فصلنامه زیست شناسی میکروارگانیسم ها
پیاپی 13 (بهار 1394)

Nep1 is a natural bio-herbicide protein which has an effective necrosis stimulant in dicotyledonous weeds. This protein was first purified in 1995 form fermentation broth of Fusarium oxysporum. In this study, the nep1gene was isolated form F. oxysporum, after removal of signal peptide from the beginning of the gene; final pET16b-nep1 construct was cloned and transformed into E. coli BL21 (DE3). The recombinant Nep1 was produced inE. coli. Recombinant protein was purified, diluted and prepared for biological assay on weed. Two ml of recombinant Nep1 with 60 µg/ ml final concentrations was sprayed on Convolvulus arvensis. Our results showed necrosis and chlorosis symptoms were started on the plant leaves after 2 days, also significant necrosis on the leaves of C. arvensis was seen after 5 days. No necrosis lesion was seen in the negative control plants that sprayed only with buffer and denatured recombinant Nep1 in 100 ºC for 15 min. Discussion and Our results introduced recombinant Nep1 as a biological potent agent for biocontrol of C. arvensis.

The use of hydrolysis process in order to release reducing sugars of Polysaccharide materials for ethanol production, especially in acid hydrolysis process, causes the produce growth inhibitors compounds such as furfural and hydroxymethyl. Existing these compounds in culture medium, in addition to decrease microorganism growth and glucose consumption they cause the decrease efficiency of ethanol production. In order to investigate the effect of both inhibiting compounds hydroxymethyl furfural and furfural on growth, glucose consumption and ethanol production by Saccharomycescerevisiae yeast, four field experiments with different concentrations include 0orcontrol0.5, 1 and 2gl-1of each compound was prepared, and their inhibitory effects on yeast growth and ethanol production was studied in during 36 h. The results showed that with increasing concentration hydroxymethyl furfural and furfural, both compounds have inhibitory effect on growth yeast, glucose consumption and ethanol production, so that the maximum efficiency of ethanol production for two compounds (hydroxymethylfural and furfural), were observed at concentrations of 0 or control and 0.5 gl-1.The results showed that the furfural compound in comparison to hydroxymethylfural have more inhibitory effect on Saccharomycescerevisiae growth and ethanol production by it.Discussion and In general decrease in glucose consumption by increasing concentrations of these compounds showed the negative effects of these compounds on the growth and metabolism yeast, and therefore the ethanol production is reduced.

The enterotoxigenic Staphylococcus aureus is considered as one of the most common agents of food poisoning, gastroenteritis, diarrhea and vomiting that the presence of bacterial virulence genes can be as the most important cause of this foodborn illness. The present study was conducted to determine the presence of S. aureus and its virulence factors in chicken nugget and ready to eat foods in Iran. In summer 2012 a total of 420 samples chicken nuggets were collected from randomly selected retail stores in Isfahan provinces, Iran. Samples were cultured and the positive results were studied using PCR for detection of sea-sej virulence genes. Results showed that 24 of 420 samples (5.7 %) were found to be contaminated with S. aureus. Also 23 of 24 isolated S. aureus were positive for one or more entrotoxin genes. The most detected genes were sea (25 %), sec (12.5 %), sed + sej (4.16 %), sed + seg (4.16 %), seg + sei (4.16 %), sea + seg (8.33 %), seb + sej + sei (4.16 %), sea + sed (12.5 %), sea + sec + sej (12.5 %). The most commonly detected genes were sea and sec.Discussion and The chicken nugget can be easily contaminated by the S. aureus and usually this contamination causes Staphylococcal food poisoning. Therefore, some food safety and quality standards need to be applied and performed in most of food units to control prevalence of S. aureus and its virulence factors. Nowadays, consumption of fast foods like chicken nuggets is very popular. Therefore, it is important to study the prevalence of enterotoxigenic S. aureus and its virulence genes in chicken nugget that this shows the important public health issue.

