فصلنامه زیست شناسی میکروارگانیسم ها
پیاپی 17 (بهار 1395)

Microbial β - 1, 4 glucanase is one of the key enzymes in glucan hydrolysis. Among bacteria, Bacillus species is a main source for industrial glucanse production. According to interaction between environmental factors on the enzymatic catalytic properties, in this study the potential of β-glucanase production by native strain B. subtilis B5d isolated from apple phyllosphere orchards and determination and modeling the optimum conditions of enzyme activity was surveyed.
In this study, the potential of β-glucanase production by B. subtilis B5d was confirmed using qualitative method. The B. subtilis B5d isolate was identified through biochemical and molecular methods. Determination of optimum condition for beta glucanase activity was evaluated by response surface method and Central Composite Design. Investigated variables included temperature (20- 80 °C), pH (3.5- 8) and concentration of substrate (0.1- 1%).
According to the molecular and biochemical analysis, the strain B. subtilis B5d belonges to B.subtilis species. Based on the Central Composite Design results, the quadratic model was the most suitable model for predicting β-glucanase activity in experimental temperature, pH and substrate concentration range. Furthermore, the optimization of enzyme activity showed that the β-glucanase produced by strain B5d has highest catalytic activity at pH (5.5- 7) and temperature (50- 65°C) and the high concentration of lechinan. However, the enzyme activity is reduced in 80 and 20 °C as well as low concentration of substrate. Validation test of the prediction model of optimization obtained by response surface methodology showed a good agreement between experimental and predicted data.
Discussion and According to the optimum catalytic characteristics, the β-glucanase produced by B. subtilis B5d has a potential to be used as a feed additive.

Daily increasing of bacteria resistance (specially Staphylococcus aureus) to various antibiotics in particular penicillin and methicillin has always led the scientists to look for new medicines.
600 g of Fulgensia fulgens was collected from KaneGonbad mountains in Ilam province, the methanol extract was prepared by soxhle. In vitro antimicrobial activity of the extract against two gram-positive bacteria (S. aureus and Entrococcus faecalis) and two gram-negative bacteria (Pseudomonas aeruginosa and Escherchia coli) was tested by the use of disc diffusion method and microdilution (with determination of MIC and MBC). Wound was made on the dorsal surface of therat and wound infections caused by S.aureus for determination of in vivo antibacterial effect. Than rats were randomly divided into three groups; control, treated with tetracycline ointment and treated with 10% ointment of F. fulgens extract. Finally, wound areas wear measured on days 3, 5, 7, 9 and 11.
Average inhibitory zone diameter of methanolic F. fulgens extract against S. aureus ranged between 11.21 mm to 33.01 mm. According to the wound area on 11th day, it could be concluded that there was a statistically significant difference between the control group (0.63 cm2) and two treatment groups (0) (pDiscussion and According to the results, the methanol extract of F. fulgens in the treatment of infections as S. aureus can be replaced by chemical antibiotics.

Raw ingredients used in confectionary carry high risk of infection with Escherichia coli. Since confectionaries are offered in the market in quite varied forms and types and there is a great difference in the sanitary status of the confectionaries, this study aimed at evaluation of E.coli frequency distribution in different types of confectionery products in Isfahan market. In addition, the effect of moisture content, products category and the sanitary level of the confectionaries as well as product types (Industrially or traditionally produced) on the contamination level were studied.
A total of 200 samples were randomly collected from confectioneries in Isfahan city through simple random sampling method. Preparation and dilution procedures were conducted under sterile conditions. Samples were cultured on EMB agar medium. Later, some of the positive isolates were randomly selected and confirmed by TSI and IMVIC test. Data analysis was performed using descriptive indices. Also, one way ANOVA and LSD test or independent t test were applied to determine the statistically significant difference between the mean E.coli cell numbers in the categorized groups of sweets.
It was found that 19% of the total tested samples were contaminated with E.coli strains. The mean, median and maximum of contamination were (35±.5), (0) and (3.4) CFU/gr, respectively. Moisture content, products category and being traditionally or industrially produced have significant effects on the level of contamination; while, the sanitary status of the traditional confectionaries as graded in this study has no impact on the average E.coli cell count.
Discussion and Regarding the microbial quality, at least about 25% of the sweets in the market do not meet the national standards of confectionary products. Implementation of strict hygiene regulation in the traditional confectionaries is in need to provide the public with safe, guaranteed products.

