM), sterilized CSM inoculated with Br (Br + sCSM), and sterilized CSM without any inocula (sCSM). Samples were taken at various times during the fermentation and the contents of free gossypol (FG), crude protein (CP), and amino acids were measured. The polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) method was used to analyze the changes in bacterial community structure during the fermentation. After the Bacillus. cereus Br was inoculated into CSM, the FG concentrations decreased to the minimum level of detection at 12 h, while CP and amino acids concentrations reached maxima at 18 h. The initial content of FG and CP decreased significantly when the CSM was pre-sterilized, and decreased to minima after 12 h fermentation. The changes in bacterial community structure mirrored the changes in FG and CP. B. cereus Br was dominant in the CSM + Br group during the first 12 h of fermentation, after which Br growth was inhibited by native bacteria in CSM. In contrast, B. cereus Br was always dominant during fermentation of autoclaved CSM. These results suggest that the native microbes of CSM significantly influenced the detoxification rate, and inhibited B. cereus Br growth during fermentation. But pre-sterilization of CSM significantly affects the FG content and quality of CSM. The B. cereus Br was capable of detoxifying FG from CSM, and the optimal time of fermentation was 18 h. Key words: Bacterial community, free gossypol, detoxification, fermentation, cottonseed meal.

The Gut Bacteria Associated with Camponotus japonicus Mayr with Culture-Dependent and DGGE Methods

The bacterial composition and distribution in the different gut regions of Camponotus japonicus were investigated using both culture-dependent method and culture-independent method of polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE). Five different bacterial strains were isolated using culture-dependent method, and they all belong to the phylum Firmicutes, including three genera of bacteria Bacillus, Paenibacillus, and Enterococcus. Bacillus cereus and Enterococcus mundtii were found in the midgut; Paenibacillus sp. was isolated from the hindgut; and the other two Bacillus spp. were isolated from the crop. Twelve distinct DGGE bands were found using PCR-DGGE method, and their sequences blasting analysis shows that they are members of the Proteobacteria and the Firmicutes, respectively, including three genera (Pseudomonas, Candidatus Blochmannia, Fructobacillus) and one uncultured bacterium, in which Pseudomonas was the most dominant bacteria group in all the three gut regions. According to the DGGE profile, the three gut regions had very similar gut communities, and all the DGGE bands were presented in the midgut and hindgut, while just two bands representing Blochmannia were not present in the crop. The results of our study indicate that the gut of C. japonicus harbors several other bacteria besides the obligate endosymbionts Blochmannia, and more work should be carried on to verify if they are common in the guts of other Camponotus ants.

Temperature effect on nitrogen removal performance and bacterial community in culture of marine anammox bacteria derived from sea-based waste disposal site

Anaerobic ammonium oxidation (anammox) bacteria have been detected in variety of marine environment in recent years, however, there have been only a few studies on their characteristics in the culture. The aim of this study is to reveal the effect of temperature on nitrogen removal ability and bacterial community in a culture of marine anammox bacteria (MAAOB). The MAAOB were cultured from the sediment of a sea-based waste disposal site at the North Port of Osaka Bay in Japan. The maximum nitrogen removal rate (NRR) was observed at 25°C in the MAAOB culture, and it decreased both at below 20°C and over 33°C. The activation energy of the MAAOB culture was calculated to be 54.6 kJ mol(-1) in the 5°C to 30°C range. No significant change in bacterial community according with temperature (5-37°C) was confirmed in the results of polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE). Meanwhile, a number of bacteria related to the oxidation-reduction reaction of sulfur were confirmed and it is speculated that they involved in the activity of MAAOB and nitrogen removal ability in the culture.

