فصلنامه زیست شناسی میکروارگانیسم ها
پیاپی 15 (پاییز 1394)

Lipase of the yeast Yarrowia lipolytica used in detergents, cosmetics, pharmaceuticals and food industries. This enzyme production depends on medium composition, especially carbon source. The purpose of this study was to investigate the effect of olive oil different feeding strategies on Y. lipolytica lipase production. The yeast strains Y. lipolytica FDY1390 was cultured in media with different feeding strategies. Three different models were used to fed-batch feeding method. The yeast growth is monitored by direct counting method with neubauer chamber. Lipase activity was measured using titration method. All three models of fed-batch feeding increased lipase toward the batch culture. Fed3 was the best model of fed-batch feeding, in which the model leads to the production of lipase activity with 526 U/ml after 120 hours and the productivity reached to 4.38 U/ h.Discussion and The results showed that feeding patterns of fed-batch culture increase production rate of lipase production towards batch culture. The best strategy is to add olive oil as the carbon source induction substrate in medium after 48 hours of inoculation.

Hordeum spontanum is one of the most important crop weeds, especially for wheat. The aim of this study was screening of phytopathogenic fungi for biocontrol of H. spontanum. Firstly, 150 fungal isolates were obtained from different soil sources of Iran. The isolates were subjected under primary and secondary screening on the sunflower and H. spontanum leaves. In these steps, 200 µl of centrifuged fermentation broth of cultivated fungi in PDB medium was sprayed on the leaves of sunflower and H. spontanum. Also, the ability of selected isolates to induce disease and necrosis on H. spontanum leaves in pot, preventing seed germination and root growth repression were studied. Finally, the host range of selected isolate was investigated. The results of primary and secondary screening showed that Aspergillus westerdijkiae UTMC 5040 produced the highest lesion halo on H. spontanum leaves. In pot test, treatment with 1.5 ml of fermentation broth produced huge necrosis on mature H. spontanum plant after 3 weeks. H. spontanum seed germination decreased from 90 to 25% and root emersion decreased from 60 to 35 mm 10 days after treatment by the fermentation broth. Host range study revealed that among 25 different plant strains, A. westerdijkiae UTMC 5040 caused disease onH. spontanum, Eremopyrum bonaepartis and Convolvulus arvensis leaves.Discussion and This study was the first report of biological control ofH. spontanum with fermentation broth of A.westerdijkiae UTMC 5040. The obtained results can be useful to finding a new fungal bio-herbicide.

Pseudomonas aeruginosa is an aerobic gram-negative bacteria, which causes hospital infections. Bacteria under stress, such as lack of food, pH and osmotic pressure change and antibiotic stress transforms its morphology to coccoid form. In the bacill form due to changes in the peptidoglycan cell wall, membrane lipids and decreased metabolic activity, bacteria resistant to antibiotics. Due to an increase in mortality in burn patients and important problem of antibiotic resistance in P.aeruginosa the researcher decided to study the factors affecting on morphologic change to coccoid form. In this study P.aeruginosa strains obtained from clinical samples of burned patients (8 samples were taken from the wound by Infectious Disease Specialist) and standard strain ATCC 27853 were used. Samples were confirmed by biochemical tests and PCR by 16srDNA primer. Then bacteria were put under lack of food and antibiotic stress invitro. After that bacterial morphology was examined on different days by digital DP 72-BX 51 microscope to 60 days. After induction coccoid forms, bacterial viability was confirmed by flow cytometry. Bacteria begin to change morphology from 5 days for antibiotic stress and 10 days for other stress. Changing morphology was initially elongate bacilli, U shape and finally the coccoid form was seen.Discussion and Changing morphology of bacilli to coccoid bacteria that are the result of stress on the bacteria which enter the body can lead to bacterial resistance to antibiotics and have grave consequences for the patient.