Plant probiotic bacteria like fluorescent pseudomonads are worldly used against soil-borne pathogens through mechanisms such as production of bacterial metabolites and enzymes. These bacteria can also help plants to tolerate environmental stresses. Ectoine is a compatible solution which plays an important role in environmental osmotic stresses.
Tolerance of 24 bacterial strains to salt and heat was tested and 6 tolerant strains were chosen. Then dual culture of Fusarium solani and bacteria grown in presence of ectoine and hydroxy ectoine with 3 NaCl concentrations (0, 150 and 300 mM) was done and Pseudomonas fluorescens UTPF5 was selected. Finally the effect of ectoine, hydroxyectoine and NaCl on bacterial population, lipase, protease, siderophore and hydrogen-cyanide production and biofilm formation was investigated.
In 300 mM NaCl, the bacterium grown in presence of ectoines, increased the inhibition percentage 5- times more than control. NaCl had a positive effect on bacterial population in water and ectoine, hydrogen-cyanide production in all treatment and biofilm formation in ectoine and hydroxyectoine and negative effect on bacterial population in hydroxyectoine, protease and siderophore production in all treatments and biofilm formation in water. On the other hand, ectoine increased lipase and hydrogen-cyanide production and biofilm formation in 300 mM NaCl and siderophore production in 150 mM. Hydroxyectoine had similar effects with little differences.
Discussion and Ectoine and hydroxyectoine moderate the biocontrol reduction in presence of NaCl through positive effect on lipase and hydrogen-cyanide production and biofilm formation.

Arachidonic acid is an important essential fatty acid in human nutrition. The filamentous fungus Mortierella alpina has been identified as a promising producer of arachidonic acid. Mortierella alpine can accumulate up to 40% (w/w) lipid, of which up to 40% can be arachidonic acid.
Mortierella alpina CBS 754.68 was cultivated in low cost substrate such as glucose syrup, brown sugar and starch for lipid and arachidonic acid production. The reduced sugar, total lipids and content of ARA were determined by dinitrosalicylic acid method, soxhlet and Gas chromatography–mass spectrometry (GC-MS) respectively.
The carbon sources were applied at 70 g/l and nitrogen source (soybean powder) at 10 g/lit. The results showed that lipid in dry biomass in glucose syrup, starch and brown sugar media were obtained 32, 25 and 13 % w/w respectively. The arachidonic acid contents of lipid in the glucose syrup, starch and brown sugar media were 41, 33 and 31 % w/w respectively.
Discussion and Lipid fatty acid compositions are affected by the growth of microorganism. Cell membrane fatty acids such as stearic acid and oleic acid increased substantially concomitant with increases in the amount of biomass. Biomass and oil production efficiency fell due to inappropriate brown sugar medium.

Biodegradation is a good alternative rather than chemical and physical methods for cleaning oil contaminated areas. Several factors like crude oil concentration, biosurfactant production, salinity and incubation time affect the biodegradation.
In this study, seawater sample and gastropod were collected from Persian Gulf. To isolate oil degrading bacteria from collected samples, ONR7a medium was used. The strains that had more growth and higher oil removal were selected and identified. The factors such as the effect of different concentrations of oil, incubation time, mixed cultures and salinity on the biodegradation were investigated.
Six crude oil degrading bacteria were isolated. Between these bacteria 2 strains were selected based on higher oil removal. These strains belonged to the genus Vibrio and Halomonas. Strains with higher Emulsification activity produce more biosurfactant and have higher oil biodegradation. Growth and oil degradation have increment pattern by prolonging the incubation time. Mixed culture of Vibrio and Halomonas strains have higher rates of degradation rather than culturing with one of them. Increase in crudeoil concentration to 2.5% caused reduction in growth of bacteria and degradation of oil.
Discussion and The results of this study show that crude oil degrading bacteria have high diversity in Persian Gulf. These bacteria have higher capability for oil degradation thus they can be used for remediation of oil contaminated areas.