Lipases are a class of hydrolases which catalyze the hydrolysis of triglycerides to glycerol and free fatty acids. Lipases have become an integral part of the modern food, detergents, cosmetics and pharmaceutical industries. The aim of this study was investigation of the effect of olive oil with different commercial purity as a substrate on lipase production by Yarrowia lipolytica. One percentage of olive oil with three different commercial grades Extra-virgin, Virgin and Ordinary was used as cheap substrate to produce lipase by wild-type strain Y. lipolytica DSM3286 and mutant strain Y. lipolytica FDY139. Maximum lipase activity was observed 72 hours after inoculation. The wild-type strain Y. lipolytica DSM3286 produced lipase with 56 U/ml activity on Virgin olive oil, but mutant strain Y. lipolytica FDY1390 produced lipase with 300 U/ml activity on Ordinary olive oil. Discussion and The Y. lipolytica FDY1390 produced lipase about 5.4-fold higher than the wild-type strain on Ordinary olive oil. Whereas other expensive type of olive oil, Ordinary olive oil is a cheap oil which could be used to lipase production and optimization.

Biofilms are communities of microorganisms embedded in a self-produced extracellular polymeric matrix. Bacterial cells are protected from antimicrobial agents in biofilm structure. Biofilms formation cause many problems in industry, medicine and microbial drug resistance; thus it is essential to find new techniques for removing and inhibiting biofilms. This study aimed to examine the antimicrobial effect of Tamarix hispida alcoholic extracts against six pathogenic bacteria in planktonic and biofim forms. Antimicrobial activities of the plant extracts against the planktonic form of bacteria were evaluated by using the disc diffusion method. MIC and MBC values were determined by a macrobroth dilution technique. Anti-biofilm effects were assessed by microtiter plate method. The results of this study confirmed high ability of T. hispida extracts against the biofilm of tested bacteria and their free-living forms. Methanolic extracts better inhibited bacterial growth and biofilm formation than ethanolic extracts. Anti-biofilm effects of plant extracts were depended on the solvent and extract concentration. The maximum inhibitory effects of T. hispida extracts on biofilm formation, demolish of biofilm structure and inhibition of metabolic activity of bacteria in the biofilm were observed for S. aureus (87/35 %), S. pneumonia (74/49 %) and B. ceruse (88/34 %) respectively. Discussion and In this study the capacity of herbal extracts to encountering whit biofilm was demonstrated and it was confirmed that these extracts have interesting potentiality to become active agents of new drugs.

Self- assemble structures play an important role in many aspects of nanotechnology field such as biosesnsors, drug delivery, tissue engineering and targeting activity. S-layers proteins are an example of such molecules. These molecules possess the ability of re-crystallization after isolation from the surface of bacteria and therefore could be used for fabrication of assembled functional groups. To this regard in this work S- Layers were isolated from Caulobacter and were assembled on mica and silicon wafers. S-Layers re-crystallization were characterized by means of AFM. In order to evaluate the production of assembled functional groups Immunological methods were used. S-Layer with molecular weight of 96 KDa were successfully isolated from the surface of Caulobacter and crystallized on solid silicone and mica support. AFM results indicated assembled domains on mica and silicone. IgG immobilization was induced in the presence of S-Layer structures. Discussion and The results confirmed the effect of S-Layer crystallization on fabrication of assembled functional groups.

In the recent years, application of microorganisms as green biocatalysts for removing caffeine pollution from industrial wastes and food caffeinated have been extensively considered. This investigation reports on optimization of bio-decaffeination process under growing cells of Saccharomyces cerevisiae by using the Taguchi statistical approach. Five variables, i.e. caffeine, Zn+2, glucose, peptone concentrations and time incubation, which have significant effects on bio-decaffeination process, were selected and L16 (44× 13) orthogonal array was determined for experimental trials. Caffeine degradation was estimated by HPLC (High Performance Liquid Chromatography) analysis. Use of Taguchi approach for optimization of design parameters resulted in about 82.8 % reduction of caffeine in 48 h incubation when 3g/l peptone, 5mM Zn+2 ion and 5 g/l of caffeine are present in the designed media. Under the optimized conditions, the yield of degradation of caffeine (5 g/l) by the growing cells of yeast strain TFS9 has been increased from 25.5 to 82.8 % which is 3.2 fold higher than the normal yield. The improvement of caffeine removal after best conditions were made shows the efficiency of Taguchi experimental design in such studies. Discussion and The current investigation is the first report for successful application of the Taguchi experimental approach to the bio-decaffeination process. According to the analysis of experimental results, the present study proposes the potentiality of the Taguchi approach to enhance the bio-decaffeination performance with the native strain of Saccharomyces cerevisiae.