Pectinase is one of the most important industrial enzymes which was isolated from a wide variety of microorganisms such as bacteria and filamentous fungi. This enzyme has been usually used in the juice 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 have been done by plate culture on pectin medium and staining with Lugol's iodine solution. The best strain was identified by method of Pitt and Hocking as Aspergillus fumigates. The enzyme production was optimized by application of the factorial design which involves five factors, each at three levels. Five factors were carbon sources (whey, sugar, stevia) and ammonium sulfate, manganese sulfate, temperature, and pH). Pectinase concentration was measured by the Miller method. The results showed that the optimum condition for enzyme production was at 32 °C, PH = 6, 3g / L manganese sulfate, 2.75g / L of ammonium sulfate, 10g / L of each carbon source (whey, stevia, and glucose). Optimum of enzyme production was observed in the presence of 1.328 mg / ml of glucose. Molecular weight of enzyme was obtained about 40 kDa by SDS-PAGE.Discussion and The results demonstrated that this strain could grow in a wide range of carbon sources, PH and temperature. This study indicates that this strain is a good candidate for use in industrial application.

Fusarium head blight (FHB), is the most destructive disease of wheat, producing the mycotoxin deoxynivalenol, a protein synthesis inhibitor, which is harmful to humans and livestock. dsRNAmycoviruses-infected-isolates of Fusariumgraminearum, showed changes in morphological and pathogenicity phenotypes including reduced virulence towards wheat and decreased production of trichothecene mycotoxin (deoxynivalenol: DON). Previous studies indicated that over expression of yeast acetyl transferase gene (ScAYT1) encoding a 3-O trichothecene acetyl transferase that converts deoxynivalenol to a less toxic acetylated form, leads to suppression of the deoxynivalenol sensitivity in pdr5 yeast mutants. To identify whether ScAYT1 over-expression in transgenic tobacco plants can deal with mycotoxin (deoxynivalenol) in fungal extract and studying the effect of dsRNA contamination on detoxification and resistance level, we have treated T1 AYT1 transgenic tobacco seedlings with complete extraction of normal F. graminearum isolate carrying dsRNA metabolites. First, we introduced AYT1into the model tobacco plants through Agrobacterium-mediated transformation in an attempt to detoxify deoxynivalenol. In vitro tests with extraction of dsRNA carrying and cured isolates of F. graminearum and 10 ppm of deoxynivalenol indicated variable resistance levels in transgenic plants.Discussion and The results of this study indicate that the transgene expression AYT1 and Fusarium infection to dsRNA can induce tolerance to deoxynivalenol, followed by increased resistance to Fusarium head blight disease of wheat.

Fusarium head blight (FHB), is the most destructive disease of wheat, producing the mycotoxin deoxynivalenol, a protein synthesis inhibitor, which is harmful to humans and livestock. dsRNAmycoviruses-infected-isolates of Fusariumgraminearum, showed changes in morphological and pathogenicity phenotypes including reduced virulence towards wheat and decreased production of trichothecene mycotoxin (deoxynivalenol: DON). Previous studies indicated that over expression of yeast acetyl transferase gene (ScAYT1) encoding a 3-O trichothecene acetyl transferase that converts deoxynivalenol to a less toxic acetylated form, leads to suppression of the deoxynivalenol sensitivity in pdr5 yeast mutants. To identify whether ScAYT1 over-expression in transgenic tobacco plants can deal with mycotoxin (deoxynivalenol) in fungal extract and studying the effect of dsRNA contamination on detoxification and resistance level, we have treated T1 AYT1 transgenic tobacco seedlings with complete extraction of normal F. graminearum isolate carrying dsRNA metabolites. First, we introduced AYT1into the model tobacco plants through Agrobacterium-mediated transformation in an attempt to detoxify deoxynivalenol. In vitro tests with extraction of dsRNA carrying and cured isolates of F. graminearum and 10 ppm of deoxynivalenol indicated variable resistance levels in transgenic plants.Discussion and The results of this study indicate that the transgene expression AYT1 and Fusarium infection to dsRNA can induce tolerance to deoxynivalenol, followed by increased resistance to Fusarium head blight disease of wheat.