Nitriles are toxic and hazardous compounds for all organisms which are produced enormously by human being and cause environment pollution. Biodegradation is the best method for Nitrile elimination in sewage. The aim of this study was isolation and identification of native bacteria Acetonitrile-degrading from the sewage of the city of Kerman.
The enrichment and screening of Acetonitrile degrading bacteria was performed in a specific medium containing 1 % Acetonitrile as sole carbon and nitrogen source. Enzyme activity and ammonium production was determined in growth medium using a modification of the phenol/hypochlorite method. Identification of the isolates was undertaken using microbial - biochemical and molecular tests. The optimization of enzyme production was examined. The amount of nitrile and acid were also determined by gas chromatography.
Among three isolated nitrile hydrolyzing producing species (FA3, FA8 and AB19), FA8 isolate showed maximum enzyme productivity. The results of characteristic tests showed that these bacteria belonged to the Pseudomonas sp. Moreover, 16S rRNA sequencing and phylogenetic analyses exhibited that FA11, FA8 and AB19 strains were similar to Pseudomonas Otitidis and Pseudomonas geniculate with 99, 100% homology, respectively. Results of medium optimization of Pseudomonas FA8 strain showed that glucose (10 gL-1) and yeast extract (5 gL-1) in neutral medium strongly supported enzyme production. Gas chromatography results showed that FA8 produced 58% acetic acid at 48 h of incubation.
Discussion and The results demonstrated that Pseudomonas FA8 isolated bacterium is a suitable candidate for degradation of Acetonitrile. This bacterium could be used for treatment of industrial wastewater and sites that contaminated with Acetonitrile.

Liposomes are spherical vesicles composed of concentric phospholipid bilayers that can entrap hydrophilic, hydrophobic drugs. Liposomes can be prepared from natural phospholipids, synthetic lipids or bacterial lipids. The aim of this study was to formulate liposome from bacterial lipids and evaluate physicochemical properties.
This study was performed experimentally on E.coli. The lipids were extracted from E.coli. using chloroform and methanol. Film method was used for preparing nano-systems and methylene blue was used as a drug model. Then their particle sizes were determined using particle sizer. The release methylene blue was carried out using dialysis membrane. Also, trailing them in cancer cells was evaluated by using carboxyfluorescein.
The average particle size of E.coli. liposomal was 338 nm. Encapsulation efficiency was 53.33 ± 2.88% and the value of release after 24 h was 97.54% ± 0.00. Liposomes could deliver the carboxyfluorescein to cancer cells.
Discussion and The results of this study demonstrated that bacterial liposome has probably a suitable nano-particle such as particle size and desirable loading and it is possible to use them as drug delivery system.

Biosurfactants are unique amphipathic molecules with extensive application in removing organic and metal contaminants. The purpose of this study was to investigate production of biosurfactant and determine optimal conditions to produce biosurfactant by Bacillus pumilus 1529 and Bacillus subtilis WPI.
In this study, effect of carbon source, temperature and incubation time on biosurfactant production was evaluated. Hemolytic activity, emulsification activity, oil spreading, drop collapse, cell hydrophobicity and measurement of surface tension were used to detect biosurfactant production. Then, according to the results, the optimal conditions for biosurfactant production by and Bacillus subtilis WPI was determined.
In this study, both bacteria were able to produce biosurfactant at an acceptable level. Glucose, kerosene, sugarcane molasses and phenanthrene used as a sole carbon source and energy for the mentioned bacteria. Bacillus subtilis WPI produced maximum biosurfactant in the medium containing kerosene and reduced surface tension of the medium to 33.1 mN/m after 156 hours of the cultivation at 37°C. Also, the highest surface tension reduction by Bacillus pumilus 1529 occurred in the medium containing sugarcane molasses and reduce the surface tension of culture medium after 156 hours at 37°C from 50.4 to 28.83 mN/m.
Discussion and Bacillus pumilus 1529 and Bacillus subtilis WPI had high potential in production of biosurfactant and degradation of petroleum hydrocarbons and Phenanthrene. Therefore, it could be said that these bacteria had a great potential for applications in bioremediation and other environmental process.

