فصلنامه زیست شناسی میکروارگانیسم ها
پیاپی 14 (تابستان 1394)

Greenhouse effect problems، environmental pollution، global increase in oil demand، and reduced fossil fuel resources have boosted research on the production of renewable energies such as bioenegries in recent years. Amongst various biofuels، biobutanol has been recently introduced as a replacement liquid fuel for gasoline and gas oil. Anaerobic bacteria such as Clostridium acetobutylicum are able to produce acetone، butanol، and ethanol using different sugar sources. This bacterium exhibits amylolytic activity and therefore is able to hydrolyzes starch to glucose and use it directly as carbon source. In this study، three substrates including glucose، treated starch and starch were used in different concentrations to produce butanol by Clostridium acetobutylicum PTCC 1492. For all three carbon sources، 60 g/ l of substrate was found as the optimum concentration. The results revealed that this bacterium is capable of producing butanol using all three carbon sources without any treatment. Butanol concentrations of 6. 45، 5. 81، and 4. 64 g/ l were obtained using non-treated starch، treated starch، and glucose as carbon sources، respectively. Discussion and The results suggested the possibility of using non-hydrolyzed starch as carbon source for butanol production. Also it was shown that non-treated starch produces more butanol compared to the treated starch.

Omega 3 fatty acids play an important role in human health. Docosahexaenoic acid is the most important of polyunsaturated fatty acids that is supplied through the consumption of fish oil. Consequently، there is a need for new alternative sources like as single cell oils، because of possibility of heavy metal contamination in fish oil. In this research، the samples were collected from mangrove forests in the Persian Gulf and Oman Sea. The strains of marine protist were isolated in GYP medium. Oil-producing strains were screened by fluorescence microscopy. Selected strain was identified by sequencing 18s rRNA gene and polyunsaturated fatty acids profile was determined by gas chromatography. More than 20 marine protist strains were isolated. Triacylglycerol granules were detected in strain TA4 by orange fluorescent. Dyad، tetrad، octad cells and zoospore were observed in cell cycle. 18s rRNA gene sequence of this strain (1730 bp) was similar more than 97 % to Aurantiochytrium strain (Accession number: KJ938302). Its oil content is 46 % of CDW. The quantity of DHA، DPA and EPA fatty acids was 105، 46 and 26 mg per liter، respectively. Their contents were 16، 7 and 4 % of the total fatty acids، respectively. Discussion and Thraustochytrids strains have a unique ability to produce polyunsaturated fatty acids، especially DHA and can be an alternative to these valuable compounds. These strains can be used for production of omega 3 oils due to their potential for production of DHA.

Phenolic compounds are toxic environmental pollutants. Removal of phenol from wastewaters is very important. Phenol biodegradation is generally preferred to common physico- chemical methods. The aim of this study is to enrich and isolate haloalkaliphilic phenol-degrading bacteria and evaluate their potential for caustic wastewater treatment in consortium and pure cultures. Soil and water samples were collected from hydrocarbon polluted areas. Samples were enriched in the mineral medium with phenol as the only carbon and energy source. Phenol degrading strains were isolated and identified using morphological and phylogenic techniques. Isolates were screened based on phenol removal ability and key parameters such as pH، temperature and various salt (NaCl، NaNO3، NH4Cl and KCl) and phenol concentration were evaluated for optimizing biodegradation activity of the selected strain. Three phenol-degrading strains were isolated from Yadavaran oilfield sample، which belong to bacterial genera Halomonas، Janibacter and Pseudomonas. The strain Janibacter sp. YF3 was selected due to its high phenol-degrading rate. The strain was gram positive coccus whose optimal growth condition was 30 ºC، Nacl 5 % (w/v)، pH 8. 5 and phenol 400 ppm. The results showed that this strain could thrive in highly saline conditions and preferred NaNO among other salts. Discussion and Isolate entitled Janibacter sp. strain YF3 was a polyextremophile which could tolerate harsh conditions (NaCl concentration up to 10 % and pH 9. 5) and could utilize phenol at high concentration up to 1000 ppm. These results showed that this strain could be potentially applicable for phenol removal from haloalkaline industrial wastewaters such as spent caustic.