Excess activated sludge contains large amounts of components such as phosphorus, nitrogen, and sulfur which can theoretically be used as a nutrient source in fermentation processes to produce value added materials. In the present study, the possibility to grow Saccharomyces cerevisiae and produce ethanol on pretreated and untreated activated sludge as a nutrient source was investigated. In this article, Saccharomyces cerevisiae, CEN.PK 113-7D, was used as an ethanol producer microorganism. In order to release the organic matter of the sludge and increase the efficiency of ethanol production, four pretreatments, i.e. acidic, alkaline, thermal and ultrasonic were performed on the sludge. The effectiveness of each pretreatment on biomass growth at aerobic conditions was measured by CFU method. Moreover, the yield of ethanol at anaerobic conditions was measured using gas-chromatography. The highest biomass yield of yeast on the wet sludge at aerobic conditions was obtained after alkaline pretreatment, in which the biomass concentration increased from 1.2 105 (CFU/ml) to 1.5 106 (cell/ml) after 36 hour of cultivation. At anaerobic conditions, the yield of ethanol on alkaline pretreated sludge was 0.11 g ethanol/g initial glucose. Additionally, by drying the sludge and performing the alkaline pretreatment, the ethanol production was increased up to 0.41 g ethanol/g initial glucose. Discussion and The results showed that the excess activated sludge from wastewater treatment plants can be used as nutrient source for ethanol production. However, it needs to be treated and alkaline pretreatment of dried sludge is suggested to produce a high yield of ethanol.

The intestinal microbiota of fish belongs to different species compare to terrestrial animals, mainly Gram-negative bacteria are predominant. Despite importance as probiotic, gram-positive bacteria like Lactobacillus Sp. are minor constitutes of gut microbiota. The aim of this study was to investigate the possibility of modulation of Kutum intestinal microbiota and elevation of lactic acid bacteria (LAB) levels through administration of yeast based prebiotic. The study was performed as randomized design with 5 treatments and 3 replications in which kutum were fed different levels, 0, 0.5, 1, 1.5 and 2 % of yeast based prebiotic for 60 days. At the end of the trial, culture based analysis of intestinal microbiota include lactic acid bacteria levels, total bacteria and obligatory anaerobic bacteria were performed by using MRS, TSA, SPS media. Also, the isolated lactic acid bacteria species were determined by biochemical test. The highest and lowest lactic acid bacteria levels observed in 0.5 % treatment and control treatment, respectively (P value > 0.05). Also, administration of 0.5 % yeast based prebiotic significantly increased total viable bacteria (P value > 0.05). The highest and lowest obligatory anaerobic bacteria were recorded in control and 0.5 and 1 % treatment (P value> 0.05). The biochemical test revealed isolation of three species Lactobacillus plantarum, L. caseie, L. acidophilous in the intestinal microbiota after administration of yeast based prebiotic. Discussion and The results of this study determined that administration of 0.5 % yeast based prebiotic significantly modulates the intestinal microbiota of kutum fry toward potentially beneficial communities (i.e LAB).

