Open Access Original Research Article

Isolation and Characterization of Phosphate Solubilizing Bacteria from the Rhizosphere of Faba Bean (Vicia faba L.) in Meknes Region, Morocco

Abderrazak Rfaki, Laila Nassiri; Jamal Ibijbijen

Microbiology Research Journal International, Page 247-254
DOI: 10.9734/BMRJ/2015/14379

Aims: The objective of this research was to isolate phosphate solubilizing bacteria (PSB) from faba bean rhizosphere in Meknes region and evaluate in vitro their potential for P solubilization as well as their response to the effect of temperature, NaCl, pH and antibiotics.
Study Design: Rhizosphere soil and root nodules from faba bean crop in different sites from Meknes region were collected for the study.
Place and Duration of Study: Department of Biology (Soil & Environment Microbiology Unit) Faculty of Sciences, Moulay Ismail University, Meknes, Morocco; between February and August 2014.
Methodology: Four strains, which retained their P-solubilizing ability after repeated subculturing, from a total of seventy eight isolates were purified, characterized and identified by 16S rRNA gene sequencing.
Results: The isolates were closely related to Pseudomonas cedrina (PT19), Rahnella aquatilis HX2 (P24), Rhizobium nepotum (BSP30) and Rhizobium tibeticum (Rh8). P solubilization index of these isolates ranged from 1, 57-2, 88 in tricalcium phosphate amended National Botanical Research Institute's Phosphate (NBRIP) agar medium. Amount of phosphate solubilized ranged from 62.45 to 119.95 mg P L-1 and drop in pH of the medium ranged from 7 to 3.5. Besides, these strains exhibited resistance to several antibiotics.
Conclusion: The ability of the PSB to solubilize insoluble P and their adaptation to environmental conditions add value to these strains, which could lead to improved inoculants to increase the available phosphorus in the soil for faba bean plant growth and to promote sustainable agriculture.

Open Access Original Research Article

Response Surface Methodology for the Optimization of Proteases Production by a Novel Egyptian Isolate Bacillus amyloliquefaciens 35s

Fatma Refaat Nassar, Ahmed Abdelwahab Abdelhafez, Tarek Said El-Tayeb, Samah Hashem Abu-Hussein

Microbiology Research Journal International, Page 255-269
DOI: 10.9734/BMRJ/2015/15495

Aims: The present work aimed to optimize proteases production by Bacillus amyloliquefaciens 35s using the response surface methodology (RSM).
Study Design: Variables affecting proteases production were screened using a Plackett–Burman design. Face Centered Central Composite Design (FCCCD) of RSM was adopted for the augmentation of total proteases production assessed at three coded levels (–1, 0, +1). All obtained data were analyzed by ANOVA with post hoc multiple comparison analysis performed using Tukey’s HSD.
Place and Duration of Study: Department of Agricultural Microbiology, Faculty of Agriculture, Ain Shams University, between March 2014 and September 2014.
Methodology: Bacillus amyloliquefaciens 35s was used for proteases production. Modified TGY (Tryptone gluscose yeast extarct) medium was the basal medium. Impacts of nutritional factors (carbon and nitrogen and mineral salts) were studied using Plackett-Burman design with fold over augmenting method. “Design Expert®” Stat-Ease was used to analyze the experimental Plackett–Burman design. Temperature, pH and agitation rate (using shake flask) were optimized statistically by the factorial FCCCD of the RSM. Validation of statistical model of physical factors was done by carrying out the experiment at optimum conditions of the process parameters as determined from the model. Optimum conditions obtained through RSM in terms of FCCCD were examined and verified in a 5 L bench top continuous stirred tank bioreactor and production process was scaled-up in a batch process with controlled and non-controlled pH. Fermented medium was centrifuged to collect cells and determination of biomass and protease concentration.
Results: Among the significant media components, peptone and starch showed to have significant effects on the response as for protease production, with confidence level > 98% and were further optimized using FCCCD. Conditions promoted proteases production were different from those enhanced cell growth. Physical parameters indicated that production of proteases by Bacillus amyloliquefaciens is non-growth dependent. Maximum proteases production predicted (992.12 u/ml) was observed near the mid-point (0) values (concentrations) of both peptone (10 g/l) and starch (10 g/l) and the experimental value 935 u/ml was very close to the predicted value validating the model. The final proteases production in the bioreactor reached 1530 u/ml obtained within 12-14 h at 0.6 vvm aeration and 120 rpm of agitation speed.
Conclusion: Instead of conventional method of one variable at time approach, Response Surface Methodology, as statistical approach, showed to be adequate and efficient to optimize protease production by Bacillus amyloliquefaciens.