Nicotine is a toxic plant alkaloid and it has been designated as hazardous by the United States Environmental Protection Agency (USEPA) since 1994. The present work was directed to screen nicotine resistant bacteria, that is used as biocatalyst in the biodegradation of nicotine from contaminated sites. Collected soil samples from 12 tobacco farms were selected as target sites for sampling. Enrichment nicotine-degrading bacteria were performed in minimal salt media containing nicotine as the sole carbon and nitrogen sources. Agar dilution plate method was performed for determining intrinsic tolerance of bacterial isolates to nicotine. Phenotypic characterization and phylogenetic analysis were used to identify the selected bacterial isolate able to degrade nicotine. To determine the optimal conditions for the bio-removal of nicotine, the effects of initial nicotine concentration, incubation time and the addition of carbon and nitrogen sources in the selected strain were tested. The quantification of residual nicotine in the culture media was measured by high performance liquid chromatography (HPLC). Among 20 bacterial isolates for degradation of nicotine, the strain TPS2 showed a high level of resistance and degradation efficiency. Results of phenotypic identification and phylogenetic analysis showed the native strain TPS2 belongs to the Citrobacter sp. strain TPS2 (GeneBank accession no. KM110046). According to the results of de-nicotination experiment, the native strain TPS2 is able to remove 100% of nicotine with an initial concentration 2.5 g/l in the presence of 2.5 g/l fructose.Discussion and The results showed that the screened Citrobacter sp. was suitable candidate for degradation of nicotine from wastewater and sites that contaminated with nicotine. It is seemed by using of the microbial biocatalyst the ecosystem contamination of toxic nicotine can be decreased. The present work is the first report on the degradation of nicotine by native microorganisms.

The aim of present study was isolation of polyhydroxybutyrate producing Bacillus species from oil refinery waste water, Isfahan, Iran and primarily optimization of production condition. Petroleum wastes are rich of carbon sources and have low amounts of nitrogen and phosphorus sources. AS the most important factor in production of intracellular inclusions is increasing the C/N ratio, it seemed that polyhydroxybutyrate producing microorganisms will be found in these wastes. Bacillus species were isolated and purified from oil refinery wastewater. The polymer was verified using different staining procedures. Polymer was extracted by digestion method and the optimum production conditions were investigated in minimal salt medium with the organic carbon source by submerged fermentation. Production of polyhydroxybutyrate was studied using dry weight and optical density measurement. Between various isolated Bacillus strains, two of them (B1 and B2) were polyhydroxybutyrate producers. Maximum PHA production based on dry weight and concentration were obtained for strain B1 after 72 hours incubation, at 31°C, in the presence of glucose as carbon source and yeast extract as nitrogen source, pH=7, and aeration in 120 rpm and for strain B2 in the same condition, except optimal temperature which was 32°C. The most production amounts were 367 mg.ml-1 for B1 and 473 mg.ml-1 for B2 isolates. Also the most polymer percentage was 52/16 and 58.43 for B1 and B2 isolates respectively.Discussion and The results showed that the production of polyhydroxybutyrate was increased by optimization of the conditions in both isolates. Using petroleum wastes as well as production of biodegradable plastics, leads to decontamination of theses wastes.

Amylases are among the most important enzymes and have great significance in present-day biotechnology. Amylase with commercial applications is mainly derived from the genus Bacillus. The main purpose of this study is identification and isolatation amylase enzyme producer Bacillus, determining the amylase enzyme activity and affecting a number of culture medium on amylase enzyme production. Soil, water and wastewater samples were collected from agricultural area, choghakhor lake in chahar mahal e bakhtiari province and from food factory in Esfahan. Bacillus isolates were screened for amylolytic properties by starch hydrolysis test on starch agar plate. Amylase producing Bacillus were identified biochemical tests and molecular experiments. Amylase enzyme activity of isolates was measured using di-nitro salicylic acid (DNS) method. Enzyme production was studied in variose medium culture TSB, NB, Yeast extract, molases and milk medium. The enzyme amylase-producing strains, one sample showed was the highest amylase activity. The Bacillus has been detected as a member of Bacillus subtilis according to Bergey's Manual of Systematic Bacteriology and molecular recognition. The enzyme activity of Bacillus subtilis was measured 7/21 (U/ml) in production media. Trough medium culture maximum amylase production for Bacillus subtilis was achieved in molases medium.Discussion and In this study, Bacillus subtilis strains isolated from wastewater of a significant amount of enzyme producing 7/21 (U/ml) as indicated. Among the medium-amylase from Bacillus subtilis highest enzyme activity was observed in beet molasses. According to this study, the use of Bacillus strains is an efficient way to achieve the amylase enzyme.