In recent years، substitution of soy milk with cow milk in the individuals'' diet suffering from inflammatory diseases have been considered in clinical studies. However، soy milk is more prone to oxidation compared to cow milk due to its unsaturated fatty acid. Oxidative stability of soy milk fat and its comparison with cow milk fat has been barely discussed in the literature. Present study aimed at comparing the oxidative stability of commercial pasteurized milk and soy milk samples and their fermented counterparts by L. plantarum A7 during 14 days refrigerated period. Commercial pasteurized milk and soy milk with the same concentration of fatty compound were subjected to fermentation using a native lactobacillus strain، Lactobacillus plantarum A7. Peroxide number and thiobarbitoric acid in the four trials including milk، soymilk، fermented milk and fermented soy milk were investigated during 14 days of cold storage in the controlled condition. The results showed that milk and soy milk fermentation by Lactobacillus plantarum significantly reduced the amounts of both oxidative indices. In non fermented products، peroxide value was found to increase by a similar trend، reaching to the pick after five days of refrigeration. No significant difference was observed between such an increase between milk and soy milk. Instead، TBA value was measured at higher level in soy milk samples all through the study period. Discussion and Despite the higher non saturated fatty acid، soy milk appeared to be as sensitive as milk regarding oxidative damage، However، comparing the great impact of fermentation on retarding oxidation، the difference between no fermented products can be regarded as negligible.

Identification of the optimum culture conditions of unicellular algae in different culture media is one of the most important steps required for industrial cultivation requirements of algae. Two microalgae Haematococcus sp. and Desmodesmus cunaetus, as valuable microalgae families, were newly isolated from inland of West Azerbaijan Province. In order to determine the best medium for these algae five different media (BM, RM, OHM, BBM and CHU) were studied for their growth rate and fatty acid content in a period of 12 days. To determine the growth rate, cell density and doubling time, counted daily done by Neubauer haemocytometer. The cells were harvested in the stationary phase to determine the fatty acid and protein content. The results of present study revealed the highest density (18.9 ± 0.68 ×105 cell.mL-1) and maximum specific growth rate (0.16 ± 0.06 day-1) in the microalgae Haematococcus sp. while using in OHM medium. The current study also depicted the maximum cell density (21.31 ± 1.11×105 cell.mL-1) and growth rate (0.17±0.06 day-1) of D.cunaetus in RM medium. In addition, the maximum content of PUFA (polyunsaturated fatty acid) and n-3/n-6 of both microalgae was found in the culture medium of BBM.Discussion and On the basis of our experimental results, we conclude that no clear parallel could be established between good nutritional characteristics of the biomass and mechanism of algae growth hence further research is demanded in this area. However, a compromise between the nutritional properties and growth of both microalgae could be achieved by the use of BBM medium, which provides good fatty-acid composition and adequate protein content as well as relatively high specific growth rate.

Proteases are the most important industrial enzymes. These enzymes have gained much attention due to their industrial applications in eliminating the environmental pollutions and improving the industrial processes. The aims of this research were the isolation and identification of native yeasts that were able to produce the alkaline protease. Several samples of soil, water and wastewater were collected from various places. The culture media used for screening of yeast spp. with potential of alkaline protease production were alkaline peptone agar and alkaline milk agar. The alkaline protease activity in potential spp. was measured using Lowry method. The effects of different culture media on the production of alkaline protease by selected yeast isolates were determined. For molecular identification of the best isolate, the DNA extraction was performed and the PCR followed using universal 18s rDNA primers. Among 27 isolates with the capability of alkaline protease production, one isolate with the most enzyme activity, 525 u/ ml, was selected for further processing. The best culture medium for alkaline protease production was YPG broth. The comparison of 18s rDNA sequence from isolated yeast with the highest enzyme activity, with the genomic database available in Gen Bank, using BLAST software showed that our yeast isolate had the highest similarity with Yarrowia lipolytica.Discussion and According to numerous applications of alkaline proteases industrially and the lack of their production in our country, the native strains could be beneficial for their production. Regarding to non-pathogenicity and high productivity of alkaline protease by our isolated yeast, Yarrowia lipolytica, this isolate could be introduced as an amenable strain for industrial production of alkaline protease.

