فصلنامه زیست شناسی میکروارگانیسم ها
پیاپی 16 (زمستان 1394)

Xylan is the main hemicellulosic polymer in a number of lignocelluloses which can be hydrolyzed by xylanolytic enzymes. One of the main ways for enzymes production is solid state fermentation (SSF). The ability of three fungal strains (Mucor indicus, Mucor hiemalis, and Rhizopus oryzae) for xylanase production on wheat bran by SSF was investigated.
The effects of cultivation temperature, medium moisture content, and cultivation time on the enzyme production were investigated. Experiments were designed with an orthogonal central composite design on three variables using response surface methodology (RSM). Analysis of variance was applied and the enzyme production was expressed with a mathematical equation as a function of the three factors. The optimum operating conditions for the enzyme production was obtained.
For xylanase production by M. indicus, M. hiemalis and R. oryzae the optimum temperatures were 40.0, 43.4 and 43.4ºC respectively. These values were 49.8, 54.2 and 71.8% for moisture percent and 51.3, 53.2 and 53.5 h for cultivation time. The highest enzyme activities per g of dry substrate (gds) were 43.1, 43.8 and 25.9 U/gds for M. indicus, M. hiemalis and R. oryzae respectively.
Discussion and All the fungi were able to produce xylanase. Maximum xylanase production was predicted by M. indicus and M. hiemalis at similar optimum conditions, while R. oryzae produced relatively lower xylanase activity even at the best condition.

L- asparaginase is in an excessive demand in medical applications and in food treating industries, the request for this therapeutic enzyme is growing several folds every year.
In this study, a L- asparaginase gene from Pseudomonas aeruginosa strain SN4 was sequenced and cloned in E. coli. Primers were designed based on L- asparaginase from P. aeruginosa DSM 50071, which show high similarity to SN4 strain, according to 16S rRNA sequence. The L- asparaginase gene was exposed to restriction digestion with NdeI and XhoI enzymes and then ligated into pET21a plasmid. The ligated sample was transformed into competent E. coli (DE3) pLysS DH5a cells, according to CaCl2 method. The transformed E. coli cells were grown into LB agar plate containing 100 µg/ml ampicillin, IPTG (1 mM).
Recombinant L- asparaginase from E. coli BL21 induced after 9 h of incubation and showed high L- asparaginase activity about 93.4 IU/ml. Recombinant L- asparaginase sequencing and alignments showed that the presumed amino acid sequence composed of 350 amino acid residues showed high similarity with P. aeruginosa L- asparaginases about 99%. The results also indicated that SN4 L- asparaginase has the catalytic residues and conserve region similar to other L- asparaginases.
Discussion and This is the first report on cloning and expression of P. aeruginosa L- asparaginases in Escherichia coli. These results indicated a potent source of L- asparaginase for in vitro and in vivio anticancer consideration.

g-linolenic acid is an essential fatty acid in human nutrition. In the present study, production of g-linolenic acid by Mucor hiemalis PTCC 5292 was evaluated in submerged fermentation.
The fermentation variables were chosen according to the fractional factorial design and further optimized via full factorial method. Four significant variables, glucose, peptone, ammonium nitrate and pH were selected for the optimization studies. The design consisted of total 16 runs consisting of runs at two levels for each factor with three replications of the center points.
The analysis of variance and three-dimensional response surface plot of effects indicated that variables were regarded to be significant for production of g-linolenic acid by Mucor hiemalis. Results indicated that fermentation at the optimum conditions (100 g/l glucose concentration; 1 g/l peptone; 1 g/l ammonium nitrate, and pH of 4.5) enhanced the g-linolenic acid production up to 709 mg/l.
Discussion and The results of this study indicated that higher g-linolenic acid yield can be achieved in a simple medium at high glucose and ammonium nitrate, low peptone concentrations and acidic pH by Mucor hiemalis PTCC 5292. This simple and low cost optimization condition of culture media can be applied for g-linolenic acid production at higher scale for pharmaceutical and nutritional industries.