An important issue in microbial biotechnology is linkage between screened beneficial strains in laboratory and industry. Therefore, to develop beneficial microbial biocontrol agents optimization of nutritional and physiological condition for high level production and selection of carrier for final formulation are necessary. In this research we tried to find the best growth condition and suitable formulation for biocontrol Streptomyces rimosus strain C-2012. For optimization of growth condition of strain C-2012, utilization of carbon sources, growth in different media, effect of temperature, pH and NaCl were investigated. Then the effect of different carriers and additives in final formulation and shelf life of microbial community were studied. Study on utilization of carbon sources showed that glucose, fructose and mannitole were suitable carbon sources for growth and the best initial pH and temperature were 7 and 28°C, respectively. Results showed that the culture medium containing glucose, yeast extract and malt extract was the best medium. Investigation on NaCl effect showed that from 0 up to 300 mM sodium chloride could increase microbial community and salinity more than this range decreased microbial community. Based on the results we found that sand is suitable as microbial carrier comparing hydrogel polymers. Viability test during 36 months showed that formulation with NaCl content could keep 200 times (8*106 cfu/g) more than samples without salinity at last month.Discussion and Using suitable carbon sources such as glucose at 28 °C and pH at 7 are important items in optimum growth and preparing final formulation with good level of microbial community. Capability in secondary volatile and liquid metabolites production and fungal pathogen control by Streptomyces rimosus in the presence of NaCl showed that this strain has high potentiality to apply in both normal and saline area. Long shelf life (3 years) without any saprophytic contamination of this strain in final formulation is important in industry and it can prepare a lucrative business.

The chemical fungicides are used widely in the world. To reduce the application of synthetic fungicides in treating plant diseases, biological methods are considered as an alternative way to control plant diseases. Many actinomycetes, particularly Streptomyces species are biological agents against a broad spectrum of fungal plant pathogens. The purpose of this study was using the kitinolitik actinomycetes isolated from soil of Eastern Azerbaijan province In order to produce biological pesticides. Soil samples were taken from different areas of Eastern Azerbaijan province. According to Streptomyces morphological features, single colonies were isolated. To identify the bacteria by molecular characteristic, the genomic DNA was extracted and then the sequences of 16S rDNA were replicated. By using specific primers the bacterial isolates containing chitinase gene were screened. The isolates consisted Chitinase enzyme and were antagonistically cultured with Alternaria genus which is a fungal plant pathogen. Out of 60 soil collected samples, 31 Streptomyces bacterial isolates were separated. Four isolates showed positive results to selectivity action of the chitinase enzyme. Treatment of 3 bacterial isolates with 2 pathogenic fungi showed that AE09 is the most effective anti-fungal isolates.Discussion and Soils in Eastern Azerbaijan province are rich of Streptomyces bacteria which generate antifungal compounds. Obtaining the Streptomyces bacteria which have chitinase gene, can lead to identification of very effective strains as anti-fungal.

Modulation of intestinal microbiota toward potentially beneficial communities (probiotics) positively affects fish physiology and health status. Different prebiotics showed contradictory effects on intestinal microbiota. The present study investigates the effects of different levels of two prebiotics, galacto- and fructooligosaccharide on intestinal microbiota of Caspian roach fry which is a commercially valuable species of Caspian sea. The study was performed as a randomized design with 5 treatments and 3 replications in which Caspian roach were fed different levels, 0, 1, and 2% of galacto- and fructooligosaccharide prebiotics for 6 weeks. At the end of the trial culture, analysis of intestinal microbiota include lactic acid bacteria levels, total bacteria as well as proportion of LAB were performed by using MRS agar, Plate count agar media. Administration of different levels of galacto- and fructooligosaccharide had no significant effects on total bacteria of intestinal microbiota (P > 0.05). The lactic acid bacteria levels significantly increased compared to control group following prebiotics administration in diet (P > 0.05). LAB levels in galactooligosaccharide treatment were higher than those of fructooligosaccharide treatment. The highest LAB proportion in intestinal microbiota was observed in roach fed diet which contains 2% galactooligosaccharide (P > 0.05).Discussion and The results of the present study revealed that prebiotics can be used for modulation of Caspian roach intestinal microbiota toward beneficial bacterial communities. Also, the results showed that galactooligosaccharide was more efficient than fructooligosaccharide in case of modulation of intestinal microbiota and elevation of LAB levels.