Oil is one of the most important energy sources that contain variety of organosulfur compounds that are combustible and can produce sulfur dioxide which will cause pollution over the atmosphere and the soil. Dibenzothiophene (DBT) is often used as a model for biodesulfurization studies and surfactant Tween80 increases the solubility of DBT in water that leads to higher consumption by microorganisms.
DBT specific UV spectrophotometry at a wavelength of 323 nm was used to evaluate the ability of isolated Exophiala spinifera fungus in removal of DBT. The effect of various concentrations of surfactant Tween80 on the growth of the fungus and DBT utilization was studied.
Exophiala spinifera was able to remove 100% DBT after 7 days of incubation at 30 ° C and 180 rpm shaking. The effect of different concentrations of surfactant Tween80 on growth and DBT utilization by this fungus was examined and it was observed that the presence of surfactant in the culture medium increased the growth and removal of DBT, therefore the amount of DBT utilized with 0.4% concentration of the surfactant was about 30% more than that utilized without surfactant. However, higher concentrations of surfactant Tween80 decreased the growth and consumption of DBT by fungi.
Discussion and Exophiala spinifera was isolated from oil contaminated soil and able to utilize toxic compound DBT as a sulfur source in the presence of other carbon sources such as glucose. So this isolated strain could be a good candidate for the petroleum desulfurization and it is the first report about desulfurization of DBT by fungus Exophiala spinifera. Growth and removal of DBT by fungus increased in the presence of surfactant Tween80. It can be concluded that the surfactant increases the total DBT transfer between the organic and aqueous phases and has a potential application in DBT bioremediation system by the studied fungus biocatalyst.

Pectinase enzyme is one of the most important industrial enzymes which isolated from a wide variety of microorganisms such as bacteria and filamentous fungi. This enzyme has been usually used in the fruit and textile industry. In this study, the isolation and optimization of pectinase-producing fungi on decaying rotten fruits were studied.
Isolation and screening of pectinase producing fungi performed through plate culture on pectin medium and staining with Lugol's iodine solution. The best strains were identified by ITS1, 4 sequencing as Aspergillus fumigatus, Rhizopus oryzae, Penicilium chrysogenum. The enzyme production was optimized by application of the five factorial design, each at three levels. These factors are carbon sources (whey, glucose and stevia), ammonium sulfate, manganese sulfate, temperature, and pH. Pectinase concentration was measured by the Miller method.
The results indicate that optimum condition for enzyme production for three fungi strains was obtained at 32 °C, pH = 6, 3g / L manganese sulfate, 2.75g / L of ammonium sulfate and 10g / L of each carbon source. The best experiment in obtaining the optimum enzyme contained 1.328 mg / ml of glucose for Aspergillus niger 1.284 and 1.039 mg / ml of whey for Rhizopus oryzae and Penicilium chrysogenum. Molecular weight of enzyme was about 40 and 37 kDa which was obtained by SDS- PAGE.
Discussion and The results indicate that three strains could grow in a wide range of carbon source, pH and temperature, which could be a good candidate for industrial application.

Travertine results in the accumulation of calcium carbonate on karst springs, hot springs, small rivers and marshes. Badab Sourt is the travertine-maker spring that located in Mazandaran province and is the second natural place in Iran. It has two springheads with different characteristics and different colors and different sediments that give unique beauty to spring. Travertine is a good model to study the relationship between environment and microorganisms. This study centered on the influence of microorganisms on precipitation of calcium carbonate and isolation of microorganisms that have precipitation potential.
Here, to assess effects of microorganisms on mineralization were used microscopic and culture methods; from stone samples, photos have gotten with SEM and polarizing microscope and from water sample bacteria were isolated. Mineralization function of bacteria was investigated with culturing them on B4 medium and mineralization potential was approved by polarizing microscope.
SEM photos showed microorganisms have effect on mineralization and can act as a nucleus in beginning of crystal production. Finally, five strains Bss-3, Bsw-28d, Bss-11a, Bsw-1c1, and Bsw-39b were isolated, which have ability to precipitate calcium carbonate. In between strain Bss-3 that has 99.6% similarity with taxon Labrenzia aggregate IAM 12614T was the first report that has the ability to precipitate calcium carbonate and among the strains, strain Bsw-28d with precipitation of 45.6 mg / ml CaCO3 was the best strain.
Discussion and Microorganisms are important in the formation and evolution of their surrounding environments, thereby native microbes must be concerned for conserving and restoring the environments.

