فصلنامه زیست شناسی میکروارگانیسم ها
پیاپی 27 (پاییز 1397)

This study evaluated the effect of dietary administration Lactobacillus brevis MF01 on survival rate of total bacteria, lactic acid bacteria intestinal tract fish and changes in gut bacterial communities Sander lucioperca. Lactobacillus brevis was isolated and identified from intestine of Sander luciopercaby biochemical and molecular tests.Fish were fed with dietary administration containing A1 (L.brevis MF01 10 10 CFU / g), A2 (L. brevis MF01 10 8CFU / g) and the control group (without Lactobacillus) for 45 days. At the end of the feeding period, fish were challenged with 4.5× 10 8CFU /ml Aeromonas hydrophila. The intestinal micro biota includes lactic acid bacteria and total bacteria at different times (15,30, 45 days) and 15 days after stopping feedings with probiotics were performed by using MRS agar, Plate count agar media. The lactic acid bacteria levels were significantly increased compared to the control group following probiotics administration in diet (P,< 0.05). The highest number of intestinal micro biota was observed in Lactobacillus brevis 1010Cfu /g treatment (A1)(P < 0.05). Any lactic acid bacteria of the intestine, was not detected in the control group after 15 days. After the end of feeding, the number of total bacteria and Lactic acid bacteria in all groups was decreased. The highest and lowest survival rate, respectively were treatments A1 (86%) and the control group (60%). Discussion and conclusion: The results showed that addition of Lactobacillusbrevis as an additive in feeding of sander lucioperca, significantly increased the lactic acid bacteria, the intestinal bacteria and the survival rate was compared to the control group (injected).

Salmonella is a member of the Enterobacteriaceaefamily causing infectious disease in human and animals. The diseases caused by this bacterium are typhoid, bacteremia, enterocolitis, salmonellosis which are a health problem worldwide. The aim of this study was to evaluate the changes in expression of ahpF and tviAgenes in Salmonella enterica PTCC 1230 exposed to osmotic and oxidative stresses. For osmotic stress, S.enterica PTCC 1230 was subjected to 6, 8, 10, 12, and 14% (W/V) NaCl concentrations and for the oxidative stress, H2O2at concentrations of 1200, 2400, 4800, 6000 and 7200 ppm were used. Change in expression of ahpF and tviA genes were quantified using Real-time PCR method. Based on the obtained results, NaCl at concentrations of 14% and 10% and H2O2atconcentrations of 7200 (ppm) and 6000 (ppm) had maximum effects on the bacterial growth, respectively. In concentration of 7200 ppm, the expression of ahpFgene increased about 1.8 and 2.5-fold more thanH2O2at 6000 ppm concentration and reference gene (16S rRNA).Also, in 14% (W/V) sodium chloride concentration the expression of tviAgene increased about1.3 and 1.5 fold more than 10% and the reference gene (16S rRNA), respectively. Discussion and Conclusion: In this study, changes in expression level of ahpF and tviAgenes upon oxidation and osmotic stresses were studied in Salmonella entericaPTCC 1230. The overall results of this study showed that the expression level of both ahpF and tviAgenes were increased in stress condition and we recommend the study of the other genes incorporate in responses to environmental stresses.

Khabr national park is one of the invaluable natural sources in our country and its microbial variety has not been investigated. Cyanobacteria are of those organisms that are present in all terrestrial and water ecosystems and some minerals are limiting factors in their growth. The goal of this study is to investigate the present cyanobacteria in the soils of the grazed and non-grazed plots in two cold and hot steppes of Khabr Park in the autumn season by means of classic and molecular methods. 32 different regions of Khabr Park were sampled in autumn and were cultured in BG11 medium. Isolation of cyanobacteria was conducted by various sub-cultures. Then the pure colonies were morphologically studied by valid taxonomic keys. Molecular identification of mentioned isolates was performed by amplification and sequencing of 16srRNA gene. The features of mentioned soils were analyzed physically and chemically. The results showed that for the soil texture of sandy loam, the average electrical conductivity was 99.12 µSiemens/cm and the average acidity of soil was 8.29. The isolates of Cyanobacteria belonged to Chroococcidiopsis, Leptolyngbya, Synechococcus, Nodularia. The results of DNA sequencing were compared to NCBI and its results were subjected to BLAST alignment. Discussion and conclusion: The present work revealed that the variety and the number of the Cyanobacteria in this desert area are dependent on different parameters such as weather, humidity, source of nutrients, the amount of light absorbed by the surface and the temperature. The results of molecular identification validated the results of morphological tests.