Open Access Original Research Article

Role of Chronic Viral Hepatitis B and C as Risk Factors for Celiac Disease

Marwa Salah Mostafa, Hisham Abdel-Sadek Ismail, Khaled Mohamed Hassanein

Microbiology Research Journal International, Page 270-276
DOI: 10.9734/BMRJ/2015/15794

Aims: Celiac disease (CD) is an autoimmune disease and hepatitis B virus (HBV) is suspected to trigger immunologic gluten intolerance in susceptible individuals. The aim of this study is to assess the role of chronic HBV and HCV infection in adult patients as risk factors of celiac disease.
Study Design: Case-control study.
Place and Duration of Study: Department of Medical Microbiology and Immunology and Department of Clinical Pathology, College of Medicine, Qassim University, between September 2012 and November 2014.
Methodology: This study included 30 chronic HBV patients and 30 chronic HCV patients, thirty normal individuals were included as controls. Anti-tissue transglutaminase (Anti-tTG) IgA was assessed in the three groups, non-organ specific autoantibodies (NOSA) including antimitochondrial (AMA), antinuclear (ANA) and anti-smooth muscle (ASMA) antibodies were also assessed.
Results: Anti-tTG IgA revealed significantly higher levels in both HBV (mean = 41.72, SE = 1.70) and HCV (mean = 46.93, SE = 5.46) groups compared to the controls (mean = 29.60, SE = 1.27), (P = .01, < .001 respectively). The positivity of ASMA was significantly higher in HBV patients than in the controls (46.7%, 16.7%, P = .01). ANA and AMA showed insignificant difference between the three groups.
Conclusion: The higher levels of anti-tTG autoantibodies in adult patients with chronic HBV and HCV infections reflect higher incidence to develop CD than normal population. Screening for CD in such patients is recommended.

Open Access Original Research Article

Determination of Proteolytic Activities of Bacillus species Isolated From Traditional Fermented Oil Bean Seed (UGBA: Pentaclethra macrophylla, Benth)

V. Orji, C. N. Obi, J. N. Ogbulie

Microbiology Research Journal International, Page 277-285
DOI: 10.9734/BMRJ/2015/15950

Eighty samples of traditional fermented “ugba” (Pentaclethra macrophylla, Benth) wraps collected from eight markets in Umuahia were analyzed for proteolytic activities (by zone of inhibition in mm) of Bacillus species using two protein-based substrates: Skim Milk Agar and Nutrient Agar supplemented with 1% gelatin. 51 isolates were identified as B. subtilis, 18 as B. licheniformis and 11 as B. pumilus with Ariam Market having the highest mean count (mm) of 1.93±0.16. B. subtilis had the highest proteolytic activity (28.00±0.55) on Skim Milk Agar (P=.05) but together with B. licheniformis had the same level of proteolytic activity (26.00±0.00 and 26.00±0.55 respectively at P=.05) which is higher than B. pumilis (13.00±0.55) on Nutrient agar supplemented with 1% gelatin. However, B. licheniformis and B. pumulis had better proteolytic activities (26.00±0.55 and 13.00±0.55: P=.05) respectively on Nutrient agar supplemented with 1% gelatin than on Skim Milk Agar. The two potential protease producers that showed higher proteolytic activity were incubated at high temperature and result showed that B. subtilis proteolytic activity was optimum at (29.00±0.71) at 50°C while that of B. licheniformis was optimum (28.00±0.00) at 40°C (P=.05). Antimicrobial susceptibility of the Bacillus species showed that the three Bacillus species were most sensitive to Chloramphenicol (21.00±0.51) without any statistical difference followed by Gentamycin (20.00±0.31) but completely resistant to Rifampicin (0.00±0.0; P=.05). B. subtilis showed better proteolytic activity at a higher temperature and will be better suited for production processes in industries where thermophilic temperatures are needed.