One of the most important factors in Urinary Tract Infection caused by Uropathogenic Escherichia coli, is the attachment of bacteria to the host cell surface. Thus, inhibition of bacterial attachment is the appropriate action to prevent infection. The aim of this study was to investigate the antimicrobial and especially anti adhesive characteristics of probiotic bacteria against Escherichia coli by using microbial techniques.
In this study two strains of Lactobacillus acidophilus PTCC 1643 and Lactobacillus casei PTCC 1608 were used .40 Uropathogenic Escherichia coli were collected from Semnan province hospitals.20 samples with the more capability of biofilm production were selected for microbial tests. To evaluate the antimicrobial activity of complete culture and supernatant of probiotic lactobacilli, modified double layer method and dilution of supernatant were used, respectively. The mechanism of co- aggregation of lactobacilli with pathogens was examined. The microtitre plate method was used to detect anti-adhesive activity of Lactobacilli supernatant.
The antimicrobial and anti-adhesive effects of probiotic lactobacilli on Uropathogenic Escherichia coli were confirmed in all tests. In this study, Lactobacillus casie with the growth inhibitory (42/7 mm) and anti-adhesive (46/7mm) effects were reported as a proper probiotic bacterium.
Discussion and According to the results, the probiotic lactobacilli have spectacular effects to prevent attachment, biofilm formation and pathogenicity of UPEC, so using them to prevent and treat Urinary tract infection is a practical, reasonable and acceptable method.

Bacterial strains present in food products undergo different thermal processes such as coldness and warmth. Such cases cause a shock in bacteria and force the bacteria to produce proteins and partly, develop a change in the production of enzyme. This can give the strain a special characteristic, knowledge of this characteristic will contribute to a timely and more precise identification.
During this time more than 100 samples have been examined, out of which, 48 Indol positive isolation samples were examined by phenotypic tests and sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS- PAGE).
– The results of numerical analysis of phenotypic characteristics and protein patterns showed that only 79% of the collected isolates (phenon 1 and 2) could be identified as E.coli compared with reference strains. E.coli strains from ice creams were showed some Variation in banding patterns. Major differences were observed in protein bands between 23.59 - and 20.79 -kDa molecular mass range which the isolates were compared with reference strains.
Discussion and Our study concluded that food’s bacterial strains are influenced by temperatures in different processes and also it could stimulate the production of proteins or change the enzymes. Therefore, The reason of taking care of the issues is that changes in the proteins’ structures can lead to change in the biochemical properties, and finally this change can misguide us. Further research is being performed to characterize these atypical strains by molecular methods.

Selenium is an element with antioxidant activities that plays roles in thyroid hormone homeostasis, immunity and also fertility. Nevertheless, selenium toxicity (selenosis) causes problems for humans such as abnormalities of the nervous system, gastrointestinal problems and hair loss. Thus, this study was performed with the aim of bacterial biosorbent isolation in order to remove selenium contaminant from wastewater.
In this research, at first using modified Luria- Bertani agar (mLBA) medium with certain concentration of sodium selenate salt, isolation of bacterial isolates was done from three collected wastewater and sludge samples from Khouzestan industrial factories. After determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), the sorption capacity and the percentage of metal removal efficiency (%RE) were investigated by atomic absorption spectrophotometer using metabolically active and inactive samples belonging to an efficient isolate. Identification was performed by morphological, biochemical and molecular methods.
Among 73 attained bacterial isolates at the first stage, 8 selenate oxyanion resistant isolates were gathered. Among these, AMS1-S8 isolate with MIC= 600­mM and MBC= 1200­mM were selected for more studies. Attained results in sorption mechanism determination stage showed that the sorption capacity in metabolically active sample is more than the inactive samples. Based on the identification results, it is revealed that this isolate belongs to the Enterobacter genus. This isolate is deposited as accession JQ965667 in the GeneBank database.
Discussion and The results showed that active biomass of selected isolate, have most sorption capacity and %RE and among the other isolates, have high partial resistance against selenate. Therefore, it can be a relatively ideal option for the bioremediation of polluted environments.