One of the best methods to remove pollutions resulted from heavy metals in industrial wastewater is biological removing. The first step in this way is identifying and clustering microorganisms especially, resistant bacteria to these metals. Material and methods: In this research, 17 non-fermenting gram-negative bacilli were isolated and identified from industrial wastewater. Their MIC was determined by different concentrations of Cu and Cd. With CTAB and Dellaporta, methods were extracted bacterial DNA. Then evaluated genetic diversity by 7 RAPD, 3 ISSR markers. The similarity matrix was calculated by using NTSYS-pc software based on Jaccard’s coefficient for genotypes and dendrogram was drawn in UPGMA method. 133 bands were identified by using 10 markers that they included 122 polymorphic bands and 11 monomorphic bands. This markers show high level of polymorphism among the bacteria studied. Discussion and Conclusion: In the study cluster analysis and dendrogram drawn based on RAPD and ISSR markers together, there was a significant match between genetic diversity of markers and genetic diversity based on the MIC values. The combined use of both markers for genetic clustering based on resistance to copper and cadmium was more accurate. Principal component analysis was performed to complete the results of cluster analysis and 2-D, 3-D plots were drawn. The results of comparing these two methods showed that these markers had been properly selected for studying genetic diversity, and it was recommended using cluster analysis.

Replication and recombination are two fundamental cellular processes. GyrB and RecF are two proteins that are involved in these processes, respectively. GyrB is a subunit of DNA gyrase, the enzyme maintains negative supercoil in genomic DNA. RecF helps to load RecA in a single-strand gap, which is then repaired by RecA. Genes encoding these proteins are located in the same operon. Besides the general promoter, which is used in logarithmic phase, RecF has its own promoter, which is activated in stationary phase. The aims of this study were to investigate the expression of these genes and to estimate the pattern of RecF expression in the presence of ciprofloxacin. RNAs of the cells were first extracted in logarithmic phase and after cDNA synthesis, the relative expression of these genes was determined in the E. coli mutant with low resistance to ciprofloxacin by Real-Time PCR. Results showed that both genes are overexpressed similarly in the mutant cell treated with a low amount of ciprofloxacin in the exponential phase of growth. The present work revealed that the variety and the number of the Cyanobacteria in this desert area are dependent on different parameters such as weather, humidity, source of nutrients, the amount of light absorbed by the surface and the temperature. The results of molecular identification validated the results of morphological tests.

Probiotics refers to a group of living microorganisms that have beneficial effects on host health. Lactobacilli are one of the most important probiotic bacteria. The stimulatory effects of the immune system, besides reduction of gastrointestinal infections and the risk of inflammation of intestines in humans and animals, are some of Lacobacillus benefits. The effect of rhizospher bacteria, like Lactobacillus, on production of plant hormones, dissolving plant nutrient, facilitates the absorption of nutrients (like nitrogen), and the reduction of plant diseases were reported. Isolation of Lactobacillus has been reported of various sources of dairy and non-dairy sources. The purpose of this study was to investigate the presence of L. plantarum, one of most important probiotic bacteria, in rice rhizosphere, along with examination of the morphological, molecular, biochemical and probiotic properties of the isolated microorganisms. Sampling of rhizosphere rice variety was carried out in Lenjan (Lenjan, Isfahan) carried out. Dillutions of samples were transferred to MES medium. Biochemical test, as sugar fermentation and enzyme activity, performed on gram-positive isolate bacilli. The ability of bacteria to grow in presence of Bile salt, and different pH and temperatures was investigated. Antibiotic resistance of isolates was examined. Specific primers of L. plantarum was used for molecular identification. Of 16 microorganisms, isolated from the rice's rhizosphere, there were 10 bacillus-shaped bacteria, which eight gram-positive isolates were oxidase and catalase negative (characteristic of Lactobacillus species). Sugar fermentation experiments identified six isolates with L. plantarum characteristics. Molecular experiments with the specific primers of L. plantarum confirmed the results. The bacteria grew very well (similar to control samples) in acidic medium at pH 3.5, 40ºC, and in presence of 0.3% bile. Resistance to seven of 12 antibiotics was detected. Discussion and conclusion: The present study showed that rhizosphere of rice is the natural source for isolationof L. plantarum.

L-Asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia. This enzyme is used for the treatment of patients with acute lymphoblastic leukemia, melanosarcoma and lymphosarcoma. It is also used in the production of acrylamide-free foods. Considering the important applications of this enzyme, the aim of this study was to introduce a new microbial source for the production of eukaryotic L-asparaginase. The quality and quantity production of L-asparaginase was investigated in four strains of yeast Yarrowia lipolytica. The quality assessment of L-asparaginase production was done on asparagine minimal agar. Quantitative assay of the enzyme was done in Czapex Dox’s medium by spectrophotometric method using Nessler’s reagent. All of four strains show that they are able to produce asparaginase making a purple halo around the colony. Y. lipolytica DSM3286, DSM70562, DSM3218 and CBS6303 were produced 17/14, 11, 6/98 and 5/61 U/mL of asparaginase in Czapex Dox’s medium after 24 h, respectively. Discussion and Conclusion: Y. lipolytica DSM3286 was selected as the best asparaginase producer strain with the largest halo and the highest asparaginase production. The Y. lipolytica could be used as a potential source of L-asparaginase production.