Mycobacterium marinum is a slow growing nontuberculosis mycobacterium. This organism can cause disseminated granulomatous infections in fish, called ‘fish tuberculosis’. Changes in nutritional components in culture medium and environmental conditions may lead to some changes in growth patterns and biofilm formation having new characteristics. The aim of this study was the evaluation of changes in medium components and environmental factors on the growth pattern and biofilm formation of M. marinum CCUG 20998. M. marinum CCUG 20998 was grown in 7H9 broth medium containing different concentrations of Tween 80 (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 % v/v), Oleic Albumin Dextrose Catalase supplement (OADC), (4, 8, 12, and 16 % v/v) and glycerol (30, 35, 40, 45, and 50 % v/v). The changes in growth patterns and biofilm formation were determined by using measurement of optical density and staining by crystal violet. The obtained results showed that maximum growth was at 0.4 % of Tween 80. Oleic Albumin Dextrose Catalase supplement at different concentrations did not show any significant effect on the growth pattern. Decreasing the glycerol concentration significantly decreased the growth rate. The maximum biofilm formation was obtained in 35 % glycerol without Tween 80 and OADC in culture medium. Discussion and According to the obtained results, Tween 80 and glycerol in the formulation of Middlebrook 7H9 Broth are very critical for M. marinum CCUG 20998 growth. In contrast, Oleic Albumin Dextrose Catalase supplement can be omitted from the medium formulation due to showing no significant effect on the growth pattern.

Polyhydroxyalkanoate (PHA) is counted biodegradable polymer, produced by bacteria, which is employed instead of petrochemical plastics, to reduce environment pollution. The purpose of this study is the isolation and identification of the PHA producing bacteria in Masjed-Soleiman oil fields, determination of PHA produced by the bacteria, determine the optimal conditions of temperature, pH, concentration and type of carbon and nitrogen sources on the level of PHA synthesis and the use of waste oil as a cheap substrate of carbon for the synthesis of PHA by isolated bacteria. At first 9 samples of soil contaminated with crude oil, from 9 points at 4 well sites in Masjed-Soleiman oil field were taken. The initial screening was done by MSM at the temperature of 28 °C. Isolates from the view point of producing PHA, were confirmed, by using Sudan black stain and were identified by using phenotypical tests and 16S rRNA sequence determination. Also, optimization of temperature, pH, carbon and nitrogen sources were made for bacteria growth. In this study eleven isolates were identified as PHA producers and among them the highest range belongs to MIS-1 strain by the range of 58.522 percent of the cellular dry waight, wich produces the maximum PHA. This isolate is recognized as the species of pseudomonas aeroginosa according to the phenotypic tests and sequence analysis of 16S rRNA with 95 % similarity. In addition, the results of optimization tests showed that, the isolates in the temperature of 37 °C, pH7, glucose as the source of carbon and ammonium sulphate as the source of nitrogen and finally, concentration of 10 g/lit of the carbon source and 0.5 g/lit of nitrogen source, the highest range of PHA was synthesized. Discussion and The results of this research show that the soil polluted with crude oil of Masjed- Soleiman oil field, in long term and also due to high rate of carbon, provides a suitable environment for screening of beneficial bacteria such as PHA producing bacteria.

Clostridiumbifermentans is a non pathogenbacteria and first isolated by Tissier and Martelli in 1992. This bacteria with a view to taxonomy has a close relationship with the Clostridiumsourdilli and is able to produce a wide range of metabolites such as acetic acid, lactic acid, formic acid, ethanol, butanol, acetone, carbon dioxide, nitrogen and specially hydrogen as clean source energy. The aim of this study was to isolate and molecular identify Clostridium bifermentans. With aim to isolation of Clostridium bifermentans, the anaerobic wastewater samples from Mahdishahr sewage treatment laggons cultured on asynthetic complex medium and incubated anaerobically at room temperature. Chromosomal DNA isolated from grown bacteria and the 16s rRNA gene amplified with PCR. The PCR product was sequenced and purified. Isolated bacteria from a macroscopic view on the complex media has white colonies and from a microscopic view was rod-shaped Gram-positive and had spore. of the 16s rRNA gene sequencing, isolated bacteria belong to the species Clostridium bifermentans which is called IRI2 strain and were recorded in gene bank with acception number KC525132. Discussion and In this study a strain of Clostridium bifermentans was separated and identified with 16S rRNA sequencing method. With respect to the ability of this bacteria to produce various industrial products, this bacteria is to be used especially for production of hydrogen as biogas.