Acinetobacter baumannii is a gram-negative coccobacill that has a great distribution in the nature and is considered as one of the important causes of hospital infections. Due to the antibiotic resistances in this bacterium several problems in the successful treatment of patients and subsequently their mortality have been occurred. The present study was carried out in order to detect the most prevalent antibiotic resistance genes in Acinetobacter baumannii strains isolated from the hospital infections. This descriptive study was carried out on 121 strains of Acinetobacter baumannii isolated from clinical samples of two hospitals of Tehran city. After identifying isolates using the biochemical tests, the antibiotic resistance pattern of them was done using the disk diffusion and molecular (in order to identify the antibiotic resistance genes) methods. Of 121 strains of Acinetobacter baumannii isolated from infectious samples, the most commonly detected antibiotic resistance was against tetracycline and the most sensitivity was achieved against chloramphenicol, nitrofurantoin and meropenem by distribution of 1.65 %. The distribution of dfrA1, tet(A), aac(3)-IV, sul1, tet(B), aadA1, qnr, CITM, blaSHV, sim, vim, oxa-58-like. Oxa-24-like, imp, oxa-51-like, oxa-23-like, cat1 and cmlA were 79 (65.27 %), 71 (58.67 %), 68 (56.19 %), 67 (55.37 %), 44 (36.36 %), 41 (33.88 %), 29 (23.96 %), 28 (23.14%), 24 (19.83 %), 17 (14.04 %), 16 (13.22 %), 14 (11.57 %), 12 (9.91 %), 10 (8.26 %), 9 (7.43 %), 8 (6.61 %), 5 (4.13 %) and 3 (2.47 %), respectively.Discussion and Multiple antibiotic resistance in studied isolates the use of molecular techniques in the antibiogram test to select the most effective antibiotic treatment of infections and the importance of continued antibiotic surveillance that will provide succession in the efforts of infection control programs for the future.

Biofilms are population of bacteria cells that cause irreversible binding to the surfaces by producing extracellular polymers. Biofilm formation in bacteria causes resistance to antimicrobial agents and can lead to severe problems in this ground. The purpose of this study was to evaluate biofilm formation in some isolates of Pseudomonas aeruginosa (13 case), Staphylococcus aureus (13 case), Enterobacter (13 case) and Acinetobacter (13 case) which were collected from human infections of Alzahra hospital in Isfahan. Also the minimum inhibitory concentration of chlorhexidine and its impact on the growth of planktonic and biofilm formation for these isolates were determined. Statistical analysis and graphing have been carried out by using SPSS software (version 20) and Excel. All isolates (52 isolates) have produced biofilm. The mean of MIC of chlorehexidine antiseptic for the p.aeruginosa, S.aureus, Enterobacter and Acinetobacter were 0/001, 0/00013, 0/001, 0/0003 g/ml respectively. Planktonic bacterial growth inhibition from p.aeruginosa and Enterobacter in 1/4 MIC and 1/8 MIC respectively was seen in 40 and 60 % cases. Acinetobacter and Staphylobacter aureus, have been controlled in 40 % of cases in 1/4 MIC and 60 % of cases in 1/8 MIC. Biofilm has not been produced in any of MIC and 2MIC dilution, and the power of biofilm formation had been increased significantly by reducing concentration of chlorhexidine dilution.Discussion and The results indicate that the use of chlorhexidine in appropriate concentrations (MIC) can prevent bacterial growth and biofilm formation in different species causing hospital infections, but doses of chlorhexidine that are less than the MIC can stimulate biofilm formation.

Environmental factors and diet are of the most important factors that affect the microbial population of the gastrointestinal tract and there are many antimicrobial systems in honey bees and their foods naturally. The purpose of this study was to investigate the effect of dietary acetic acid and concentrated mixture of probiotic and antibiotics of oxytetracycline and neomycin on population coliform bacteria and lactobacilli changes in honey bee pupae. The experimental treatments consisted of different levels of acetic acid 5 % (10, 20 and 30 g/l), protexin probiotic, Oxytetracycline 20 % and Neomycin 20 % antibiotics with levels of 0.1, 0.2 and 0.3 g/l and sugar syrup 50 % and honey syrup 50 % was performed in a completely randomized design with four replications for 20 days. After feeding the experimental groups, one gram of 18 day old worker bee's pupae samples of each colony was poured into 9 ml of physiological solution. After preparing different dilutions, each sample was inoculated on MRS agar and MacConkey agar medium. Lactobacillus and coliform colonies were counted after finishing incubation period. The results showed that the highest population of coliform bacteria treated with sugar syrup (2.58 log CFU/g) and the lowest in treatments 0.2 and 0.3 g Neomycin, Oxytetracycline, and probiotic that were equal to zero (P value <0.05). The highest population of Lactobacillus bacteria belonged to 0.2 g probiotic treatment (4.05 log CFU/g) and the lowest was for 0.2 and 0.3 g Neomycin treatment which was equal to zero (P value<0.05).Discussion and This study showed that the addition probiotic in sugar syrup for feeding larvae resulted in increasing population of Lactobacilli in the body of the bee pupae than the control groups.