Open Access Original Research Article

Purification, Characterization and Applications of Proteases Produced by Bacillus amyloliquefaciens 35s Isolated from Soil of the Nile Delta of Egypt

Fatma Refaat Nassar, Ahmed Abdelwahab Abdelhafez, Tarek Said El-Tayeb, Samah Hashem Abu-Hussein

Microbiology Research Journal International, Page 286-302
DOI: 10.9734/BMRJ/2015/15504

Aims: The current study aimed at purifying and determining general characteristics of proteases produced by Bacillus amyloliquefaciens 35s isolated from the soil of Nile Delta of Egypt and to study its applications in environmental and industrial purposes.
Study Design: The produced proteases were purified by ammonium sulfate precipitation method followed by dialysis. The effects of some physical and chemical factors on the activity and stability of the partially purified proteases were determined. The produced proteases were tested in various environmental and industrial applications.
Place and Duration of Study: Department of Agricultural Microbiology, Faculty of Agriculture, Ain Shams University, between July 2014 and September 2014.
Methodology: Proteases produced in optimized medium and conditions were extracted by centrifugation, purified by ammonium sulfate precipitation and followed by dialysis. The effects of pH, temperature, metal ions, inhibitors, organic solvents, H2O2 and surfactants on enzyme’s activity and stability were determined. Protease’s ability to hydrolyze gelatin was employed to recover silver from used X-ray films, and used a detergent additive for stain removal from cloths. Both proteases and the producing isolate were used to digest chicken feathers to produce amino acids, peptides and soluble proteins. All generated data were analyzed using one-way ANOVA with post hoc multiple comparison analysis using Tukey’s HSD.
Results: Ammonium sulfate at 70% had the best effect on precipitating the produced protease increasing specific activity to 5143 (u/mg), a 2.6 fold of that of the crude enzyme (1408.5 u/ml). Characterization experiments showed optimum conditions for activity to be pH 9 and 60ºC. Ca2+, Mn2+, Pb2+, Mg2+, Co2+ , Zn2+ and Ba2+ negatively affected the enzyme activity at 5mM. The enzyme was stable at low concentrations of oxidizing agent and surfactants and retained nearly 100% and 65% activity in presence of 0.1% and 0.5% of SDS, respectively. Purified proteases was 23 fold active than the commercial Savinase® under the standard assay conditions. As a detergent additive, it was effective in removing blood and egg stains from cotton fabric when combined with detergent. In digesting chicken feather, the isolate showed maximum caseinase activity of 645 u/ml in the initial screening process and an ability to degrade raw feather to free protein hydrolysates under optimal conditions (after 96 h of fermentation in 30 g/l chicken feather and pH 8.0).
Conclusion: Optimum pH indicated alkalinity class and optimum indicated thermal classification. Purified proteases was 23 fold more active than the commercial Savinase® under the standard assay conditions. Applications of the partially purified proteases proved it to be a good profitable industrial tool.

Open Access Original Research Article

Anaerobic Microbial Activities of a Nigerian Deep Offshore Oil Production Facility that Uses High Sulfate Sea Water for Injection

Okoro Chuma Conlette

Microbiology Research Journal International, Page 303-314
DOI: 10.9734/BMRJ/2015/13588

Aim: To evaluate the Anaerobic microbiological activities and souring potential of a Nigerian deep water offshore oil production facility with special emphasis on functional group activities.
Methodology: CSB-K medium was used for the anaerobic microbial activity assay such as the ability to reduce sulfate by sulfate reducing bacteria (SRB), oxidize sulfide and reduce nitrate by the sulfide oxidizing, nitrate reducing bacteria (soNRB) and reduce nitrate by the heterotrophic nitrate reducing bacteria (hNRB) while API-RP-38 and ZPRA-5 broth media were used to quantify the presence of SRBs and acid producing bacteria respectively.
Results: Results on the activities of anaerobic microorganisms indicated that sample 3N1(treated produced water) had the highest concentration of SRBs and Acid producing bacteria (105 and 107 cells/ml respectively), considerable concentrations of heterotrophic nitrate reducing bacteria and sulphide oxidizing nitrate reducing bacteria were also present in the same sample. The partially treated produced water samples (3N2 and 3N4) showed relatively lower concentrations of SRBs and acid producing bacteria. Expectedly very low microbial activity was recorded in the biocide treated injection water (3N3). Comparatively, the highest microbial activity on sulfide oxidation, nitrate reduction, sulfate reduction and production of hydrogen sulfide was recorded in sample 3N1 while the lowest microbial activity was recorded in sample 3N3.
Conclusion: Anaerobic microbiological activities of the oil field under investigation indicate that the field have high potential for souring and corrosion due to the availability of sulfate, organic nutrients and SRBs but the operators of the field have taken measures to control souring and bio-corrosion which include periodic biocide treatment and nitrate injection.