The genus Nocardia belongs to aerobic Actinomycetes. They are a large group of soil-dwelling bacteria that are distributed worldwide. Using various key conventional and molecular diagnostic tests, several Nocardia isolates were identified, characterized and distinguished.
In our study, soil isolation method was Slip-buried type and DNA extraction was obtained through Microwave oven method and Nocardia asteroides DSM 43757-type strain as standard was tested to approve the above method. Following this study, Polymerase Chain Reaction was carried out using universal primers.
Phenotypic and molecular data analysis, particularly 16S rRNA gene sequencing, provided evidence on Nocardia cyriacigeorgica KC577151, Nocardia asteroides KC577152, Nocardia cummidelens KC577153, Nocardia asteroides KC577154, Nocardia asteroides KC577155, Nocardia coubleae KC577156 involvement in Iran soil in Isfahan and these isolates were eventually registered in NCBI gene bank.
Discussion and In this research, six Nocardia species were isolated and identified some of which isolated species were novel in Iran soil in Isfahan, at the time of isolation and detection. To identify more species of Nocardia in subsequent studies, proliferation and sequencing of other genes of the bacteria such as hsp65, ITS, secA, rpoB and sod can be applied.

Gram-negative bacteria are the most pathogenic bacteria for marine organisms including ornamental fish.
In the present study Vibrio species isolated from ornamental guppy fish in Kashan, Isfahan, Iran fish ponds and were detected according to molecular detection and genetic alignment. Liver, kidney, skin, brain and gill samples were taken from ornamental guppy fish in Kashan, Isfahan, Iran. Samples were cultured on enriched culture media and purification steps were performed based on microbiological methods. Primary identification was done using biochemical characterization of the isolated bacteria. Molecular detection was done based on amplification of 16SrDNA sequence of Vibrio cholera genome containing ITS (internal transcribed spacer); and sequence alignment of the amplified nucleotides.
The isolated bacteria detected as Vibrio spp., including Vibrio cholera (99% sequence similarity), Vibrio alginolyticus, Vibrio mimicus and Vibrio parahaemolyticus (up to 90% similarity in the genome sequence). The aquaculture ponds had alkaline water and the amount of five-day BOD was not in a safe range, which are favorable conditions for Vibrio species.
Discussion and Aquatic organisms in Iran can be carriers of human pathogens such as Vibrio species. The results obtained in the present study and similar investigations should be mentioned in aquaculture healthcare systems.

Companion animals, such as cat and dog, are potential sources of transmissible diseases to humans, especially children. They harbor zoonotic agents in gastrointestinal tracts as carriers which are capable of infecting their owners. Salmonella and Yersinia bacteria are considered as frequent causes of illness in children. This study was aimed at finding out the prevalence rate of infection in apparently healthy dogs and cats in Tehran, Iran.
A total of 100 rectal swabs from dogs and cats were analyzed by a multiplex PCR method with specific primers for detection of Yersinia and Salmonella species.
Fifteen samples (4 cats and 11 dogs) were positive for Yersinia and 20 samples (9 cats and 11 dogs) were positive for Salmonella. So the prevalence rate of Yersinia was 8% in cats and 22% in dogs and the prevalence rates of Salmonella were 18 and 22% in cats and dogs respectively.
Discussion and According to the results, Yersinia and Salmonella were detected in 8- 22% of pet animals without any clinical signs. The contaminated animal foods may be the main source of infection. These results may be useful in planning control and preventive programs.