Treatment with microorganisms is a new developing method that in this field, probiotic bacteria, especially lactic acid bacteria have a significant role. Olive is a productive and useful Iranian native plant which is rich in lactic acid bacteria. In this study, a number of Lactobacillus plantarum strains were isolated from Iranian native olives and some of their probiotic properties were investigated. At first, different strains of lactic acid bacteria were isolated from Iranian native olive on MRS agar medium, and after biochemical and molecular identification of bacteria, some probiotic properties such as acid and bile salt tolerance; and antimicrobial effects against two pathogenic species of Enterobacteriaceae, Escherichia coli (PTCC1399) and Shigella dysenteriae (PTCC1188) were studied by disk and well diffusion agar methods. According to error reduction, each test was performed in triplicate. Average zone of inhibition for each strain was compared by one-way ANOVA. According to biochemical tests, 57 percent of 28 isolated lactic acid bacteria were identified as Lactobacillus plantarum. Among these strains, 75 percent and 81.5 percent were acid and bile tolerance respectively. In the molecular analysis, 6 strains were shown specific bands in PCR reaction which performed by specific primers designed for Lactobacillus plantarum. The results described antimicrobial effects of Lactobacillus plantarum isolated from Iranian native olives on two enteric pathogens, Escherichia coli and Shigella dysenteriae. Discussion and According to probiotic properties of isolated Lactobacillus plantarum from Iranian native olives, using them is acceptable as an important and practical approach to prevent infectious disease and its treatment due to Escherichia coli and Shigella dysenteriae.

Pseudomonas aeruginosa is an opportunistic human pathogen which causes serious problems especially in people who have immunodeficiency. Recently, metallo-β-lactamase (MBLs) resistance in this bacterium has led some difficulties in treating bacterial infections. MBL gene family, including blaSPM-1 caused resistance to beta-lactam antibiotics especially carbapenem (eg, imipenem) and have been reported with high prevalence worldwide. The aim of this study is detection of metallo-β-lactamase gene blaSPM-1 in Imipenem-resistant Pseudomonas aeruginosa strains isolated from hospitalized patients in Isfahan hospitals. In this study, 252 samples were isolated from various nosocomial infections. These isolates were identified as Pseudomonas aeruginosa by using biochemical tests. Disk diffusion method (Kirby-Bauer) was used to determine the bacterial drug resistance pattern. All imipenem-resistant isolates were screened for the presence of MBLs by using the Combine disk (IMP-EDTA). The minimal inhibitory concentration (MIC) of imipenem was determined by E-test on Mueller-Hinton agar. To detect blaSPM-1 gene, the isolates were subjected to polymerase chain reaction (PCR). In total, 106 isolates of Pseudomonas aeruginosa were collected. Of all isolates, 62 (58.5 %) were found to be imipenem-resistant Pseudomonas aeruginosa. MIC levels in all strains of imipenem-resistant were MIC≥32μg/ml. Twenty-six (48 %) of imipenem-resistant Pseudomonas aeruginosa isolates were MBL positive. None of the isolates carried blaSPM-1 gene by PCR assay. Discussion and Results of this study demonstrate that the rate of imipenem resistance due to MBL is increased dramatically. Early detection and infection-control practices are the best antimicrobial strategies for this organism. None of MBL-producing isolates in this study carry blaSPM-1 gene; therefore another gene in MBLs family should be investigated.

Today the cells immobilization is a suitable method for reusing microorganisms and protecting them from direct changes in the physico-chemical factors in the environment. In this study, Bacillus cells were immobilized on calcium alginate. Among the methods immobilization ways, entrapment was used and on the other hand the drop method was used from the immobilization ways. Also, after producing fine Bacillus containing alginate, a packed bed reactor, was used to remove the metal nickel. This study showed that uptake of Nickel by Bacillus fixing calcium alginate was more compared with free Bacillus cell and bacteria-free calcium alginate. The percentage of the nickel uptake from the solution by calcium alginate, free bacillus and calcium alginate containing Bacillus are respectively 17.5, 29.5 and 47.5 percent. Discussion and By accepting that active groups of Bacillus have a major role in the uptake of toxic metals and most notably are the alginate carboxylic groups and the organisms containing these groups or used directly free of macromolecules containing these groups can be used.