Contamination caused by pesticides is considered as one of the environmental problems. Bioremediation is exploiting the ability of microorganisms to remove pollutants. Trichoderma species are free-living fungi that exist naturally in the environment. These fungi have the ability to uptake some contaminants biologically. The aim of this study is to evaluate the effect of Confidor, as an environmental contaminant, on the growth ability of Trichoderma sp. as a contaminant absorber. Five species of Trichoderma fungi were cultured in PDA media. Then the fungi were adapted with 3 different concentrations of Confidor gradually (5, 10 and 20 mg/l). The diameter of the fungal colonies growing in different concentrations of the toxin, were measured after 24 hr and were compared with the control samples (medium without toxin). Results showed that in all species of fungi the colony diameters increased significantly with increasing toxin concentrations. The largest colony diameter was related to T.tomentosum, T.asperellum and T.harzianum (88.88, 87.5 and 86.95%, respectively) at the concentration of 20 mg of toxic. Also, in all studied fungal species, in the medium containing 20 (mg/ l) of toxic, the aerial hyphae expanded much thicker and faster than other concentrations.Discussion and The results indicate a significant increase in the growth ability of Trichoderma strains with increasing Confidor concentration. Therefore it could be concluded that Trichoderma fungi have a high potentiality for biodegradation of Confidor.

Lactobacilli are a group of lactic acid bacteria that their final product of fermentation is lactic acid. The objective of this research is selection of local Lactobacilli producing L (+) lactic acid. In this research the local strains were screened based on the ability to produce lactic acid. The screening was performed in two stages. The first stage was the titration method and the second stage was the enzymatic method. The superior strains obtained from titration method were selected to do enzymatic test. Finally, the superior strains in the second stage (enzymatic) which had the ability to produce L (+) lactic acid were identified by biochemical tests. Then, molecular identification of strains was performed by using 16S rRNA sequencing. In this study, the ability of 79 strains of local Lactobacilli in terms of production of lactic acid was studied. The highest and lowest rates of lactic acid production was 34. 8 and 12. 4 mg/g. Superior Lactobacilli in terms of production of lactic acid ability of producing had an optical isomer L (+), the highest levels of L (+) lactic acid were with 3. 99 and the lowest amount equal to 1. 03 mg/g. The biochemical and molecular identification of superior strains showed that strains are Lactobacillus paracasei. Then the sequences of 16S rRNA of superior strains were reported in NCBI with accession numbers KF735654، KF735655، KJ508201and KJ508202. Discussion and The amounts of lactic acid production by local Lactobacilli were very different and producing some of these strains on available reports showed more products. The results of this research suggest the use of superior strains of Lactobacilli for production of pure L (+) lactic acid.

Easy access and wide use of antimicrobial compounds led to the emergence of resistance among microorganisms. Therefore, screening and identifying antimicrobial compound with high effect of microorganisms in different environments is necessary and vital. Using microorganisms for biological aims change them to an important tool to control pathogens. Streptomyces griseus is one of them. The aim of this study is isolation of marine bacteria with antimicrobial effect against gram positive and negative bacteria. Finally, molecular identification of strains with antimicrobial activity. In this study, 162 strains were isolated from the Caspian Sea. The strains were cultured on special medium and finally antimicrobial activity on references strains as measured. Among them four strains with remarkable antimicrobial activity were identified and selected. The strains were subjected to 16S rDNA PCR sequencing. The strains were submitted to NCBI as new Streptomyces griseus strains. Among 162 strains, 4 strains had the most antimicrobial activity. The result showed, the strains were the most effective on Bacillus subtilis and Staphylococcus aureus (Gram positive bacteria) and the least effect were observed on Escherichia coli and Pseudomonas aeruginosa (Gram negative bacteria). After sequencing, the strains were classified to sterptomyces griseus genu.Discussion and In this study, 4 strains with antimicrobial activity were identified. According to the strength of these bacteria for controlling pathogenic bacteria resistant to antibiotic, we can have more pure microorganisms in optimized and controlled conditions for using in pharmaceutical industries and also for the treatment of dangerous pathogenic bacteria.