In the last decade, biology and microorganisms is built to clean up environmental pollutants such as heavy metals. Isolation of metal resistant strains to achieve the goal is very important and the aim of this study was isolate the chrome resistant strains. Five samples of oil contaminated soils of Khuzestan zones were collected under sterile conditions and transferred to laboratory immediately. The soil samples were homogenized and diluted by sterile saline up to 10-10 and cultured on Luria Bertani agar containing 5ppm potassium dichromate. Resistant strains were isolated after 24 hours of incubation, and then for isolation of appropriate strains, cultured on Macconkey agar. Isolated bacteria were identified by biochemical tests. Then, Luria Bertani agar was used for screening of resistant strains in form of MIC test. The best conditions for bacterial growth were found in the presence of chromium in various temperatures, rate of shaking and pH values by spectrophotometry at 600 nm in the overnight of cultivation. Absorption tests of metal were started under optimal conditions. From total of 24 strains of isolated Pseudomonads, 14 strains were resistant to chromium. The best strain (Mac-2) had removed 35.60 % of chromium from aqueous culture mediums under optimal conditions (pH: 8, Temp: 40°C and Shaking rate: 200rpm).Discussion and Since heavy metals in various physicochemical forms are considered as environmental pollutants in most parts of the world, thus, the contaminated regions must be detoxified. The isolated strain can be used for studies of bioremediation of chromium contaminated sites.

Cyanobacteria have been identified as a new and rich source of bioactive compounds. Attention to the properties of cyanobacteria as a good source of secondary metabolites in the past was very low but today has been shown that these microorganisms have many applications in the medical field and pharmaceutical products. In this experimental study, the cyanobacteria of Synechococcus elangatus ISC 106, Fischerella ambigua ISC67 and Schizothrix vaginata ISC108‎ were obtained from the algal culture collection of research institute of applied science, ACECR, Tehran, Iran. Extraction was performed by adding the solvent to cyanibacteria biomass and then filtering and drying the mixture. Disk diffusion method was used to study the effect of antimicrobial and broth microdilution method was used to determine the minimum inhibitory concentration. The results showed that the methanol extract of Synechococcus elangatus had no significant effect on the bacteria, but the methanol extract of Fischerella ambigua showed antibacterial activity. Aqueous extract of Fischerella ambigua had significant effect on Gram-positive bacteria, so that maximum antibacterial activity was against Staphylococcus aureus (PTCC 1112) which the average zone diameter around it was 33.33 mm. Among tested extracts, only methanol extract of Fischerella ambigua ‎had inhibitory effects against four plant pathogenic fungi. The effect of aqueous extract Fischerella ambigua on Staphylococcus aureus (PTCC1112) and Staphylococcus epidermidis (PTCC1114) was more effective than all of the affecting antibiotics. The highest antibacterial activity of cyanobacteria was related to aqueous extract of Synechococcus elangatus and maximum effect of antifungal activity was related to methanol extract of Fischerella ambigua.Discussion and Among the three species of cyanobacteria, two species of cyanobacteria Fischerella ambigua, and Synechococcus elangatus had antimicrobial activity, thus it can be a good candidate for the extraction of antimicrobial compounds and many compounds found in these extracts can be used to monitor and inhibit many diseases.

Integrons are mobile genetic elements able to acquire and integrate the antibiotic resistance gene cassettes and play an important role in the development of the drug resistant. This study assessed the contribution of class 1 integron in drug resistant diarrheagenic Escherichia coli strains, in children under 5 years. 164 diarrheagenic E. coli strains isolated from children under 5 were evaluated to investigate intI1 gene and gene cassette. Furthermore the antibiotic resistance was determined using CLSI critria. The rate of intI1 gene and gene cassette in Escherichia coli isolates were 70.73 % and 64.63 %, respectively. In these samples, gene cassette sizes varied from 750 to 2000 bp. There was a significant correlation between the presence of class 1 integron and resistance to streptomycin, gentamicin, kanamycin, amikacin, chloramphenicol, tetracycline, nalidixic acid and cotrimoxazole.Discussion and The present study elucidates that the rampancy of class 1 integron and the incorporated gene cassettes is quite high among diarrheagenic E. coli in our area of research. Hence, considering the prospect of widespread break out of the drug-resistant strains of E.coli, further molecular analysis in different regions of the country is essential.