 Using fungi as sources of enzymes that can catalyze specific reactions conducing to inorganic nanoparticles, is being developed. Aspergillus flavus has a great potential in extracellular biosynthesis of silver nanoparticles. Since this fungus can grow on simple media quickly and adequately, it is cheaper to use it in silver nanoparticles biosynthesis than other methods. Extracellular biosynthesis of silver nanoparticles was carried out by adding 1mM silver nitrate to A. flavus cell filtrate. Nanoparticles production was confirmed by visual observation of color change in the cell filtrate and UV-vis spectrophotometry. More investigation was conducted using XRD, FTIR and TEM. The stability of nanoparticles and the effect of culture media and silver nitrate concentration on nanoparticles’ production were studied too. The color change of cell filtrate from pale to brown confirmed the extracellular production of silver nanoparticles. The absorption spectra indicated a peak at 420nm attributable to the sliver nanoparticles. X-ray diffraction showed crystalline nature of the biosynthesized nanoparticles. Characterization of sliver nanoparticles’ shape and size using TEM showed that they were spherical and 18.2 nm in diameter. These nanoparticles were stable for 11 months and FTIR demonstrated the presence of proteins as capping agents which lead to their stability. More silver nitrate caused more nanoparticles production and PDB was the best media in this process. Biosynthesized nanoparticles were crystalline in nature, small in size and monodispersed. Besides, they were very stable due to capping by fungal secreted proteins; this stability is reported for the first time in the world. The fungal-mediated green and ecofriendly approach towards the synthesis of nanoparticles can be used as an alternative to the more popular physical and chemical methods for the synthesis of nanoparticles

The increasing use of uranium as a suitable source of energy in various industries has led to the depletion of high-grade uranium mines in different countries. Today, the uranium bioleaching process has been used in different countries for easy and cheap access to uranium. In this process, microorganisms are used to extract uranium from low-grade mines. The Acidithiobacillus sp. FJ2 bacterium was exposed to UV radiation. Then, the uranium bioleaching process was conducted in the presence of bacteria exposed to UV and non-exposed bacteria. In followings, this gene was amplified by PCR technique after DNA extraction from bacterial species and coxB gene primer design. Subsequent to gene sequencing and editing with bioedit software, the final sequence of the coxB gene was determined from both bacterial species. Later than, the sequences were examined and compared to prove the presence or absence of the mutation in the radiation sample. The amount of uranium extraction in the presence of bacteria exposed to UV reached to 100% on the second day at the 5% pulp density, whereas the 96.36% extraction yield was obtained on the thirteenth day in pulp density of 50%. This amount was recorded in an unexposed bacterium, in the third and thirteenth days at 5& 50% pulp densities, respectively. The coxB gene sequence was identical in both bacterial specimens. Discussion and conclusion: In this study, UV irradiation to Acidithiobacillus sp. FJ2 increased the rate of uranium bioleaching in the pulp density of 5%, whereas uranium extraction yield was sustained in the 50% pulp density. These effects were independent to the coxB gene.

Nowadays, biodegradation of oil pollution using native bacteria isolates is considered in different research programs. The purpose of this study was to investigate biosurfactant production and degradation of oil spills on Caspian Sea coastlines using native bacterial isolates. Sediment and oil spills samples were isolated from seven stations in the coasts of Bandar Anzali and Kiah Shahr. Biosurfactant production was evaluated using quantitative and qualitative methods such as hemolysis on blood agar, oil spreading method, droplet disintegration, emulsification activity and surface tensile test. Also, total hydrocarbon of oil was estimated. Biodegradation of compounds in sediments and petroleum analyzed using gas chromatography mass spectrometry (GC-MS) method. Identification of strains was performed using different morphological, biochemical and sequencing of 16S rRNA genes. Amongst 115 isolates, 57 isolates showed biosurfactant activity based on qualitative methods, and 15 strains with highest biological removal efficiency were selected for further studies. The spectrophotometry results showed the J3 isolate could remove 94.6% of oil within 7 days, was the most potent isolate in removal of hydrocarbons. Gas chromatography analysis indicated that 90% of aliphatic compounds were removed byJ3 strain in 21 days. The isolates J3, J5, and J12, which showed highest biosurfactant activity, were identified as strains of Bacillus cereussenso latu, Staphylococcus aureus and Pseudomonas aeruginosa, respectively. Discussion and conclusion: The results of this study showed that the isolated strains showed suitable decreasing hydrophobicity and removal of oil hydrocarbons. So, it can be concluded that isolated J3, J5 and J12 are potent microorganisms in cleaning of contaminated waters by oil and other hydrocarbons.