The microbial communities in ethanol-methane coupling fermentation reactors were studied. The community structure variation was monitored at the genus and phylum levels using PowerSoil® DNA Isolation ...Kit with 16S ribosomal RNA (16S rRNA). The distribution of microbial communities in the ethanol reactor was higher than the methane reactor at the phylum and genus level, indicating the influence of coupling fermentation. Clostridia (hydrogen producer) was the dominant species throughout the process at genus and phylum levels. This result indicates the efficient degradation of organic acids. In addition, Archaea methanogen species (aceticlastic methanogens) utilize both acetate and hydrogen to produce methane. The dominance of Methanosaeta rather than Methanosarsina in the anaerobic digestion reactor (R1) of coupling fermentation added further valuable information on food waste treatment. Moreover, lactic acid bacteria species (Lactococcus) was dominant in the ethanol reactor (R2), suggesting the efficient conversion of food waste to lactic acid, which could continue its conversion to ethanol. Interestingly, the high amount of ammonia, salts, and volatile fatty acids (VFAs) (including high acetate) could promote the SAO pathway in the coupling fermentation system.
Genomic DNA from WBCs is widely often used for PCR. Although kits for DNA isolation are in common use, there is scarce information about their performance and about the PCR quality of the genomic DNA ...obtained. Hence, three different kits, QIAamp blood mini kit, High Pure PCR Template Preparation Kit and the Puregene DNA isolation kit were evaluated on these aspects. Genomic DNA was isolated from whole blood samples with WBC counts ranging from 0.5 to 20*109 WBC/L. The WBC count was used to calculate the amount of genomic DNA. The actual amount of genomic DNA isolated, was determined spectrophotometrically. The DNA extraction efficiency was obtained by dividing the actual amount of DNA by the calculated amount yielded. PCR quality was analysed by measuring Cycle threshold (Ct) values with a quantitative real-time PCR β-globin assay. The extraction efficiency of the three DNA isolation kits was 20% to 40%. Spectrophotometrically determined DNA concentrations correlated inversely with Ct values, regardless of the DNA isolation kit applied, whereas the strongest correlation was obtained for the Puregene DNA isolation kit. WBC counts also correlated inversely with Ct values and here the strongest correlation was found for the QIAamp blood mini kit. In conclusion, the overall performance of the DNA isolation kits was quite comparable (DNA recoveries of 20-40%), albeit the QIAamp blood mini kit yielded the most reproducible extraction efficiencies and lowest Ct values within every WBC count category.
Cyclospora cayetanensis is a protozoan parasite that causes foodborne outbreaks of diarrheal illness (cyclosporiasis) worldwide. Contact with soil may be an important mode of transmission for C. ...cayetanensis and could play a role in the contamination of foods. However, there is a scarcity of detection methods and studies for C. cayetanensis in soil. Traditional parasitology concentration methods can be useful for the detection of C. cayetanensis, as found for other protozoa parasites of similar size. The present study evaluated a concentration method using flotation in saturated sucrose solution, subsequent DNA template preparation and qPCR following the Bacteriological Analytical Manual (BAM) Chapter 19b method. The proposed flotation method was compared to three commercial DNA isolation kits (Fast DNATM 50 mL SPIN kit for soil (MP Biomedicals, Irvine, CA, USA), Quick-DNATM Fecal/Soil Microbe Midiprep kit (Zymo Research, Irvine, CA, USA) and DNeasy® PowerMax® Soil Kit (Qiagen, Hilden, Germany)) for the isolation and detection of DNA from experimentally seeded C. cayetanensis soil samples (5−10 g with 100 oocysts). Control unseeded samples were all negative in all methods. Significantly lower cycle threshold values (CT) were observed in the 100 oocyst C. cayetanensis samples processed via the flotation method than those processed with each of the commercial DNA isolation kits evaluated (p < 0.05), indicating higher recovery of the target DNA with flotation. All samples seeded with 100 oocysts (n = 5) were positive to the presence of the parasite by the flotation method, and no inhibition was observed in any of the processed samples. Linearity of detection of the flotation method was observed in samples seeded with different levels of oocysts, and the method was able to detect as few as 10 oocysts in 10 g of soil samples (limit of detection 1 oocyst/g). This comparative study showed that the concentration of oocysts in soil samples by flotation in high-density sucrose solutions is an easy, low-cost, and sensitive method that could be implemented for the detection of C. cayetanensis in environmental soil samples. The flotation method would be useful to identify environmental sources of C. cayetanensis contamination, persistence of the parasite in the soil and the role of soil in the transmission of C. cayetanensis.