Dynamics of fungal diversity in different phases of Pinus litter degradation revealed through denaturing gradient gel electrophoresis (DGGE) coupled with morphological examination

Fungal diversity in Pinus sylvestris var. mongolica litter was investigated by PCR-DGGE coupled with a traditional cultivation method. Twenty-one fungal strains were isolated by traditional cultivation methodology, most being filamentous fungi. Total DNA was extracted directly from the L, F1, F2 and H litter layers respectively using the bead-beating method. About 460 bp rDNA fragments were obtained by Nested-PCR and analyzed by denaturing gradient gel electrophoresis (DGGE). PCR-DGGE analysis recovered seven operational taxonomic units (OTUs) from the different decomposing litter layers. Six OTUs belonged to Ascomycetes and one to Basidiomycota. Sporobolomyces inositophilus was revealed by both methods, but there was no overlap in other species detected. A major shift of fungal communities on litter occurred during decomposition. The Shannon-Weaver index, which measures diversity in categorical data, was maximal in the F1 layer, then decreased with substratum degradation and reached it lowest value in the H layer. Key words: Fungal diversity, polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE), Traditional culture, Litter.

Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens

BackgroundThe influence of diet on intestinal microflora has been investigated mainly using conventional microbiological approaches. Although these studies have advanced knowledge on human intestinal microflora, it is imperative that new methods are applied to facilitate scientific progress. Culture-independent molecular fingerprinting method of Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR-DGGE) has been used to study microbial communities in a variety of environmental samples. However, these protocols must be optimized prior to their application in order to enhance the quality and accuracy of downstream analyses. In this study, the relative efficacy of four commercial DNA extraction kits (Mobio Ultra Clean® Fecal DNA Isolation Kit, M; QIAamp® DNA Stool Mini Kit, Q; FastDNA® SPIN Kit, FSp; FastDNA® SPIN Kit for Soil, FSo) were evaluated. Further, PCR-DGGE technique was also assessed for its feasibility in detecting differences in human intestinal bacterial fingerprint profiles.MethodTotal DNA was extracted from varying weights of human fecal specimens using four different kits, followed by PCR amplification of bacterial 16S rRNA genes, and DGGE separation of the amplicons.ResultsRegardless of kit, maximum DNA yield was obtained using 10 to 50 mg (wet wt) of fecal specimens and similar DGGE profiles were obtained. However, kits FSp and FSo extracted significantly larger amounts of DNA per g dry fecal specimens and produced more bands on their DGGE profiles than kits M and Q due to their use of bead-containing lysing matrix and vigorous shaking step. DGGE of 16S rRNA gene PCR products was suitable for capturing the profiles of human intestinal microbial community and enabled rapid comparative assessment of inter- and intra-subject differences.ConclusionWe conclude that extraction kits that incorporated bead-containing lysing matrix and vigorous shaking produced high quality DNA from human fecal specimens (10 to 50 mg, wet wt) that can be resolved as bacterial community fingerprints using PCR-DGGE technique. Subsequently, PCR-DGGE technique can be applied for studying variations in human intestinal microbial communities.

Degradation of detergent (linear alkylbenzene sulfonate) in an anaerobic stirred sequencing-batch reactor containing granular biomass

This study aimed to determine the efficiency of an anaerobic stirred sequencing-batch reactor containing granular biomass for the degradation of linear alkylbenzene sulfonate (LAS), a surfactant present in household detergent. The bioreactor was monitored for LAS concentrations in the influent, effluent and sludge, pH, chemical oxygen demand, bicarbonate alkalinity, total solids, and volatile solids. The degradation of LAS was found to be higher in the absence of co-substrates (53%) than in their presence (24–37%). Using the polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE), we identified populations of microorganisms from the Bacteria and Archaea domains. Among the bacteria, we identified uncultivated populations of Arcanobacterium spp. (94%) and Opitutus spp. (96%). Among the Archaea, we identified Methanospirillum spp. (90%), Methanosaeta spp. (98%), and Methanobacterium spp. (96%). The presence of methanogenic microorganisms shows that LAS did not inhibit anaerobic digestion. Sampling at the last stage of reactor operation recovered 61 clones belonging to the domain bacteria. These represented a variety of phyla: 34% shared significant homology with Bacteroidetes, 18% with Proteobacteria, 11% with Verrucomicrobia, 8% with Fibrobacteres, 2% with Acidobacteria, 3% with Chlorobi and Firmicutes, and 1% with Acidobacteres and Chloroflexi. A small fraction of the clones (13%) were not related to any phylum.