Human growth hormone (hGH) is a single-chain polypeptide derived from pituitary gland that participates in a wide range of biological functions and has therapeutic applications. The hormone consists of 191 amino acid residues (22kD) which folds into a four-helix bundle structure with two disulfide bridges. Due to the complication of internal cell protein extraction, several methods have been established for this purpose that most of them need special instruments. Thus, in the preliminary studies, scientists need a quick and simple method for cell disruption and protein extraction. The aim of the present study was production of hGH in E.coli and comparison between the freeze/thaw and Sonication method for protein extraction from cells. The competent cells of E. coli Origami (DE3) were transformed using plasmid containing human growth hormone gene and then cultured in LB medium. After IPTG induction, the hGH was extracted using various methods including bacterial lysing, freeze/thaw and sonication methods and then extracted protein was assessed using ELISA, Bradford, dot blot and Western blotting. According to our findings, bacterial transformation showed high efficiency of transformation. Furthermore, the results of dot blot and Western blot showed production of recombinant hGH at high levels. Protein extraction measurement using ELISA and Bradford methods indicated that the proportion of the extracted protein in the slow freeze/thaw method was higher than the fast freeze/thaw method and was almost equal to sonication method.Discussion and The extracted protein levels in both of the slow freeze/ thaw and ultrasonic methods was equal, which introduces the slow freeze/thaw method as an excellent substitution for ultrasonic method when special instruments are not available.

Bacillus thuringiensis is the most important biological agent and by producing parasporal body it acts as a key role against agricultural pests. Furthermore, it produces Surface layer (S-layer) which is a protein or glycoprotein crystalline structure and has wide applications in nanobiotechnology. Accordingly it is significant to study more about this surface layer and toxin. In this study, purified parasporal body and mixed crystal/ spore were stained with coomasie blue G250 and were observed with light microscope. With a point mutation in CRY4Ba, protein stabilization was predicted and the location of protein cavities were predicted by Molegro software. Finally, the surface layer was extracted and its molecular weight and morphology were determined. To compare the surface layer and parasporal body, some features of them were estimated by the Protparam server. The results confirm the presence of polyhedral crystal proteins which were accumulated to form larger crystals after releasing spores. Also, it is predicted that in this research replacing the aspartic acid position- 451 with isoleucine, a more stable CRY4Ba pesticide protein is probably produced. This protein with three subunits contains 59 cavities and like the surface layer in this strain, it comprises low percent of methionine, histidine, cysteine and tryptophan.Discussion and In sporulation phase, Bacillus thuringiensis produces insecticidal crystals that integrate to form larger crystals. It is predicted that replacing the aspartic­ acid position- 451 with isoleucine would ameliorate the stability of CRY4Ba pesticide protein. This bacterium in vegetative phase produces a surface 100 KD protein which is similar to parasporal body in a shape and the percentage of some of amino acid.

Brucellosis which is an important zoonotic disease, exists in our country. Camel is an animal that is frequently imported to Iran and doesnt have any inspection for its brucellosis. The objective of this study was determination of active and passive infection by serologic and genomic detection of brucellosis in one-humped camel that was slaughtered in central part of Iran during 2012- 2013. For this purpose, 150 blood samples were collected from camels that were slaughtered in Najaf-Abad abattoir and they transported to laboratory in cool box. Initially, Samples were tested by serological methods include: Rose Bengal plat test, tube agglutination test and 2-mercaptoethanol test. Samples which showed anti-brucella antibodies titer equal or more than 1/80 in Wright test and equal or more than 1/40 in 2-ME test were considered as positive. Nucleic acid of samples were extracted and tested by polymerase chain reaction. Results showed that the infection rates were12, 8 and 6 % in RBPT, tube agglutination and 2-ME tests respectively. However 1.3 % of samples were positive in PCR test.Discussion and According to 2ME titers, results of serological tests indicated that animals were chronically infected. In present study sequence of pb26 gene was detected, that is common in most brucella species. Present study shows that camel can be a potential carrier; therefore importing camel to the country is a way of bacterial spread to central part of Iran.