Polyhydroxyalkanoates (PHAs) are natural polyesters and biodegradable plastics that are stored as intracellular inclusion bodies by a great variety of bacteria. The aim of this study was to extract polyhydroxyalkanoate from native Sinorhizobium meliloti in Iran.
Sinorhizobium meliloti isolates were collected from roots of alfalfa plants and were identified by Gram staining, biochemical experiments and amplification of 1500 bp fragment of 16Sr DNA gene. PHA granules were detected by microscopic examination. PHA production was evaluated in nutrient deficient medium and its amount was determined by conversion of PHA into crotonic acid by sulphuric acid treatment. The effect of various temperatures, agitation rate and carbon source (sucrose, mannitol, and maltose) were evaluated on dry cell weight and polyhydroxybutyrate (PHB) production.
The maximum amount of polymer production (43.10%) was seen in basal mineral medium at 29°C, pH~7 and 215 revolutions per minute (rpm). The results of this research showed that the S5 isolate was capable to produce maximum poly3- hydroxybutyrate. The produced polymer was analyzed for its purity by GC- mass (gas chromatography- mass spectroscopy) and confirmed to be PHB compared with the standard polymer.
Discussion and Native strains of Sinorhizobium can be used in the production of biodegradable plastics and the results of present study showed that S. meliloti S5 was capable to produce maximum PHB at 29°C, agitation rate of 215 rpm, and pH~7.

Some rhizobacteria by various mechanisms influence plant growth as they are called plant growth promoting rhizobacteria (PGPR). Scientists identified some PGPR characters involved in promoting plant growth, while all these characters are not able to study. The aim of this study was to evaluate PGP activities of bacterial isolates, (45 isolates belonged to rhizobium and 2 bacterial isolates belonged to Pseudomonas fluorescens), which were isolated from alfalfa (Medicago sativa) rhizosphere and root nodules grown around Zanjan.
These bacteria were isolated from alfalfa roots grown around Zinc industries in Zanjan province. After bacterial isolation and purification from root and soil samples, isolates were screened in vitro for plant growth promoting traits such as IAA (Indole Acetic Acid), ACC- deaminase (Amino Cyclopropan Carboxylate), HCN (Hydrogen Cyanide), siderophore, chitinase production and mineral and organic phosphate solubilization activities.
The results indicated that 43 bacterial isolates produced IAA (4.04- 4.95 μg/ml) and 15 isolates produced ACC- deaminase (0.23- 1.05 μg/ml). Only one isolate (Rm66) produced high amount of HCN. Qualitative siderophore production was observed in 9 isolates. None of the isolates produced chitinase. Solubilization of mineral phosphate was commonly detected in 19 isolates (4.33- 5.86 μg/ml), and 15 isolates solubilized organic phosphate (1.66- 144.28 μg/ml).
Discussion and This study shows that most of the bacterial strains which isolated from alfalfa cultivated lands had PGP activities and also a good potential to increase plant growth after inoculation with to seeds as eco- friendly fertilizers.

Production of an endogenous α-amylase from Bacillus amyloliquefaciens ATCC 23350 was studied and enhanced.
Protein and carbon sources were analyzed for free and immobilized bacterial cells and number of beads was considered for immobilized cells via one factor at a time methodforα-amylase production by Bacillus amyloliquefaciens. Subsequently, optimization condition was employed solely for immobilized bacterial cells by response surface methodology (RSM).
Peptone and rice starch showed to improve the α-amylase production in immobilized Bacillus cells. RSM generated a mathematical model explaining the optimum concentration of the efficient nutrients (139.35 g/l of rice starch and 80.00 g/l of peptone) leading to an optimum amylase production (205 U/ml).
Discussion and The statistical advance displayed significant outcomes to optimize the process parameters for maximal α-amylase production using Bacillus amyloliquefaciens and gave permission to rapid screening of variables. RSM led to find out an immense improvement in enzyme activity (more than 90%: from 25 to 225 U/ml) for the first time.

Phosphorus and potassium are major essential macronutrients for biological growth and development. Application of soil microorganisms is one approach to enhance crop growth. Some bacteria are efficient in releasing K and solubilizing P from mineral sources but their behavior was not studied more in presence together.
In this study the ability of seven bacterial strains, including Pseudomonas putida P13, P. putida Tabriz, P. fluorescens Tabriz, P. fluorescens Chao, Pantoea agglomerans P5, Azotobacter sp. and Bacillus megaterium JK3 to release mineral K from muscovite and biotite with application of insoluble (Ca3(PO4)2) or soluble (Na2HPO4) P-sources was investigated. Nutrient Broth was used to prepare an overnight culture of bacteria to inoculate in Aleksandrov medium, which was used to study the dissolution of silicate minerals. It should be mentioned that Aleksandrov medium was used to determine the amount of released P from tricalcium phosphate (TCP) while muscovite was added to the medium as a sole source of potassium. Concentration of P was determined spectrophotometrically by ammonium-vanadate-molybdate method and K was determined by flame photometry.
The insoluble P-source led to a significantly increased released K into assay medium (66%), and the net release of K from the biotite was significantly enhanced. Among bacterial strains, the highest mean of released K was observed with P. putida P13 which released more K (27%) than the control. The amounts of released K from micas in the presence of insoluble and soluble phosphate by P. putida P13 were 8.25 and 4.87 mg/g, respectively.
Discussion and Application of insoluble phosphate could increase K release from mica minerals. The enhanced releasing of mineral K might be attributed to the release of organic acids from the bacteria, a mechanism which plays a pivotal role in solubilizing phosphate from inorganic source of phosphate.

Probiotics are non- pathogen living microorganisms which express beneficial effects on host, when are administered in adequate amounts. Traditional dairy products are regarded as good sources of probiotics. Present study aims to isolate Lactic Acid Bacteria (LAB) from Siahmezgi cheese, a traditional cheese produced in the north of Iran, and to evaluate their probiotic potential for the first time.
LAB was isolated by culturing cheese samples on MRS agar. The isolates were screened for their probiotic potential using in-vitro tests, including tolerance to acid and bile, survival in simulated gastrointestinal tract conditions,
β- galactosidase and hemolytic activity as well as antibiotic susceptibility. In addition, antibacterial activity of the isolated strains against E. coli O157 and Salmonella enterica serovar typhimurium ATCC 14028 was determined.
One strain, labeled as Lb3 showed the highest tolerance to low pH, bile and simulated gastrointestinal tract conditions. This strain exhibited resistance to Streptomycin, Vancomycin and Polymixin B as well as effective antibacterial activity against two Gram negative pathogens, lacking hemolytic activity as well as high β- galactosidase activity. Finally, the strain Lb3 was identified as Lactobacillus plantarum CJLP55 using biochemical characterization and 16S rRNA sequencing assay.
Discussion and In the present work, a potentially probiotic Lactobacillus plantarum CJLP55 was isolated from traditionally produced Siahmezgi cheese. The bacterium displayed good probiotic properties and could be used in dairy industry.

Microbial biofilms have attracted interest in recent years because they have become the most important cause of nosocomial infections. This study was aimed to examine the antibacterial activities of Peganum harmala extracts on the development of microbial biofilms and planktonic form of six pathogenic bacteria which include Staphylococcus aureus, Bacillus cereus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae.
Antimicrobial activities of the crude extracts against the planktonic form of bacteria were evaluated by using disc diffusion method, minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) values were determined by a macrobroth dilution technique. Anti- biofilm effects of the extracts were assessed by microtiter plate method.
According to the results, P. harmala extracts could inhibit test bacteria in planktonic form. To inhibit biofilm formation, biofilm metabolic activity and eradication of established biofilms, efficiency of the extracts depended on concentration. The highest inhibitory effects of P. harmala extracts were observed on biofilm formation of S. aureus (90.28%) also, the greatest demolish were observed on S. pneumonia biofilm (77.76%). These extracts cause dramatically decrease the metabolic activity of bacteria in biofilm structures, in this case the decrement of B. cereus were highest (69.98%) compared to other tested bacteria.
Discussion and Therefore, it can be suggested that P.harmala extracts applied as antimicrobial agents against testing bacteria particularly in biofilm forms.